WO2007135426A2

WO2007135426A2 - Treatment of neurodegeneratives diseases

Info

Publication number: WO2007135426A2
Application number: PCT/GB2007/001900
Authority: WO
Inventors: Richard Wade-Martins; Tim Fountaine
Original assignee: Isis Innovation Limited
Priority date: 2006-05-23
Filing date: 2007-05-23
Publication date: 2007-11-29
Also published as: GB0610183D0; WO2007135426A3

Abstract

The present invention is directed to the use of an agent that down regulates α-synuclein to down regulate α-synuclein in a subject with normal physiological levels of α-synuclein expression. In particular, for the treatment of neurodegenerative disease, such as sporadic Parkinson's disease.

Description

TREATMENT OF NEURODEGENERATIVE DISEASES

The present invention relates to compositions and methods for use in the treatment of one or more neurodegenerative disease. In particular, for the treatment of sporadic Parkinson's disease.

Neurodegenerative diseases, and the resulting illness and dementia they cause, represent a growing public health concern. Each year 150,000 people in the UK develop cognitive impairment and memory loss associated with a neurodegenerative disease; within 5 years half will have dementia. The prevalence of dementia rises from 5% to over 30% between the ages of 65 and 85 years old. Thus, in the aging populations of the Western world, the socio-economic impact of neurodegenerative diseases is set to increase dramatically given that the greatest risk factor is age.

Parkinson's disease is the second most common neurodegenerative disease after Alzheimer's disease, with a lifetime risk of 2% for men and 1.3% for women (Elbaz et al., 2002 J Clin Epidemiol 55:25-31) . About 3% of the cases of Parkinson's disease are defined as familial Parkinson's disease, and about 97% are defined as sporadic Parkinson's disease (Klein, 2006 Arch Neurol 63:328-334) . The risk of developing Parkinson's disease is known to be 2-3 times higher when relatives have had Parkinson's disease.

Parkinson's disease is progressive and is characterised by bradykinesia, resting tremor, rigidity and postural instability. Patients may also develop autonomic, cognitive and psychiatric symptoms. The main causative pathological defect is selective and catastrophic loss of the pigmented dopaminergic neurons of the substantia nigra pars compacta (the midbrain) , which is almost invariably associated with the accumulation of concentric eosinophilic intracytoplasmic inclusions (protein aggregations) called Lewy bodies (Takahashi and Wakabayashi, 2001 Neuropathology 21:315-322) .

Lewy bodies have been shown to be mainly composed of the protein α-synuclein (Spillantini et al. , 1997 Nature 388:839-840) . α-synuclein is a 140 amino acid protein which is enriched in presynaptic terminals and is highly expressed throughout the brain.

The human α-synuclein is sometimes also called alpha-synuclein isoform NACP 140, non A-beta component of AD amyloid or non A4 component of amyloid precursor, α-synuclein is encoded by the SNCA gene, also sometimes referred to as MGCl 10988, NACP, PARKl, PARK4, or PDl. The human SNCA gene is located on chromosome 4 at location 4q21. The SNCA gene has NCBI GenelD: 6622; OMIM ID: * 163890 and NCBI referenced sequences: NMJ300345, and NM_007308.

Mouse (Mus musculus) α-synuclein is sometimes referred to as NACP or alphaSYN. The mouse Snca gene is located on chromosome 6 at location 6 29.OcM. The mouse Snca gene has NCBI GenelD: 20617.

Rat (Rattus norvegicus) α-synuclein is sometimes referred to as MGC 105443. The rat Snca gene is located on chromosome 4 at location 4q24 and has NCBI GenelD: 29219.

Parkinson's disease can be defined as familial or sporadic.

Familial Parkinson's disease is genetic and may be the result of an individual having multiple copies of the SNCA gene and/or point mutations in the genetic code. Having multiple copies of the gene will increase the expression of α-synuclein, and having point mutations results in a toxic gain of function of the protein. In addition to SNCA, mutations in at least six other genes have been shown to cause Parkinson's disease. These genes include Parkin, PINKl, DJ-I, UCHLl, LRRK2, and PARK2.

Missense mutations (Polymeropoulos et al. , 1997 Science 276:2045-2047; Kruger et al. , 1998 Nat Genet 18:106-108; Zarranz et al. , 2004 Ann Neurol 55:164-173) and gene multiplication mutations (Singleton et al. , 2003 Science 302:841; Chartier-Harlin et al. , 2004 Lancet 364: 1167- 1169) of the α-synuclein (SNCA) gene have been shown to cause rare inherited (familial) forms of Parkinson's disease. Three known familial forms of Parkinson's disease are caused by autosomal dominant (AD) missense mutations in the SNCA gene; these missense mutations are A53T, A30P or E46K. Studies on families with duplication or triplication of the SNCA locus have shown this to be an autosomal dominant form of familial Parkinson's disease.

The term "familial Parkinson's disease" is used herein to refer to a disease caused by a mutation or duplication which leads to a monogenic form of Parkinson's disease that is inherited in a Mendelian pattern. This must be distinguished from disease susceptibility contributed to by allelic sequence variation. The accepted definition of allelic sequence variation is a DNA polymorphism that occurs in a human population with a frequency greater than 0.01 (1%) (Strachan and Read, 2004 Human

Molecular Genetics 3, 3rd Edition. London: Garland Science) . It may be that these common variations contribute to a subject's propensity to develop Parkinson's disease but they are not considered mutations and are not part of familial Parkinson's disease. By contrast, in sporadic, or idiopathic, Parkinson's disease there is no evidence of mutations in the α-synuclein gene or of over-expression of the α-synuclein protein. There is no apparent genetic predisposition to this form of the disease.

