WO2010112034A2

WO2010112034A2 - Compositions and methods for treatment and diagnosis of synucleinopathies

Info

Publication number: WO2010112034A2
Application number: PCT/DK2010/050077
Authority: WO
Inventors: Poul Henning Jensen; Christine Lund Kragh
Original assignee: Aarhus Universitet
Priority date: 2009-04-02
Filing date: 2010-04-06
Publication date: 2010-10-07
Also published as: WO2010112034A3

Abstract

A method of treating a neurodegenerative disorder is provided, wherein compound, which inhibits or down-regulates the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji, Ccl2, Ccl7, Clcfl, Csfl, CxcH, Cxcl2, CxcH 0, IL6, Bhlhb2, Camkk2, Duspi, Lphn2, RiI, Hmoxi, Hspal a, Myd1 16, Srxni, Egr1, Fos, FosL, Hes1, KIfI O, Myc, Kpnai, SId 5a4, Slc35b2, Stx1 1, and Nudt18. Pharmaceutical compositions for use in such methods of treatment are also provided, as well as diagnostic kits comprising a detection member for detecting the expression of the genes involved in neurodegenerative disorders. Additionally, a method for identifying novel compounds for treatment of a neurodegenerative disorder is provided, which is based on detection of expression levels of the causative genes.

Description

Compositions and methods for treatment and diagnosis of synucleinopathies.

Field of invention

The present invention relates to methods and composition for use in treatment and diagnosis of neurodegenerative disorders in particular synucleinopathies, for example Parkinson's and Alzheimer's diseases or Lewy body dementia. Also provided is a method for screening for novel compounds for treatment of said clinical conditions.

Background of invention

The α-synucleinopathies comprise the neurodegenerative disorders, Parkinson's disease (PD), Dementia with Lewy bodies (DLB), multiple system atrophy (MSA) as well as neurodegeneration with brain iron accumulation type I and Lewy body variant of Alzheimer's disease. The common feature of these disorders is the presence of intracellular inclusions containing aggregated α-synuclein. The inclusions are deposited in populations of neurons and glia, which varies among the disorders.

PD is the most common movement disorder in the elderly and is clinically characterized by tremor, rigidity, and bradykinesia. It is a progressive disorder that affects various neuronal populations in the human nervous system, particularly dopaminergic neurons of the substantia nigra pars compacta. Affected neurons develop inclusions termed Lewy bodies (LBs) in their perikarya and Lewy neurites (LNs) in their processes. LBs are proteinaceous cytoplasmic inclusions, which in brainstem are characterized by a dense eosinophilic core and a clear surrounding halo. The major component of LBs is aggregated α-synuclein, though more than 70 different molecule constituents have been identified in these inclusions. Moreover, the majority of LBs are stained for ubiquitin by immunohistochemistry.

The presence of proteins in LBs suggests that protein aggregation and accumulation play a prominent role in the pathogenesis of both familial and sporadic PD. Genetic screening of families with PD has demonstrated that mutations in specific genes are responsible for a small number of disease cases whereas the vast majority of cases are sporadic. A combination of environmental factors or toxins, genetic susceptibility, and the aging process may account for many sporadic cases. The first gene to be associated with disease was α-synuclein (SNCA) in which three pathogenic mutations leading to amino acid substituitions (A30P, E46K, and A53T) have been linked to autosomal dominant PD and DLB. Additionally, genomic multiplications of the SNCA gene have been found in patients with early-onset PD and DLB. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene also cause autosomal dominant PD and can be associated to α-synuclein pathology. Additional genes and genetic loci have been implicated in recessive forms of PD including parkin, PINK1 , and DJ-1. The recessive genes may trigger PD in an alternative fashion as their relation to α-synuclein cytopathology is uncertain. Shortly after the α-synuclein gene was linked to PD and DLB, a series of studies demonstrated that α-synuclein is the major constituent of the filaments that form the amyloid inclusions characteristic of the α-synucleinopathies.

α-synuclein is a 140-amino acid protein and belongs to a family of closely related members, which include α-synuclein, β-synuclein, and γ-synuclein. Among the members of the synuclein family, α- and β-synuclein share the highest degree of sequence identity. They are 62% identical in their amino acid sequence with greatest homology in the N-terminal region. Both α- and β-synuclein are expressed within the central nervous system (CNS) and are mainly located in presynaptic terminals, v- synuclein mainly localizes to the cell bodies and axons of primary sensory neurons, sympathetic neurons, and motor neurons of the peripheral nervous system. Of the three synucleins, only α-synuclein is believed to be involved in disease pathology. The α-synuclein protein can be divided into two functional regions: i) The N-terminal region (residues 1-95), which contains seven 11 -amino acid repeats with a highly conserved hexameric motif (KTKEGV) and the aggregation-prone non-amyloid component (NAC) sequence (residues 60-95). The sites of the three disease-causing mutations are located within the N-terminal region, ii) The unstructured C-terminal region (96-140), which is rich in acidic residues. The C-terminus can bind metal ions and proteins able to affect the aggregation process (for reviews, see {49;50)).

α-synuclein is expressed in a number of neuronal and non-neuronal cell types including dopaminergic neurons, cortical neurons, noradrenergic neurons, endothelial cells, platelets. It is especially abundant in neural tissue and is enriched in presynaptic terminals. During development, α-synuclein is initially expressed in the cell body, but after synthesis it is axonally transported to the synapse. The redistribution is believed to occur through the association of α-synuclein with vesicles targeted to synapses. During the pathological development of α-synucleinopathies, a part of the α-synuclein protein is found in the cell bodies and neurites of degenerating neurons.

α-synuclein is an abundant protein in brain, but its precise function still remains unknown. However, several putative functions have been ascribed to α-synuclein including lipid binding, regulation of certain enzymes, transporters, and neurotransmitter vesicles, as well as roles in neuronal survival.

The disease mechanisms behind α-synucleinopathies are unknown. However, several lines of evidence suggest that misfolding and aggregation of α-synuclein play a key role. Aggregation of proteins is observed in a range of other neurodegenerative diseases including Alzheimer's disease (AD) and other tauopathies such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Huntington's disease (HD) and prion disease are also characterized by protein aggregation. The discoveries of missense mutations and gene multiplications in α- synuclein that are linked to early-onset PD and the finding that α-synuclein is the prime component of inclusions clearly implicates α-synuclein in pathogenesis. Mechanisms by which abnormal accumulation of α-synuclein disrupt cellular functions have been investigated intensively in several different model systems. In a yeast model, α- synuclein accumulation leads to a blockade of vesicular transport from endoplasmatic reticulum (ER) to the Golgi apparatus. Moreover, activation of an ER stress response, also called the unfolded protein response (UPR), has been demonstrated in SHSY5Y cells overexpressing α-synuclein and in post-mortem brain tissue from PD patients. Mitochondrial dysfunction may also be a consequence of α-synuclein accumulation since transgenic mice expressing human A53T α-synuclein develop mitochondrial pathology. Several lines of evidence implicate a dysfunction of the ubiquitin- proteasome system in the pathogenesis of α-synucleinopathies. Studies show that LBs contain ubiquitinated proteins and that the proteasomal activity is decreased in the substantia nigra of PD patients.

Initially, α-synuclein was described as a neuronal protein localized to the nucleus and presynaptic terminals. However, subsequent studies have focused mainly on α- synuclein within nerve terminals. Interestingly, intranuclear inclusions containing α- synuclein are found in MSA patients. Thus, nuclear localization of α-synuclein may play an important role in the neurotoxicity in α-synucleinopathies. α-synuclein aggregates spontaneously into β-folded amyloid structures in vitro. The transformation from a monomeric state to an amyloid state proceeds through soluble oligomeric intermediates. Incubation of purified recombinant α-synuclein above a certain threshold concentration leads to the formation of amyloid fibrils. The α-synuclein fibrils formed in vitro resemble those extracted from disease-affected brain, thus permitting the use of in vitro formed fibrils in modelling aspects of α-synuclein pathology. The kinetic of α-synuclein aggregation is consistent with a nucleation-dependent mechanism, which is known for other aggregation prone proteins such as the Aβ peptide. A nucleation-dependent process is characterized by 1 ) a slow nucleation phase (lag phase) in which the protein undergoes a series of association steps to form ordered oligomeric nuclei, 2) a growth phase (elongation phase) where the nuclei rapidly grow to form large polymers, and 3) a steady state phase where insoluble filamentous aggregates are at equilibrium with the monomers. The lag phase is dramatically reduced by the addition of pre-aggregated α-synuclein or pro-aggregatory factors.

The PD-associated mutations have been extensively investigated both in vitro and in vivo, providing important insights into the mechanisms underlying α-synucleinopathies. All three mutations have been shown to accelerate α-synuclein aggregation in vitro. The monomeric forms of wild-type, A30P, and A53T α-synuclein proteins possess identical structural properties and conformational behaviour. The A53T mutation clearly accelerates aggregate formation relative to both wild-type and the A30P mutation, whereas the A30P variant forms oligomers more rapidly than wild-type α-synuclein. The A30P mutation reduces the binding of α-synuclein to vesicles.

α-synuclein contains several phosphorylation sites e.g. Ser87, Ser129, and Tyr125. Though predominantly found in a non-phosphorylated state in vivo, α-synuclein is phosphorylated at Ser129 in inclusions in all α-synucleinopathies. The role of S129 phosphorylation in promoting α-syn aggregation is currently unclear. It also also not clear, which cellular kinases and phosphatases control the level of Ser129 phosphorylation of α-synuclein. However, several kinases have been proposed, including casein kinase 1 (CK1 ), CK2, and members of the G-protein coupled receptor kinase (GRK) family, particularly GRK2, and GRK5. Most recently, polo-like kinase 2 (PLK2) has been demonstrated to phosphorylate α-synuclein in cell culture and by in vitro reactions with the purified kinase. In addition, inhibitor and knockout studies in mouse brain support a role for PLK2 as an α-synuclein kinase in vivo. Identification of the kinase(s) responsible for phosphorylation of α-synuclein at Ser129 may help to clarify the role of phosphorylation in α-synucleinopathies.

α-synuclein is known to interact with a large variety of proteins (reviewed in Dev, K. K., Hofele, K., Barbieri, S., Buchman, V. L., and van der Putten, H. (2003) Part II: alpha- synuclein and its molecular pathophysiological role in neurodegenerative disease, Neuropharmacology 45, 14-44) perhaps owing to its flexible and dynamic structure.

Some of these proteins have been shown to stimulate the aggregation process in vitro at substociometric concentrations as well as to colocalize with α-synuclein in inclusions in α-synucleinopathies. The identification of proteins stimulating aggregation of α- synuclein in vitro suggests that dysregulation of the expression of such proteins can trigger α-synuclein aggregation in vivo.

The mode of cell death in neurodegenerative disorders remains a matter of controversy but both apoptotic and non-apoptotic modes of cell death are likely to participate. In PD, the level of active caspase-3 is increased in the substantia nigra. Moreover, other markers of apoptosis such as Bax and p53 have been associated with degenerating neurons in PD patients and in MPTP-treated mice. Overexpression of wild-type α-synuclein induces apoptosis in neurons and glia and classic markers such as chromatin condensation, nuclear fragmentation, and cytochrome c release have been observed in yeast expressing wild-type and mutant forms of α-synuclein (266). Finally, overexpression of mutant α-synuclein has been demonstrated to increase the sensitivity of cells to toxin-induced apoptosis.

Inflammation is also thought to contribute to PD pathogenesis, in part through upregulation of inflammatory cytokines such as TNFα (Hirsch, E. C, Hunot, S., Damier, P., and Faucheux, B. (1998) Glial cells and inflammation in Parkinson's disease: a role in neurodegeneration?, Ann. Neurol. 44, S115-S120; and Nagatsu, T., Mogi, M., lchinose, H., and Togari, A. (2000) Changes in cytokines and neurotrophins in Parkinson's disease, J. Neural Transm. Suppl 277-290). Studies of post-mortem brain tissue have suggested that Fas (Ferrer, I., Blanco, R., Cutillas, B., and Ambrosio, S. (2000) Fas and Fas-L expression in Huntington's disease and Parkinson's disease, Neuropathol. Appl. Neurobiol. 26, 424-433; and Mogi, M., Harada, M., Kondo, T., Mizuno, Y., Narabayashi, H., Riederer, P., and Nagatsu, T. (1996) The soluble form of Fas molecule is elevated in parkinsonian brain tissues, Neurosci. Lett. 220, 195-198), FADD (Hartmann, A., Mouatt-Prigent, A., Faucheux, B. A., Agid, Y., and Hirsch, E. C. (2002) FADD: A link between TNF family receptors and caspases in Parkinson's disease, Neurology 58, 308-310), and caspase-8 (Hartmann, A., Troadec, J. D., Hunot, S., Kikly, K., Faucheux, B. A., Mouatt-Prigent, A., Ruberg, M., Agid, Y., and Hirsch, E. C. (2001 ) Caspase-8 is an effector in apoptotic death of dopaminergic neurons in Parkinson's disease, but pathway inhibition results in neuronal necrosis, J. Neurosci. 21 , 2247-2255) may contribute to neuronal loss. An upregulation of apoptotic proteins such as Bax, FasL, and TNFα has been demonstrated in oligodendrocytes in MSA, whereas neuronal apoptosis was not observed (Probst-Cousin, S., Rickert, C. H., Schmid, K. W., and Gullotta, F. (1998) Cell death mechanisms in multiple system atrophy, J. Neuropathol. Exp. Neurol. 57, 814-821 ). Fas is involved in motorneuron death in a transgenic mouse model of ALS (Locatelli, F., Corti, S., Papadimitriou, D., Fortunato, F., Del, B. R., Donadoni, C, Nizzardo, M., Nardini, M., Salani, S., Ghezzi, S., Strazzer, S., Bresolin, N., and Comi, G. P. (2007) Fas small interfering RNA reduces motoneuron death in amyotrophic lateral sclerosis mice, Ann. Neurol. 62, 81- 92) and enhanced levels of TNFα, TNFR1 , and TNFR2 have been detected in plasma from ALS patients (Cereda, C, Baiocchi, C, Bongioanni, P., Cova, E., Guareschi, S., Metelli, M. R., Rossi, B., Sbalsi, I., Cuccia, M. C, and Ceroni, M. (2008) TNF and sTNFRI/2 plasma levels in ALS patients, J. Neuroimmunol. 194, 123-131 ). In addition, a recent report claimed that perispinal injections of TNFα antagonists relieved symptoms in AD patients (Frid, P., Anisimov, S. V., and Popovic, N. (2007) Congo red and protein aggregation in neurodegenerative diseases. Brain Research Reviews 53, 135-160).

Summary of invention

The present invention provides methods, compositions and kits for treatment and diagnosis of a neurodegenerative disorder in particular synucleinopathies, such as

Parkinson's or Alzheimer's diseases. The invention relates to a number of interrelated aspects. The aspects are herein below divided into a first and a second set of aspects. First set of aspects:

In a broad aspect, the present invention relates to a method of treating, preventing or ameliorating a neurodegenerative disorder comprising administering to a subject in need thereof a compound, wherein said compound i) inhibits or down-regulates the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudt18, and/or ii) inhibits or down-regulates the activity of a gene product of a gene according to i) or part thereof. In a more specific embodiment, the gene is any one of Gadd45a, Gadd45b, Gadd45g, or Nfkbia.

The genes are for example identified by a sequence or comprise a sequence selected from the group consisting of SEQ ID NO: 29-59, or part thereof and any sequence which is at least 90% identical to any of SEQ ID NO: 29-59, or part thereof. In a specific embodiment, the gene comprises a sequence identifed by SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or part thereof, or any sequence which is at least 70%, at least 80%, such as at least 90% identical to said sequences or part thereof.

The subject is preferably a human being, and the neurodegenerative disorder is preferably a synucleinopathy, such as a neurodegenerative disorder selected from the group consisting of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and multiple system atrophy, preferably Parkinson's disease or alzheimer's disease.

The method is directed to inhibit or reduce the expression and/or the activity of a gene or gene product, in order to neutralize a deleterious overexpression of a gene of the invention in a neurodegenerative disorder. The relevant tissues, wherein the genes are overexpressed are typically brain tissue, cerebral cortex tissue, liver tissue, skeletal muscle tissue, and intestinal tissue. Thus, by the method of the invention, the expression of the gene and/or the activity of said gene product is reduced in a specific tissue of said human being, said tissue is for example brain tissue, cerebral cortex tissue, liver tissue, skeletal muscle tissue, and intestinal tissue, such as specifically cerebral cortex tissue. By employing the provided method, the expression of the gene and/or the activity of the gene product, for example in a human being, is reduced by any amount ranging from a subtle reduction to less than 99% or less than 95% of the previous expression and/or activity to almost complete abolishment of gene expression and/or activity. In general, the expression and/or said activity is reduced to less than 90%, such as less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, such as less than 30%, such as less than 20%, such as less than 15%, such as less than 10%, such as less than 5%, for example 0% of said expression and/or said actitivty in said human being before administration.

The compound for inhibiting gene expression and/or inhibiting or down-regulating the activity of a gene product of according to the provided method is selected from any compound suitable for specific genetic inhibition or knockdown. The compound is for example selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affi body-antibody chimeras, and non-immunoglobulins. In a preferred embodiment, the compound is an siRNA. The siRNA is then designed against a target sequence of any gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx11 , and Nudti 8. The siRNA for example comprise any sequence selected from any sequence selected from the group consisting of SEQ ID NO: 29-59, or part thereof, or any sequence which is at least 90% identical to any of SEQ ID NO: 29-59, or part thereof. In a specific embodiment, the siRNA comprises or targets a sequence selected from SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or part thereof or the complement thereof, or any sequence which is at least 70%, at least 80%, such as at least 90% identical to said sequences or part thereof or the complement thereof. In a more specific embodiment, the siRNA is designed against a target sequence identified by any of SEQ ID NO: 21-28, SEQ ID NO: 60-63 or part thereof, and/or the siRNA comprises or consists of a sequence identified by any of SEQ ID NO: 21-28, SEQ ID NO: 60-63, or part thereof.

In a preferred embodiment of the provided method the inhibited gene or inhibited or downregulated gene product is the Gadd45a gene or gene product, and the compound used for inhibition and/or downregulation is an siRNA, which comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 21 , 22, 23, and 24, or part thereof. In another preferred embodiment of the provided method the inhibited gene or inhibited or downregulated gene product is the Gadd45g gene or gene product, and the compound used for inhibition and/or downregulation is an siRNA, which comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 and 28, or part thereof.

In a further preferred embodiment of the provided method the inhibited gene or inhibited or downregulated gene product is the Nfkbia gene or gene product, and the compound used for inhibition and/or downregulation is an siRNA, which comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 60, 61 , 62, and 63, or part thereof.

The treatment method of the present invention may also be combined with any other conventional treatment or treatment regime against a neurodegenerative disorder, and thus, the method in one embodiment further comprises administering at least one additional synucleinopathy therapeutic, such as at least one additional Parkinson's or Alzheimer's disease therapeutic.

In another aspect, the invention relates to a specific compound for use in the provided method of treating, preventing and/or ameliorating a neurodegenrative disorder. Thus, in another aspect, the invention relates to a compound capable of i) inhibiting or down-regulating the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudti 8, or part thereof and/or ii) inhibiting or down-regulating the activity of a transcriptional and/or translational product of a gene according to i) or part thereof. The compound should be capable of interacting with a gene or gene product of the invention directly or indirectly in order to modulate the expression and/or activity thereof. The compound is therefore in one embodiment capable of selectively binding said gene, and/or a transcriptional and/or translational product of said gene.

Many compounds are known, which may be used for downregulation or inhibition of a specific gene or gene product. Accordingly, the compound is for example selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non- immunoglobulins. In a specific embodiment, the compound is an antibody, antigen binding fragment or recombinant protein thereof, for example an antibody, antigen binding fragment or recombinant protein thereof, which selectively binds Gadd45a, Gadd45g and/or Nfkbia.

In a preferred embodiment, the compound is an siRNA, in particular an siRNA comprising any sequence selected from the group consisting of SEQ ID NO: 29-59, or part thereof, or any sequence which is at least 90% identical to any of SEQ ID NO: 29- 59, or part thereof. In a specific embodiment, the claimed siRNA compound comprises or targets a sequence selected from SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or part thereof or the complement thereof, or any sequence which is at least 70%, at least 80%, such as at least 90% identical to said sequences or part thereof or the complement thereof. In a more specific embodiment, the claimed siRNA compound is designed against a target sequence identified by any of SEQ ID NO: 21-28, SEQ ID NO: 60-63, or part thereof, and/or the siRNA compound comprises or consists of a sequence identified by any of SEQ ID NO: 21-28, SEQ ID NO: 60-63, or part thereof. In a preferred embodiment, the compound is an siRNA, which comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 21 , 22, 23, and 24, or part thereof. In another preferred embodiment, the compound is an siRNA, which comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 and 28, or part thereof. In a further preferred embodiment, the compound is an siRNA, which comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 60, 61 , 62, and 63, or part thereof.

In one embodiment, the compound is an siRNA, which consists of or comprise 17-25, such as 19-22, consecutive nucleotides selected from a region of a sequence selected from SEQ ID NO: 1-20 or the complement thereof. In a more specific embodiment, the compound is an siRNA, which siRNA comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 21-28, SEQ ID NO: 60-63, or part thereof.

In a further aspect, the invention relates to a pharmaceutical composition comprising at least one compound provided herein. The pharmaceutical composition typically comprises at least one additional component, such as an adjuvant, an excipient and/or a carrier. The carrier is for example selected from the group consisting of keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.

The invention also in one aspect provides a use of a compound as provided herein, and/or a composition as provided herein for the manufacture of a medicament for treatment, amelioration and/or prevention of a neurodegenerative disorder.

In yet another aspect, the invention relates to a compound as provided herein, and/or a composition as provided herein for treatment, amelioration and/or prevention of a neurodegenerative disorder. In a related aspect, the invention relates to a pharmaceutical composition for treatment, amelioration and/or prevention of a neurodegenerative disorder, said composition comprising a compound as provided herein and/or a composition as provided herein.

The neurodegenerative disorder is preferably a synucleinopathy, such as a neurodegenerative disorder selected from the group consisting of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and multiple system atrophy, preferably

Parkinson's disease or Alzheimer's disease.

Another aspect of the invention relates to a diagnostic method, in particular a method for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said method comprising providing a biological sample isolated from said subject and detecting in said biological sample i) at least one polymorphism or mutation of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudt18, and/or ii) at least one transcriptional and/or translational product of said gene.

The isolated sample is selected from any biologicallly derived sample, which is suitable for determining gene expression and/or gene product activity, for example, the biological sample is a blood sample, a tissue sample, a secretion sample, semen, ovum, hairs, nails, tears, and urine.

Specifically, in the provided diagnostic method, the level of gene product is increased in a sample isolated from a subject suffering from or being predisposed for said neurodegenerative disorder relative to a subject not suffering from said neurodegenerative disorder. Any increase in gene product and/or gene product activity is indicative of a neurodegenerative disorder and/or the predisposition therefore, but preferably, the level should be increased by at least 25%, such as 50%, such as at least 100%, for example at least 200%, such as at least 300% relative to the median for healty people. Polymorphisms or other mutations of a gene of the invention may also affect, the level of gene expression and/or affect the acticvity of a gene product thereof, and therefore, the provided diagnostic method also comprise detecting a polymorphis and/or a mutation in any of the genes or their regulatory sequences, in those situation where the at least one polymorphism leads to increased expression of said gene in a subject relative to a subject not carrying said polymorphism.

The tested subject is in a preferably a human being, and the neurodegenerative disorder is preferably a synucleinopathy, such as a neurodegenerative disorder selected from the group consisting of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and multiple system atrophy, preferably Parkinson's disease or alzheimer's disease.

In a specifically preferred embodiment of the provided diagnostic method, the gene is Gadd45a, Gadd45g or Nfkbia.

An important aspect of the present invention relates to a diagnostic kit for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said kit comprising at least one detection member for detecting in a biological sample isolated from said subject i) at least one polymorphism of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clcfi , Csf1 , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala,

Myd1 16, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudt18, and/or ii) at least one transcriptional and/or translational product of said gene.

46. The kit according to claim 45, said kit further comprising at least one reference sample comprising i) At least one nucleic acid sequence comprising at least one gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji ,

Ccl2, Ccl7, Clcfi , Csf1 , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI,

Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudti 8 or part thereof, and/or ii) at least one transcriptional and/or translational product of said gene or part thereof.

The detection member of the diagnostic kit is in principle the same as the detection members described herein in relation to the diagnostic method. Thus, the detection member is for example selected from the group consisting of oligonucleotide primers, oligonucleotide probes, nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins.

In a specific embodiment of the kit, the detection member is an antibody, antigen binding fragment or recombinant protein thereof, in particular, such as preferably an antibody, antigen binding fragment or recombinant protein thereof, which selectively binds Gadd45a, Gadd45g and/or Nfkbia. In another specific embodiment of the kit, the detection member is an oligonucleotide primer and/or an oligonucleotide probe. The oligonucleotide oligonucleotide primer or probe for example consists of or comprises at least 5 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 1-20, SEQ ID NO: 29-63 and the complement thereof. In another embodiment, the detection member is an oligonucleotide primer or probe with a sequence selected from the group consisting of SEQ ID NO: 1-20, or any sequence at least 90% identical thereto. For validating the test result provided by the use of the kit, the diagnostic kit in one embodiment further comprises at least one reference sample. The reference sample is designed as a positive or a negative control with respect to presence of a gene product or polymorphism or mutation of a gene of the present invention. The control sample for example comprises i) at least one nucleic acid sequence comprising at least one gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, or part thereof, and/or ii) at least one transcriptional and/or translational product of said gene or part thereof.

In a more specific embodiment, the kit comprises i) at least one oligonucleotide primer or probe consisting of or comprising at least 5 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 1-2, SEQ ID NO: 32 and the complement thereof, and ii) at least one reference sample comprising at least one nucleic acid sequence comprising the Gadd45a gene or part thereof.

In another specific embodiment, the kit comprises i) at least one oligonucleotide primer or probe consisting of or comprising at 5 least 5 consecutive nucleotides of a sequence selected from the group consisting of

SEQ ID NO: 5-6, SEQ ID NO: 34 and the complement thereof, and ii) at least one reference sample comprising at least one nucleic acid sequence comprising the Gadd45g gene or part thereof.

In another example, the comprises O i) at least one oligonucleotide primer or probe consisting of or comprising at least 5 consecutive nucleotides of a sequence selected from the group consisting of

SEQ ID NO: 13-14, SEQ ID NO: 30 and the complement thereof, and ii) at least one reference sample comprising at least one nucleic acid sequence comprising the Nfkbia gene or part thereof. 5 In addition, the provided kit may be supplied with reagents and buffers for detection, and/or instructions for performing the detection method and interpretation of the result.

The invention also relates to the specific use of the diagnostic kit provided herein for the diagnosis of a neurodegerative disorder 0 The neurodegerative disorder is preferably diagnosed in a human being, and the neurodegenerative disorder is preferably a synucleinopathy, such as a neurodegenerative disorder selected from the group consisting of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and multiple system atrophy, preferably Parkinson's disease or Alzheimer's disease. Moreover, in the use of the provided kit, a5 neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject is preferably diagnosed according to any method provided herein for determining a neurodegenerative disorder.

The invention also relates to a screening method for identifying new compounds for the0 treatment of a neurodegenerative disorder, and the invention also relates to a method of treating a neurodegenerative disorder comprising administering a compound identified by a screening method of the invention.

Thus, in one aspect, the present invention relates to a method of identifying a drug for treating, preventing or ameliorating a neurodegenerative disorder, said method5 comprising the steps of i) providing a biological sample, ii) determining in said biological sample the expression of a gene or the activity of a gene product, wherein said gene is selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a,

Myd1 16, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudt18, iii) providing a drug to said biological sample and determining the expression of said gene or the activity of a product of said gene, and iv) comparing the expression of said gene and/or the activity of said gene product in said biological sample in the presence and absence of said drug, wherein in the presence of said drug in said biological sample said expression of said gene and/or said activity of said gene product is inhibited or down-regulated relative to the expression of said gene and/or activity of said gene product in said sample in the absence of said drug.

The drug is selected from any sort of conventional and unconventional compound, which may be applicable in the treatment of a neurodegenerative disorder. For example, the drug is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins. Functional drugs are identified on the basis of their ability to induce a down-regulation or inhibition of gene espression and/or down- regulation or inhibition of the activity of a transcriptional or translational product thereof. The expression is for example reduced or down-regulated to less than 90%, such as less than 80% such as less than 70% for example less than 60%, for example less than 50%, such as less than 40%, such as less than 30% such as less than 20% for example less than 10%, for example less than 5%, such as completely inhibited (0%) relative to the expression or activity in the absence of that drug compound..

Second set of aspects:

In a further aspect, the present invention relates to a method of treating, preventing or ameliorating a neurodegenerative disorder comprising administering to a subject in need thereof a compound, wherein said compound i) inhibits or down-regulates the expression of a gene selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcf1 , Csf1 , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnal , Slc15a4, Slc35b2, Stx11 , and Nudti 8, and/or ii) inhibits or down-regulates the activity of a gene product of a gene selected from the group set out in i).

In another aspect, the invention relates to a compound capable of a. inhibiting or down-regulating the expression of a gene selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnal , SId 5a4, Slc35b2, Stx11 , and Nudt18, and/or b. inhibiting or down-regulating the activity of a transcriptional and/or translational product of a gene according to a.

In another aspect, the invention relates to a pharmaceutical composition comprising at least one compound of the present invention.

In a further aspect, the invention relates to a kit-of-parts comprising a composition of the present invention, and at least one additional active ingredient.

In yet another aspect, the present invention relates to a use of a compound, a composition and/or a kit-of-parts of the present invention for the manufacture of a medicament for treatment, amelioration and/or prevention of a neurodegenerative disorder.

The invention also in an aspect relates to a compound, a composition and/or a kit-of- parts of the present invention for treatment, amelioration and/or prevention of a neurodegenerative disorder.

In a further aspect, the present invention relates to a pharmaceutical composition for treatment, amelioration and/or prevention of a neurodegenerative disorder comprising a compound, a composition and/or a kit-of-parts of the present invention. In another aspect, the present invention relates to a method for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said method comprising the steps of a. providing a biological sample isolated from said subject b. detecting in said biological sample of step a. i) at least one polymorphism or mutation of a gene, and/or ii) at least one transcriptional and/or translational product of a gene, wherein said gene is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfl , Csfl , CxcM , Cxcl2, CxcM 0, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18.

In a yet a furhter aspect, the present invention relates to a diagnostic kit for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said kit comprising at least one detection member for detecting in a biological sample isolated from said subject a. at least one polymorphism of a gene, and/or b. at least one transcriptional and/or translational product of a gene, wherein said gene is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfl , Csfl , CxcM , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18.

In a final aspect, the present invention relates to a method of identifying a drug for treating, preventing or ameliorating a neurodegenerative disorder, said method comprising the steps of a. providing a biological sample, b. determining in said biological sample the expression of a gene or the activity of a gene product, wherein said gene is selected from the group consisting of

Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfl , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudt18, c. providing a drug to said biological sample and determining the expression of a gene or the activity of a gene product, wherein said gene is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csf1 , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudt18, d. comparing the expression of said gene and/or the activity of said gene product in said biological sample in the presence and absence of said drug, wherein in the presence of said drug in said biological sample said expression of said gene and/or said activity of said gene product is inhibited or down-regulated relative to the expression of said gene and/or activity of said gene product in said sample in the absence of said drug.

Description of Drawings Figure 1. Degeneration involves activation of caspase-8, Fas, and TNF receptors. A, OLN-AS cells were treated with peptide aldehyde inhibitors (20 μM) against caspase-3 (Ac-DEVD-CHO), caspase-8 (Ac-IETD-CHO), and caspase-9 (Ac-LEHD- CHO) prior to transfection with p25α. Bars represent the mean ± 1.S. D. from five microscopic fields from one of three representative experiments. Inhibition of caspase- 3 and caspase-8 but not caspase-9 caused a significant reduction in MT retraction as compared to the control (p < 0.05 with respect to untreated cells). B, OLN-AS cells were pretreated with Fas-blocking antibody (ZB4) (1 μg/ml), anti-TNFα antibody (Infliximab) (10 μg/ml), or soluble TNFα receptor (Eternacept) (10 μg/ml) for 1 h prior to transfection with p25α. Bars represent the mean ± 1.S. D. from five microscopic fields from one of three representative experiments. Inhibition of Fas and TNFR signalling caused a significant reduction in the number of p25α-positive cells displaying MT retraction (p < 0.05 with respect to untreated cells).

Figure 2. Effect of p25α-expression on TNF-R1 , Fas, and FasL mRNA expression in OLN-AS cells. Cells were transfected with mock or p25α for 24 h, total RNA was extracted, and TNF-R1 , Fas, and FasL mRNA levels were analyzed by RT-PCR. Total RNA extracted from PC12 cells was used as positive control. p25α-expression caused a 20% increase in the TNFR1 mRNA level whereas the Fas and FasL mRNA levels remained unchanged. Molecular weight markers are shown to the left. Figure 3. Fas, TNFR1 , and TNFR2 are upregulated on myelin sheets in MSA. A, Paraffin sections from putamen and external capsule from normal control (left) or MSA cases (middle and right) were immunostained for Fas (upper row), TNFR1 (middle row), or TNFR2 (lower row). Sections shown in the right column were similarly stained but with omitting primary antibodies. Scale bar, 50 μm. B, Confocal laser scanning microscopical analysis of basal ganglia from MSA tissue stained for TNFR2 and MBP (top and middle rows) or TNFR2 and neurofilament (lower row). Top row demonstrates longitudinally sectioned myelin sheets and middle and lower rows transversely sectioned myelin sheets. Note the colocalization between MBP and TNFR2 and the presence of TNFR2 surrounding neurofilament. Scale bar, 10 μm.

Figure 4. Functional cluster of genes involved in biological processes categorized according to Gene Ontology. The pie chart shows the distribution of regulated genes in cells coexpressing α-syn and p25α compared to control cells. The number of altered genes in the different groups is indicated (the total number of genes is 104). Each gene was assigned to a single group to avoid overrepresentation of the true size of each functional group.

Figure 5. The chemokines display an early expression profile. RNA isolated from OLN- AS cells transfected with p25α for different time points was subjected to microarray analysis. The symbols represent the average fold increase in chemokine expression levels from two independent microarray experiments. Cxcl, chemokine (C-X-C motif) ligand; CcI, Chemokine (C-C motif) ligand.

Figure 6. Comparison of microarray analysis with real-time qPCR analysis. Expression analysis is shown for five selected genes (filled circles represent fold changes in the microarray analysis, open circles represent fold changes in the real-time qPCR analysis). Microarray fold changes were calculated using MAS 5.0 software (Affymetrix) and represent the average from two independent experiments. Real-time qPCR fold changes were determined from triplicate measurements and normalized to the NADH gene.

Figure 7. Gadd45a and Gadd45g are involved in α-synuclein dependent degeneration. OLN-AS cells were transfected with siRNA targeting rat GADD45a, Gadd45g, or a non- targeting siControl for 72 h. A. Silencing of Gadd45a and g was confirmed by semi- quantitative RT-PCR. B. Following siRNA transfection for 72 h, OLN-AS cells were transfected with p25α for 24 h and the number of cells with MT retraction was quantified. Bars represent the mean ± 1 S. D. from five microscopic fiels in one of two representative experiments. RNAi-mediated silencing of Gadd45a and g causes a significant reduction in the amount of degenerating cells (p < 0.05 with respect to siControl-treated cells).

Figure 8. Proposed model of the role of NF-κB inhibition in α-synuclein dependendent degeneration, α-syn aggregate formation stimulated by p25α leads to an upregulation of lκBα, an inhibitor of the transcription factor NF-κB. Inhibition of NF-κB leads to the induction of Gadd45a and g presumably though repression of NF-κB target genes. Gadd45a and g induction leads to apoptotic cell death through activation of the JNK pathway. The genes shown in blue are upregulated in this study.

Figure 9. Quantification of cellular degeneration measured by microtubule retraction. Bars represent the mean± 1 S. D. from five microscopic fields in one of two representative experiments. The diagram shows that RNAi-mediated silencing of Gadd45a and Nfkbia protects against α-synuclein dependent degeneration.

Detailed description of the invention

The present invention broadly relates to methods, compounds, compositions and uses thereof for treatment, amelioration and/or prevention of neurodegenerative disorders, in particular synucleinopathies such as Parkinson's disease. In order to facilitate the understanding of the invention, important terms are defined initially herein below.

Terms and definitions Amino acids and nucleic acids

Throughout the description and claims the three letter code for natural amino acids are used. Where the L or D form has not been specified it is to be understood that the amino acid in question has the natural L form, cf. Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so that the peptides formed may be constituted of amino acids of L form, D form, or a sequence of mixed L forms and D forms. Where nothing is specified it is to be understood that the C-terminal amino acid of a polypeptide of the invention exists as the free carboxylic acid, this may also be specified as "-OH". The N-terminal amino acid of a polypeptide comprise a free amino- group, this may also be specified as "H-".

Where nothing else is specified amino acid can be selected from any amino acid, whether naturally occurring or not, such as alfa amino acids, beta amino acids, and/or gamma amino acids. Accordingly, the group comprises but are not limited to: Ala, VaI, Leu, lie, Pro, Phe, Trp, Met, GIy, Ser, Thr, Cys, Tyr, Asn, GIn, Asp, GIu, Lys, Arg, His, Aib, NaI, Sar, Orn, Lysine analogues DAP and DAPA.

The term "nucleic acid" is meant to encompass DNA and RNA as well as derivatives thereof such as peptide nucleic acids (PNA) or locked nucleic acids (LNA) throughout the description.

The term "biological sample" as used herein refers to any suitable biological sample comprising genetic material, such as RNA or DNA, and/or proteins. The biological sample is in a preferred embodiment, isolated from the subject, such as a human being. In a preferred embodiment the sample is a blood sample, a tissue sample, a secretion sample, semen, ovum, hairs, nails, tears, and urine. The most convenient sample type is a blood sample; however, the choice of sample depends on the specific disorder or clinical condition as well as detection method and will be evident for those of skill in the art.

"Treatment," "treating," and the like, as used herein, refer to obtaining a desired pharmacologic and/or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse affect attributable to the disorder. That is, "treatment" includes (1 ) preventing the disorder from occurring or recurring in a subject who may be predisposed to the disorder but has not yet been diagnosed as having it, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or at least symptoms associated therewith, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and/or protein aggregation or formation of Lewy bodies.

Thus, the terms " ameliorate" or "amelioration" refers to relieving, alleviating or reducing symptoms associated with a clinical conditino, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and/or protein aggregation or formation of Lewy bodies.

The term "agonist" refers to a substance that mimics the function of an active molecule.

The term "antagonist" refers to a molecule that competes for the binding sites of an agonist, but does not induce an active response. Antagonists include, but are not limited to, drugs, hormones, antibodies, and neurotransmitters, as well as analogues and fragments thereof.

The term "ligand" refers to any molecule that binds to a specific site on another molecule.

The term "modulate" encompasses an increase or a decrease, a stimulation, inhibition, or blockage in the measured activity when compared to a i suitable control. "Modulation" of expression levels includes increasing the level and decreasing the level of an mRNA or polypeptide encoded by a polynucleotide of the invention when compared to a control lacking the agent being tested. In some embodiments, agents of particular interest are those which inhibit a biological activity of a subject polypeptide, and/or which reduce a level of a subject polypeptide in a cell, and/or which reduce a level of a subject mRNA in a cell and/or which reduce the release of a subject polypeptide from a eukaryotic cell. In other embodiments, agents of interest are those that increase a biological activity of a subject polypeptide, and/or which increase a level of a subject polypeptide in a cell, and/or which increase a level of a subject mRNA in a cell and/or which increase the release of a subject polypeptide from a eukaryotic cell. A "pharmaceutically acceptable carrier," "pharmaceutically acceptable diluent," or "pharmaceutically acceptable excipient", or "pharmaceutically acceptable vehicle," used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. For example, the carrier for a formulation containing polypeptides would not normally include oxidizing agents and other compounds that are known to be deleterious to polypeptides. Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the formulation. Adjuvants of the invention include, but are not limited to Freunds's, Montanide ISA Adjuvants [Seppic, Paris, France], Ribi's Adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT), I Hunter's TiterMax (CytRx Corp., Norcross, GA), Aluminum Salt Adjuvants (Alhydrogel - Superfos of Denmark/Accurate Chemical and Scientific Co., Westbury, NY), Nitrocellulose-Adsorbed Protein, Encapsulated Antigens, and Gerbu Adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany/C-C Biotech, Poway, CA). Topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate

(5%) in water. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents can be added as necessary. Percutaneous penetration enhancers such as Azone can also be included.

"Pharmaceutically acceptable salts" include the acid addition salts (formed with the free amino groups of the polypeptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, mandelic, oxalic, and tartaric. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, and histidine.

Compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses, or powders. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an "effective amount," that is, a dosage sufficient to produce the desired result or effect in association with a pharmaceutically acceptable carrier. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed, the host, and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

The term "antibody" refers to protein generated by the immune system that is capable of recognizing and binding to a specific antigen. Antibodies, and methods of making antibodies, are commonly known in the art. The term "antibody" as referred to herein includes whole antibodies and/or any antigen binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical complement system.

The term "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR), (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker and (viii) the Unibody technology from Genmab making use of fragmented antibodies with prolonged half-lives. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. A further example is binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Such binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The term "discontinuous epitope", as used herein, means a conformational epitope on a protein antigen which is formed from at least two separate regions in the primary sequence of the protein.

The term "bispecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities. For example, the molecule may bind to, or interact with, (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" or "heterospecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities. For example, the molecule may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules which are directed to a gene or a translcriptional or translational product thereof, wherein said gene is selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudt18. Specifically, Gadd45a, Gadd45b, Gadd45g are also referred to as Gadd45α, Gadd45β, Gadd45γ

As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, e.g., by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library, and wherein the selected human antibody is at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences, more preferably, no more than 5, or even more preferably, no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further in Section I, below), (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

As used herein, a "heterologous antibody" is defined in relation to the transgenic non- human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.

An "isolated antibody", as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a translational product of the present invention is substantially free of antibodies that specifically bind antigens other than translational products of the present invention). An isolated antibody that specifically binds to an epitope, isoform or variant of a translational product of the present invention may, however, have cross-reactivity to other related antigens, e.g., from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies having different specificities is combined in a well defined composition.

As used herein, "specific binding"' refers to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity corresponding to a KD of about

10^"7 M or less, such as about 10^"8 M or less, such as about 10^"9 M or less, about 10^"10 M or less, or about 10^"11 M or even less, when measured as apparent affinities based on IC50 values in FACS, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.

Affinity: the strength of binding between receptors and their ligands, for example between an antibody and its antigen.

Avidity: The functional combining strength of an antibody with its antigen which is related to both the affinity of the reaction between the epitopes and paratopes, and the valencies of the antibody and antigen

Antibody Classes: Depending on the amino acid sequences of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. lgG-1 , lgG-2, lgG-3 and lgG-4; lgA-1 and lgA-2. The heavy chains constant domains that correspond to the different classes of immunoglobulins are called alpha (α), delta (δ), epsilon (ε), gamma (v) and mu (μ), respectively. The light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino sequences of their constant domain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Antibody Combining Site: An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen. The term immunoreact in its various forms means specific binding between an antigenic determinant- containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof. Alternatively, an antibody combining site is known as an antigen binding site.

Chimeric antibody: An antibody in which the variable regions are from one species of animal and the constant regions are from another species of animal. For example, a chimeric antibody can be an antibody having variable regions which derive from a mouse monoclonal antibody and constant regions which are human.

Complementarity determining region or CDR: Regions in the V-domains of an antibody that together form the antibody recognizing and binding domain.

Constant Region or constant domain or C-domain: Constant regions are those structural portions of an antibody molecule comprising amino acid residue sequences within a given isotype which may contain conservative substitutions therein. Exemplary heavy chain immunoglobulin constant regions are those portions of an immunoglobulin molecule known in the art as CH1 , CH2, CH3, CH4 and CH5. An exemplary light chain immunoglobulin constant region is that portion of an immunoglobulin molecule known in the art as CL.

Diabodies: This term refers to a small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11 161 ; and Hollinger et al. (1993), Proc. Natl. Acad Sci. USA 90: 6444-6448.

Fv: dual chain antibody fragment containing both a VH and a VL. Human antibody framework: A molecule having an antigen binding site and essentially all remaining immunoglobulin-derived parts of the molecule derived from a human immunoglobulin.

Humanised antibody framework: A molecule having an antigen binding site derived from an immunoglobulin from a non-human species, whereas some or all of the remaining immunoglobulin-derived parts of the molecule is derived from a human immunoglobulin. The antigen binding site may comprise: either a complete variable domain from the non-human immunoglobulin fused onto one or more human constant domains; or one or more of the complementarity determining regions (CDRs) grafted onto appropriate human framework regions in the variable domain. In a humanized antibody, the CDRs can be from a mouse monoclonal antibody and the other regions of the antibody are human.

Immunoglobulin: The serum antibodies, including IgG, IgM, IgA, IgE and IgD.

Immunoglobulin isotypes: The names given to the Ig which have different H chains, the names are IgG (IgGI ,2,3,4), IgM, IgA (IgAI ,2), slgA, IgE, IgD.

Immunologically distinct: The phrase immunologically distinct refers to the ability to distinguish between two polypeptides on the ability of an antibody to specifically bind one of the polypeptides and not specifically bind the other polypeptide.

Monoclonal Antibody: The phrase monoclonal antibody in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific monoclonal antibody.

Polyclonal antibody: Polyclonal antibodies are a mixture of antibody molecules recognising a specific given antigen, hence polyclonal antibodies may recognise different epitopes within said antigen. Single Chain Antibody or scFv: The phrase single chain antibody refers to a single polypeptide comprising one or more antigen binding sites. Furthermore, although the H and L chains of an Fv fragment are encoded by separate genes, they may be linked either directly or via a peptide, for example a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain antibody, sAb; Bird et al. 1988 Science 242:423-426; and Huston et al. 1988 PNAS 85:5879-5883) by recombinant methods. Such single chain antibodies are also encompassed within the term "antibody", and may be utilized as binding determinants in the design and engineering of a multispecific binding molecule.

Valency: The term valency refers to the number of potential antigen binding sites, i.e. binding domains, in a polypeptide. A polypeptide may be monovalent and contain one antigen binding site or a polypeptide may be bivalent and contain two antigen binding sites. Additionally, a polypeptide may be tetravalent and contain four antigen binding sites. Each antigen binding site specifically binds one antigen. When a polypeptide comprises more than one antigen binding site, each antigen binding site may specifically bind the same or different antigens. Thus, a polypeptide may contain a plurality of antigen binding sites and therefore be multivalent and a polypeptide may specifically bind the same or different antigens. V-domain: Variable domain are those structural portions of an antibody molecule comprising amino acid residue sequences forming the antigen binding sites. An exemplary light chain immunoglobulin variable region is that portion of an immunoglobulin molecule known in the art as VL.

VL: Variable domain of the light chain.

VH: Variable domain of the heavy chain.

As used herein, the term "antibody" encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, e. g. , Winter et al., Nature 349: 293 (1991 ) and U. S. Patent No. 4,816, 567); F (ab¹) 2 and F (ab) fragments; Fv molecules (noncovalent heterodimers (see, e. g. , lnbar et al., Proc Natl Acad Sci USA 69: 2659 (1972) and Ehrlich et al., Biochem 19 : 4091 (1980) ) ; single-chain Fv molecules (sFv) (see, e. g. , Huston et al., Proc Natl Acad Sci USA 85: 5879 (1980) ) ; dimeric and trimeric antibody fragment constructs; minibodies (see, e. g., Pack et al., Biochem 31 : 1579 (1992) and Cumber et al., J. Immunology 149B : 120 (1992)) ; humanized antibody molecules (see, e. g., Riechmann et al., Nature 332: 323 (1988) and Verhoeyan et al., Science 239: 1534 (1988) ) ; and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding.

An "antigen" is a substance that provokes an immune response.

An "agonist antibody" is one that mimics, enhances, stimulates, or activates the function of a molecule with which the agonist interacts.

An "antagonist antibody" is one that competes, inhibits, or interferes with the activity of a molecule with which the antagonist interacts. For example, an antagonist antibody may bind to the receptor without inducing an active response.

The "constant region" of an antibody is its effector region, and determines the functional class of the antibody. The constant region of a heavy or light chain is located at or near the carboxyl terminus. The "variable region" of an antibody is the region that binds to the antigen; it provides antibody specificity. The variable region of a heavy or light chain is located at or near the amino terminus. A"VH"fragment contains the variable region of a heavy chain; a"VL"fragment contains the variable region of a light chain.

An "immunoglobulin" is an antibody molecule, i. e. , a polypeptide that can respond to a foreign molecule of invading organism, e. g. , by binding to it, marking it for destruction, and/or inactivating it.

A "heavy chain" is the larger of the two classes of polypeptide chains that combine to form immunoglobulin molecules. The class of the heavy chain determines the class of the immunoglobulin, e. g. , IgG, IgA, IgE, IgD, or IgM.

A "light chain" is the smaller of the two classes of polypeptide chains that combine to form immunoglobulin molecules. Light chains are generally classified into two classes, kappa and lambda, on the basis of structural differences in their constant regions. A "humanized" antibody is an antibody that contains mostly human immunoglobulin sequences. This term is generally used to refer to a non-human immunoglobulin that has been modified to incorporate portions of human sequences, and may include a human antibody that contains entirely human immunoglobulin sequences.

Methods of making polyclonal and monoclonal antibodies are known in the art. Polyclonal antibodies are generated by immunizing a suitable animal, such as a mouse, rat, rabbit, sheep or goat, with an antigen of interest, such as a stem cell transformed with a gene encoding an antigen. In order to enhance immunogenicity, the antigen can be linked to a carrier prior to immunization. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Furthermore, the antigen may be conjugated to a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc. , in order to enhance the immunogenicity thereof. The term "binds specifically", in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific polypeptide, or more accurately, to an epitope of a specific polypeptide. Antibody binding to such epitope on a polypeptide can be stronger than binding of the same antibody to any other epitopes, particularly other epitopes that can be present in molecules in association with, or in the same sample as the polypeptide of interest.

For example, when an antibody binds more strongly to one epitope than to another, adjusting the binding conditions can result in antibody binding almost exclusively to the specific epitope and not to any other epitopes on the same polypeptide, and not to any other polypeptide, which does not comprise the epitope. Antibodies that bind specifically to a subject polypeptide may be capable of binding other polypeptides at a weak, yet detectable, level (e. g. , 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to a subject polypeptide, e. g. , by use of appropriate controls. In general, antibodies of the invention bind to a specific polypeptide with a binding affinity of 10^"7 M or greater (e. g., 10⁸ M, 10⁹ M, 10¹⁰ M, 10¹¹M, etc. ). The term "gene product" as used herein refers to any transcriptional or translational product of a gene. A transcriptional product comprises any RNA-species, which is transcribed from the specific gene, such as pre-RNA, mRNA, tRNA, miRNA, spliced and nonspliced RNA. Thus, a transcriptional gene product of the present invention comprise any RNA-species encoded by or comprising a sequence selected from any one of the genes Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudti 8. For example, a transcriptional gene product of the present invention comprises any RNA-species encoded by or comprising a sequence selected from any of SEQ ID NO: 29-59. The transcript may be bound by RNA-binding proteins and, thus, packaged into a ribonucleoprotein (RNP), for example an mRNP molecule.