Sporadic Parkinson's disease refers to Parkinson's disease which is not caused by a monogenic mutation or gene duplication inherited in a Mendelian fashion.

The clinical features of familial Parkinson's disease are quite different compared to patients in whom Parkinson's disease occurs without a monogenic basis (i.e. sporadic Parkinson's disease) . Familial Parkinson's disease usually has an earlier onset and characteristic clinical features depending on the particular gene which is involved. These differences have led to most investigators and clinicians drawing a distinction between sporadic Parkinson's disease and familial Parkinson's disease - they are essentially different diseases with different natural histories, prognosis, and treatment. Presently, there are seven different proteins with identified monogenic mutations which cause familial Parkinson's disease. One of the seven proteins implicated in familial Parkinson's disease is α-synuclein in which three autosomal dominant mutations and gene multiplication mutations have been described.

The function of α-synuclein remains unknown, its physiological and pathological role are still unclear. The protein is highly conserved in all vertebrates, α-synuclein has been shown to be upregulated during song learning and synaptic plasticity in birds (George et al. , 1995 Neuron

15:361-372) . The accumulating evidence suggests that α-synuclein plays an important role in all aspects of dopamine homeostasis through involvement in regulating synaptic vesicle function, dopamine synthesis, and re-uptake of dopamine via the dopamine transporter (DAT) . Dopamine is known to be toxic to cells, and levels of dopamine within dopaminergic neurons must be tightly regulated. A major determinant of dopamine accumulation is the dopamine transporter (DAT) which is selectively expressed in dopaminergic neurones (Mortensen and Amara, 2003 Eur J Pharmacol 479:159-170) . α-synuclein is known to interact with DAT and this may represent a mechanism by which α-synuclein can alter dopamine entry into cells and, in so doing, affect cellular viability (Lee et al. , 2001 Faseb J 15:916-926; Wersinger and Sidhu, 2003 Neurosci Lett 340:189-192) .

Accumulating evidence suggests that a change in α-synuclein levels may be important in the pathogenesis of Parkinson's disease. Observations in cellular and animal models show that over-expression of, or excess levels of, α-synuclein is toxic (Junn and Mouradian, 2002 Neurosci Lett 320:146-150; Zhou et al., 2002 Brain Res 926:42-50; Maries et al. , 2003 Nat Rev Neurosci 4:727-738; Orth et al. , 2003 Neurosci Lett 351 :29-32; Kalivendi et al. , 2004 J Biol Chem 279: 15240-15247; Moussa et al. , 2004 Biochemistry 43:5539-5550; Yamada et al. , 2004 J Neurochem 91:451- 461) . This is consistent with the observed relationship between α- synuclein levels and disease severity in familial Parkinson's disease patients with duplications and triplications of the SNCA locus (Singleton et al. , 2003 Science 302:841; Chartier-Harlin et al. , 2004 Lancet 364:1167-1169; Farrer et al. , 2004 Ann Neurol 55:174-179) .

Aside from the accepted view that over expression or increased levels of α-synuclein protein correlates with the occurrence of Parkinson's disease, it is generally believed in the art that normal levels of α-synuclein are required to promote cellular survival by preventing or reducing apoptosis (programmed cell death) and/or by modulating dopamine levels within cells to reduce dopamine toxicity.

α-synuclein is believed to reduce the expression of pro-apoptotic proteins and its expression is known to be upregulated during stress which has been associated with neuroprotection (Gomez-Santos et al. , 2002 Brain Res 935:32-39; Sidhu et al. , 2004 Ann N Y Acad Sci 1035:250-270) . In addition, α-synuclein overexpression has been suggested to reduce levels of dopamine within the cell cytoplasm thereby reducing toxicity (Lotharius and Brundin, 2002 Hum MoI Genet 11 :2395-2407) . These observations have promoted the idea that reducing α-synuclein levels below normal physiological levels would be deleterious (Lotharius and Brundin, 2002 Hum MoI Genet 11 :2395-2407; Perez and Hastings, 2004 J Neurochem 89:1318-1324; Sidhu et al. , 2004 Ann N Y Acad Sci 1035:250-270) .

The aim of this invention is to provide a novel therapeutic and/or prophylactic treatment for neurodegenerative diseases such as Parkinson's disease, and in particular sporadic Parkinson's disease. It is shown herein that reducing normal physiological levels of α-synuclein is not harmful to human neurons and protects them from compounds known to cause Parkinson's disease. This is contrary to the prevailing opinion which believes that normal physiological levels of α-synuclein are necessary for cell survival, and that reduced levels would actually cause Parkinson's disease.

According to one aspect the invention provides the use of an agent that down regulates α-synuclein to down regulate α-synuclein in a subject with normal physiological levels of α-synuclein expression. Reference herein to the down regulation of α-synuclein refers to a reduction in the level of α-synuclein in a cell or population of cells. An agent that down regulates α-synuclein therefore refers to a substance that reduces the level of α-synuclein in a cell or population of cells. Down regulation of α-synuclein may be achieved at the transcriptional, translational or post translational level. For example, the agent may prevent or reduce transcription or translation of the gene encoding α-synuclein. Alternatively, or additionally, the agent may act on the α-synuclein protein once produced to reduce protein activity levels. Preferably reference herein to down regulation of α-synuclein refers to a down regulation in the expression of α-synuclein at the transcriptional and/or translational level such that less α-synuclein is produced.

This use is preferably to treat one or more neurodegenerative diseases.

According to another aspect the invention provides the use of an agent that down regulates α-synuclein to treat sporadic Parkinson's disease.

Preferably the down regulation of α-synuclein reduces the level and/or activity of α-synuclein protein from normal physiological levels.

Preferably a subject with sporadic Parkinson's disease has normal physiological levels of α-synuclein, that is, has levels of α-synuclein that would be observed in a subject who does not have Parkinson's disease.

That is, they do not have elevated levels of α-synuclein.

Sporadic Parkinson's disease may be characterised in that there is not a duplication of, or mutation in, the α-synuclein gene.

According to a further aspect the invention provides the use of an agent that down regulates α-synuclein in the preparation of a medicament for the treatment of a neurodegenerative disease in a subject with normal physiological levels of α-synuclein.

According to a yet further aspect the invention provides the use of an agent that down regulates α-synuclein in the preparation of a medicament for the treatment of sporadic Parkinson's disease.

According to another aspect the invention provides a method of treating a neurodegenerative disease in a subject with normal physiological levels of α-synuclein comprising down regulating α-synuclein in the subject.

According to another aspect the invention provides a method for treating sporadic Parkinson's disease comprising down regulating α-synuclein. Preferably α-synuclein is down regulated to reduce the level and/or activity of α-synuclein protein from normal physiological levels to less than normal physiological levels.

Normal levels and/or activity of α-synuclein may be determined in a relative fashion by comparing levels and/or activity in an individual with the normal two copies of the SNCA gene, with levels in an individual with additional copies of the SNCA gene and therefore higher levels of the α-synuclein protein. Normal is defined as the levels found in people with two non-mutated copies of the SNCA gene + /- 95% confidence intervals.

Surprisingly, down-regulation of α-synuclein to below normal physiological levels and/or activity protects cells from neurotoxic insult without affecting cell viability. Preferably reference to a neurodegenerative disease refers to a disease selected from the group comprising diseases characterised by a set of symptoms called Parkinsonism which include ridgity, postural instability and bradykinesia, including Parkinson's disease, dementia with Lewy bodies, multiple systems atrophy, neurodegeneration with iron accumulation in the brain, post-encephalytic parkinsonism, frontotemporal dementia with parkinsonism, progressive supranuclear palsy, corticobasal degeneration, Lytico-bodig of Guam, X-linked dystonia and parkinsonism, rapid-onset dystonia and parkinsonism, Wilson's disease, fragile X tremor ataxia syndrome, vascular parkinsonism and drug induced parkinsonism.

Preferably the neurodegenerative disease is sporadic Parkinson's disease.

In the uses and methods of the invention the level and/or activity of α-synuclein in a subject is preferably reduced from a normal physiological level and/or activity to a level lower than a normal physiological level and/or activity.

Preferably the subject to be treated does not have elevated levels of α-synuclein before treatment, that is, they do not have levels of α- synuclein greater than normal physiological levels.

Preferably α-synuclein is down regulated so that the level and/or activity of α-synuclein protein in a cell or population of cells is reduced by between about 1% and about 100%, more preferably by about 10%, 20%, 30%, 40%, 50%, 60%, 70% 80% 90%, 95% or about 99% when compared to an untreated subject. Most preferably the levels and/or activity of α-synuclein are reduced between about 40% and about 90%. Preferably levels and/or activity of α-synuclein are reduced in cells of the midbrain. Levels and/or activity of α-synuclein in the midbrain may be reduced by between about 1% and about 100%, more preferably by about 10%, 20%, 30%, 40%, 50%, 60%, 70% 80% 90%, 95% or about 99% when compared to an untreated subject. Most preferably the levels and/or activity of α-synuclein in the midbrain are reduced between about 40% and about 90%. Levels and/or activity of α-synuclein may be reduced less in other cells. Levels and/or activity of α-synuclein may be reduced only, or substantially only, in cells in the midbrain.

Preferably the level and/or activity of α-synuclein is reduced by down regulating expression of the α-synuclein gene SNCA. Preferably expression of the SNCA gene which is down regulated is the wild type gene, and does not include a mutation associated with a neurodegenerative disease. The wild type gene may however contain common polymorphisms, but preferably does not contain monogenic mutations associated with a neurodegenerative disease and with Parkinson's disease in particular.

Preferably the subject is a human or a non-human animal, or cells derived therefrom. Preferably if the subject is a non-human animal it is a mammal, for example, a cow, horse, sheep, mouse, rat, dog, pig, goat, or a primate. In a preferred embodiment, the subject is a human.

The subject may have been diagnosed as having, or as being at risk of developing, a neurodegenerative disorder.

The treatment may be prophylactic (preventative) or therapeutic. Preferably the agent to down regulate α-synuclein works by down regulating expression of α-synuclein. Down regulation may occur at the level of transcription and/or translation and/or post-translation. Down regulation may involve preventing or reducing the level of α-synuclein protein produced, or it may involve blocking the activity of α-synuclein protein. For example, down regulation of α-synuclein may be achieved using siRNA which targets translation and down regulates SNCA expression by reducing the amount of α-synuclein protein produced. However, in an alternative embodiment, normal levels of the α-synuclein protein may be produced but the function/activity of the protein may be blocked, for example by including an antagonist of α-synuclein, the antagonist may be a small molecule.

Preferably the down regulation of α-synuclein is sufficient to reduce protofibril formation, preferably protofibril formation is reduced by at least about 25%, 30%, 40%, 50%, 60%, 70%, 80% or more.

Preferably the down regulation of α-synuclein is sufficient to decrease an MPP driven increase in NOS activity, preferably a decrease of at least about 25%, 30%, 40%, 50%, 60%, 70%, 80% or more is observed.