A translational gene product of the present invention comprises any peptide or polypeptide encoded by the gene or a fragment thereof. Thus, a "polypeptide encoded by a gene of the present invention" is comprised in the terms "gene product", or "translational gene product". A translational gene product of the present invention comprise any polypeptde-species encoded by a sequence selected from any one of the genes Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csf1 , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudti 8. For example, a gene product or translational gene product of the present invention comprises any polypeptide-species encoded by a sequence selected from any of SEQ ID NO: 29-59, or the complement thereof or part thereof or any sequence which is at least 70%, such as at least 80%, for example at least 90% identical to any of said sequences or part thereof.

The term "downregulation" or "down-regulated" as used herein in respect of a transcriptional or translational gene product refers to a reduction of said transcriptional or translational gene product; i.e. the level of a given parameter is lower compared to the average level for example in a population of after a given treatment. In respect of a transcriptional product of the present invention, such as an RNA transcript encoded by any of SEQ ID NO: 29-59, the level of transcript may for example be determined by quantitative or semiquantitative reverse transcriptase polymerase chain reaction (RT- PCR). For example, the level of transcript may be determined by RT-PCR using an oligonucleotide primer, as disclosed herein (e.g. any of SEQ ID NO: 1-20). The level of transcript may be normalized according to an endogenous transcript, such as illustrated in the example herein below.

A down-regulated actitivity of a transcriptional product is for example observed by a reduction or downregulation of the level of a specific RNA transcript, as determined for example by RT-PCR. In a preferred embodiment the level of RNA is determined by RT- PCR, for example using at least one oligonucleotide primer selected from any one of SEQ ID NO: 1-20. The choice of primer is illustrated in table 3; e.g. in case of Gadd45a transcripts at least one oligonucleotide primer selected from SEQ ID NO: 1 or SEQ ID NO: 2 is used.

Down-regulation of the actitivity of a translational product comprises both a reduction in the amount/level of polypeptide, and/or reduced enzymatic actitiv of said polypeptide and/or reduced abililty of the polypeptide to interact with other polypeptides and signal cascades. The level of polypeptide may be determined by any suitable method available to those of skill in the art, for example by western blotting, or ELISA.

The terms "fragment thereof or "part thereof as used herein refers to a fragment. piece, or sub-region of a nucleic acid or protein molecule whose sequence is disclosed herein, such that tho fragment comprising 5 10, 15 20 or more amino acids, or 5, 10, 15 30,45, 80 or more nucleotides that are contiguous in the parent protein or nucleic acid compound When referπng to a nucleic acid sequence, "fragment thereof" or "part thereof refers to 5. 10, 15. 30,45, 60 or more contiguous nucleotides, derived from the parent nucleic acid sequence, and also, owing to the genetic code, to the compiomontary sequonco For example, if tho fragment entails tho sequence 5'- AGCTAG-3' then '"fragment thereof would also include the complementary sequence, 3"-TCGATC-5¹. Thus, the terms "fragment thereof or "part thereof as used herein in relation to an amino acid sequence refers to any portion of the given amino acid sequonco which has the same activity as the complete amino acid sequence.

Fragments will suitably comprise at least 10 and preferably at least 20 consecutive ammo acids from the basic sequence. Fragments or parts of the polypeptide include deletion mutants and polypeptides where small regions of the polypeptides are joined together. Tho fragments should contain an epitope, and preferably contain at loast one antigenic region The terms '"fragment thereof" or "part thereof as used herein in relation to a nucleic acid or polynucleotide sequence refers to any portion of the given polynucleotide sequence which serves a relavant purpose. In an oligonucleotide primer or probe comprising a fragment or part of a given basic sequence, the fragment or part should comprise enough nucleotides to support specific binding of the oligonucleotide primer or probe to its target. Such fragments typically comprise or consists of at least 5 nucleotides, such as at least 10, 15, or at least 20 consecutive nucleotides. With respect to a nucleic acid sequence encoding a polypeptide, wherein the nucleic acid sequence comprise or consists of a fragment or part of a basic nucleic acid, the fragment or part should comprise or consist of a nuclaic acid sequence, which encodes s polypeptide with an acitivity which corresponds to the activity of the basic protein. Such fragments or parts will typically comprise at least 15, preferably at least 30 and more preferably at least 60 consecutive bases from the basic sequence.

Treatment

The present invention broadly relates to the treatment and diagnosis of neurodegenerative disorders, in particular synucleinopathies such as Parkinson's disease and/or alzheimers.

In one aspect, the present invention relates to a method of treating, preventing or ameliorating a neurodegenerative disorder in a subject. The method of the invention comprises the steps of a. providing a subject, b. administering a compound, wherein said compound i) inhibits or down-regulates the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA,

Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcM , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfI O, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18, and/or ii) inhibits or down-regulates the activity of a gene product of a gene selected from the group set out in b.

Thus, in one embodiment, the method of the invention comprises providing a compound which inhibits or down-regulates the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, CIcM , Csf1 , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnal , Slc15a4, Slc35b2, Stx11 , and Nudti 8. In another embodiment, the compound inhibits or down- regulates the activity of a gene product of a gene selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfi , Csf1 , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnal , Slc15a4, Slc35b2, Stx1 1 , and Nudt18. However, in yet another embodiment, the compound inhibits or down- regulates the expression of a gene selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfi , Csf 1 , CxcM , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfI O, Myc, Kpnal , SId 5a4, Slc35b2, Stx11 , and Nudt18, and inhibits or down-regulates the activity of a gene product of said gene.

The expression of the specific gene and/or the activity of the gene product may be reduced globally, i.e. in the whole subject, such as the whole human being; however, in one embodiment, the expression of the gene or the activity of the gene product is reduced in a specific tissue of said human being. The expression of the gene and/or activity of the gene product may be reduced in any tissue of the subject. In one embodiment, the expression of the gene and/or activity of the gene product is inhibited or down-regulated in a tissue selected from the group consisting of brain tissue, cerebral cortex tissue, liver tissue, skeletal muscle tissue, and intestinal tissue. In a preferred embodiment, the expression of the gene and/or activity of the gene product is inhibited or down-regulated in the cerebral cortex tissue.

The activity of a gene product includes any parameter, which reflects the function of the polypeptide encoded by the gene or part thereof. For example, the activity of an RNA transcript is reduced if the level of polypeptide translated from said RNA is reduced. Thus, reduced level/amount both absolute and relative of an RNA transcript, increased degradation, shorter lifespan of an RNA transcript corresponds to a downregulation of the activity of said RNA as a transcriptional gene product. The level of RNA transcripts may be measure by any method available to those of skill in the art; examples are provided elsewhere herein. However, in a preferred embodiment the level of RNA is determined by RT-PCR, for example using at least one oligonucleotide primer selected from any one of SEQ ID NO: 1-20. In terms of translational gene products, the activity is reduced if the polypeptide is degraded or bound by antagonists, which serve to inhibit one or more functional domains, for example domains involved in kinase activity, DNA binding or protein- protein interaction.

In one embodiment, the expression of a gene and/or the activity of said gene product in said human being is reduced to less than 90%, such as less than 80%, for example less than 70, such as less than 60, for example less than 50%, such as less than 40%, for example less than 30, such as less than 20, for example less than 10%, for example 0% or the normal expression level or activity level. The normal expression or activity level in one embodiment corresponds to the expression or activity level before treatment according to the present invention.

The genes targeted by the method of the present invention are defined more specifically herein below; however, in a preferred embodiment, the gene is Gadd45a, Gadd45b, Gadd45g or Nfkbia.

In one embodiment, the method of treating, preventing or ameliorating a neurodegenerative disorder, such as a synucleinopathy for example Parkinson's or

Alzheimer's diseases in a subject according to the present invention further comprises administering at least one additional synucleinopathy therapeutic, such as at least one additional Parkinson's or Alzheimer's disease therapeutic.

Neurodegenerative disorders

The present invention relates to methods, compounds, compositions, kits, kit-of-parts, and uses thereof for treatment, amelioration and/or prevention of a neurodegenerative disorder. The invention also relates to diagnostic methods and kits for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject or for assisting in determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder. Furthermore, screening methods are provided for identifying a drug for treating, preventing or ameliorating a neurodegenerative disorder.

A neurodegenerative disorder of the present invention is in one embodiment a disorder related to α-synuclein, designated herein as an α-synucleinopathy or a synucleinopathy. The α-synucleinopathies comprise the neurodegenerative disorders, Parkinson's disease (PD), Dementia with Lewy bodies (DLB), multiple system atrophy (MSA) as well as neurodegeneration with brain iron accumulation type I and Lewy body variant of Alzheimer's disease (Table a). The common feature of these disorders is the presence of intracellular inclusions containing aggregated α-synuclein. The inclusions are deposited in populations of neurons and glia, which varies among the disorders.

Parkinson's disease (PD) is the most common movement disorder in the elderly and is clinically characterized by tremor, rigidity, and bradykinesia. It is a progressive disorder that affects various neuronal populations in the human nervous system, particularly dopaminergic neurons of the substantia nigra pars compacta. Affected neurons develop inclusions termed Lewy bodies (LBs) in their perikarya and Lewy neurites (LNs) in their processes. LBs are proteinaceous cytoplasmic inclusions, which in brainstem are characterized by a dense eosinophilic core and a clear surrounding halo (76). The major component of LBs is aggregated α-synuclein, though more than 70 different molecule constituents have been identified in these inclusions. Moreover, the majority of LBs are stained for ubiquitin by immunohistochemistry.

The presence of proteins in LBs suggests that protein aggregation and accumulation play a prominent role in the pathogenesis of both familial and sporadic PD. Several lines of evidence suggest that LBs in themselves are not harmful to the cells and may even have a cytoprotective role. A PD staging procedure using LB and LN pathology has been proposed, which states that the pathological process spreads from the lower brain stem and olfactory bulb and progresses in a predictable sequence. Nuclei in the substantia nigra have been shown to be particularly vulnerable and have received particular attention during the past decades. Lesions in the substantia nigra are probably responsible for the majority of motor dysfunctions occurring in PD. However, the pathology of PD cannot be completely described unless changes in the extranigral system are taken into account. Severe lesions also occur in the amygdala, in nuclei projecting to the cerebral cortex, and in nuclei regulating endocrine and autonomic functions.

Multiple System Atrophy (MSA) is a sporadic neurodegenerative disorder characterized clinically by parkinsonism, autonomic failure, and cerebellar ataxia. The neuropathological hallmarks are neuronal loss, gliosis, and myelin pathology particularly in the striatonigral system. In affected white matter, oligodendroglia contain glial cytoplasmic inclusions (GCIs) with deposited, filamentous α-synuclein as the main component, α-synuclein is a neuronal protein; hence the presence of α-synuclein immunopositive inclusions in glia raises a question regarding its source. It may be endogenously expressed in glia or, alternatively, transmitted from neurons. Oligodendrocytes were found to express α-synuclein during development suggesting that mature oligodendrocytes possess the ability to express the protein.

Thus, in one embodiment of the methods, compounds, compositions, kits, kit-of-parts, and uses thereof, the neurodegenerative disorder is a synucleinopathy, and in a more specific embodiment, the neurodegenerative disorder is selected from the group consisting of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and Multiple system atrophy. In a preferred embodiment, the neurodegenerative disorder is Parkinson's disease, and in another preferred embodiment, the neurodegenerative disorder is Alzheimer's disease or Lewy body dementia.

Genetic markers and genes

The present invention relates to methods, compounds, compositions, kits, kit-of-parts, and uses thereof for treatment, amelioration, prevention and/or diagnosis of a neurodegenerative disorder as defined herein, wherein a specific gene or product thereof is targeted by a compound, which recognizes, inhibits or downregulates the expression of said gene and/or the activity of said gene product. In one embodiment, the gene of the present invention is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfi , Csf1 , CxcM , Cxcl2, CxcM O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx11 and Nudt18. In one embodiment, the gene of the present invention is selected from the group consisting of Bbc3/PUMA, Nfkbia, and Ninji . In one embodiment, the gene of the present invention is selected from the group consisting of Gadd45a, Gadd45b, and Gadd45g. In another embodiment, the gene of the present invention is selected from the group consisting of Ccl2, Ccl7, Clef 1 , and Csf1. In another embodiment, the gene of the present invention is selected from the group consisting of CxcH , Cxcl2, CxcH O, and IL6. In another embodiment, the gene of the present invention is selected from the group consisting of Bhlhb2, Camkk2, Duspi , Lphn2, and RiI. In another embodiment, the gene of the present invention is selected from the group consisting of Hmoxi , Hspal a, Myd1 16, Srxni , and EgM . In another embodiment, the gene of the present invention is selected from the group consisting of Fos, FosL, Hes1 , KIfIO, Myc, and Kpnai . In yet another embodiment, the gene of the present invention is selected from the group consisting of SId 5a4, Slc35b2, Stx1 1 and Nudt18. However, in a preferred embodiment, the gene is Gadd45a, Gadd45b, Gadd45g or Nfkbia.

The genes of the methods, compounds, compositions, kits, kit-of-parts, and uses thereof according to the present invention is in separate embodiments selected from any one of the genes Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , or Nudt18. Thus, In a particular embodiment, the gene of the present invention is Gadd45a (also called Gadd45α). In another specific embodiment, the gene of the present invention is Gadd45b (also called Gadd45β). In another specific embodiment, the gene of the present invention is Gadd45g (also called Gadd45γ). In yet another embodiment, the gene is IL-6. In another embodiment, the gene is Egr-1 , Myc, Nfkbia, or Hspai b. In another embodiment, the gene is NADH or GADH.

The genes of the present invention comprise upstream and downstream regulatory regions of the coding region of the specific gene, such as upstream 5'-UTR, promoter regions, transcription initiation cis-acting elements, and downstream 3'-UTR, 3'- processing signals, polyadenylation signal, transcriptional termination signals. Moreover, any mutation such as a polymorphism, deletion, substitution, or inversion in the gene is comprised in the present invention.

In one specifc embodiment, the gene is selected from the group consisting of SEQ ID NO: 29-59.

Thus, in one embodiment, the transcriptional gene product of the present invention is encoded by or consists of or comprises a nucleic acid sequence idenitified as any one of SEQ ID NO: 29-59, or part thereof, or the complement of said sequence or part thereof.

Furthermore, a translational gene product of the present invention is in one embodiment encoded by a nucleic acid sequence idenitified as any one of SEQ ID NO: 29-59, or part thereof, or the complement of said sequence or part thereof.

Compounds

In one aspect, the present invention relates to a compound capable of a. inhibiting or down-regulating the expression of a gene selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudt18, and/or b. inhibiting or down-regulating the activity of a transcriptional and/or translational product of a gene according to a.

The compound may interact indirectly with the gene or gene product of the present invention, for example via interaction with a polypeptide or other cellular components, which affects the expression or activity, and thereby reduce the expression and/or activity of the gene or gene product. However, the compound may also directly interact with the gene or gene product of the present invention. Thus, in one embodiment, the compound is capable of selectively binding said gene, and/or a transcriptional and/or translational product of said gene. In one embodiment the compounds competitively inhibit the binding or complexing of a translational gene product (polypeptide) of the present invention with a native interaction partner. Such a compound could for example be a compound that specifically interacts with a protein, which binds a translational gene product of the present invention, in a way that sterically inhibits further association with either the translational gene product or the native interaction partner.

Specifically, the invention relates to a compound capable of binding to a gene or transcriptional or translational gene product of the present invention, thereby inhibiting or down-regulating the activity of a translational product of said gene.

The compounds of the present invention is for example selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small organic molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affi body-antibody chimeras, and non-immunoglobulins.

In one embodiment of the present invention, the compound is a polypeptide. For example such polypeptides could be selected from the group consisting of steroid hormone binding protein receptor domains and fragments thereof, steroid hormone binding protein co-receptor domains and fragments thereof, natural steroid hormone binding protein receptor ligands, modified steroid hormone binding proteins or fragments thereof, fragments of steroid hormone binding proteins, steroid hormone binding protein receptor antagonists, and functional homologues of any of these. In one embodiment, the compound is an antibody, antigen binding fragment or recombinant protein thereof, for example an antibody, antigen binding fragment or recombinant protein thereof, which selectively binds Gadd45b and/or Gadd45g.

In another embodiment, the compound is an siRNA. The choice of siRNA depends on the gene to target. I one embodiment, the siRNA targets a transcriptional gene product of the present invention, wherein said gene is identified as any of SEQ ID NO: 29-59. Thus in one embodiment, a compound of the present invention is an siRNA consisting or comprising of 17-25, such as 19-22 consecutive nucleotides selected from a region of a sequence selected from SEQ ID NO:29-59 or the complement thereof.

In a specific embodiment, the compound is an siRNA selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 28. Thus, in one embodiment, the compound is an siRNA corresponding to, comprising or consisting of SEQ ID NO: 21 , 22, 23, 24, 25, 26, 27, 28, 60, 61 , 62, or 63. Therefore, the present invention also in one aspect relates to an siRNA corresponding to, comprising or consisting of SEQ ID NO: 21 , 22, 23, 24, 25, 26, 27, 28, 60, 61 , 62, 63, 64, 65, 66, or 67. The term "corresponding" as used in connection with siRNAs, merely implies that the siRNA is designed on the basis of that sequence, and that the specific siRNA may be optimized with respect to 5'-end and/or 3'-end nucleotides as well as total length.

The present invention encompass compounds, which are capable of binding to at least one region of a gene or a polypeptide or part thereof of the present invention, thereby inhibiting or reducing the expression of said gene and/or the activity of a gene product of said gene.

In one embodiment of the present invention, the compound is capable of specifically recognizing and binding to a region of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a,

Myd116, Srxni , Egr1 , Fos, FosL, Hes1 , KIfI O, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudt18. Specifically, a compound according to the present invention is capable of specifically recognizing and binding to a region comprising or consisting of 3 to 100 nucleic acid residues, such as 5 to 50 nucleic acid residues, such as 5 to 30 nucleic acid residues selected from a region of a gene of the present invention.

In one embodiment of the present invention, the compound is capable of specifically recognizing and binding to a region of a translational gene product of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudt18. Specifically, a compound according to the present invention is capable of specifically recognizing and binding to a region of said translational gene product comprising or consisting of 3 to 10 amino acid residues, such as 3 to 8 amino acid residues, such as 3 to 6 amino acid residues selected from a region of a translational gene product of the present invention. Specifically, the compound according to the present invention is capable of specifically recognizing and binding to an epitope comprising or consisting of 3 to 10 amino acid residues, such as 3 to 8 amino acid residues, such as 3 to 6 amino acid residues, selected from any of the residues of a translational gene product of the present invention. Peptides

In some embodiments of the present invention, the compound is a peptide. Suitable peptides include peptides of from about 5 amino acids to about 50, from about 6 to about 30, or from about 10 to about 20 amino acids in length. In some embodiments, a peptide has a sequence of from about 7 amino acids to about 45, from about 9 to about 35, or from about 12 to about 25 amino acids of corresponding naturally-occurring protein. In some embodiments, a peptide exhibits one or more of the following activities: inhibits binding of a subject polypeptide (a translational gene product) to an interacting protein or other molecule; inhibits subject polypeptide binding to a second polypeptide molecule; inhibits a signal transduction activity of a subject polypeptide; inhibits an enzymatic activity of a subject polypeptide; or inhibits a DNA binding activity of a subject polypeptide.

Peptides can include naturally-occurring and non-naturally occurring amino acids. Peptides can comprise D-amino acids, a combination of D-and L-amino acids, and various"designer"amino acids (e. g., P-methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids, etc.) to convey special properties.

Additionally, peptides can be cyclic. Peptides can include non-classical amino acids in order to introduce particular conformational motifs. Any known non-classical amino acid can be used. Non-classical amino acids include, but are not limited to, 1 ,2, 3, 4- tetrahydroisoquinoline-3-carboxylate ; (2S, 3S)-methylphenylalanine, (2S, 3R)- methyl- phenylalanine, (2R, 3S)-methyl-phenylalanine and (2R, 3R)-methyl- phenylalanine ; 2- aminotetrahydronaphthalene^-carboxylic acid; hydroxy-1 ,2, 3,4- tetrahydroisoquinoline-3-carboxylate ; p-carboline (D and L); HIC (histidine isoquinoline carboxylic acid) ; and HIC (histidine cyclic urea). Amino acid analogs and peptidomimetics can be incorporated into a peptide to induce or favor specific secondary structures, including, but not limited to, LL-Acp (LL-3-amino-2- propenidone- 6-carboxylic acid), a P-tum inducing dipeptide analog ; p-sheet inducing analogs; p- tunn inducing analogs; a-helix inducing analogs; y-turn inducing analogs ; GIy-AIa turn analogs; amide bond isostere; or tetrazol, and the like.

In addition to the foregoing N-terminal and C-terminal modifications, a peptide or peptidomimetic can be modified with or covalently coupled to one or more of a variety of hydrophilic polymers to increase solubility and circulation half-life of the peptide. Suitable nonproteinaceous hydrophilic polymers for coupling to a peptide include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran, and dextran derivatives. Generally, such hydrophilic polymers have an average molecular weight ranging from about 500 to about 100,000 daltons, from about 2,000 to about 40,000 daltons, or from about 5,000 to about 20,000 daltons. The peptide can be derivatized with or coupled to such polymers using any of the methods set forth in Zallipsky, Bioconjugate Chem. 6: 150 (1995); Monfardini et al., Bioconjugate Chez. 6: 62 (1995); U. S. Pat. Nos. 4,640, 835; 4,496, 689; 4,301 , 144; 4,670, 417; 4,791 , 192; 4,179, 337, or WO 95/34326.

Also, the present invention relates a peptide fragment as defined herein, for example an immunogenic peptide fragment containing the epitope recognizable by an antibody as defined by the present invention. Accordingly, the invention also relates to use of a peptide fragment as defined above for the production of an antibody, in particular for the production of an antibody, as defined in the present invention.

In one embodiment, the compound of the present invention is a polypeptide comprising or consisting of a polypeptide encoded by a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudt18, and any fragment thereof. In one embodiment, the compound is a polypeptide comprising at least consecutive 5 amino acids, such as at least 10, 20, 30, 40, 50 consecutive amino acids, for example at least 100 consecutive amino acids, wherein said consecutive amino acids are encoded by a nucleic acid sequence selected from any of the genes Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx11 , Nudti 8 or fragments thereof; or the compound is a polypeptide comprising at least consecutive 5 amino acids, such as at least 10, 20, 30, 40, 50 consecutive amino acids, for example at least 100 consecutive amino acids, wherein said consecutive amino acids are encoded by a nucleic acid sequence selected from any of SEQ ID NO: 29-59.

Moreover, the present invention relates to use of a peptide fragment as defined herein for the manufacture of a medicament for treatment of a clinical condition, such as a neurodegenerative disorder as defined herein.

Natural single domain antibodies

Heavy-chain antibodies (HCAbs) are naturally produced by camelids (camels, dromedaries and llamas). HCAbs are homodimers of heavy chains only, devoid of light chains and the first constant domain (Hamers-Casterman et al., 1993). The possibility to immunise these animals allows for the cloning, selection and production of an antigen binding unit consisting of a single-domain only. Furthermore these minimal- sized antigen binding fragments are well expressed in bacteria, interact with the antigen with high affinity and are very stable.

New or Nurse Shark Antigen Receptor (NAR) protein exists as a dimer of two heavy chains with no associated light chains. Each chain is composed of one variable (V) and five constant domains. The NAR proteins constitute a single immunoglobulin variable- like domain (Greenberg, A. S., Avila, D., Hughes, M., Hughes, A., McKinney, E. C. & Flajnik, M. F. (1995) Nature (London) 374, 168-173.) which is much smaller than an antibody molecule.

Non-immonoglobulin binding members In one preferred embodiment, the present invention relates to binding members derived from a naturally occurring protein or polypeptide; said protein or polypeptide may for example be designed de novo, or may be selected from a library. The binding member may be a single moiety, e.g., a polypeptide or protein domain, or it may include two or more moieties, e.g., a pair of polypeptides such as a pair polypeptides. The binding member may for example, but exclusively, be a lipocalin, a single chain MHC molecule, an Anticalin™ (Pieris), an Affibody™, or a Trinectin™ (Phylos), Nanobodies (Ablynx). The binding member may be selected or designed by recombinant methods known by people well known in the art.

Affibody Affibody: A recombinant immunologically active molecule, selected from a library constructed by combinatorial variegation of the Fc binding surface of of a protein that is not an antibody, preferably the 58 residue staphylococcal protein A (SPA).

Affibodies are produced recombinantly by methods well known to those skilled in the art of recombinant DNA technology. Phage display techniques may be used to identify affibodies capable of specifically recognising a particular antigen. Affibodies can be produced in any suitable host, as for example, but not exclusively E. coli or S. cerevisiae (se below) (Hansson M et al., Jmmunotechnology. 1999 Mar; 4(3-4): 237- 52.)