Preferably the down regulation of α-synuclein is sufficient to protect cells from one or more of MPP + , rotenone and dopamine. Preferably the down regulation of α-synuclein is sufficient to decrease the effect on cell viability of one or more of MPP + , rotenone and dopamine by about 25%, 30%, 40%, 50%, 60%, 70%, 80% or more.

Preferably the down regulation of α-synuclein is sufficient to reduce dopamine uptake into cells. Preferably the uptake of dopamine is reduced by at least about 25%, 30%, 40%, 50%, 60%, 70%, 80% or more. Preferably the level and/or activity of α-synuclein is reduced (that is, α-synuclein is down regulated) using an agent selected from the group comprising: an RNAi agent, such as a siRNA molecule; an antisense RNA molecule; an antisense DNA molecule; a ribozyme; an antibody; and any other small molecule that down regulates α-synuclein expression. Alternatively a combination of agents may be used.

Preferably RNAi (RNA interference) techniques are used to down regulate the expression of α-synuclein. The term RNAi was coined by Fire and co-workers in 1998 to describe the observation that double- stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al. 1998, Nature 391 :806-811) .

RNAi refers to sequence-specific post-transcriptional gene silencing mediated by small interfering RNAs (siRNAs) . RNAi techniques are well known in the art (Carthew et al 2001 Current Opinions in Cell Biology 12, 244-248; Elbashir et al 2001 Nature 411, 494-498)

RNAi arises when double stranded RNA (dsRNA) is introduced into an organism and causes the degradation of mRNA complementary to one of the strands of the RNA. The strand of the siRNA complementary to the mRNA is usually referred to as the antisense strand. Typically, the mechanism of RNAi works as follows. Long dsRNA is introduced into a cell where it is cleaved into short, say 15 to 30 nucleotide pairs long, double stranded interfering RNAs (siRNAs) by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein into an RNA-induced silencing complex (RISC) . The siRNA then unwinds to form activated RISC, which then comprises a single stranded RNA molecule. The activated RISC then binds to complementary mRNA by base pairing between the single stranded siRNA, known as the antisense strand, and the mRNA. The bound mRNA is then cleaved and sequence specific degradation of the mRNA occurs causing gene silencing. RNAi can be achieved by using a long dsRNA which is cleaved in a cell to form an siRNA, or by using an siRNA directly which is already short. Preferably the siRNA has an antisense stand which is complementary to at least part of a target mRNA, or is sufficiently complementary to at least part of a target mRNA to cause degradation of the mRNA.

Preferably an siRNA is used to down regulate α-synuclein. Preferably the siRNA used comprises an antisense strand of RNA which is complementary to a part of the mRNA transcribed from the SNCA gene, or is sufficiently complementary to a part of the SNCA mRNA such that it serves to reduce or eliminate expression of the α-synuclein protein.

The siRNA may be double stranded, comprising a sense strand and a complementary anti-sense strand. The strands may not be perfectly complementary; they may be just sufficiently complementary to form a stable double stranded molecule. Preferably, the anti-sense strand sequence is complementary to part of the nucleotide sequence of mRNA derived from the SNCA gene, or is sufficiently complementary to part of the nucleotide sequence of the mRNA to bind thereto and cause its degradation.

Preferably the siRNA molecule includes a 3'dTdT overhang. The 3' overhang may be on one or both strands of the siRNA.

The siRNA may be prepared for administration to a subject by one of the following methods, chemical synthesis; in vitro transcription; or digestion of long dsRNA by, for example, RNaseIII or Dicer. Alternatively, the siRNA may be produced within the cells where it is needed, for example by expression from a plasmid or viral vector inside the cell. The siRNA may be an isolated molecule.

Preferably the siRNA is about 10 to about 100 nucleotides in length. Preferably the siRNA comprises about 15 to about 30 nucleotide base pairs. Preferably the siRNA comprises about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs. The siRNA may also include a 3'dTdT overhang on one or both strands.

The siRNA may include one or more modifications that stabilise it in a biological sample, or in vivo . For example, the siRNA may include phosphorothioate derivatives which have increased resistance to nucleases.

The siRNA may include unmodified RNA, modified RNA or a combination of both. Unmodified RNA refers to components of the nucleic acid, namely sugars, bases, and phosphate moieties, which are the same or essentially the same as those that occur in nature, preferably as occur naturally in the human body. Modified RNA includes components which are synthetic, are synthetically modified or which are rare or unusual.

The siRNA may be delivered as a siRNA, or as a larger RNA molecule which can be processed into a siRNA, or it may be delivered as a DNA molecule which encodes a siRNA.

The siRNA may be produced synthetically, naturally or recombinantly.

Administration of the siRNA may be carried out using the various mechanisms known in the art, including naked administration and administration in a pharmaceutically acceptable lipid carrier. The siRNA, for example, may be administered by injection (e.g. , intravenous or intramuscular, intrathecally, or directly into the brain) , or may be inhaled, or may be topically applied.

The siRNA molecules may be expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siRNA expressing viral vectors may be constructed based on, but not limited to the Herpes Simplex Virus- 1 , Lentivirus, retrovirus, Adeno Associated Virus and Adenovirus. Recombinant vectors capable of expressing the siRNA molecules may be delivered to target cells, for example in the brain. Viral vectors may be used that provide for transient expression of siRNA molecules. Delivery of siRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

The siRNA may be administered at a dose of less than about 10, 5, 2, 1 , 0.5, 0.1, 0.05, 0.01 , 0.005, 0.001, 0.0005, 0.0001 , 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. , about 4.4 x lO¹⁶ copies) per kg of bodyweight, or less than 1500, 750,

300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015,

0.00075, 0.00015 nmole of RNA agent per kg of bodyweight. Particularly preferred dosages are less than 2, 1 , or 0.1 mg/kg of body weight. The dosage used is preferably in an amount effective to treat or prevent a particular neurodegenerative disease.

The siRNA may be administered daily, or more than once a day, or less frequently than once a day, e.g. , less than every 2, 4, 8 or 30 days. The siRNA may include the sequence of Sequence ID No: 1 , 2 or 3 and or the sequence complementary thereto, or include a sequence which is a variant of Sequence ID No. 1, 2 or 3 and/or a sequence complementary thereto .

Preferably the siRNA includes the sequence of sequence ID No: 1 or 2 and/or the sequence complementary thereto or a sequence which is a variant thereof.

Preferably the variant sequence has at least 50% identity to the sequence of Sequence ID No. 1, 2 or 3, and/or a sequence complementary thereto. The nucleic acid sequence preferably has at least 60%, 65%, 70%, 75% or 80% identity to Sequence ID No. 1 , 2 or 3, and/or a sequence complementary thereto. Even more preferably, the nucleic acid sequence has 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to Sequence ID No. 1, 2 or 3, and/or a sequence complementary thereto.

The term "identity" in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about six nucleotides, usually at least about 10 nucleotides, more usually at least about 15 nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG) , Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (1990) , Methods Enzymol. 183:63-98) . For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al. , 1990 J. MoI. Biol. 215:403-410; Gish and States, 1993 Nature Genet. 3:266-272; Madden et al. , 1996 Meth. Enzymol. 266:131- 141; Altschul et al. , 1997 Nucleic Acids Res. 25:3389-3402; Zhang and Madden, 1997 Genome Res. 7:649-656), especially blastp or tblastn (Altschul et al. , 1997 Nucleic Acids Res. 25:3389-3402) .

According to another aspect the invention provides a siRNA comprising a nucleotide sequence identical to the sequence of sequence ID No: 1 , 2, or 3, No. 1 or 2, and/or a sequence complementary thereto, or a nucleotide sequence which is a variant of Sequence ID No. 1, 2 or 3 and/or a sequence complementary thereto.

According to another aspect the invention provides a vector comprising a promoter, a nucleotide sequence of sequence ID No 1, 2 or 3 and/or a sequence complementary thereto, or a nucleotide sequence which is a variant of Sequence ID No. 1, 2 or 3 and/or a sequence complementary thereto, operatively linked to said promoter, wherein the nucleotide sequence encodes a siRNA that is capable of down regulating expression of α-synuclein.

Most preferably, the siRNA or vector comprises the sequence of SEQ ID No: 1 or 2.

Operative linking of the promoter to the nucleotide sequence ensures that the promoter can regulate the expression of the nucleotide sequence. Usually this requires the promoter to be located upstream, but in close proximity, to the nucleic acid sequence. According to another aspect the invention provides a method of treating a neurodegenerative disorder in a subject with normal physiological levels of α-synuclein comprising administering to the subject a therapeutically or prophylactically effective amount of an agent for down regulating α-synuclein.

According to another aspect the invention provides a method of treating sporadic Parkinson's disease comprising administering to a subject a therapeutically or prophylactically effective amount of an agent for down regulating α-synuclein.

According to another aspect the invention provides a method of treating a neurodegenerative disorder in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of an siRNA comprising the sequence of Sequence ID No: 1, 2 or 3 and/or a sequence complementary thereto, or a nucleotide sequence which is a variant of Sequence ID No. 1 , 2 or 3 and/or a sequence complementary thereto.

Preferably, the siRNA comprises the sequence of SEQ ID No: 1 or 2.

According to another aspect the invention provides a method of reducing α-synuclein in a cell with normal physiological levels of α-synuclein comprising introducing into the cell an effective amount of an agent to down regulate expression of the α-synuclein gene.

According to a further aspect the invention provides a pharmaceutical composition for down regulating α-synuclein in a subject with normal physiological levels of α-synuclein comprising one or more agents capable of down regulating α-synuclein and a pharmaceutically acceptable carrier. According to a yet further aspect the invention provides a pharmaceutical composition for down regulating α-synuclein in a subject with sporadic Parkinson's disease comprising one or more agents capable of down regulating α-synuclein and a pharmaceutically acceptable carrier.

Preferably the agent capable of down regulating α-synuclein is an RNAi agent, or a ribozyme, or an antisense molecule which targets SNCA RNA, or an antibody, or a naturally occurring or synthetic polypeptide, or a small molecule.

A pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, intranasal, transdermal) , oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

According to another aspect the invention provides a method for protecting human neuronal cells with normal physiological levels of α-synuclein from neurotoxic insult by down regulating α-synuclein. Preferably α-synuclein is down regulated by using RNA interference (RNAi) to down regulate α-synuclein expression. According to a further aspect the invention provides a method for treating sporadic Parkinson's disease, comprising determining whether a subject has sporadic Parkinson's disease then administering to the subject a therapeutically effective amount of an agent to down regulate α-synuclein.

To determine whether a subject has sporadic Parkinson's disease the DNA of a subject with symptoms of Parkinson's disease could be analysed to determine whether any monogenic indicators of familial Parkinson's disease are present, if there are no monogenic indicators then the subject may be diagnosed as having sporadic Parkinson's disease.

The skilled man will appreciate that any of the preferable features discussed above can be applied to any of the aspects of the invention.

Embodiments of the present invention will now be described, by way of example only, with reference to the following figures.