Affibody-antibody chimeras

In another embodiment of the present invention, said binding member is an affibody- antibody chimera (Ronnmark J et al,., J Immunol Methods. 2002 Mar 1 ; 261 (1-2): 199- 211 ). According to the invention affibody-antibody chimeras can be constructed by several methods, for example by fusion of nucleotide sequences or fusion of polypeptide sequences. The nucleic acid sequence of an affibody maybe fused to a nucleic acid sequence of an antibody by DNA recombinant technology for the production of the binding member in a suitable host. The affibody nucleotide sequences may for example be fused to an antibody light chain nucleotide sequence or an antibody heavy chain nucleic acid sequence. In an embodiment of the invention the affibody sequence may be fused with a fragment of an antibody sequences. The affibody sequence may for example, but not exclusively, be fused with an Fc fragment of an antibody, thus potentially allowing dimers to form by homo-dimerisation. The affibody antibody chimeras may contain multiple affibody sequences, such as at least two, three, four of at least six affibody sequences. In an embodiment of the invention a fusion of two affibodies may be fused with an Fc fragment resulting in a tetravalent binding member upon dimerisation.

Alternatively the chimeras may be obtained by linking of the two protein/polypeptide molecules together by methods known to people skilled in the art.

Peptide aptamers

Peptide aptamers are peptides or small polypeptides that act as dominant inhibitors of protein function. Peptide aptamers specifically bind to target proteins, blocking their functional ability (Kolonin et al. (1998), Proc. Natl. Acad. Sci. USA 95: 14266). Due to the highly selective nature of peptide aptamers, they can be used not only to target a specific protein, but also to target specific functions of a given protein, such as a kinase activity or protein-protein interaction. Further, peptide aptamers can be expressed in a controlled fashion by use of promoters that regulate expression in a temporal, spatial or inducible manner. Peptide aptamers act dominantly; therefore, they can be used to analyze proteins for which loss-of-function mutants are not available.

Peptide aptamers that bind with high affinity and specificity to a target protein can be isolated by a variety of techniques known in the art. Peptide aptamers can be isolated from random peptide libraries by yeast two-hybrid screens (Xu et al. (1997), Proc. Natl.

Acad. Sci. USA 94 : 12473. They can also be isolated from phage libraries

(Hoogenboom et al. (1998), lmmunotechnology 4: 1 ) or chemically generated peptides/libraries.

Specifically, the present invention encompasses peptide aptamers, which are capable of binding to a translational gene product of a gene of the present invention.

Also, the present invention relates a peptide aptamer as defined herein, which is an immunogenic peptide aptamer containing the epitope recognizable by an antibody as defined by the present invention. Accordingly, the invention also relates to use of a peptide aptamer as defined above for the production of an antibody, in particular for the production of an antibody, as defined in the present invention.

Moreover, the present invention relates to use of a peptide aptamer as defined herein for the manufacture of a medicament for treatment of a clinical condition, such as a neurodegenerative disorder, such as Parkinson's disease.

Antibodies It is one aspect of the present invention to provide antibodies or functional equivalents thereof, such as antigen binding fragments or recombinant proteins specifically recognising and binding a polypeptide encoded by a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudti 8. In one embodiment, the antibody, antigen binding fragment, recombinant protein or functional homologue thereof is capable of inhibiting binding of said polypeptide to a native cellular interaction partner. The antibody, or functional homologue thereof, specifically recognizes an epitope or a functional homologue thereof. The epitope may be any of the epitopes mentioned herein below.

The antibody or functional equivalent thereof may be any antibody known in the art, for example a polyclonal or a monoclonal antibody derived from a mammal or a synthetic antibody, such as a single chain antibody or hybrids comprising antibody fragments. Furthermore, the antibody may be mixtures of monoclonal antibodies or artificial polyclonal antibodies. In addition functional equivalents of antibodies may be antibody fragments, in particular epitope binding fragments. Furthermore, antibodies or functional equivalent thereof may be small molecule mimetics, mimicking an antibody. Naturally occurring antibodies are immunoglobulin molecules consisting of heavy and light chains. In preferred embodiments of the invention, the antibody is a monoclonal antibody.

Monoclonal antibodies (Mab's) are antibodies, wherein every antibody molecule are similar and thus recognises the same epitope. Monoclonal antibodies are in general produced by a hybridoma cell line. Methods of making monoclonal antibodies and antibody-synthesizing hybridoma cells are well known to those skilled in the art. Antibody producing hybridomas may for example be prepared by fusion of an antibody producing B lymphocyte with an immortalized B-lymphocyte cell line. Monoclonal antibodies according to the present invention may for example be prepared as described in Antibodies: A Laboratory Manual, By Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, 1988. Said monoclonal antibodies may be derived from any suitable mammalian species, however frequently the monoclonal antibodies will be rodent antibodies for example murine or rat monoclonal antibodies. It is preferred that the antibodies according to the present invention are monoclonal antibodies or derived from monoclonal antibodies.

Polyclonal antibodies is a mixture of antibody molecules recognising a specific given antigen, hence polyclonal antibodies may recognise different epitopes within said antigen. In general polyclonal antibodies are purified from serum of a mammal, which previously has been immunized with the antigen. Polyclonal antibodies may for example be prepared by any of the methods described in Antibodies: A Laboratory Manual, By Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, 1988. Polyclonal antibodies may be derived from any suitable mammalian species, for example from mice, rats, rabbits, donkeys, goats, sheeps, cows or camels. The antibody is preferably not derived from a non-mammalian species, i.e. the antibody is for example preferably not a chicken antibody. The antibody may also for example be an artificial polyclonal antibody as for example described in US 5,789,208 or US 6,335,163, both patent specifications are hereby incorporated by reference into the application in their entirety.

The antibodies according to the present invention may also be recombinant antibodies. Recombinant antibodies are antibodies or fragments thereof or functional equivalents thereof produced using recombinant technology. For example recombinant antibodies may be produced using a synthetic library or by phage display. Recombinant antibodies may be produced according to any conventional method for example the methods outlined in "Recombinant Antibodies", Frank Breitling, Stefan Dϋbel, Jossey- Bass, September 1999.

The antibodies according to the present invention may also be bispecific antibodies, i.e. antibodies specifically recognising two different epitopes. Bispecific antibodies may in general be prepared starting from monoclonal antibodies, or from recombinant antibodies, for example by fusing two hybridoma's in order to combine their specificity, by Chemical crosslinking or using recombinant technologies. Antibodies according to the present invention may also be tri-specific antibodies.

Functional equivalents of antibodies may in one preferred embodiment be a fragment of an antibody, preferably an antigen binding fragment or a variable region. Examples of antibody fragments useful with the present invention include Fab, Fab', F(ab') 2 and Fv fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc'). Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')2 fragments.

Preferred antibody fragments retain some or essential all the ability of an antibody to selectively binding with its antigen or receptor. Some preferred fragments are defined as follows:

(1 ) Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule. A Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.

(2) Fab' is the fragment of an antibody molecule and can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region.

(3) (Fab')2 is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction. F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds. (4) Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

In one embodiment of the present invention, the antibody is a single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also refered to as "single-chain Fv" or "scFv" antibody fragments. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. The antibody may also be selected for useful properties, for example it may be desirable to control serum half life of the antibody. In general, complete antibody molecules have a very long serum persistence, whereas fragments (<60-80 kDa) are filtered very rapidly through the kidney. Glycosylation on complete antibodies in general, prolongs serum persistence. Hence, if long term action of the antibody is desirable, the antibody against a translational product of the present invention is preferably a complete antibody, whereas if shorter action of the MASP-2 antibody is desirable, an antibody fragment might be preferred. In another embodiment of the present invention the functional equivalent of an antibody is a small molecule mimetics, mimicking an antibody.

In one embodiment of the present invention the antibody or functional equivalent thereof comprises specific hypervariable regions, designated CDR. Preferably, the CDRs are CDRs according to the Kabat CDR definition. CDRs or hypervariable regions may for example be identified by sequence alignment to other antibodies. Preferably, the antibody or funtional equivalent thereof comprises at least one, more preferably at least 2, even more preferably all three heavy chain CDRs.

Human Antibodies

Human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of human antibody genes.

In a preferred embodiment, human monoclonal antibodies directed against a translational product (polypeptide) of a gene of the present invention can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice." The HuMAb mouse contains a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and v) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and K chain loci (Lonberg, N. et al. (1994) Nature 368 (6474):856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or K and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG, K monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 1 13:49-101 ; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536- 546). The preparation of HuMAb mice is described in detail in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg et al., (1994) Nature 368(6474):856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 1 13:49-101 ; Taylor, L. et al. (1994) International Immunology 6:579-591 ; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851. See further, US Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661 ,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, as well as US 5,545,807 to Surani et al.; WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.

The KM mouse contains a human heavy chain transchromosome and a human kappa light chain transgene. The endogenous mouse heavy and light chain genes also have been disrupted in the KM mice such that immunization of the mice leads to production of human immunoglobulins rather than mouse immunoglobulins. Construction of KM mice and their use to raise human immunoglobulins is described in detail in WO 02/43478.

Immunizations

To generate fully human monoclonal antibodies to a translational product (polypeptide) of a gene of the present invention, transgenic or transchromosomal mice containing human immunoglobulin genes (e.g., HCo12, HCo7 or KM mice) can be immunized with an enriched preparation of polypeptide antigen and/or cells expressing said polypeptide, as described, for example, by Lonberg et al. (1994),; Fishwild et al. (1996), , and WO 98/24884. Alternatively, mice can be immunized with DNA of a gene of the present invention. Preferably, the mice will be 6-16 weeks of age upon the first infusion. For example, an enriched preparation (5-50 μg) of the antigen derived from a translational product of the present invention can be used to immunize the HuMAb mice intraperitoneal^. In the event that immunizations using a purified or enriched preparation of the antigen do not result in antibodies, mice can also be immunized with cells expressing a polypeptide encoded by a gene of the present invention, e.g., a cell line, to promote immune responses.

Cumulative experience with various antigens has shown that the HuMAb transgenic mice respond best when initially immunized intraperitoneally (i.p.) or subcutaneously (s.c.) with expressing cells a polypeptide encoded by a gene of the present invention in complete Freund's adjuvant, followed by every other week i.p. immunizations (up to a total of 10) with said polypeptide expressing cells in PBS. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by FACS analysis, and mice with sufficient titers of anti- peptide human immunoglobulin can be used for fusions. Mice can be boosted intravenously with peptide-expressing cells for example 4 and 3 days before sacrifice and removal of the spleen.

Generation of Hybridomas Producing Human Monoclonal Antibodies to translational products of the present invention.

To generate hybridomas producing human monoclonal antibodies to a polypeptide encoded by a gene of the present invention, splenocytes and lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to SP2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581 ) with 50% PEG (w/v). Cells can be plated at approximately 1 x 10⁵ per well in flat bottom microtiter plate, followed by a two week incubation in selective medium containing besides usual reagents 10% fetal Clone Serum, 5-10% origen hybridoma cloning factor (IGEN) and 1X HAT (Sigma). After approximately two weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human kappa- light chain containing antibodies and by FACS analysis using cells expressing a translational product of the present invention for specificity for said translational product. Once extensive hybridoma growth occurs, medium can be observed usually after 10-14 days. The antibody secreting hybridomas can be replated, screened again, and if still positive for human IgG, anti-peptide of the present invention, for example anti-Gadd45b or anti-Gadd45g monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization.

Generation of Transfectomas Producing Human Monoclonal Antibodies to a translational product of a gene of the present invention.

Human antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art, see e.g. Morrison, S. (1985) Science 229:1202.

For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification, site directed mutagenesis) and can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40

(SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or β-globin promoter.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., US 4,399,216, US 4,634,665 and US 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE- dextran transfection, lipofectin transfection and the like.

In one embodiment the antibodies are expressed in eukaryotic cells, such as mammalian host cells. Preferred mammalian host cells for expressing the recombinant antibodies of the invention include CHO cells (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) MoI. Biol. 159:601-621 ), NS/0 myeloma cells, COS cells, HEK293 cells and SP2.0 cells. In particular for use with NS/0 myeloma cells, another preferred expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Further Recombinant Means for Producing Human Monoclonal Antibodies to a polypeptide encoded by a gene of the present invention. Alternatively the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g. E. coli for the production of scFv antibodies, algi, as well as insect cells. Furthermore, the antibodies can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or eggs from hens, or in transgenic plants. See e.g. Verma, R., et al. (1998) "Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems", J. Immunol. Meth. 216:165-181 ; Pollock, et al. (1999) "Transgenic milk as a method for the production of recombinant antibodies", J. Immunol. Meth. 231 :147-157; and Fischer, R., et al. (1999) "Molecular farming of recombinant antibodies in plants", Biol.Chem. 380:825-839. Use of Partial Antibody Sequences to Express Intact Antibodies Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature

332:323-327; Jones, P. et al. (1986) Nature 321 :522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody which contains mutations throughout the variable gene but typically clustered in the CDRs. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy-terminal portion of framework region 4. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/45962). Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons. The nucleotide sequences of heavy and light chain transcripts from hybridomas are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak's rules (Kozak, 1991 , J. Biol. Chem. 266:19867- 19870); and Hind III sites are engineered upstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimized coding and corresponding non-coding, strand sequences are broken down into 30 - 50 nucleotides approximately at the midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into overlapping double stranded sets that span segments of 150 - 400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150 - 400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the Bbsl site of the kappa light chain, or the Agel site of the gamma heavy chain) in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs.

The reconstructed heavy and light chain variable regions are then combined with cloned promoter, leader, translation initiation, constant region, 3' untranslated, polyadenylation, and transcription termination, sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains.

In another aspect of the invention, the structural features of the human antibodies of the invention are used to create structurally related human antibodies that retain at least one functional property of the antibodies of the invention, such as binding to a polypeptide encoded by a gene of the present invention, or parts thereof as defined elsewhere herein. More specifically, one or more CDR regions of 2C6 can be combined recombinantly with known human framework regions and CDRs to create additional, recombinantly-engineered, human antibodies of the invention.

Accordingly, in another embodiment, the invention provides a method for preparing an antibody directed against a polypeptide encoded by a gene of the present invention comprising: preparing an antibody comprising (1 ) human heavy chain framework regions and human heavy chain CDRs; and (2) human light chain framework regions and human light chain CDRs; wherein the antibody retains the ability to bind to a polypeptide encoded by a gene of the present invention.

Monovalent antibodies

The monospecific binding member may be monovalent, i.e. having only one binding domain. For a monovalent antibody, the immunoglobulin constant domain amino acid residue sequences comprise the structural portions of an antibody molecule known in the art as CH 1 , CH2, CH3 and CH4. Preferred are those binding members which are known in the art as CL. Preferrred CL polypeptides are selected from the group consisting of Ckappa and Clambda.

Furthermore, insofar as the constant domain can be either a heavy or light chain constant domain (CH or CL, respectively), a variety of monovalent binding member compositions are contemplated by the present invention. For example, light chain constant domains are capable of disulfide bridging to either another light chain constant domain, or to a heavy chain constant domain. In contrast, a heavy chain constant domain can form two independent disulfide bridges, allowing for the possibility of bridging to both another heavy chain and to a light chain, or to form polymers of heavy chains.

Thus, in another embodiment, the invention contemplates a composition comprising a monovalent polypeptide wherein the constant chain domain C has a cysteine residue capable of forming at least one disulfide bridge, and where the composition comprises at least two monovalent polypeptides covalently linked by said disulfide bridge. In preferred embodiments, the constant chain domain C can be either CL or CH. Where C is CL, the CL polypeptide is preferably selected from the group consisting of Ckappa and Clambda.

In another embodiment, the invention contemplates a binding member composition comprising a monovalent polypeptide as above except where C is CL having a cysteine residue capable of forming a disulfide bridge, such that the composition contains two monovalent polypeptides covalently linked by said disulfide bridge.

Multispecificity, including bispecificity

In a preferred embodiment the present invention relates to multispecific binding members, which have affinity for and are capable of binding at least two different entities. Multispecific binding members can include bispecific binding members.

In one embodiment the multispecific molecule is a bispecific antibody (BsAb), which carries at least two different binding domains, at least one of which is of antibody origin.

A bispecific molecule of the invention can also be a single chain bispecific molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding domain, or a single chain bispecific molecule comprising two binding domains. Multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules.

The multispecific, including bispecific, antibodies may be produced by any suitable manner known to the person skilled in the art.

The traditional approach to generate bispecific whole antibodies was to fuse two hybridoma cell lines each producing an antibody having the desired specificity. Because of the random association of immunoglobulin heavy and light chains, these hybrid hybridomas produce a mixture of up to 10 different heavy and light chain combinations, only one of which is the bispecific antibody. Therefore, these bispecific antibodies have to be purified with cumbersome procedures, which considerably decrease the yield of the desired product. Alternative approaches include in vitro linking of two antigen specificities by chemical cross-linking of cysteine residues either in the hinge or via a genetically introduced C- terminal Cys as described above. An improvement of such in vitro assembly was achieved by using recombinant fusions of Fab's with peptides that promote formation of heterodimers. However, the yield of bispecific product in these methods is far less than 100%.

A more efficient approach to produce bivalent or bispecific antibody fragments, not involving in vitro chemical assembly steps, was described by Holliger et al. (1993). This approach takes advantage of the observation that scFv's secreted from bacteria are often present as both monomers and dimers. This observation suggested that the VH and VL of different chains could pair, thus forming dimers and larger complexes. The dimeric antibody fragments, also named "diabodies" by Hollinger et al., are in fact small bivalent antibody fragments that assembled in vivo. By linking the VH and VL of two different antibodies 1 and 2, to form "cross-over" chains VH 1VL 2 and VH 2-VL 1 , the dimerisation process was shown to reassemble both antigen-binding sites. The affinity of the two binding sites was shown to be equal to the starting scFv's, or even to be 10- fold increased when the polypeptide linker covalently linking VH and VL was removed, thus generating two proteins each consisting of a VH directly and covalently linked to a VL not pairing with the VH. This strategy of producing bispecific antibody fragments was also described in several patent applications. Patent application WO 94/09131 (SCOTGEN LTD; priority date Oct. 15, 1992) relates to a bispecific binding protein in which the binding domains are derived from both a VH and a VL region either present at two chains or linked in an scFv, whereas other fused antibody domains, e.g. C- terminal constant domains, are used to stabilise the dimeric constructs. Patent application WO 94/13804 (CAMBRIDGE ANTIBODY TECHNOLOGY/MEDICAL RESEARCH COUNCIL; first priority date Dec. 4, 1992) relates to a polypeptide containing a VH and a VL which are incapable of associating with each other, whereby the V-domains can be connected with or without a linker.

Mallender and Voss, 1994 (also described in patent application WO 94/13806; DOW CHEMICAL CO; priority date Dec. 1 1 , 1992) reported the in vivo production of a single- chain bispecific antibody fragment in E. coli. The bispecificity of the bivalent protein was based on two previously produced monovalent scFv molecules possessing distinct specificities, being linked together at the genetic level by a flexible polypeptide linker. Traditionally, whenever single-chain antibody fragments are referred to, a single molecule consisting of one heavy chain linked to one (corresponding) light chain in the presence or absence of a polypeptide linker is implicated. When making bivalent or bispecific antibody fragments through the "diabody" approach (Holliger et al., (1993) and patent application WO 94/09131 ) or by the "double scFv" approach (Mallender and Voss, 1994 and patent application WO 94/13806), again the VH is linked to a (the corresponding) VL.

The multispecific molecules described above can be made by a number of methods. For example, all specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the multi- specific molecule is a mAb X mAb, mAb X Fab, Fab X F(ab')2 or ligand X Fab fusion protein. Various other methods for preparing bi- or multivalent antibodies are described for example described in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881 ,175; 5,132,405; 5,091 ,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

By using a bispecific or multispecific binding member according to the invention the invention offers several advantages as compared to monospecific/monovalent binding members.

A bispecific/multispecific binding member has a first binding domain capable of specifically recognising and binding a polypeptide encoded by a gene of the present invention or part thereof, whereas the other binding domain(s) may be used for other purposes:

In one embodiment at least one other binding domain is used for a polypeptide encoded by a gene of the present invention, such as binding to another epitope on the same peptide as compared to the first binding domain. Thereby specificity for polypeptide species may be increased as well as increase of avidity of the binding member.

In another embodiment the at least one other binding domain may be used for specifically binding a mammalian cell, such as a human cell. It is preferred that the at least one other binding domain is capable of binding a neuronal cell, in order to increase the effect of the binding member in a therapeutic method. Accordingly, the present invention includes bispecific and multispecific molecules comprising at least one first binding specificity for a polypeptide encoded by a gene of the present invention and a second binding specificity for a second target epitope.

Bispecific and multispecific molecules of the invention can further include a third binding specificity, in addition to a binding specificity for a polypeptide encoded by a gene of the present invention. In one embodiment, the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one further antibody, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. in US 4,946,778. The antibody may also be a binding-domain immunoglobulin fusion protein as disclosed in US 2003/0118592 and US 2003/0133939.

While human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific or multispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies. Such murine, chimeric and humanized monoclonal antibodies can be prepared by methods known in the art. Bispecific and multispecific molecules of the present invention can be made using chemical techniques (see e.g., D. M. Kranz et al. (1981 ) Proc. Natl. Acad. Sci. USA 78:5807), "polydoma" techniques (see US 4,474,893), or recombinant DNA techniques.

In particular, bispecific and multispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, e.g., the anti-peptide binding specificities, using methods known in the art. For example, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S- acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o- phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, M. A., et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648. Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78, 1 18-132); Brennan et al. (1985) Science 229:81-83, and Glennie et al. (1987) J. Immunol. 139:2367-2375. Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein. A bispecific and multispecific molecule of the invention, e.g., a bispecific molecule can be a single chain molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for preparing bi- and multispecific molecules are described for example in US 5,260,203; US 5,455,030; US 4,881 ,175; US 5,132,405; US 5,091 ,513; US 5,476,786; US 5,013,653; US 5,258,498; and US 5,482,858.

Binding of the bispecific and multispecific molecules to their specific targets can be confirmed by enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), or a Western Blot Assay.

Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the polypeptide encoded by a gene and antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-polypeptide complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986). The radioactive isotope can be detected by such means as the use of a v counter or a scintillation counter or by autoradiography.

Humanised antibody framework It is not always desirable to use non-human antibodies for human therapy, since the non-human "foreign" epitopes may elicit immune response in the individual to be treated. To eliminate or minimize the problems associated with non-human antibodies, it is desirable to engineer chimeric antibody derivatives, i.e., "humanized" antibody molecules that combine the non-human Fab variable region binding determinants with a human constant region (Fc). Such antibodies are characterized by equivalent antigen specificity and affinity of the monoclonal and polyclonal antibodies described above, and are less immunogenic when administered to humans, and therefore more likely to be tolerated by the individual to be treated.

Accordingly, in one embodiment the binding member has a binding domain carried on a humanised antibody framework, also called a humanised antibody.

Humanised antibodies are in general chimeric antibodies comprising regions derived from a human antibody and regions derived from a non-human antibody, such as a rodent antibody. Humanisation (also called Reshaping or CDR-grafting) is a well- established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent), increasing the homology to a human immunoglobulin, and for improving their activation of the human immune system. Thus, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.

It is further important that humanized antibodies retain high affinity for the antigen and other favourable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is maximized, although it is the CDR residues that directly and most substantially influence antigen binding.

One method for humanising MAbs related to production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody are fused to constant domains derived from a second antibody, preferably a human antibody. Methods for carrying out such chimerisation procedures are for example described in EP-A-O 120 694 (Celltech Limited), EP-A-O 125 023 (Genentech Inc.), EP- A-O 171 496 (Res. Dev. Corp. Japan), E P-A-0173494 (Stanford University) and EP-A-O 194 276 (Celltech Limited). A more complex form of humanisation of an antibody involves the re-design of the variable region domain so that the amino acids constituting the non-human antibody binding site are integrated into the framework of a human antibody variable region (Jones et al., 1986).

The humanized antibody of the present invention may be made by any method capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. Winter describes a method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987), the contents of which is expressly incorporated by reference. The human CDRs may be replaced with non-human CDRs using oligonucleotide site- directed mutagenesis as described in the examples below.

As an example the humanized antibody of the present invention may be made as described in the brief explanation below. The humanized antibodies of the present invention may be produced by the following process:

(a) constructing, by conventional techniques, an expression vector containing an operon with a DNA sequence encoding an antibody heavy chain in which the CDRs and such minimal portions of the variable domain framework region that are required to retain antibody binding specificity are derived from a non-human immunoglobulin, and the remaining parts of the antibody chain are derived from a human immunoglobulin, thereby producing the vector of the invention;

(b) constructing, by conventional techniques, an expression vector containing an operon with a DNA sequence encoding a complementary antibody light chain in which the CDRs and such minimal portions of the variable domain framework region that are required to retain donor antibody binding specificity are derived from a non- human immunoglobulin, and the remaining parts of the antibody chain are derived from a human immunoglobulin, thereby producing the vector of the invention;

(c) transfecting the expression vectors into a host cell by conventional techniques to produce the transfected host cell of the invention; and

(d) culturing the transfected cell by conventional techniques to produce the humanised antibody of the invention.

The host cell may be cotransfected with the two vectors of the invention, the first vector containing an operon encoding a light chain derived polypeptide and the second vector containing an operon encoding a heavy chain derived polypeptide. The two vectors contain different selectable markers, but otherwise, apart from the antibody heavy and light chain coding sequences, are preferably identical, to ensure, as far as possible, equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used, the vector including the sequences encoding both the light and the heavy chain polypeptides. The coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both.