Figure 1 - illustrates the expression of dopaminergic markers in SH-SY5Y cells. SH-SY5Y cells display the phenotype of a dopaminergic neuron of the midbrain. A distinguishing feature of a dopaminergic cell is the presence of the dopamine transporter

DAT. Figure IA shows the results of RT-PCR performed on RNA extracted from SH-S Y5 Y cells. The primers used are able to amplify between exons 2-3 of the RNA encoding VMAT2, exons

13-14 of the RNA encoding TH, exons 2-3 of the RNA encoding

DAT and exons 1-2 of the SNCA RNA which encodes the α-synuclein protein. The predicted amplicon sizes are VMAT2 264 bp, TH 183 bp, DAT 322 bp, SNCA 198 bp. Figure IB shows western blot analysis of protein from SH-SY5Y cells using antibodies for α-synuclein, Tau, DAT and actin. Each produced a band corresponding to the predicted size for each protein.

Figure 2 - illustrates the morphological features of differentiated SH-S Y5 Y and MESC2.10 cells. Figure 2 A shows SH-S Y5 Y and

MESC2.10 cells at the end of the differentiation process, phase contrast images were taken using a 2Ox objective. Figure 2B shows the cells of Figure 2A immunostained with the neuronal marker antibodies SMI-31 and MAP2 and imaged by laser scanning confocal microscopy. The SMI-31 antibody recognises axonal cytoskeletal proteins, including MAPlB and HMW-NFP. The MAP2 antibody preferentially recognises heavy isoforms of MAP2 associated with mature neurons. In both cell models, the neuritic network is similar to that seen in primary neurons. Nuclei are counter-stained with DAPI.

Figure 3 - shows that the differentiation of SH-SY5Y and MESC2.10 cells involves up regulation of α-synuclein and the neuronal markers tau and DAT. Figure 3 A shows protein extracted from SH-SY5Y cells at the start of differentiation (day 1) , after RA treatment (day 5) and at the end of differentiation (day 10) . The protein lysate was analysed by western blotting. Figure 3B shows protein analysed from MESC2.10 cells at the start of differentiation (day 1) , half way through differentiation (day 2.5) and at the end of differentiation (day 5) .

Figure 4 - shows the efficient RNAi-mediated down regulation of α-synuclein protein in SH-S Y5 Y cells. Figure 4 A shows fluoroscene labelled siRNAs transfected into SH-SY5Y cells and imaged using confocal microscopy. The nuclei are counterstained with propidium iodide. Intracellular localisation of siRNA was confirmed using fine slice Z-stack imaging. Figure 4B shows the effect on α-synuclein expression in SH-SY5Y cells when transfected with three siRNAs targeting α-synuclein or with a scrambled control or with carrier agent only. Protein from the transfected cells was analysed by western blot analysis 72 hours after transfection. Figure 4C shows the intensity of each band in Figure 4B, the intensity was quantified using a CCD camera, α-synuclein expression was normalised to actin and the results are expressed relative to untransfected cells ( ± SE) . The results show a significant effect of treatment with the siRNA on α-synuclein expression (ANOVA, p < 0.001) . Each of the active siRNA molecules, SNCAl, SNC A2 and SNC A3, produced a significant down regulation in α-synuclein expression when compared to all the controls (p < 0.001 for each) . There was no difference between the control groups.

Figure 5 - shows the efficient RNAi-mediated down regulation of α-synuclein protein in MESC2.10 cells.

Figure 6 - shows that cells differentiate normally with α-synuclein down regulation. Figure 6 A shows SH-SY5Y cells and Figure 6B shows MESC2.10 cells transfected with SNCAl (knockdown) or a scrambled version of SNCAl (control) . After differentiation, immunofluorescence studies were performed using the axonal marker antibody SMI-31 and an anti-α-synuclein antibody. Nuclei are counter stained with DAPI. Successful down regulation was confirmed by western blot in cells treated in parallel with those used in these studies. Figure 7 - shows that α-synuclein down regulation does not alter tau expression. Figure 7A shows SH-S Y5 Y cells and Figure 7B shows MESC2.10 cells transfected with SNCAl (knockdown) or a scrambled version of SNCAl (control) . After differentiation the protein was analysed by western blot to assess expression of the neuronal marker tau. Expression levels were the same in down regulated, control and untransfected cells, α-synuclein was also assayed to confirm down regulation and actin is used as a loading control.

Figure 8 - shows the effect of α-synuclein suppression on baseline cell viability. Figure 8 A shows the effect on cell growth of transfection with the siRNAs. Cell growth was assessed at time points 0, 24, 48, 72, 96 and 120 hours after transfection using the MTT assay. No significant difference between cells treated with the three different siRNAs was observed. Figure 8B shows the effect of transfection with the siRNAs on α-synuclein expression over time. Protein was extracted from cells 48, 72, 96 and 120 hours after transfection and evaluated by western blot analysis.

Figure 9 - further illustrates that down regulation of α-synuclein does not alter neuronal viability. Viability of differentiated SH- SY5Y (Figure 9A) and MESC2.10 cells (Figure 9B) was assessed by staining live cells with calcein AM and dead cells with ethidium homodimer. Six fields of view were counted for differentiated

SH-SY5Y (Figure 9C) and MESC2.10 (Figure 9D) transfected with either SNCAl (knockdown) or a scrambled version of SNCAl (control) siRNAs. No significant difference was found in the numbers of neurons for either group in either SH-SY5Y (unpaired Student's t-test, p = 0.60) or MESC2.10 cells (unpaired Student's t-test, p = 0.67) . Viability was also measured using the MTT assay which measures mitochondrial activity for differentiated SH-SY5Y cells (Figure 9E) and MESC2.10 (Figure 9F) cells treated in the same way. MTT absorbance was measured in triplicate and is expressed relative to control cells. No difference was found in

MTT absorbance between groups for SH-S Y5 Y (unpaired Student's t-test, p = 0.81) and MESC2.10 cells (unpaired Student's t-test, p = 0.80) . Results are the mean ± SEM for three separate experiments. In each experiment, down regulation was confirmed by western blot in cells treated in parallel with those used for viability determinations.

Figure 10 - shows the function of endogenous DAT in SH-S Y5 Y cells. In Figure 1OA confocal microscopy shows DAT distribution in an SH-S Y5 Y cell. The nucleus is counterstained with propidium iodide. Figure 1OB shows the amount of uptake into SH-S Y5 Y cells of ³HDA with and without Mazindol. Cells were incubated with 20 mM ³HDA for time periods from 0-10 minutes either with or without 10 μM Mazindol. Uptake was quantified using scintillation counting and normalised to cellular protein in each sample ( ± SE) .

Figure 11 - shows the effect of α-synuclein suppression on DAT kinetics. In Figure HA dopamine uptake velocity was measured in untransfected cells or 72 hours after transfection with SNCAl ,

SNC A2 and scrambled siRNAs with 15 nM - 1 μM of dopamine.

Data was fitted to the Michaelis-Mentin equation using non-linear regression with Poisson weighting. Data is plotted ± SE.

Figure HB shows the results of an Eadie-Hofstee transformation of the data, this was performed to determine K_m and V_max values.

There was a significant effect of siRNA treatment on V_max (ANOVA, p < 0.001). Cells treated with SNCAl had a 50% decrease in V_raax compared to scrambled control cells (p < 0.01) while cells treated with SNCA2 had a 38% decrease compared to scrambled control cells (p < 0.05). There was no significant difference in V_max between scrambled siRNA and untransfected cells. There was no effect of treatment on K_m. Figure HC shows the results of protein analysis by Western blot in a parallel experiment, the results demonstrate that RNAi-mediated down regulation of α-synuclein does not affect DAT expression.

Figure 12 - illustrates that down regulation of α-synuclein protects neurons in models of sporadic PD. Figure 12A shows representative pictures of control and down regulated (knockdown) neurons treated with ImM MPP+ . Live cells are stained green with calcein AM. Dead cells are stained red with ethidium homerdimer-1. Figure 12B shows viability curves of differentiated SH-SY5Y treated with different concentrations of MPP + . Control cells are shown as triangles and a dotted line. Down regulated cells are shown as squares with a solid line. Down regulation caused a significant reduction in cell death (two-way ANOVA, p < 0.001) .

Figure 12C shows viability curves of MESC cells treated with different concentrations of MPP + . Down regulation caused a significant reduction in cell death (two-way ANOVA, p < 0.001) . Figure 12D shows viability curves for differentiated SH-S Y5 Y cells treated with rotenone. While there was a trend to increased survival in the down regulated groups this was not statistically significant (two-way ANOVA, p = 0.07) . Figure 12E shows viability curves of MESC cells treated with different concentrations of rotenone. Down regulation caused a significant reduction in cell death (two-way ANOVA, p < 0.001) . Figure 12F shows viability curves of SH-SY5Y cells treated with 5μM dopamine for 48 hours. Down regulation caused a significant reduction in cell death (two- way ANOVA, p < 0.001) . Figure 12G shows viability curves of MESC cells treated with 5μM dopamine for 48 hours. Down regulation causes a significant reduction in cell death (two-way ANOVA, p < 0.05) . All results are the mean ± SEM of three separate experiments.

Figure 13 shows the effect of α-synuclein level on the susceptibility of cells to MPP + . Forty-eight hours after treatment with siRNA, cells were treated for 12 hours with 5 mM of MPP + in media without serum. Control cells were only exposed to medium without serum. Cell viability was assessed by MTT assay and cell survival normalised to control cells which were not exposed to MPP + ( ± SE) . There was no difference in baseline cell viability between the three siRNAs tested (data not shown) . There was a significant effect of MPP+ exposure on cell survival (ANOVA, p < 0.001) with a 40% reduction in survival in cells treated with scrambled siRNA. This was entirely prevented by treatment with siRNAs to reduce α-synuclein expression (p < 0.001 for both SNCAl and SNC A2) .

Figure 14 shows the effect of α-synuclein level on the susceptibility of cells to dopamine (DA) .

Figure 15 - shows that down regulation of α-synuclein reduces formation of toxic protofibrillar species of α-synuclein. Figure 15 A illustrates a prevailing hypothesis in the field of PD research that α-synuclein protofibrils, which are an intermediate in the formation of Lewy bodies, are toxic due to a number of factors. Figure 15B shows analysis of triton soluble α-synuclein species by western blot. Figure 15C shows quantification of soluble α-synuclein oligomers and multimers (protofibrils) from western blots. MPP+ caused a siginificant increase in protofibril formation in control neurons (p < 0.01) which was prevented by α- synuclein down regulation.

Figure 16 - shows that down regulation of α-synuclein prevents MPP+ driven increases in NOS activity. NOS activity was assayed by the conversion of arginine to citrulline. NOS activity was significantly increased by MPP+ treatment in control neurons (two way ANOVA, p < 0.001) was prevented by α-synuclein down regulation (two-way ANOVA, p < 0.05) .