The host cell used to express the altered antibody of the invention may be either a bacterial cell such as Escherichia coli, or a eukaryotic cell. In particular a mammalian cell of a well defined type for this purpose, such as a myeloma cell or a Chinese hamster ovary cell may be used. The general methods by which the vectors of the invention may be constructed, transfection methods required to produce the host cell of the invention and culture methods required to produce the antibody of the invention from such host cells are all conventional techniques. Likewise, once produced, the humanized antibodies of the invention may be purified according to standard procedures as described below. Human antibody framework

In a more preferred embodiment the invention relates to a binding member, wherein the binding domain is carried by a human antibody framework, i.e. wherein the antibodies have a greater degree of human peptide sequences than do humanised antibodies. Human mAb antibodies directed against human proteins can be generated using transgenic mice carrying the complete human immune system rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741 ; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21 ; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81 :6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720- 3724; Bruggeman et al. 1991 Eur J Immunol 21 :1323-1326). Such transgenic mice are available from Abgenix, Inc., Fremont, Calif., and Medarex, Inc., Annandale, NJ. It has been described that the homozygous deletion of the antibody heavy-chain joining region (IH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993); and Duchosal et al. Nature 355:258 (1992). Human antibodies can also be derived from phage-display libraries (Hoogenboom et al., J. MoI. Biol. 227: 381 (1991 ); Marks et al., J. MoI. Biol. 222:581-597 (1991 ); Vaughan, et al., Nature Biotech 14:309 (1996)).

Antibody targets

The present invention relates to any antibody, antigen binding fragment or recombinant protein thereof, which is specific for a translational gene product of/polypeptide encoded by a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcM , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18, or a part or functional homolog of said polypeptide. In a preferred embodiment, the antibody, antigen binding fragment or recombinant protein thereof is capable of specifically binding Gadd45a, Gadd45b, Gadd45g and/or Nfkbia. In one embodiment, the antibody, antigen binding fragment or recombinant protein thereof is capable of specifically inhibiting binding of a native protein interaction partner to a polypeptide or part thereof encoded by a gene of the present invention. Preferably, said inhibition of binding is suitable for treatment of a neurodegenerative disorder, such as Parkinson's disease.

In one embodiment of the present invention, the antibody, antigen binding fragment or recombinant protein thereof is capable of specifically recognizing and binding Gadd45a, Gadd45b, Gadd45g and/or Nfkbia.

Specifically, the antibody, antigen binding fragment or recombinant protein thereof according to the present invention, is capable of specifically recognizing and binding to an epitope consisting of 3 to 10 amino acid residues, such as 3 to 8 amino acid residues, such as 3 to 6 amino acid residues selected from a polypeptide sequence of Gadd45b or Gadd45g, and even more specifically selected from any of residues 24- 307, 1016-1351 , 2697-3113 and/or 351 1-3978 of said sequence. Preferably, the antibody, antigen binding fragment or recombinant protein thereof according to the present invention, is capable of specifically recognizing and binding to an epitope consisting of 3 to 10 amino acid residues, such as 3 to 8 amino acid residues, such as 3 to 6 amino acid residues selected from any of residues 1016-1351 of the polypeptide sequence of Gadd45a, Gadd45b, Gadd45g and/or Nfkbia, e.g. SEQ ID NO: 30, 32, 33, 34.

Another aspect of the present invention relates to antibodies and functional homologues thereof, which are able to specifically recognize and bind, and modulate the activity of a polypeptide encoded by a gene of the present invention.

Importantly, the present invention encompasses use of an antibody as defined herein, for the manufacture of a medicament for the treatment of a neurodegenerative disorder.

Also, the present invention encompasses methods of treatment of a neurodegenerative disorder comprising administration of an antibody as described herein to a person in need thereof. The invention also relates to an antibody as defined herein for treatment of a neurodegenerative disorder. Specific examples of neurodegenerative disorder are provided elsewhere herein, and comprise synucleinopathies, such as Parkinson's and Alzheimer's diseases.

Functional homologues Functional homologues of polypeptides according to the present invention is meant to comprise any polypeptide sequence which displays a similar activity as the basic protein. Functional homologues according to the present invention comprise polypeptides with an amino acid sequence, which are sharing at least some homology with the predetermined polypeptide sequences as outlined herein above. For example such polypeptides are at least about 40 percent, such as at least about 50 percent homologous, for example at least about 60 percent homologous, such as at least about 70 percent homologous, for example at least about 75 percent homologous, such as at least about 80 percent homologous, for example at least about 85 percent homologous, such as at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 percent homologous, for example at least 99 percent homologous with the predetermined polypeptide sequences as outlined herein above.

The homology between amino acid sequences may be calculated using well known algorithms such as for example any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.

Functional homologues may comprise an amino acid sequence that comprises at least one substitution of one amino acid for any other amino acid. For example such a substitution may be a conservative amino acid substitution or it may be a non- conservative substitution.

A conservative amino acid substitution is a substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids within predetermined groups exhibit similar or substantially similar characteristics. Within the meaning of the term "conservative amino acid substitution" as applied herein, one amino acid may be substituted for another within groups of amino acids characterised by having i) polar side chains (Asp, GIu, Lys, Arg, His, Asn, GIn, Ser, Thr, Tyr, and Cys,) ii) non-polar side chains (GIy, Ala, VaI, Leu, lie, Phe, Trp, Pro, and Met) iii) aliphatic side chains (GIy, Ala VaI, Leu, lie) iv) cyclic side chains (Phe, Tyr, Trp, His, Pro) v) aromatic side chains (Phe, Tyr, Trp) vi) acidic side chains (Asp, GIu) vii) basic side chains (Lys, Arg, His) viii) amide side chains (Asn, GIn) ix) hydroxy side chains (Ser, Thr) x) sulphor-containing side chains (Cys, Met), and xi) amino acids being monoamino-dicarboxylic acids or monoamino- monocarboxylic-monoamidocarboxylic acids (Asp, GIu, Asn, GIn).

Non-conservative substitutions are any other substitutions. A non-conservative substitution leading to the formation of a functional homologue would for example i) differ substantially in hydrophobicity, for example a hydrophobic residue (VaI, lie, Leu,

Phe or Met) substituted for a hydrophilic residue such as Arg, Lys, Trp or Asn, or a hydrophilic residue such as Thr, Ser, His, GIn, Asn, Lys, Asp, GIu or Trp substituted for a hydrophobic residue; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or GIy by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as GIu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, GIy or Ser (and vice versa).

Functional homologues according to the present invention may comprise more than one such substitution, such as e.g. two amino acid substitutions, for example three or four amino acid substitutions, such as five or six amino acid substitutions, for example seven or eight amino acid substitutions, such as from 10 to 15 amino acid substitutions, for example from 15 to 25 amino acid substitution, such as from 25 to 30 amino acid substitutions, for example from 30 to 40 amino acid substitution, such as from 40 to 50 amino acid substitutions, for example from 50 to 75 amino acid substitution, such as from 75 to 100 amino acid substitutions, for example more than 100 amino acid substitutions.

The addition or deletion of an amino acid may be an addition or deletion of from 2 to 5 amino acids, such as from 5 to 10 amino acids, for example from 10 to 20 amino acids, such as from 20 to 50 amino acids. However, additions or deletions of more than 50 amino acids, such as additions from 50 to 200 amino acids, are also comprised within the present invention.

The polypeptides according to the present invention, including any variants and functional homologues thereof, may in one embodiment comprise more than 5 amino acid residues, such as more than 10 amino acid residues, for example more than 20 amino acid residues, such as more than 25 amino acid residues, for example more than 50 amino acid residues, such as more than 75 amino acid residues, for example more than 100 amino acid residues, such as more than 150 amino acid residues, for example more than 200 amino acid residues.

Additional factors may be taken into consideration when determining functional homologues according to the meaning used herein. For example functional homologues may be capable of associating with antisera which are specific for the polypeptides according to the present invention.

In a further embodiment the present invention relates to functional equivalents which comprise substituted amino acids having hydrophilic or hydropathic indices that are within +/-2.5, for example within +/- 2.3, such as within +/- 2.1 , for example within +/- 2.0, such as within +/- 1.8, for example within +/- 1.6, such as within +/- 1.5, for example within +/- 1.4, such as within +/- 1.3 for example within +/- 1.2, such as within +/- 1.1 , for example within +/- 1.0, such as within +/- 0.9, for example within +/- 0.8, such as within +/- 0.7, for example within +/- 0.6, such as within +/- 0.5, for example within +/- 0.4, such as within +/- 0.3, for example within +/- 0.25, such as within +/- 0.2 of the value of the amino acid it has substituted.

The importance of the hydrophilic and hydropathic amino acid indices in conferring interactive biologic function on a protein is well understood in the art (Kyte & Doolittle, 1982 and Hopp, U.S. Pat. No. 4,554,101 , each incorporated herein by reference). The amino acid hydropathic index values as used herein are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4 ); threonine (-0.7 ); serine (-0.8 ); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5) (Kyte & Doolittle, 1982).

The amino acid hydrophilicity values are: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1 ); glutamate (+3.0.+-.1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1 ); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4) (U.S. 4,554,101 ).

Substitution of amino acids can therefore in one embodiment be made based upon their hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like. Exemplary amino acid substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

In addition to the polypeptide compounds described herein, sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be employed to modify the amino terminus of, e.g., a di-arginine peptide backbone, to mimic a tetra peptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.

Peptides with N-terminal alkylations and C-terminal esterifications are also encompassed within the present invention. Functional equivalents also comprise glycosylated and covalent or aggregative conjugates, including dimers or unrelated chemical moieties. Such functional equivalents are prepared by linkage of functionalities to groups which are found in fragment including at any one or both of the N- and C-termini, by means known in the art.

Functional equivalents may thus comprise fragments conjugated to aliphatic or acyl esters or amides of the carboxyl terminus, alkylamines or residues containing carboxyl side chains, e.g., conjugates to alkylamines at aspartic acid residues; O-acyl derivatives of hydroxyl group-containing residues and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g. conjugates with Met-Leu- Phe. Derivatives of the acyl groups are selected from the group of alkyl-moieties (including C3 to C10 normal alkyl), thereby forming alkanoyl species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species. The reactive groups preferably are difunctional compounds known per se for use in cross-linking proteins to insoluble matrices through reactive side groups.

Homologues of nucleic acid sequences within the scope of the present invention are nucleic acid sequences, which encodes an RNA and/or a protein with similar biological function, and which is either a) at least 50% identical, such as at least 60% identical, for example at least 70% identical, such as at least 75% identical, for example at least 80% identical, such as at least 85% identical, for example at least 90% identical, such as at least 95% identical b) or able to hybridise to the complementary strand of said nucleic acid sequence under stringent conditions.

Stringent conditions as used herein shall denote stringency as normally applied in connection with Southern blotting and hybridisation as described e.g. by Southern E. M., 1975, J. MoI. Biol. 98:503-517. For such purposes it is routine practise to include steps of prehybridization and hybridization. Such steps are normally performed using solutions containing 6x SSPE, 5% Denhardt's, 0.5% SDS, 50% formamide, 100 μg/ml denaturated salmon testis DNA (incubation for 18 hrs at 42⁰C), followed by washings with 2x SSC and 0.5% SDS (at room temperature and at 37⁰C), and a washing with 0.1 x SSC and 0.5% SDS (incubation at 68⁰C for 30 min), as described by Sambrook et al., 1989, in "Molecular Cloning/A Laboratory Manual", Cold Spring Harbor), which is incorporated herein by reference. Homologous of nucleic acid sequences also encompass nucleic acid sequences which comprise additions and/or deletions. Such additions and/or deletions may be internal or at the end. Additions and/or deletions may be of 1-5 nucleotides, such as 5 to 10 nucleotide, for example 10 to 50 nucleotides, such as 50 to 100 nucleotides, for example at least 100 nucleotides.

Compositions

In an important aspect, the present invention relates to compositions, preferably a pharmaceutical composition, comprising at least one compound of the present invention, wherein in its broadest aspect, said compound is capable of a. inhibiting or down-regulating the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd1 16, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudti 8, and/or b. inhibiting or down-regulating the activity of a transcriptional and/or translational product of a gene according to a.

Specifically, the invention relates to compositions comprising at least one compound capable of binding to a gene or gene product of the present invention, thereby inhibiting or down-regulating the activity of a translational product of said gene.

The compositions of the present invention are to be understood as pharmaceutical compositions. The subject to receive treatment is any animal, however, preferably the subject is a human being. Thus, in on aspect, the invention relates to compositions comprising at least one compound of the present invention for the treatment of a neurodegenerative disorder. Thus, the composition of the present invention is in a preferred embodiment, a pharmaceutical composition, said composition comprising at least one compound of the present invention. Specific compounds are disclosed and defined elsewhere herein, however, broadly said at least one compound is capable of a. inhibiting or down-regulating the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudti 8, and/or b. inhibiting or down-regulating the activity of a transcriptional and/or translational product of a gene according to a.

The composition of the present invention comprise in one embodiment at least one additional component. For example the at least one additional component is an adjuvant, an excipient and/or a carrier. A carrier of the present invention is for example selected from the group consisting of keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.

The composition of the present invention is preferably provided in a formulation, which is suitable for pharmaceutical use. Formulations of the compositions of the present invention, routes of administration and the medical use thereof is described in further detail herein below.

Kit-of-parts

The present also provides in one aspect, a kit-of-parts, which comprises a composition of the present invention, and at least one additional active ingredient, wherein said composition in its broadest aspect comprises at least one compound capable of a. inhibiting or down-regulating the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clcfi , Csf1 , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudt18, and/or b. inhibiting or down-regulating the activity of a transcriptional and/or translational product of a gene according to a.

Examples of such additional active ingredients are provided elsewhere herein, when describing formulations of the composition and kits-of-parts of the present invention. In one embodiment, the at least one additional active ingredient is an antibiotic, such as defined elsewhere herein. In one example, the antibiotic is selected from: amoxicillin, penicillin, acyclovir and /or vidarabine. The components of the kit-of-parts of the present invention may be administered by any suitable method of administration, and by any suitable administration regime. Thus, the components or ingredients are to be administered simultaneously or sequentially. Further, the kit-of-parts of the present invention in one embodiment further comprises or contains instructions for combining the components so as to formulate a pharmaceutical composition suitable for administration to a human being. Examples of methods for formulating pharmaceutical compositions are provided elsewhere herein.

Medical use

The present invention provides a number of therapeutic applications. Thus, in one aspect, the present invention relates to a use of a compound, a composition, and/or a kit-of-parts of the present invention for the manufacture of a medicament for treatment, amelioration and/or prevention of a neurodegenerative disorder, in particular for treatment, amelioration and/or prevention of a synucleinopathy, such as Parkinson's or Apzheimer's diseases, Lewy body dementia and/or multiple system atrophy. Thus, one embodiment relates to a use of a compound, such as an siRNA, antibody, peptide, or peptide aptamer of the present invention for the manufacture of a medicament for the treatment of a clinical condition, preferably a neurodegenerative disorder, such as a synucleinopathy, Parkinson's or Alzheimer's diseases. However, a number of clinical conditions may be treated by a composition and/or binding member of the present invention. Accordingly, the compounds, compositions and/or kits-of-parts of the invention may be useful in treatment of said clinical conditions. Hence, it is an object of the present invention to provide use of a compound, composition and/or kit-of-parts according to the invention for the preparation of a pharmaceutical composition for the treatment of a clinical condition associated α-synclein aggragation.

In one aspect, the present invention relates to a compound, a composition, and/or a kit- of-parts of the present invention for treatment, amelioration and/or prevention of a neurodegenerative disorder, as defined herein above.

The present invention provides compositions, compounds, antibodies, peptides and peptide aptamers for treatment of a neurodegenerative disorder, as described elsewhere herein. In a preferred embodiment, the invention provides an antibody as defined herein for treatment of a neurodegenerative disorder. Also provided are compounds as defined elsewhere herein for the treatment of a neurodegenerative disorder, such as a syncleinopathy. The present invention relates to a method of treatment, amelioration and/or prevention of a neurodegenerative disorder comprising administration of a compound, composition or kit-of-parts of the present invention to a person in need thereof.

In one embodiment, the invention provides a method of treatment of a neurodegenerative disorder comprising administration of an antibody, peptide or peptide aptamer of the present invention to a person in need thereof. Also, provided are methods of treatment of a neurodegenerative disorder comprising administration of compound as defined elsewhere herein. The clinical conditions associated with a syncleinopathy may for example be a condition characterised α-synuclein aggregation or Lewy bodies, for example parkinsons or alzheimers disease..

Hence, in one embodiment of the present invention the clinical condition may be characterised by the undesirable presence of aggregated α-synuclein or Lewy bodies. In a preferred embodiment, the clinical condition is a neurodegenerative disorder, such as Parkinson's or Alzheimer's diseases, multiple system atrophy or Lewy body dementia.

The compounds, compositions and/or kits-of-parts according to the present invention that may be used to treat a clinical condition as defined herein may be used alone or in combination with one or more other suitable therapies said clinical condition. Such therapies include but are not limited to surgery, chemotherapy, radiotherapy, gene therapy, therapy with cytokines and immunotherapy. In a specific embodiment, such other therapies are treatment with Parkinson's disease therapeutics and/or Alzheimer's disease therapeutics.

In one preferred embodiment the compounds of the invention may be administered either simultaneously or sequentially in any order in combination with one or more other therapeutic or active ingredient.

Formulation and administration

The main routes of administering a compound, composition, or kit-of-parts of the present invention are parenteral injections, oral, and topical, as will be described below. Other drug-administration methods, such as subcutaneous injection, which are effective to deliver the drug to a target site or to introduce the drug into the bloodstream, are also contemplated.

The mucosal membrane to which the compounds, compositions and kits-of-parts of the invention is administered may be any mucosal membrane of the mammal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum.

Compounds, compositions or kits-of-parts of the invention may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques. The compounds, compositions and kits-of-parts may also be administered by inhalation that is by intranasal and oral inhalation administration. In a preferred embodiment, the compounds, compositions or kits-of-parts of the present invention are delivered by intravenous, subcutaneous, and/or intra-muscular administration.

The compounds, compositions and kits-of-parts according to the invention may be administered with at least one other compound. The compounds, compositions and kits-of-parts may be administered simultaneously, either as separate formulations or combined in a unit dosage form, or administered sequentially.

The dosage requirements will vary with the particular drug composition employed, the route of administration and the particular individual being treated. Ideally, an individual to be treated by the present method will receive a pharmaceutically effective amount of the compound, composition or kit-of-parts in the maximum tolerated dose, generally no higher than that required before drug resistance develops.

For all methods of use disclosed herein for the compounds, compositions or kits-of- parts, the daily oral dosage regimen will preferably be from about 0.01 to about 80 mg/kg of total body weight. The daily parenteral dosage regimen about 0.001 to about 80 mg/kg of total body weight. The daily topical dosage regimen will preferably be from 0.1 mg to 150 mg, administered one to four, preferably two or three times daily. The daily inhalation dosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kg per day. It will also be recognised by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal individuals, each unit containing a predetermined quantity of a compound, alone or in combination with other agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound or compounds employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dose administered should be an "effective amount" or an amount necessary to achieve an "effective level" in the individual patient.

Since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on interindividual differences in pharmacokinetics, drug distribution, and metabolism. The "effective level" can be defined, for example, as the blood or tissue level desired in the individual that corresponds to a concentration of one or more compounds according to the invention.

Pharmaceutical compositions containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.

Pharmaceutical acceptable salts of the compounds according to the present invention should also be considered to fall within the scope of the present invention.

Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent.

The compounds, compositions or kits-of-parts of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount.

Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.

Whilst it is possible for the compositions and compounds or salts of the present invention to be administered as the raw chemical, it is preferred to present them in the form of a pharmaceutical formulation. Accordingly, the present invention further provides a pharmaceutical formulation, for medicinal application, which comprises a compound of the present invention or a pharmaceutically acceptable salt thereof, as herein defined, and a pharmaceutically acceptable carrier therefore, as well as optionally ingredients, such as pharmaceutically acceptable excipients.

The compositions and compounds of the present invention may be formulated in a wide variety of oral administration dosage forms. The pharmaceutical compositions and dosage forms may comprise the compounds of the invention or its pharmaceutically acceptable salt or a crystal form thereof as the active component. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilisers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material. Preferably, the composition will be about 0.5% to 75% by weight of a compound or compounds of the invention, with the remainder consisting of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably containing from one to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration.

Drops according to the present invention may comprise sterile or non-sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the compound or active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100⁰C for half an hour. Alternatively, the solution may be sterilised by filtration and transferred to the container aseptically. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01 %). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Also included are solid form preparations, which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilisers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.

Other forms suitable for oral administration include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilising and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavours, stabilisers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.

The compounds, compositions or kits-of-parts of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions and kits-of-parts may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.

Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides; (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-. beta. -aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimise or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

The compounds, compositions or kits-of-parts of the invention can also be delivered topically. Regions for topical administration include the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat. Compositions for topical administration via the skin and mucous membranes should not give rise to signs of irritation, such as swelling or redness.

The topical composition may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition and kits-of-parts may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry.

The compounds, compositions or kits-of-parts of the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic acid together with an alcohol such as propylene glycol or a macrogel. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturiser such as glycerol or an oil such as castor oil or arachis oil.

The pharmaceutical active compounds, compositions or kits-of-parts described herein can be administered transdermally. Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like. Transdermal delivery is accomplished by exposing a source of the active compound to a patient's skin for an extended period of time. Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modifier complex to the body. See Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery of Drugs, VoIs. 1-3, Kydonieus and Berner (eds.), CRC Press, (1987). Such dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical active compound in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

The compounds, compositions or kits-of-parts of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active compound is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.

The active compound may be formulated into a suppository comprising, for example, about 0.5% to about 50% of a compound of the invention, disposed in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%].

The compounds, compositions or kits-of-parts of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.

Pharmaceutical compositions usually comprise a carrier. Illustrative solid carrier include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. A solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions, and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Illustrative liquid carriers include syrup, peanut oil, olive oil, water, etc. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilisers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carders are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurised compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilised by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The compound can also be administered orally either in liquid or solid composition form.

The carrier or excipient may include time delay material well known to the art, such as glyceryl monostearate or glyceryl distearate along or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like. When formulated for oral administration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentrate with 1 ,2-propylene glycol, A. Nattermann & Cie. GmbH) has been recognised as providing an acceptable oral formulation for other compounds, and may be adapted to formulations for various compounds of this invention.

Diagnostic method and kit

In one aspect, the present invention provides diagnostic methods and kits for determining in a subject, such as a human being, a neurodegenerative disorder or a predisposition of said neurodegenerative disorder, or for assisting in determining a neurodegenerative disorder or a predisposition of a neurodegenerative disorder. Thus in one aspect, the present invention relates to a method for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said method comprising detecting in a biological sample isolated from said subject a. at least one polymorphism of a gene, and/or b. at least one transcriptional and/or translational product of a gene, wherein said gene is selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcM , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18. In another aspect, the invention relates to a method for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said method comprising providing a biological sample isolated from said subject and detecting in said biological sample i) at least one polymorphism or mutation of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfI O, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , Nudt18 and functional homologes thereof, and/or ii) at least one transcriptional and/or translational product of said gene a functional homologes thereof.

The subject is preferably a mammal, such as most preferably a human being. The isolated sample is selected from any biologicallly derived sample, which is suitable for determining gene expression and/or gene product activity, for example, the biological sample is a blood sample, a tissue sample, a secretion sample, semen, ovum, hairs, nails, tears, and urine. Specifically, in the provided diagnostic method, the level of gene product is increased in a sample isolated from a subject suffering from or being predisposed for said neurodegenerative disorder relative to a subject not suffering from said neurodegenerative disorder. Any increase in gene product and/or gene product activity is indicative of a neurodegenerative disorder and/or the predisposition therefore, but preferably, the level should be increased by at least 25%, such as 50%, such as at least 100%, for example at least 200%, such as at least 300% relative to the median for healty people. Polymorphisms or other mutations of a gene of the invention may also affect, the level of gene expression and/or affect the acticvity of a gene product thereof, and therefore, the provided diagnostic method also comprise detecting a polymorphis and/or a mutation in any of the genes or their regulatory sequences, in those situation where the at least one polymorphism leads to increased expression of said gene in a subject relative to a subject not carrying said polymorphism. The tested subject is in a preferably a human being, and the neurodegenerative disorder is preferably a synucleinopathy, such as a neurodegenerative disorder selected from the group consisting of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and multiple system atrophy, preferably Parkinson's disease or alzheimer's disease. In a specifically preferred embodiment of the provided diagnostic method, the gene is Gadd45a, Gadd45g or Nfkbia.

In one embodiment, the level of gene product is increased in a sample isolated from a subject suffering from or being predisposed for said neurodegenerative disorder relative to a subject not suffering from said neurodegenerative disorder, for example said level is increased by at least 10%, 20%, 30%, 40%, 50%, such as at least 100%, for example at least 200%, such as at least 300%, for example at least 400%, such as at least 500%. The relative increase may for example be detected by determining the level of transcript by reverse transriptase quantitative PCR using an oligonucleotide primer, for example a primer as disclosed herein, cf. table 3. The level of transcript may be normalized according to an endogenous transcript, such as illustrated in the example herein below. In one embodiment, the at least one polymorphism leads to increased expression of said gene in a subject relative to a subject not carrying said polymorphism. The neurodegenerative disorder is preferably a synucleinopathy, such as Parkinson's disease. Other neurodegenerative disorders are described elsewhere herein.

In one aspect, the present invention relates to a diagnostic kit for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said kit comprising at least one detection member for detecting in a biological sample isolated from said subject a. at least one polymorphism of a gene, and/or b. at least one transcriptional and/or translational product of a gene, wherein said gene is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csfl , CxcM , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18. The subject is preferably a mammal, such as most preferably a human being.

The genes of the method and diagnostic kit is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudt18. In one embodiment, the genes are selected from any one of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csfl , CxcM , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18. The sequence of the genes is disclosed as SEQ ID NO: 29-59. In a preferred embodiment, the gene is Gadd45a, Gadd45b, Gadd45g, or Nfkbia.

In one embodiment of the kit of the present invention, the detection member is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins. Thus, the detection member is selected from any one of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non- immunoglobulins.

In a specific embodiment, the detection member is an antibody, antigen binding fragment or recombinant protein thereof, such as an antibody, antigen binding fragment or recombinant protein thereof, which selectively binds any translational gene product or polypeptide of the present invention, such as Gadd45a, Gadd45b and/or Gadd45g.

In one embodiment, the detection member is an oligonucleotide primer or probe, such as an oligonucleotide primer or probe, which is linked to a detectable label. In a specific embodiment, the detection member is an oligonucleotide primer or probe consisting of or comprising a sequence selected from the group consisting of SEQ ID NO: 1-20 or SEQ ID NO: 29-59 or the complement or part thereof. In another species embodiment, the detection member is an oligonucleotide primer with a sequence selected from the group consisting of SEQ ID NO: 1-20.

The kit of the present invention also ine one embodiment further comprising reagents and buffers for detection, and/or instructions for performing the detection method and interpretation of the result.

Method of screening

In one aspect, the present invention provides a method for screening pharmaceuticals suitable for treatment of a neurodegenerative disorder as defined herein. Thus in one aspect, the present invention relatest to a method of identifying a drug for treating, preventing or ameliorating a neurodegenerative disorder, said method comprising a. providing a biological sample, b. determining in said biological sample the expression of a gene or the activity of a gene product, wherein said gene is selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a,

Myd1 16, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudt18, c. providing a drug to said biological sample and determining the expression of a gene or the activity of a gene product, wherein said gene is selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clcfl , Csfl , CxcM , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnal , Slc15a4, Slc35b2, Stx11 , and Nudt18, d. comparing the expression of said gene and/or the activity of said gene product in said biological sample in the presence and absence of said drug, wherein in the presence of said drug in said biological sample said expression of said gene and/or said activity of said gene product is inhibited or down-regulated relative to the expression of said gene and/or activity of said gene product in said sample in the absence of said drug.

The method may be used for screening any kind of drug, which may be suitable for treatment of a neurodegenerative disorder. In one example, the screened drugs are selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affi body-antibody chimeras, and non- immunoglobulins.

In the screening method of the present invention, the identified drugs suitable for treatment lead to downregulation of the expression or activity to less than 90%, such as less than 80% such as less than 70% for example less than 60%, for example less than 50%, such as less than 40%, such as less than 30% such as less than 20% for example less than 10%, for example less than 5%. The level of downregulation is compared with the level of expression and/or activity in the biological sample, wherein said drug has not been provided. In one embodiment, the expression or activity is completely inhibited, i.e. downregulated to 0%.

Sequences

SEQ ID NO: 1-20 Oligonucleotide primers or probes, see table 3.

SEQ ID NO: 21-28

Examples of compounds of the present invention, siRNAs targeting Gadd45a and Gadd45g SEQ ID NO: 21 ON-TARGETplus SMARTpool siRNA J-094525-09, GADD45A Target Sequence: CGUGCUUUCUGUUGCGAGA MoI. Wt. Ext. Coeff. 13.444.9 (g/mol) 362,408 (L/mol^*cm)

SEQ ID NO: 22

ON-TARGETplus SMARTpool siRNA 5-094525-1 0, GADD45A Target Sequence: UGUUGCUACUGGAGAACGA MoI. Wt. Ext. Coeff. 13,429.8 (g/mol) 370,151 (L/mol^*cm)

SEQ ID NO: 23

ON-TARGETplus SMARTpool siRNA J-094525-1 I, GADD45A Target Sequence: UCGAAAGGAUGGACACGGU MoI. Wt. Ext. Coeff.

1 3,444.8 (g/mol) 368,104 (L/mol^*cm)

SEQ ID NO: 24

ON-TARGETplus SMARTpool siRNA J-094525-12, GADD45A Target Sequence: ACUGUGUGCUGGUGACGAA MoI. Wt. Ext. Coeff. 13,444.8 (g/mol) 364,900 (L/mol^*cm)

SEQ ID NO: 25 ON-TARGETplus SMARTpool siRNA J-090148-09, LOC291005, GADD45G

Target Sequence: AGAGGUAAGCAAUCGGAAU MoI. Wt. Ext. Coeff.

13.414.8 (g/mol) 377,271 (L/mol^*cm)

SEQ ID NO: 26

ON-TARGETplus SMARTpool siRNA J-090148-10, LOC291005, GADD45G Target Sequence: CCAAUGGAGUGGUUAACGA MoI. Wt. Ext. Coeff.

13.429.9 (g/mol) 371 ,308 (L/mol^*cm) SEQ ID NO: 27

ON-TARGETplus SMARTpool siRNA J-090148-11 , LOC291005, GADD45G Target Sequence: GAGAGCAGUUUCACGUCUA MoI. Wt. Ext. Coeff. 13,429.9 (g/mol) 370,685 (L/mol^*cm)

SEQ ID NO: 28

ON-TARGETplus SMARTpool siRNA J-090148-12, LOC291005, GADD45G Target Sequence: CAUUAAAGUCCCACGCACU MoI. Wt. Ext. Coeff.

13,429.9 (g/mol) 370,507 (L/mol^*cm)

SEQ ID NO: 29-59

Genomic sequences of representative genes of the present invention SEQ ID NO: 29: Genomic sequence of human Bbc3/PUMA - M_014417 SEQ ID NO: 30: Genomic sequence of human Nfkbia - M_020529 SEQ ID NO: 31 : Genomic sequence of human Ninji - M_004148 SEQ ID NO: 32: Genomic sequence of human Gadd45a - M_001924 SEQ ID NO: 33: Genomic sequence of human Gadd45b - M_015675 SEQ ID NO: 34: Genomic sequence of human Gadd45g - M_006705 SEQ ID NO: 35: Genomic sequence of human Ccl2 - NM_002982 SEQ ID NO: 36: Genomic sequence of human Ccl7 - NM_006273 SEQ ID NO: 37: Genomic sequence of human Clcfl - NM_013246 SEQ ID NO: 38: Genomic sequence of human Csfl - NM_00075 eller NM_000757 SEQ ID NO: 39: Genomic sequence of human CxcM - NM_00151 1 SEQ ID NO: 40: Genomic sequence of human Cxcl2 - NM_002089 SEQ ID NO: 41 : Genomic sequence of human CxcHO - NM_001565 SEQ ID NO: 42: Genomic sequence of human IL6 - NM_000600 SEQ ID NO: 43: Genomic sequence of human Camkk2 - M_153500 SEQ ID NO: 44: Genomic sequence of human Duspi - M_004417 SEQ ID NO: 45: Genomic sequence of human Lphn2 - M_012302 SEQ ID NO: 46: Genomic sequence of human RiI - M_003687 SEQ ID NO: 47: Genomic sequence of human Hmoxi - M_002133 SEQ ID NO: 48: Genomic sequence of human Hspala - M_005345 SEQ ID NO: 49: Genomic sequence of human Srxni - M_080725

Examples

Example 1 α-synuclein dependent cell death in oligodendroglial cells is mediated through activation of Fas and TNF receptors

Multiple system atrophy is a parkinsonistic neurodegenerative disorder. It is cytopathologically characterized by the accumulation of the protein p25α in the cell bodies of oligodendrocytes followed by the accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a potent stimulator of α-synuclein aggregation in vitro. Coexpression of α-synuclein and p25α in an oligodendroglial model elicits cellular degeneration, which involves α-synuclein aggregation and phosphorylation at Ser129. The present example provides that the signalling pathway comprises autocrine signalling via extracellular death receptors as demonstrated by the antagonising effect of a Fas blocking antibody and TNFα scavenging drugs (Infliximab and Eternacept). Activation of the Fas and TNFR systems was corroborated by immunohistochemical analysis of human MSA brain tissue that demonstrated a robust upregulation of Fas, TNFR1 , and TNFR2 on myelin sheets as compared to tissue from age-matched controls.

α-synuclein (α-syn) is a natively unfolded protein, which is very abundant in brain particularly in the presynaptic terminals. It accumulates as insoluble aggregates in cytoplasmic inclusions in a subset of neurodegenerative disorders termed α- synucleinopathies, which include Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Point mutations in the α-syn gene (A30P, E46K, and A53T) as well as gene multiplications are associated with autosomal dominant PD and DLB emphasizing the crucial role of α-syn in neurodegeneration. These genetic modifications have been extensively investigated both in vitro and in vivo and have provided important insights into pathogenic mechanisms in PD and related disorders. However, the genetic variations in α-syn only account for a very small proportion of PD cases. The vast majority of disease cases are sporadic and are characterized by insoluble fibrils of wild-type α-syn. Therefore, a major task in the field of neurodegeneration has been to investigate the alterations occurring during disease, which are responsible for the conversion of normal wild-type α-syn into toxic species. Oxidative stress, Ser129 phosphorylation, and abnormal protein degradation are some of the mechanisms, which have been implicated in the aggregation of α-syn. Moreover, some proteins are able to stimulate the aggregation of α-syn in vitro and several of these proteins have been shown to co-localize with α-syn in inclusions in α- synucleinopathies. The oligodendroglial protein p25α is a potent inducer of α-syn aggregation in vitro. Recently, it was shown that p25α redistributed from the myelin sheets to the degenerating cell bodies prior to accumulation and subsequent fibrillization of α-syn in MSA.

Coexpression of α-syn and p25α in an oligodendroglial cell line results in a rapid degeneration of the cells followed by a slow development of apoptotic characteristics. Our model demonstrates pathological characteristics of α-synucleinopathies including a critical role of α-syn aggregation and phosphorylation at Ser129. In the present example is demonstrated a functional role for autocrine signalling through the death receptors Fas and TNF receptor (TNFR) in the α-syn aggregate dependent signalling pathway. This observation was corroborated in human brain tissue affected by MSA where we observed a robust upregulation of Fas, TNFR1 , and TNFR2 on myelin sheets as compared to tissue from age-matched controls. Our results suggest that specific signalling pathways involving death receptors participate in MSA.

Materials and Methods Reagents

Dulbecco's Modified Eagle's Medium (DMEM) was from Lonza (Verviers, Belgium). Fugene-6 Transfection Reagent was purchased from Roche (Mannheim, Germany). Affinity purified rabbit antibodies toward human α-syn (ASY1 ) and human p25α (p25α- 1 ) have been described previously. Monoclonal α-tubulin antibody was obtained from Sigma (Steinheim, Germany) and cleaved caspase-3 (Asp175) rabbit mAb was from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor 488-conjugated goat anti- mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG were from Invitrogen (Leiden, The Netherlands). Caspase inhibitors, Ac-Asp-Glu-Val-Asp-aldehyde (Ac- DEVD-CHO), Ac-lle-Glu-Thr-Asp-aldehyde (Ac-IETD-CHO), and Ac-Leu-Glu-His-Asp- aldehyde (Ac-LEHD-CHO) were purchased from Bachem (Weil am Rhein, Germany). Anti-Fas (clone ZB4) antibody was from Upstate (Temecula, CA, USA). Infliximab (Centocor B.V., Leiden, The Netherlands) and Etanercept (Wyeth, Pennsylvania, USA) were kindly provided by Dr. Jørgen Agnholt, Aarhus University Hospital, Denmark. Plasmids and transfection pcDNA3.1 zeo(-) plasmid expressing human wild-type α-syn was constructed by PCR with pET-1 1d vector containing the human wild-type α-syn gene as a template Similarly, pcDNA3.1 zeo(-) plasmid expressing human p25α was produced by PCR with pET-11 d vector containing the human p25α gene as a template. The products were inserted into pcDNA3.1 zeo(-) vectors, which were transformed into competent E. coli DH5α cells to select positive clones for sequencing. The chosen clones were cultured and plasmid DNA was purified. All constructs were confirmed by sequencing. Transient transfections were performed with Fugene-6 Transfection Reagent according to the manufacturer's protocol.

Cell cultures and chemicals

OLN-93 is an immortalized oligodendroglia cell line derived from primary Wistar rat brain glial cultures. These cells were engineered to stable express the longest human tau isoform (Tau40) establishing a stable cell line, OLN-t40. Additionally, a stable cell line, OLN-AS, was established by lentiviral transduction of α-syn into OLN-t40 cells (26). All cells were kept at 37°C under 5% CO₂ and grown in DMEM supplemented with 10% fetal calf serum, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. OLN-AS cells were maintained in 50 μg/ml geneticin. For inhibition of caspase activity, cells were treated with 20 μM of caspase-3, -8, and -9 inhibitors, Ac-DEVD-CHO, Ac-IETD-CHO, or Ac-LEHD-CHO, for 1 h prior to transfection with p25α. For inhibition of Fas and TNFR signalling, cells were pretreated with 1 μg/ml of ZB4 (anti-Fas antibody), 10 μg/ml of Infliximab (anti-TNFα antibody), or 10 μg/ml Eternacept (soluble TNFR) for 1 h prior to transfection with p25α.

Assessment of cellular degeneration and apoptosis

Cells were immunostained for α-tubulin and p25α, counterstained with DAPI, and analysed by fluorescence microscopy. Cellular degeneration was defined as retraction of microtubule (MT) from cellular processes to the perinucler region and quantified by counting p25α-positive cells displaying MT retraction compared to the total number of p25α-positive cells. In each experimental condition, 120-200 transfected cells localized in five randomly chosen microscopic fields were examined at 100 times magnification. Cells were counted by Christine Lund Kragh and another investigator blind to the experimental conditions. Nuclear morphology was evaluated by DAPI staining and apoptotic cells were identified by a condensed or fragmented nucleus. Apoptosis elicited by α-syn and p25α was additionally determined by cleaved caspase-3 immunostaining. Signals were analyzed by fluorescence microscopy.

Semiquantitative RT-PCR analysis

Total RNA was isolated from OLN-AS cells by using RNeasy Mini Kit including on- column DNAse treatment (Qiagen, Germany). The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis. First-strand cDNA was prepared using Protoscript Il RT-PCR Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. 1 μg RNA was reverse transcribed and the obtained cDNA was used for the subsequent PCR amplification of rat Fas, rat FasL, rat TNF-R1 , rat GAPDH, and human p25α. Primer sequences were as follows: rat Fas: 5^'- CTGCAGATATGCTGTGGATCA-3^' and δ^'-TTTGGTGTTGCTGGTTCGT-S^' (491 -bp product), rat FasL: δ^'-AAAGACCACAAGGTCCAACA-S^' and 5^'-

AGTCTCTAGCTTATCCATGA-3^' (341-bp product), rat TNF-R1 : 5^'- GGGATTCAGCTCCTGTCAAA-3^' and δ^'-ATGAACTCCTTCCAGCGTGT-S^', (399-bp product), rat GAPDH: δ^'-CCCACGGCAAGTTCAACGGCA-S^' and 5^'- TGGCAGGTTTCTCCAGGCGGC-3^' (603-bp product), human p25α: 5^'- ATGGCTGACAAGGCCAAGCC-3^'and 5^'-CTACTTGCCCCCTTGCAC CTT-3^' (660-bp product). For PCR amplification of specific cDNAs, the 50-μl reactions contained the following: 5 μl of the cDNA synthesized in the reverse transcription reaction, 200 nmol of each primer, and 25 μl Taq 2X Master Mix. Fas, FasL, p25α, and GAPDH cDNA was amplified during 35 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 30 s. For the amplification of TNFR1 cDNA, an annealing temperature of 57°C was used.

Amplification was done in a DNA thermal cycler (Eppendorf), products were analysed on a 0.8 % agarose gel in the presence of ethidium bromide and visualized with UV illumination.

RNA isolation and cRNA preparation

Isolation of total RNA from OLN-AS cells was performed using RNeasy Mini Kit including on-column DNAse treatment (Qiagen, Germany). The purity and integrity of the isolated RNA was checked spectrometrically and by agarose gel electrophoresis. Preparation of cRNA was performed according to the manufacturer^'s protocol (Gene Chip Expression Analysis, Technical Manual (Affymetrix)). In brief, first-strand cDNA synthesis was performed on 2 μg total RNA for 1 h at 42°C with Superscript Il reverse transcriptase and an oligo(dT) primer containing a T7 RNA polymerase promoter. Second-strand cDNA synthesis was performed for 2 h at 16°C using DNA polymerase I, DNA ligase, and RNase H followed by purification of the double-stranded cDNA. Biotin-labelled cRNA was generated from the cDNA by in vitro transcription with T7 RNA polymerase 16 h at 37°C using Affymetrix IVT labelling kit. Biotin-labelled cRNA was purified and fragmented for 35 min at 94°C.

Microarray analysis Biotinylated cRNAs (15 μg) generated from independent cell culture preparations were hybridized onto individual Affymetrix RAE 230 2.0 oligonucleotide arrays for 16 h at 45°C according to the manufacturer's protocol (Gene Chip Expression Analysis, Technical Manual (Affymetrix)). After hybridization, the microarrays were washed and stained with streptavidin-phycoerythrin using a fluidics station. The arrays were scanned with a Hewlett-Packard Gene Array 3000 7G Scanner (Hewlett-Packard, Palo Alto, CA, USA).

The Affymetrix RAE 230 2.0 oligonucleotide array contains approximately 31 ,000 probe sets. Each gene is represented by 1 1 pairs of 25 mer sequence probes that contain a perfectly matched probe and a mismatched probe, which serves as control for background signals. The raw image files from the quantitative scanning were analyzed by Gene Expression Analysis Software (MAS 5.0) (Affymetrix) resulting in CEL files containing background corrected probe values.

Two identical independent experiments were performed analyzing three time points in mock- versus p25α-transfected cells (8, 12, and 16 h after transfection) resulting in a total of twelve arrays. To identify genes that were differential expressed between the two conditions, the data set was filtered according to several criteria, i) The dataset was filtered by the Detection Call (Present (P), Marginal (M), or Absent (A)). Probe sets that were either increasing to an absent call or decreasing from an absent call were excluded from the dataset. ii) Data were included only if at least one of the signal intensities in a comparison was ≥ 50. iii) Genes with no name or annotation were excluded from the data set.

lmmunohistochemistry

Brain tissues from pathologically confirmed MSA cases (n = 5) and from control cases (n = 5) were obtained from the NHMRC South Australia Brain Bank (Table 1 ). Tissue was sampled from frontal, temporal, and parietal association and primary cortices, basal ganglia, basal forebrain, and brainstem. Sections were deparaffinized in xylene, rehydrated through serial changes of ethanol gradients, and boiled in citrated buffer containing 1 mM EDTA pH 8.0 for 10 min. Endogenous peroxidase was blocked by incubating sections in 1 % H₂O₂/50% methanol for 10 min. Sections were blocked with 20% normal horse serum for 1 h and incubated overnight at RT with primary antibodies diluted in TBS pH 7.4 containing 1% normal horse serum. Sections were subsequently incubated with secondary antibody (biotinylated donkey-anti-mouse antibody) for 1.5 h, Vectastain ABC (Vector Labs) for 1 h, and developed in DAB solution (SigmaFast) for 10 min. Sections were counterstained with Haematoxylene, dehydrated, and coverslipped. Primary antibodies were mouse anti-FAS (1 :40) (NovaCastra, Newcastle,

UK), mouse anti-TNFRI (1 :500) (Chemicon), and mouse anti-TNFRII (1 :250) (R&D

Systems).

For immunofluorescence staining, sections were incubated overnight with anti-Fas (1 :10), anti-TNFRI (1 :50), or anti-TNFRII (1 :250) mixed with rabbit anti-neurofilament antibody (1 :100) (Sigma) or rabbit anti-myelin basic protein antibody (1 :200) (DAKO, Glostrup, Denmark). Subsequently, sections were incubated with CY3-labelled donkey- anti-mouse antibody (1 :100) and CY2-labelled donkey-anti-rabbit antibody (1 :50) for 1 h (Jackson lmmunoresearch Laboratories). Sections were coverslipped with buffered glycerol and examined with a Leica SP5 confocal microscope.

Results

Activation of Fas and TNF receptors is involved in α-synuclein dependent degeneration

Coexpression of α-syn and p25α in the oligodendrocyte cell line OLN-93 induces cellular degeneration as demonstrated by a retraction of MT from the cellular processes to the perinuclear region within 24 h after transfection. Furthermore, the cotransfected cells develop apoptotic characteristics such as caspase-3 activation, phosphatidylserine externalization, and chromatin condensation after 24-48 h. Active caspase-3 is a classical marker of apoptosis and is activated by upstream caspases that are triggered via different pathways. Caspase-9 is activated by the apoptosome in the mitochondrial pathway and caspase-8 is activated upon ligand binding to membrane-associated death receptors including Fas and TNFR. The involvement of caspase-8 and -9 in the activation of caspase-3 was studied by treating OLN-AS cells transiently transfected with p25α with 20 μM of the peptide aldehyde inhibitors, DEVD, IETD, and LEHD, specific for caspase-3, -8, and -9, respectively. Fig. 1 A shows that both DEVD and IETD rescued the cells from degeneration as demonstrated by a significant reduction in transfected cells with MT retraction. In contrast, LEHD, did not protect the cells. These results indicate that caspase-8 and caspase-3 are involved in p25α-induced degeneration in OLN-AS cells. Caspase-8 is a downstream effector of ligand binding to the death receptors, Fas and TNFR (29). The FasL/Fas pathway was studied using the anti-Fas antibody, ZB4, which antagonizes FasL binding to Fas, thus blocking signalling through the Fas receptor. The TNFα/TNFR pathway was investigated using Infliximab (Remicade™), which is a mouse-human chimeric anti-human TNFα antibody. Fig. 1 B demonstrates that pretreatment of the cells with ZB4 or Infliximab reduced MT retraction by approximately 45%. This reduction was dose-dependent but could not be enhanced above the level demonstrated in Fig. 1 B (data not shown). Eternacept (Enbrel™), which is a recombinant fusion protein of human soluble TNFR2 coupled to the Fc portion of human IgG, was as efficient as Infliximab (Fig. 1 B). Both Infliximab and Eternacept are approved as human therapeutics and act as neutralizers of TNFα, thus preventing further interactions with TNFR. These data demonstrate that the extracellular Fas/TNFR systems are activated upon coexpression of p25α and α-syn and contribute to degeneration.

TNF receptor 1 mRNA levels are upregulated during the degenerative process.

To examine the effects of coexpression of α-syn and p25α on the mRNA levels of death receptors, OLN-AS cells was transfected with an empty control vector or p25α pcDNA3.1 for 24 h. Subsequently, total RNA was isolated and subjected to semiquantitative RT-PCR using specific primers for Fas, FasL, TNFR1 , p25α, and GAPDH. Total RNA isolated from PC12 cells was used as positive control for the RT- PCR reactions as naive PC12 cells constitutively express Fas, FasL, and TNFR1 (30). We observed an increase of approximately 20% in the TNFR1 mRNA levels upon transfection of p25α into OLN-AS cells. However, Fas and FasL mRNA levels did not change following p25α transfection (Fig. 2). Gene Chip analyses (Affymetrix RAE 230 2.0) of two independent experiments using RNA isolated from OLN-AS cells revealed inconsistent changes in Fas and TNFR1 mRNA levels in p25α-transfected cells as compared to mock-transfected cells in the two experiments (Table 2). In experiment 1 , we observed an increase of 25% in Fas mRNA levels 12 h after transfection, which was not confirmed in experiment 2. When analyzing the expression levels of TNFR1 , we observed an increase of 47% at 8 h after transfection and an increase of 20% at 16 h. In experiment 2, the observed increase in TNFR1 expression levels was 22%, 36%, and 22% at 8, 12, and 16 h, respectively. Thus, our data from the microarray analyses corroborates the semiquantitative RT-PCR analysis demonstrating that TNFR1 mRNA is upregulated in response to p25α expression in OLN-AS cells. The expression levels of FasL and TNFα are low and did not fulfill the criteria of a signal intensity of ≥ 50 (Table 2). TNFR2 was not present on the gene chip and was therefore not analysed. Thus, our results suggest that activation of the death receptors may be partly explained by an increase in TNFR1 mRNA levels. However, we did not observe an increase in Fas expression levels, which may suggest a role of posttranscriptional regulation of extracellular death receptor presentation.

Fas and TNF receptors are upregulated in human MSA tissue

The involvement of TNFR and Fas in the α-syn dependent degeneration in the oligodendroglial cell line prompted the investigation of the expression of these membrane receptors in human brain tissue affected by MSA. lmmunohistochemical analysis revealed increased Fas, TNFR1 , and TNFR2 immunoreactivity in MSA tissue compared to control tissue. The staining was associated with axons that were most abundant in deep layers of neocortex, brainstem, and forebrain major fiber tracts as demonstrated for putamen and external capsule (Fig. 3A, middle). The staining was abolished when primary antibodies were omitted (Fig. 3A, right). Among the three receptors, the increase in TNFR2 immunostaining was most obvious (Fig. 3A, lower row, middle). The increase was consistent among the five MSA cases and detectable in all examined brain regions, but most pronounced in the basal pons and fiber tracts through the basal ganglia including internal and external capsules (Fig. 3A, middle). Interestingly, GCIs which are abundant in basal ganglia and brainstem were not stained for these receptors as confirmed by double staining using α-syn or p25α as markers (data not shown). To further refine the localization of TNFR in axonal fibers, brain sections from two MSA cases containing basal ganglia regions were double immunostained for TNFR2 and neurofilament (NF) protein or myelin basic protein (MBP). Sections were examined by confocal laser scanning microscopy. It was clear that TNFR2 colocalized with MBP on nerve fibers sectioned longitudinally (Fig. 3B, upper row) and transversely (Fig. 3B, middle row). However, TNFR2 immunoreactivity was clearly wrapped around NF, which labelled the axons of nerve fibers (Fig. 3B, lower row). Thus, TNFR2 expression is associated with the oligodendroglial myelin sheets, not the neuronal component of axon fibers. In conclusion, coexpression of α-syn and p25α in the oligodendroglial cell line elicits a α-syn- dependent degenerative response. The degeneration could be attenuated by inhibitors of caspase-3 and -8, as well as antagonists of Fas and TNFR. The enhanced expression of Fas and TNFR in oligodendroglial myelin sheets in MSA tissue further corroborates the significance of the α-syn aggregate dependent signalling pathways in MSA.

Discussion

As mentioned above, coexpression of α-syn and p25α in the oligodendrocyte cell line OLN-93 caused degeneration of the cells and subsequently apoptosis. The cellular degeneration could be attenuated by inhibitors of Ser129 phosphorylation, α-syn aggregation, and caspase-3 activity. In the present example, an involvement is proposed of an extracellular signalling loop in this process. It is thus, demonstrated that the degenerative process could be significantly reduced by a peptide inhibitor of caspase-8, thus suggesting that the caspase-8/3 pathway is activated in the α-syn dependent degeneration. Caspase-8 activation has been demonstrated in post-mortem brain tissue from patients with PD, Alzheimer^'s disease (AD), and Huntington^'s disease (HD) as well as in MPTP treated mice suggesting a contribution of caspase-8 in neurodegenerative disorders. Caspase-8 activation prompted us to test compounds that blocked Fas and TNFR signalling. Indeed, blocking Fas with a monoclonal antibody and scavenging of extracellular TNFα by either a monoclonal TNFα antibody (Infliximab) or a soluble TNFα receptor (Eternacept) reduced the degeneration significantly. Thus, the model predicts autocrine signalling via FasL and TNFα in α-syn-dependent degeneration in oligodendroglial cells. Microarray demonstrated a moderate increase in TNFR1 mRNA levels, whereas the change in Fas expression levels was inconsistent in the two experiments. The upregulation of TNFR1 mRNA was confirmed by semiquantitative RT-PCR. Activation of Fas without a concomitant increase in expression levels may suggest posttranscriptional regulation of death receptor presentation. Indeed, the expression of the death receptors and their ligands has to be tightly regulated and besides transcriptional control, several posttranscriptional processes modulate their expression. Cleavage by matrix metalloproteinases (MMPs) is one important mechanism for posttranscriptional regulation of these molecules. Fas and FasL are cleaved by specific MMPs leading to shedding of soluble Fas and FasL. Similarly, MMPs cleaves pro-TNFα on the cell surface, releasing mature, soluble TNFα from the cell membrane. TNFR1 and TNFR2 are also cleaved by MMPs and shedded from the membrane.

Human MSA brain tissue demonstrated an increased expression of Fas, TNFR1 , and TNFR2 protein on the myelin sheets in white matter. The increased expression of Fas, TNFR1 , and TNFR2 was consistent in fiber tracts in white matter of all examined MSA cases. We noted a clear colocalization of TNFR2 and MBP and the presence of TNFR2 surrounding NF, thus demonstrating that TNFR2 expression is associated with the myelin sheets of axons. Fas is involved in motorneuron death in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) and enhanced levels of TNFα, TNFR1 , and TNFR2 have been detected in plasma from ALS patients. Furthermore, the Fas and TNFR signalling pathways have been associated to oligodendroglial degeneration in experimental spinal cord injury as well as experimental autoimmune encephalitis. An upregulation of apoptotic proteins such as Bax, FasL, and TNFα has been demonstrated in oligodendrocytes in MSA, whereas neuronal apoptosis has not observed suggesting that neurones are destroyed either by necrosis or by a form programmed cell death other than apoptosis. A direct relation of the death receptors to oligodendrocytes affected in MSA has previously been limited to genetic association studies, which did not reveal a consistent association. Our in vitro and in vivo data support the hypothesis that the Fas and TNFR pathways act as active signal transducers in the degenerative process in MSA. This hypothesis may be tested by the use of TNFα neutralizing drugs that previously have been used for the treatment of systemic diseases. A recent report demonstrates symptomatic relief upon perispinal injections of TNFα antagonists in patients with AD. Furthermore, thalidomide treatment to reduce TNFα expression has been shown to cause neuroprotection in a G93A superoxide dismutase 1 transgenic ALS mouse model suggesting that such experimental strategies could be used in future clinical trials of MSA.

Table 1. MSA and control cases

Brain tissue from pathologically confirmed MSA cases and from control cases were obtained from the NHMRC South Australia Brain Bank. Neuropathological diagnosis was confirmed by Prof. Peter C. Blumbergs, Department of Neuropathology, Institute of Medical and Veterinary Science, Adelaide, Australia. PMI, Postmortem interval.

Example 2

Gene expression changes induced by α-synuclein dependent degeneration in oligodendroglial cells.

Abstract

Aggregation of α-synuclein is a characteristic of α-synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, α- synuclein plays a direct pathogenic role as demonstrated by early-onset disease caused by missense mutations and multiplications of the α-synuclein gene. Coexpression of α-synuclein and the pro-aggregatory protein p25α in an oligodendroglial model causes cellular degeneration, which is dependent on α- synuclein aggregation and Ser129-phosphorylation. However, the molecular signalling pathways are currently unknown. Using microarray based gene expresssion analysis of oligodendroglial cells coexpressing α-synuclein and p25α, differentially regulated genes was investigated and genes identified which belong to different functional groups such as cell cycle regulation, apoptosis, transcription factors, and stress response. In particular, a range of genes involved in the NF-κB pathway was upregulated. The present example provides that down-regulation of growth arrest- and DNA-damage- inducible proteins, Gadd45a and Gadd45g expression by siRNA rescues the cells from degeneration, thus providing that induction of Gadd45a and g though NF-κB inhibition is involved in α-synuclein-dependent degeneration.

Introduction α-synuclein (α-syn) is a natively unfolded protein normally localized in presynaptic terminals. It has the propensity to aggregate into β-folded amyloid structures. Aggregated α-syn is a major component of pathological inclusions such as Lewy bodies (LBs) and Lewy neurites in Parkinson^'s disease (PD) and dementia with Lewy bodies (DLB) and glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA). Missense mutations in the α-syn gene leading to amino acid substitutions (A30P, E46K, A53T) or gene multiplications of the α-syn gene have been associated with familial PD and DLB. Thus, α-syn is believed to play a key role in the pathogenesis of the so-called α-synucleinopathies, which comprise PD, DLB, MSA as well as other neurodegenerative disorders characterized by α-syn inclusions. The mechanisms responsible for aggregation and subsequent degeneration are unclear but several factors have been shown to influence the aggregation process in vitro including phosphorylation at Ser129, C-terminal proteolysis, oxidation, and interaction with other proteins such as p25α and tau. p25α is normally an oligodendrocyte-specific protein but co-localizes with aggregated α-syn in LBs in PD and DLB suggesting an abnormal expression of the protein in affected neurons. Additionally, p25α colocalizes with α-syn in GCIs in MSA.

Coexpression of α-syn and p25α in the oligodendrocyte cell line, OLN-93, causes a fast degeneration of the cells as evidenced by microtubule (MT) retraction and later development of apoptotic characteristics such as caspase-3 activation, phosphatidylserine externalization, and nuclear fragmentation. The degenerative process is dependent upon α-syn aggregation and phosphorylation at Ser129. To identify molecular signalling pathways involved in α-syn-dependent degeneration in OLN cells, a microarray-based approach is employed to analyze the gene expression pattern during the cellular degeneration. Of approximately 28,000 genes present on the gene chip, 104 genes was found to be differentially regulated upon coexpression of α- syn and p25α. The identified genes belong to different functional groups such as cell cycle regulation, apoptosis, transcription factors, and stress response. Several of the identified genes are involved in the NF-κB pathway and thusprovides that inhibition of this pro-survival pathway is involved in the degenerative process. Moreover, silencing of the Gadd45 (growth arrest and DNA-damage-inducible 45) genes, Gadd45a and -g, by siRNA significantly reduces the α-syn-dependent degeneration. This indicates that NF-κB inhibition leads to transcriptional induction of the Gadd45a and -g genes, which is critical for the degenerative process.

Materials and Methods

Reagents

Dulbecco's Modified Eagle's Medium (DMEM) was from Lonza (Verviers, Belgium).

Fugene-6 Transfection Reagent was purchased from Roche (Mannheim, Germany).

Affinity purified rabbit antibody toward human p25α (p25α-1 ) has been described previously (19). Monoclonal α-tubulin antibody was obtained from Sigma (Steinheim,

Germany). Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568- conjugated goat anti-rabbit IgG were from Invitrogen (Leiden, The Netherlands).

Cell culture OLN-93 is an immortalized oligodendroglia cell line derived from primary Wistar rat brain glial cultures (22). These cells were engineered to express the longest human tau isoform (tau40) establishing a stable cell line, OLN-t40 (23). Additionally, a stable cell line, OLN-AS, was established by lentiviral transduction of α-syn into OLN-t40 cells (24). All cells were kept at 37°C under 5% CO2 and grown in DMEM supplemented with 10% fetal calf serum, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. OLN-AS cells were maintained in 50 μg/ml geneticin.

Plasmids and transfection pcDNA3.1 zeo(-) plasmid expressing human p25α was produced by PCR with pET-1 1d vector containing the human p25α gene as a template (19). The product was inserted into pcDNA3.1 zeo(-) vector, which was transformed into competent E. coli DH5α cells to select positive clones for sequencing. The chosen clones were cultured and plasmid DNA was purified. p25α pcDNA3.1 was transfected into OLN-AS cells using Fugene-6 Transfection Reagent according to the manufacturer's protocol.

RNA isolation and cRNA preparation

Isolation of total RNA from OLN-AS cells was performed using RNeasy Mini Kit including on-column DNAse treatment (Qiagen, Germany). The purity and integrity of the isolated RNA was checked spectrometrically and by agarose gel electrophoresis. Preparation of cRNA was performed according to the manufacturer^'s protocol (Gene Chip Expression Analysis, Technical Manual (Affymetrix)). In brief, first-strand cDNA synthesis was performed on 2 μg total RNA for 1 h at 42°C with Superscript Il reverse transcriptase and an oligo(dT) primer containing a T7 RNA polymerase promoter. Second-strand cDNA synthesis was performed for 2 h at 16°C using DNA polymerase I, DNA ligase, and RNase H followed by purification of the double-stranded cDNA. Biotin-labelled cRNA was generated from the cDNA by in vitro transcription with T7 RNA polymerase for 16 h at 37°C using Affymetrix IVT labelling kit. Biotin-labelled cRNA was purified and fragmented for 35 min at 94°C.

Microarray analysis

Biotinylated cRNAs (15 μg) generated from independent cell culture preparations were hybridized onto individual Affymetrix RAE 230 2.0 oligonucleotide arrays for 16 h at 45°C according to the manufacturer's protocol (Gene Chip Expression Analysis, Technical Manual (Affymetrix)). After hybridization, the microarrays were washed and stained with streptavidin-phycoerythrin using a fluidics station. The arrays were scanned with a Hewlett-Packard Gene Array 3000 7G Scanner (Hewlett-Packard, Palo Alto, CA, USA).

The Affymetrix RAE 230 2.0 oligonucleotide array contains approximately 31 ,000 transcripts representing 28,000 validated genes. Each gene is represented by 1 1 pairs of 25-mer sequence probes that contain a perfectly matched probe and a mismatched probe, which serves as control for background signals. The raw image files from the quantitative scanning were analyzed by Gene Expression Analysis Software (MAS 5.0) (Affymetrix) resulting in CEL files containing background corrected probe values. Two identical independent experiments were performed analyzing three time points in mock- versus p25α-transfected cells (8, 12, and 16 h after transfection) resulting in a total of twelve arrays. To identify genes that were differential expressed between the two conditions, the data set was filtered according to several criteria, i) The dataset was filtered by the Detection Call (Present (P), Marginal (M), or Absent (A)). Probe sets that were either increasing to an absent call or decreasing from an absent call were excluded from the dataset. ii) Data were included only if the absolute change in signal level was ≥ 1.3-fold, iii) Data were included only if at least one of the signal levels in a comparison was ≥ 50. iv) Genes with no name or annotation were excluded from the data set.

Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay

Reverse transcription was performed by adding 1 μl of the extracted mRNA to a reaction mixture consisting of 2 μl 1OxPCR buffer Il (Applied Biosystems, Naerum, Denmark) supplemented with 6.3 mM MgCI2, 0.3 mM of each of the four deoxyribonucleoside triphosphates (dATP, dTTP, dGTP, dCTP), 2.5 mM 16mer oligo dT nucleotide, 20 U RNase inhibitor, and 50 U MULV reverse transcriptase in a total volume of 20 μl (All reagents from Applied Biosystems, Naerum, Denmark). The cDNA synthesis was carried out in a GeneAmp® PCR System 9700 Thermal Cycler (Applied Biosystems, Naerum, Denmark) at 42 ⁰C for 30 min followed by 99 ⁰C for 5 min. The synthesized cDNA provided template for the real-time qPCR assay and was stored at -20 ⁰C.

Real-time RT-qPCR assays were performed in triplicate using the LightCycler® 480 (Roche Diagnostics, Hvidovre, Denmark) in a 96 multiwell plate format. The reaction volume in each well consisted of 5 μL LightCycler® 480 SYBR Green I Master (Roche Diagnostics, Hvidovre, Denmark) (containing FastStart Taq DNA Polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), SYBR Green I dye, and MgCI2), 0.5 μl_ of each primers, and 1 μl_ of synthesized sample cDNA. The volume was adjusted to 10 μl with nuclease-free H₂O. The amplification conditions consisted of an initial denaturation step of 95 ⁰C for 10 min, followed by 50 cycles of 95°C for 10 seconds, primer dependent annealing for 20 seconds and 72°C for 5 seconds. Primers and annealing conditions are listed in Table 3. A standard curve was included in each run. The standard curve used to quantify mRNA in all reactions consisted of a serial dilution of RNA from rat intestinal epithelial (RIE)-I cells (a kind gift from Dr. Kenneth D. Brown; Cambridge Research Station, Babraham, Cambridge, UK). The levels of mRNA were normalized against the NADH mRNA content. NADH was chosen as reference gene as its expression was constant in all experimental conditions as revealed by microarray analysis. Real-time RT-qPCR data was analysed using LightCycler® 480 Software 1.5.0 (Roche Diagnostics, Hvidovre, Denmark). Primer specificity was verified by gel electrophoresis of the generated product. Furthermore, amplification specificity was investigated by melting curve analysis.

Gadd45 RNAi silencing siRNA pools targeting rat Gadd45a and Gadd45g were purchased from Dharmacon (Lafayette, CO, USA), cf. SEQ ID NO: 21-28. A non-targeting siRNA pool (siControl) was included as a negative control. Transfection of siRNA into OLN-AS cells was performed using Dharmafect Transfection Reagent 1 according to the manufacturer's instructions. In brief, 100 nM siRNA was transfected into OLN-AS cells using 1 μl Dharmafect Transfection Reagent 1 for cells in each well of 24-well plates. Silencing of Gadd45a and g was confirmed by semiquantitative RT-PCR. The effect of Gadd45a and g silencing on p25α-transfected OLN-AS cells was evaluated by transfecting the cells with siRNA for 72 h followed by transient transfection with p25α. Cellular degeneration was estimated 24 h after p25α-transfection.

Semiquantitative RT-PCR

Total RNA was isolated from OLN-AS cells by using RNeasy Mini Kit (Qiagen, Hilden, Germany). The purity and integrity of the RNA was checked spectroscopically and by gel electrophoresis. First-strand cDNA was prepared using Protoscript Il RT-PCR Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. 1 μg RNA was reverse transcribed and the obtained cDNA was used for the subsequent PCR amplification of rat Gadd45a, Gadd45g, and GAPDH. Primer sequences are listed in Table 3. For PCR amplification of specific cDNAs, the 50-μl reactions contained the following: 5 μl of the cDNA synthesized in the reverse transcription reaction, 200 nmol of each primer, and 25 μl Taq 2X Master Mix. cDNA was amplified during 35 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 30 s.

lmmunocytochemistry

Cells were cultured on poly-L-lysine coated coverslips for 24 h followed by transfection with p25α. For analysis, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 30 min, and blocked in 3% BSA solution for 20 min at room temperature (RT). Cell preparations were incubated with primary antibodies for 1 h at RT and proteins were visualized by Alexa Fluor 488 or -568 conjugated secondary antibodies. Nuclei were stained by 4^',6-diamidine-2^'- phenylindole dihydrochloride (DAPI). Signals were analyzed on a fluorescence microscope (Axiovert 200M, Zeiss, Germany).

Assessment of cellular degeneration

Cells were immunostained for α-tubulin and p25α, counterstained with DAPI, and analysed by fluorescence microscopy. Cellular degeneration was quantified by counting p25α-positive cells displaying MT retraction compared to the total number of p25α-positive cells. In each experimental condition, 120-200 transfected cells localized in five randomly chosen microscopic fields were examined at 100 times magnification. Cells were counted by Christine Lund Kragh and another investigator blind to the experimental conditions.

Results Gene expression profile of oligodendroglial cells coexpressing α-synuclein and p25α As mentioned previously, coexpression of α-syn and p25α induces degeneration and apoptosis in OLN-93 cells in which α-syn aggregation and Ser129-phosphorylation play a pivotal role. To identify genes involved in the degeneration, microarray analysis of gene expression was conducted. Biotinylated cRNA from two independent transfections of OLN-AS cells with an empty vector or with p25α for 8, 12, and 16 h were hybridized to Affymetrix RAE 230 2.0 Gene Chips. By choosing these early time points it is possible to identify early cellular responses to aggregated α-synuclein. The selection of differentially expressed genes was based on the MAS 5.0 software. A gene fulfilled criteria of differential regulation with a change in signal level ≥ 1.3-fold. This was the case for 104 genes (91 up-regulated genes and 13 down-regulated genes) of approximately 28,000 genes present on the gene chip. Functional classification of the genes was based on Gene Ontology categories and was performed by using the publicly available databases: DAVID, Affymetrix, and NCBI. This enabled the identification of biological mechanisms, pathways, and functions most relevant to the genes of interest altered by coexpression of α-syn and p25α. Although many genes have multiple functions, each gene was assigned to only one functional group in order not to over-represent the data (Table 4). Genes involved in cell cycle, apoptosis, cell signalling, transcription, and response to stress represented some of the groups (Fig.

4). 36 genes (35 upregulated genes and 1 downregulated gene) were found to be differentially expressed at 8 h after transfection. These genes are particularly interesting as they represent the early intracellular response to misfolded α-syn. The genes belonging to the group of chemokines and cytokines were most predominant represented among these early expressed genes and comprised seven genes e.g. Ccl2 ((Chemokine C-C) ligand 2), Ccl7, CxcM (Chemokine (C-X-C motif) ligand 1 ),

Cxcl2, and IL6 (Interleukin 6). Several of the identified chemokines displayed a fast but transient expression profile with a strong increase in mRNA expression at 8 h after transfection followed by a decline in expression levels (Fig. 5). Chemokines belong to the cytokine family, which are secreted proteins involved in immunoregulatory and inflammatory processes. Chemokines are divided into two major subfamilies, CXC and CC, based on the arrangement of the first two of the four conserved cysteine residues (25). CxcH showed the largest increase in expression among the analyzed genes with an increase of 18-fold at 8 h after transfection. Secretion of CxcH protein from the OLN-AS cells in response to p25α expression was confirmed by Enzyme-Linked lmmunosorbant Assay (ELISA). Measurement of rat CxcH in culture media harvested 24 h after transfection demonstrated a seven-fold increase when comparing media from p25α-transfected cells to media from mock-transfected cells (565 pg/ml ± 5.75 compared to 85 pg/ml ± 0.03) (Jacek Losy, Department of Clinical Neuroimmunology, University School of Medicine, Poznan, Poland; personal communication). Early upregulation of two genes belonging to the group of apoptotic genes was observed. These were Bbc3/PUMA (Bcl-2 binding component 3, 53 upregulated modulator of apoptosis), a member of the pro-apoptotic BH3-only gene family and Nfkbia (Nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha), an inhibitor of the anti-apoptotic transcription factor NF-κB.

The Gadd45 genes, Gadd45a, Gadd45b, and Gadd45g are all upregulated in response to coexpression of α-syn and p25α although Gadd45a only at 12 and 16 h after transfection. Gadd45 proteins act as mediators of the G2/M cell cycle checkpoint. Gadd45b has been shown to possess anti-apoptotic activity, whereas the expression of Gadd45a and g is induced by NF-κB-inhibition, which ultimately leads to apoptosis. Early upregulated transcription factors include c-Fos (FBJ murine osteosarcoma viral oncogene homolog), FosL1 (Fos-like antigen 1 ), and c-Myc (Myelocytomatosis viral oncogene homolog), which are cellular proto-oncogenes belonging to the immediate early gene family of transcription factors. Expression of the stress-responsive transcriptional regulator Egr-1 (early growth response 1 ) was also significantly enhanced. Egr-1 is a zinc-finger transcription factor, which is induced as an immediate early gene during different stress conditions.

Four genes involved in cellular stress responses were upregulated at 8 h after transfection; Hmoxi (Heme oxygenase 1 ) and Srxni (Sulfiredoxin), which act to protect cells from oxidative stress; Hspala (Heat shock 7OkDa protein 1A), which encodes the heat shock protein HSP70. In conjunction with other heat shock proteins, HSP70 protects proteins against aggregation and mediates the folding of newly translated proteins in the cytosol. Finally, Myd116 (myeloid differentiation primary response gene 1 16), which is induced during stress conditions such as ischemia. Using different pathway analysis programs (e.g. Ingenuity Pathway Analysis software (Intenuity Systems)) and previously published reports, it was concluded that several of the identified genes are implicated in the NF-κB pathway (e.g. Nfkbia, the Gadd45 genes, and Egr-1 ) (Fig. 8). Therefore, these genes was used to investigate whether this pathway is involved in α-syn-dependent degeneration.

Real-Time qPCR

First, the upregulation of Gadd45a, Gadd45b, Gadd45g, Nfkbia, and Egr-1 mRNA in response to coexpression of α-syn and p25α was validated by real-time qPCR 16 h after transfection (Fig. 6). The changes in expression levels obtained by qPCR were generally lower than the values obtained by microarray analysis. However, they still met the criteria of a fold change ≥ 1.3. Additionally, changes in the expression levels of IL-6, Myc, and Hspsi a mRNA were validated by qPCR.

Silencing of Gadd45a and Gadd45g attenuates p25α-mediated degeneration To elucidate the roles of Gadd45a and g in p25α-mediated degeneration in the OLN-

AS cell line, siRNA was used to silence Gadd45a and g. A pool of four different siRNAs targeting rat Gadd45a, Gadd45g, or a non-targeting control was transfected into OLN- AS cells for 72 h prior to analysis. The silencing of α-syn was confirmed by semiquantitative RT-PCR. Transfection with siRNA targeting Gadd45a and g caused a reduction in mRNA levels of approximately 80 and 70%, respectively, compared to the non-targeting siRNA control (Fig. 7A). The effect of Gadd45a and g silencing on p25α- mediated degeneration was quantified 24 h after siRNA-treated OLN-AS cells had been transfected with p25α. Fig. 7B demonstrates that p25α caused MT retraction in approximately 60% of the transfected cells. Treatment with siRNA targeting Gadd45a and g prior to transfection with p25α reduced the degeneration to 22% and 29%, respectively. By contrast, treatment with siControl did not have any effect. The data provides that Gadd45a and g are critical and essential mediators of α-syn-dependent degeneration in the OLN-AS cells. Thus, Gadd45a and Gadd45g are suitable targets for the treatment of α-synuclein-related disorders, such as Parkinson's disease, and also suitable as biomarker for such clinical conditions for use in diagnostic methods.

Discussion

Coexpression of α-syn and p25α in oligodendroglial cells causes a rapid degeneration of the cells and a delayed onset of apoptosis. We suggest that the cellular degeneration is caused by the formation of soluble Ser129-phosphorylated oligomers and propose an involvement of Fas and TNF receptors in the degenerative process. In this example, addresses the transcriptional pathways involved in cellular toxicity mediated by coexpression of α-syn and p25α in oligodendroglial cells. In order to delineate pathways involved in cellular toxicity caused by α-syn aggregation, microarray based whole genome expression analyses was performed. The use of microarray technology for monitoring the expression of thousands of genes simultaneously may give us a better understanding of the cellular responses to aggregated α-syn. Microarray analysis was performed on OLN-AS cells transfected with empty vector or p25α expression vector for 8, 12, and 16 h. Whether the observed gene response is in fact dependent on α-syn aggregation can be examined by the addition of aggregation inhibitors e.g. ASH D peptide or baicalein prior to real-time qPCR analysis of the genes of interest. However, expression of p25α in OLN-93 cells in the absence of α-syn does not cause degeneration in the cells indicating that the strong pro-degenerative response is dependent on coexpression of the two proteins.

Significant alterations in the gene expression pattern was identified in oligodendroglial cells in response to coexpression of α-syn and p25α as compared to expression of α- syn alone (Table 4). The identified genes belong to different functional groups such as cell cycle regulation, apoptosis, transcription factors, and stress response. The gene response appears to be biphasic consisting of an early stress response peaking at 8 h after transfection followed by a later pro-apoptotic response. The early stress response is comprised of e.g. Hmoxi and chemokines such as CxcH and Cxcl2. Hmoxi is the rate-limiting enzyme in the catabolism of heme and is a crucial mediator of antioxidant and tissue-protective actions. Hmoxi has been found to be upregulated at the transcriptional level in response to noxious stimuli and several lines of evidence demonstrate that Hmoxi serves a protective function in both neuronal and glial cells. Hmoxi has been implicated in various neurodegenerative disorders and its expression is increased in brains of PD, Alzheimer^'s disease (AD), and multiple sclerosis (MS). Most chemokines are pro-inflammatory molecules that coordinate inflammatory and immune responses). Moreover, chemokines and their receptors are expressed in the CNS and their expression has been found to be increased in conditions such as AD and MS. Thus, the early stress response observed in the OLN-93 cells may be a defence mechanism aimed at protecting the cells from damage. The cellular degeneration elicited by coexpression of α-syn and p25α suggests that the early response may be overwhelmed by the late pro-apoptotic response wih a subsequent induction of apoptosis.

Several of the genes comprising the later pro-apoptotic response are involved in the NF-κB pathway (e.g. lκBα, the Gadd45 genes, and Erg-1 ) (Fig 8). NF-κB is a widely used transcription factor, which promotes the expression of a variety of target genes of which most participate in anti-apoptotic pathways. Of reported NF-κB target genes, we find an upregulation of Gadd45a, b, and g, CcxH , IL6, c-Myc, and IRF1 in response to coexpression of α-syn and p25α. Other upregulated genes known to modulate NF-κB function include lκBα, Egr-1 , and Ripk2. In non-stimulated cells, NF-κB is associated with the inhibitory protein lκBα and remains sequestered in the cytosol. lκBα is rapidly phosphorylated upon activation of NF-κB, resulting in its ubiquitination and degradation by the proteasome. NF-κB is then liberated and translocated to the nucleus to drive gene transcription.

A β-induced neurotoxicity in primary cortical neurons has been shown to cause an upregulation of lκBα mRNA and protein and a corresponding decrease in NF-κB activity. Moreover, overexpression of a mutant form of lκBα, which cannot be degraded by the proteasome, overcomes apoptosis resistance in human myeloma cells. The upregulation of lκBα as demonstrated in our model may lead to an increased sequestration of NF-κB in the cytoplasm. The present example clearly provides evidens for upregulation of the Gadd45 genes, Gadd45a, Gadd45b, and Gadd45g. Several studies have implicated Gadd45 isoforms as mediators of the G2/M cell cycle checkpoint in humane and murine cells. Gadd45a and g are involved in maintaining genomic stability and restraining cell growth through interactions with proteins such as p53, p21 , cdc2/cyclinB1 , p38, and MAP kinase kinase kinase 4 (MEKK4). Gadd45a and g was slinced in p25α-transfected OLN-AS cells by siRNA and quantified MT retraction among the transfected cells. It was found that silencing of both Gadd45a and g partly rescued the cells from degeneration suggesting that these proteins participate in the apoptotic process in synucleinopathhies. Gadd45a mRNA and protein levels have been found to be upregulated in human neuroblastoma cells in response to dopamine treatment indicating that Gadd45a participates in dopamine-induced neurotoxicity. It has been shown that apoptosis elicited by inhibition of NF-κB is due to induction of Gadd45a and g presumably through repression of certain transcription factors. Thus, Gadd45a and g have pro-apoptotic activity, whereas Gadd45b has been shown to possess anti- apoptotic effects. The Gadd45a and g proteins have been found to activate MEKK4 leading to activation of the JNK pathway and subsequently apoptosis.

In the present example discloses upregulation of Egr-1 , which is a zinc-finger transcription factor. It has been shown to be induced as an immediate early gene during stress conditions such as ischemia and tunicamycin-induced ER-stress in human SHSY5Y neuroblastoma cells, and has been linked to apoptosis in human melanoma cells. Egr-1 has been shown to interact with the ReIA subunit of NF-κB resulting in suppression of NF-κB transcriptional activity. Thus, an enhanced expression of Egr-1 may reduce the availability of active NF-κB. It has been reported that soluble and fibrillar Aβ(1 -40) and Aβ(1-42) peptides activate Egr-1 in cell culture. This is in agreement with our hypothesis stating that the increase in Egr-1 expression is caused by the formation of aggregated protein species. Several cytokines and chemokines were upregulated in response to coexpression of α- syn and p25α. Interestingly, several of the identified chemokines displayed an early expression profile with a strong increase in expression 8 h after transfection followed by a decline in expression levels. The upregulation of cytokines and chemokines suggests that inflammatory processes are involved in the cellular degeneration. This is in agreement with our previous data showing that the expression of receptors for inflammatory molecules is highly increased in human MSA tissue. Thus, these chemokines may be used as potential early biomarkers for a degenerative process elicited by aggregated α-syn species (e.g. synucleinopathies, such as Parkinson's disease or Alsheimer's disease) as their expression is significantly increased prior to any visible phenotype.

Based on the present example, it is suggested that inhibition of NF-κB is a prime event in the early MT retraction and later cell death caused by aggregated α-syn. Thus, it is proposed that enhanced expression of lκBα retains NF-κB in an inactive state in the cytosol. Moreover, the transcription factor Egr-1 may also participate in inhibition of NF- KB activity. Suppression of NF-κB activity leads to the induction of Gadd45a and g expression, which may lead to activation of the JNK pathway and subsequently apoptosis (Fig. 8).

In conclusion, microarray analysis was performed to reveal the underlying mechanisms involved in toxicity induced by aggregated α-syn. Induction of several genes related to apoptosis, cell cycle regulation, and stress responses was observed. Several chemokines showed a fast but transient increase in expression levels suggesting that these molecules can act as potential biomarkers for the degenerative process. Interestingly, the data showed upregulation of genes related to the NF-κB pathway indicating that this pathway is involved in the cellular degeneration. Silencing of Gadd45a and Gadd45g rescued the cells from degeneration indicating that inhibition of NF-κB by lκBα with concomitant induction of Gadd45 gene expression is central to the degeneration although other pathways are likely to play important roles as well.

Table 3: Primer sets used for real-time qPCR and semiquantitative RT-PCR Gene Sense primer An ti -sense primer Product Annealing length t&mp {* C) (bp)

Gadd45a δ'-ATC CAT TTC ACC CTC ATT C- 5'-TCT CAC CTC TCT CTC 474 65

Example 3

Silencing of Gadd45a and Nfkbia gene expression protects against α-synuclein dependent degeneration. α-synuclein-expressing OLN cells were transfected with siRNA targeting rat Bbc3/PUMA, Egr-1 , Gadd45a, Nfkbia or a non-targeting siControl for 72 h (SEQ ID NO: 21-28, 60-67). Silencing of the genes was confirmed by real-time PCR (knockdown efficiencies were 78-94%). Following siRNAtransfection for 72 h, the cells were transfected with p25α for 24 h. Cellular degeneration measured by microtubule retraction was quantified (figure 9). Bars represent the mean± 1 S. D. from five microscopic fields in one of two representative experiments. RNAi-mediated silencing of Gadd45a and Nfkbia protects against α-synuclein dependent degeneration.

Items 1. A method of treating, preventing or ameliorating a neurodegenerative disorder comprising administering to a subject in need thereof a compound, wherein said compound i) inhibits or down-regulates the expression of a gene selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH 0, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI,

Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4,

Slc35b2, Stx11 , and Nudt18, and/or ii) inhibits or down-regulates the activity of a gene product of a gene selected from the group set out in i). 2. The method according to item 1 , wherein said subject is a human being.

3. The method according to any of the preceding items, wherein said neurodegenerative disorder is a synucleinopathy.

4. The method according to any of the preceding items, wherein said neurodegenerative disorder is selected from the group consisting of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and multiple system atrophy. 5. The method according to item 4, wherein said neurodegenerative disorder is Parkinson's disease.

6. The method according to any of the preceding items, wherein said expression of a gene and/or the activity of said gene product is reduced in a specific tissue of said human being. 7. The method according to item 6, wherein said tissue is selected from the group consisting of brain tissue, cerebral cortex tissue, liver tissue, skeletal muscle tissue, and intestinal tissue.

8. The method according to item 6, wherein said tissue is cerebral cortex tissue.

9. The method according to any of the preceding items, wherein said expression of a gene and/or the activity of said gene product in said human being is reduced to less than 60%, such as less than 50%, such as less than 20%, for example 0% of said expression and/or said actitivty in said human being before said administration.

10. The method according to any of the preceding items, wherein said gene is selected from the group consisting of SEQ ID NO: 29-59.

1 1. The method according to any of the preceding items, wherein said gene is Gadd45a (SEQ ID NO: 32) or Gadd45g (SEQ ID NO: 34). 12. The method according to any of the preceding items, wherein said compound is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins.

13. The method according to item 12, wherein said compound is an siRNA.

14. The method according to item 13, wherein said siRNA is selected from the group consisting of SEQ ID NO: 21-28.

15. The method according to item 13, wherein said gene is Gadd45a (SEQ ID NO: 32) and said siRNA is selected from the group consisting of 21-24.

16. The method according to item 13, wherein said gene is Gadd45g (SEQ ID NO: 34) and said siRNA is selected from the group consisting of 25-28.

17. The method according to any of the preceding items, further comprising administering at least one additional synucleinopathy therapeutic, such as at least one additional Parkinson's or Alzheimer's disease therapeutic.

18. A compound capable of a. inhibiting or down-regulating the expression of a gene selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clef 1 , Csfl , Cxcl1 , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnal , Slc15a4, Slc35b2, Stx11 , and Nudt18, and/or b. inhibiting or down-regulating the activity of a transcriptional and/or translational product of a gene according to a. 19. The compound according to item 18, wherein said compound is capable of selectively binding said gene, and/or a transcriptional and/or translational product of said gene.

20. The compound according to any of items 18 and 19, wherein said compound is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins.

21. The compound according to item 20, wherein said compound is an antibody, antigen binding fragment or recombinant protein thereof.

22. The compound according to item 21 , wherein said compound is an antibody, antigen binding fragment or recombinant protein thereof, which selectively binds Gadd45b and/or Gadd45g.

23. The compound according to item 20, wherein said compound is an siRNA.

24. The compound according to item 23, wherein said siRNA consists of or comprise 17-25, such as 19-22 consecutive nucleotides selected from a region of a sequence selected from SEQ ID NO: 1-20 or the complement thereof.

25. The compound according to item 23, wherein said siRNA is selected from the group consisting of SEQ ID NO: 21-28.

26. A pharmaceutical composition comprising at least one compound as defined in any of item 18 to 25.

27. The composition according to item 26, further comprising at least one additional component. 28. The composition according to item 27, wherein said at least one additional component is an adjuvant, an excipient and/or a carrier. 29. The composition according to item 28, wherein said carrier is selected from the group consisting of keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. 30. A kit-of-parts comprising a composition as defined in any of items 26 to 29, and at least one additional active ingredient.

31. The kit-of-parts according to item 30, wherein the additional active ingredient is an antibiotic.

32. The kit-of-parts according to item 31 , wherein the antibiotic is selected from: amoxicillin, penicillin, acyclovir and /or vidarabine.

33. The kit-of-parts according to any of items 30 to 32, where the provided components are to be administered simultaneously or sequentially. 34. The kit-of-parts according to any of items 30 to 33, further contains instructions for combining the components so as to formulate an pharmaceutical composition suitable for administration to a human being.

35. Use of a compound as defined in any of items 18 to 25, a composition as defined in any of items 26 to 29, and/or a kit-of-parts as defined in any of items 30 to 34 for the manufacture of a medicament for treatment, amelioration and/or prevention of a neurodegenerative disorder.

36. The use according to item 35, wherein said neurodegenerative disorder is as defined in any of items 2 to 5.

37. A compound as defined in any of items 18 to 25, a composition as defined in any of items 26 to 29, and/or a kit-of-parts as defined in any of items 30 to 34 for treatment, amelioration and/or prevention of a neurodegenerative disorder.

38. A pharmaceutical composition for treatment, amelioration and/or prevention of a neurodegenerative disorder, said composition comprising a compound as defined in any of items 18 to 25, a composition as defined in any of items 26 to 29, and/or a kit-of-parts as defined in any of items 30 to 34.

39. The compound, composition, and/or kit-of-parts and/or pharmaceutical composition according to any of items 37 or 38, wherein said neurodegenerative disorder is as defined in any of items 2 to 5.

40. A method for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said method comprising the steps of a. providing a biological sample isolated from said subject b. detecting in said biological sample of step a. i) at least one polymorphism or mutation of a gene, and/or ii) at least one transcriptional and/or translational product of a gene, wherein said gene is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfi , Csf1 , CxcM , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18. 41. The method according to item 40, wherein said biological sample is a blood sample, a tissue sample, a secretion sample, semen, ovum, hairs, nails, tears, and urine.

42. The method according to any of items 40 and 41 , wherein the level of said gene product is increased in a sample isolated from a subject suffering from or being predisposed for said neurodegenerative disorder relative to a subject not suffering from said neurodegenerative disorder.

43. The method according to item 42, wherein said level is increased by at least 50%, such as at least 100%, for example at least 200%, such as at least 300%.

44. The method according to any of items 40 to 43, wherein said at least one polymorphism leads to increased expression of said gene in a subject relative to a subject not carrying said polymorphism.

45. The method according to any of items 40 to 44, wherein said subject is a human being.

46. The method according to any of items 40 to 45, wherein said neurodegenerative disorder is as defined in any of items 2 to 5.

47. The method according to any of items 40 to 46, wherein said gene is Gadd45a or Gadd45g.

48. A diagnostic kit for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said kit comprising at least one detection member for detecting in a biological sample isolated from said subject a. at least one polymorphism of a gene, and/or b. at least one transcriptional and/or translational product of a gene, wherein said gene is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfl , Csfl , CxcM , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18.

49. The kit according to item 48, wherein said detection member is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins. 50. The kit according to item 48, wherein said detection member is an antibody, antigen binding fragment or recombinant protein thereof. 51. The kit according to item 48, wherein said detection member is an antibody, antigen binding fragment or recombinant protein thereof, which selectively binds Gadd45a and/or Gadd45g. 52. The kit according to item 49, wherein said detection member is an oligonucleotide primer or probe, which is linked to a detectable label. 53. The kit according to any of items 48 to 52, wherein said detection member is an oligonucleotide primer or probe consisting of or comprising a sequence selected from the group consisting of SEQ ID NO: 1-20 or SEQ ID NO: 29-59 or the complement or part thereof. 54. The kit according to item 53, wherein said detection member is an oligonucleotide primer with a sequence selected from the group consisting of SEQ ID NO: 1-20. 55. The kit according to any of items 48 to 53, further comprising reagents and buffers for detection, and/or instructions for performing the detection method and interpretation of the result. 56. A method of identifying a drug for treating, preventing or ameliorating a neurodegenerative disorder, said method comprising the steps of a. providing a biological sample, b. determining in said biological sample the expression of a gene or the activity of a gene product, wherein said gene is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcfi , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx1 1 , and Nudt18, c. providing a drug to said biological sample and determining the expression of a gene or the activity of a gene product, wherein said gene is selected from the group consisting of Bbc3/PUMA, Nfkbia, Ninji , Gadd45a, Gadd45b, Gadd45g, Ccl2, Ccl7, Clcf1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudt18, d. comparing the expression of said gene and/or the activity of said gene product in said biological sample in the presence and absence of said drug, wherein in the presence of said drug in said biological sample said expression of said gene and/or said activity of said gene product is inhibited or down-regulated relative to the expression of said gene and/or activity of said gene product in said sample in the absence of said drug. 57. The method according to item 56, wherein said drug is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins. 58. The method according to any of items 56 and 57, wherein said exression or activity is down-regulated to less than 90%, such as less than 80% such as less than 70% for example less than 60%, for example less than 50%, such as less than 40%, such as less than 30% such as less than 20% for example less than 10%, for example less than 5%, such as completely inhibited (0%).

Claims

1. A method of treating, preventing or ameliorating a neurodegenerative disorder comprising administering to a subject in need thereof a compound, wherein said compound i) inhibits or down-regulates the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudti 8, and/or ii) inhibits or down-regulates the activity of a gene product of a gene according to i).

2. The method according to claim 1 , wherein said subject is a human being.

3. The method according to any of the preceding claims, wherein said neurodegenerative disorder is a synucleinopathy.

4. The method according to any of the preceding claims, wherein said neurodegenerative disorder is selected from the group consisting of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and multiple system atrophy.

5. The method according to any of the preceding claims, wherein the expression of said gene and/or the activity of said gene product is reduced in a specific tissue of said human being, said tissue being selected from the group consisting of brain tissue, cerebral cortex tissue, liver tissue, skeletal muscle tissue, and intestinal tissue.

6. The method according to any of the preceding claims, wherein said expression of a gene and/or the activity of said gene product in said human being is reduced to less than 60%, such as less than 50%, such as less than 20%, for example to 0% of said expression and/or said actitivty in said human being before said administration.

7. The method according to any of the preceding claims, wherein said gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , Egr1 , Fos, FosL, Hes1 , KIfI O, Myc, Kpnal , SId 5a4, Slc35b2, Stx11 , and Nudt18 is identified by a sequence selected from the group consisting of SEQ ID NO: 29-59, and any sequence which is at least 90% identical to any of SEQ ID NO: 29-59.

8. The method according to any of the preceding claims, wherein said gene is Gadd45a, Gadd45g or Nfkbia,

9. The method according to claim 8, wherein said gene comprises a sequence identifed by SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or part thereof, or any sequence which is at least 90% identical thereto.

10. The method according to any of the preceding claims, wherein said compound is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins.

1 1. The method according to claim 10, wherein said compound is an siRNA.

12. The method according to claim 11 , wherein said siRNA is designed against a target sequence identified by any of SEQ ID NO: 21-28 and SEQ ID NO: 60-63, and/or wherein said siRNA comprises or consists of a sequence identified by any of SEQ ID NO: 21-28 and SEQ ID NO: 60-63, or part thereof.

13. The method according to claim 11 , wherein said gene is Gadd45a and said siRNA comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 21 , 22, 23, and 24, or part thereof.

14. The method according to claim 11 , wherein said gene is Gadd45g and said siRNA comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 and 28, or part thereof.

15. The method according to claim 11 , wherein said gene is Nfkbia, and said siRNA comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 60, 61 , 62, and 63, or part thereof.

16. The method according to any of the preceding claims, further comprising administering at least one additional synucleinopathy therapeutic, such as at least one additional Parkinson's or Alzheimer's disease therapeutic.

17. A compound capable of i) inhibiting or down-regulating the expression of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csf1 , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd116, Srxni , Egr1 ,

Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18, and/or ii) inhibiting or down-regulating the activity of a transcriptional and/or translational product of a gene according to i).

18. The compound according to claim 17, wherein said compound is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins.

19. The compound according to claim 18, wherein said compound is an antibody, antigen binding fragment or recombinant protein thereof.

20. The compound according to claim 19, wherein said compound is an antibody, antigen binding fragment or recombinant protein thereof, which selectively binds Gadd45a, Gadd45g and/or Nfkbia.

21. The compound according to claim 18, wherein said compound is an siRNA.

22. The compound according to claim 21 , wherein said siRNA consists of or comprise 17-25, such as 19-22, consecutive nucleotides selected from a region of a sequence selected from SEQ ID NO: 1-20 or the complement thereof.

23. The compound according to claim 21 , wherein said siRNA comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 21- 28, SEQ ID NO: 60-63, or part thereof.

24. A pharmaceutical composition comprising at least one compound as defined in any of claim 17 to 23.

25. The composition according to claim 24, further comprising at least one adjuvant, an excipient and/or a carrier.

26. The composition according to claim 25, wherein said carrier is selected from the group consisting of keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.

27. Use of a compound as defined in any of claims 17 to 23, and/or a composition as defined in any of claims 24 to 26 for the manufacture of a medicament for treatment, amelioration and/or prevention of a neurodegenerative disorder.

28. The use according to claim 27, wherein said neurodegenerative disorder is as defined in any one of claims 3 and 4.

29. A compound as defined in any of claims 17 to 23, and/or a composition as defined in any of claims 24 to 26 for treatment, amelioration and/or prevention of a neurodegenerative disorder.

30. A pharmaceutical composition for treatment, amelioration and/or prevention of a neurodegenerative disorder, said composition comprising a compound as defined in any of claims 17 to 23, and/or a composition as defined in any of claims 24 to 26.

31. The compound and/or pharmaceutical composition according to any of claims 29 or 30, wherein said neurodegenerative disorder is as defined in any one of claims 3 and 4.

32. A method for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said method comprising providing a biological sample isolated from said subject and detecting in said biological sample i) at least one polymorphism or mutation of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia,

Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , EgM , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx11 , and Nudt18, and/or ii) at least one transcriptional and/or translational product of said gene.

33. The method according to claim 32, wherein the level of said gene product is increased in a sample isolated from a subject suffering from or being predisposed for said neurodegenerative disorder relative to a subject not suffering from said neurodegenerative disorder.

34. The method according to claim 33, wherein said at least one polymorphism leads to increased expression of said gene in a subject relative to a subject not carrying said polymorphism.

35. The method according to any of claims 32 to 34, wherein said gene is Gadd45a, Gadd45g or Nfkbia.

36. A diagnostic kit for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject, said kit comprising at least one detection member for detecting in a biological sample isolated from said subject i) at least one polymorphism of a gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , Cxcl1 , Cxcl2, CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , SId 5a4, Slc35b2, Stx1 1 , and Nudt18, and/or ii) at least one transcriptional and/or translational product of said gene.

37. The kit according to claim 36, said kit further comprising at least one reference sample comprising i) At least one nucleic acid sequence comprising at least one gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clcfi , Csf1 , CxcM , Cxcl2,

CxcH O, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspal a, Myd1 16, Srxni , Egr1 , Fos, FosL, Hes1 , KIfIO, Myc, Kpnai , Slc15a4, Slc35b2, Stx11 , and Nudti 8 or part thereof, and/or ii) at least one transcriptional and/or translational product of said gene or part thereof.

38. The kit according to any of claims 36 to 37, wherein said detection member is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins.

39. The kit according to claim 38, wherein said detection member is an antibody, antigen binding fragment or recombinant protein thereof.

40. The kit according to claim 38, wherein said detection member is an antibody, antigen binding fragment or recombinant protein thereof, which selectively binds Gadd45a, Gadd45g and/or Nfkbia.

41. The kit according to claim 38, wherein said detection member is an oligonucleotide primer and/or an oligonucleotide probe.

42. The kit according to any of claims 36 to 41 , wherein said detection member is an oligonucleotide primer or probe consisting of or comprising at least 5 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 1-20, SEQ ID NO: 29-59 and the complement thereof.

43. The kit according to claim 42, wherein said detection member is an oligonucleotide primer or probe with a sequence selected from the group consisting of SEQ ID NO: 1-20, and any sequence at least 90% identical thereto.

44. The kit according to any of claims 36 to 43, said kit comprising at least one reference sample comprising i) at least one nucleic acid sequence comprising at least one gene selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, or part thereof, and/or ii) at least one transcriptional and/or translational product of said gene or part thereof.

45. The kit according to claim 44, said kit comprising i) At least one oligonucleotide primer or probe consisting of or comprising at least 5 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 1-2, SEQ ID NO: 32 and the complement thereof ii) at least one reference sample comprising at least one nucleic acid sequence comprising the Gadd45a gene or part thereof.

46. The kit according to claim 44, said kit comprising i) At least one oligonucleotide primer or probe consisting of or comprising at least 5 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 5-6, SEQ ID NO: 34 and the complement thereof ii) at least one reference sample comprising at least one nucleic acid sequence comprising the Gadd45g gene or part thereof.

47. The kit according to claim 44, said kit comprising i) At least one oligonucleotide primer or probe consisting of or comprising at least 5 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 13-14, SEQ ID NO: 30 and the complement thereof ii) at least one reference sample comprising at least one nucleic acid sequence comprising the Nfkbia gene or part thereof.

48. The kit according to any of claims 36 to 47, further comprising reagents and buffers for detection, and/or instructions for performing the detection method and interpretation of the result.

49. Use of a diagnostic kit as defined in any one of claims 36 to 48 for the diagnosis of a neurodegerative disorder or for assisting in diagnosing a neurodegenrative disorder.

50. The use according to claim 49, wherein said neurodegenerative disorder is a synucleinopathy.

51. The use according to claim 49 for determining a neurodegenerative disorder or a predisposition for a neurodegenerative disorder in a subject according to the method as defined in any of the preceding claims 32 to 35.

52. A method of identifying a drug for treating, preventing or ameliorating a neurodegenerative disorder, said method comprising the steps of i) providing a biological sample, ii) determining in said biological sample the expression of a gene or the activity of a gene product, wherein said gene is selected from the group consisting of Gadd45a, Gadd45b, Gadd45g, Nfkbia, Bbc3/PUMA, Ninji , Ccl2, Ccl7, Clef 1 , Csfl , CxcH , Cxcl2, CxcHO, IL6, Bhlhb2, Camkk2, Duspi , Lphn2, RiI, Hmoxi , Hspala, Myd116, Srxni , Egr1 , Fos, FosL,

Hes1 , KIfI O, Myc, Kpnai , SId 5a4, Slc35b2, Stx11 , and Nudt18, iii) providing a drug to said biological sample and determining the expression of said gene or the activity of a product of said gene, and iv) comparing the expression of said gene and/or the activity of said gene product in said biological sample in the presence and absence of said drug, wherein in the presence of said drug in said biological sample said expression of said gene and/or said activity of said gene product is inhibited or down-regulated relative to the expression of said gene and/or activity of said gene product in said sample in the absence of said drug.

53. The method according to claim 52, wherein said drug is selected from the group consisting of oligonucleotide primers, oligonucleotide probes, small interfering RNAs (siRNAs), nucleic acid aptamers, small molecules, inorganic compounds, polypeptides, peptides, peptide fragments, peptide aptamers, antibodies, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immunoglobulins.

54. The method according to any of claims 52 and 53, wherein said exression or activity is down-regulated to less than 90%, such as less than 80% such as less than 70% for example less than 60%, for example less than 50%, such as less than 40%, such as less than 30% such as less than 20% for example less than

10%, for example less than 5%, such as completely inhibited (0%).