METHODS

Cell culture

Human SH-SY5Y cells (ECACC# 94030304) were obtained from the European Collection of Cell Cultures (ECACC) and used within 20 passages of the original vial. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) / F-12 (1 :1) (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Sigma, St. Louis, MO) , 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 mM L-glutamine at 37⁰C and in 5% CO₂. The protocol for cell differentiation was adapted from that used by Giminez-Cassina et al (2006 J Neurosci Res 84, 755-767) . Briefly, culture dishes were prepared by application of 0.1% poly-Lysine (70-150 kDa) (Sigma) for 24 hours. Plates were then washed thoroughly with water and cells seeded at 2 x 10⁴ per cm² in normal growth media. The next day the media was replaced with DMEM with only 5% FBS, antibiotics, 2 mM L-glutamine and 10 μM retinoic acid. The medium was changed every second day. After 4 days, the medium was changed to Neurobasal medium (Invitrogen) supplemented with 1 x B-27, 2 mM GlutaMaxI (Invitrogen) , 2 mM dibutyryl-cyclic AMP (db-cAMP), 50 ng/ml human recombinant brain derived neurotrophic factor (BDNF) and antibiotics. Cells were then allowed to differentiate for 5 days in this medium.

Human MESC2.10 cells were maintained as described by Lotharius et al (2002 J Biol Chem 277, 38884-38894) with some modifications. MESC2.10 cells are the best available model of human neurons available. Routine passage of cells was conducted in poly-L-lysine (150 - 300 kDa) (Sigma) coated 25 cm² tissue culture flasks. Cells were grown in N2 media consisting of DMEM/F12 high glucose with N2 supplement (Invitrogen) , 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 40 ng/ml human recombinant basic fibroblast growth factor (bFGF) (Sigma) . Plates were prepared for differentiating the cells by coating with 0.1% poly-L-lysine (150 - 300 kDa) (Sigma) for 24 hours and 5 μg/ml human plasma derived fibronectin (Sigma) for 2 hours. Cells were seeded at 5 x 10⁴ per cm² and allowed to adhere overnight. The next day the N2 proliferation medium was replaced by differentiation medium consisting of DMEM/F12 high glucose with N2 supplement, 2 mM L- glutamine, antibiotics, 1 mM db-cAMP, 2 ng/ml glial cell line-derived neurotrophic factor (GDNF) (Sigma) and 1 μg/ml tetracycline. Cells were differentiated for five days with the medium being changed every second day.

RT-PCR

RNA was prepared from SH-SY5Y and MESC2.10 cells cultured as described above using Trizol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. RNA (1 μg) was reverse transcribed into cDNA using Superscript First Strand Synthesis Kit (Invitrogen, Carlsbad, CA) in a 20 μL reaction as directed. PCR amplification was performed using 0.5 μL of cDNA with AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA) as directed. The primers used for the RT-PCR were chosen to amplify across an intron and were checked by BLAST searches to ensure specificity. RT-PCR primers used in this study were:

VMAT2, exons 2-3, forward 5 '-GCTCGCGGAAGCTCATCCTGTT-S ' ; and reverse 5¹-GCTGTGTGGCGGTCTGATGA-3¹ ;

TH, exons 13-14, forward 5 '-CTGTCTGAGGAGCCTGAGATT-S ' ; and reverse 5 '-CGTGTACGGGTCGAACTTCA-S ' ;

DAT, exons 2-3, forward 5 ' -ATGAGT AAGAGCAAATGCTC-S¹ ; and reverse 5 ' -TGACCATGAAGAGC AGGTAG-3 ' ;

SNCA, exons 1-2, forward 5 ' -CTTCAAGCCTTCTGCCTTTC-S ' ; and reverse 5 ' - AC ACCCTCTTTTGTCTTTCCTG-3 ' .

Western blotting

Cultured cells were washed once with phosphate buffered saline (PBS) and then scraped into PBS. The cultured cells may have been differentiated as described. Cells were pelleted by centrifugation at l,700xg for 2 minutes and lysed in WB lysis buffer (50 mM tris pH 8.0,

150 mM NaCl, 1% triton XlOO, Ix protein inhibitor cocktail (Sigma,

St. Louis, MO)) by tituration. After incubation for 30 minutes on ice, the lysate was cleared by centrifugation for 10 minutes at 1,200 xg, and the supernatant retained. Protein content was quantified using the BCA assay

(Sigma, St. Louis, MO) in accordance with the manufacturer's instructions. 10 μg of protein was boiled in Ix Laemelli buffer for

5 minutes. Protein was then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred by electrophoresis to

0.45μm PVDF membrane (Millipore, Billerica, MA) . Blots were blocked in WB blocking buffer (tris buffered saline (TBS) , 1% Tween-20, 5% milk) and probed using the primary antibodies suspended in WB hybridising buffer (TBS, 1% Tween-20, 1% milk) .

The following antibodies were used: mouse monoclonal anti-α-synuclein (Abeam, Cambridge, UK); rabbit polyclonal anti-DAT (Alpha-diagnostic, San Antonio, TX) , mouse monoclonal anti-Tau (Chemicon, Temecula, CA); mouse monoclonal anti-actin (Sigma, St Louis, MO) .

Membranes were washed three times with TBS, 1% Tween-20 and appropriate horse radish peroxidase conjugated secondary antibodies (Biorad, Hercules, CA) were applied, suspended in hybridising buffer. After washing, chemiluminescence was produced using an ECL + kit (Amersham, Piscataway, NJ) . Images were photographed using a charge couple device camera (UVP, Upland, CA). The integrated optical density of each band was measured using LabWorks software 4.6 (UVP, Upland, CA) . For comparative expression assessment, expression was normalised to actin levels in the same blot and expressed relative to untransfected cells. Each Western Blot shown is typical of at least three separate experiments .

Transfection of siRNA

In order to transfect SH-S Y5 Y cells in one well of a 6 well dish, 3 μl of Lipofectamine 2000 (LF2000) (Invitrogen) was diluted in 150 μl of OptiMEM media (OM) (Invitrogen) and incubated for 5 minutes at room temperature. At the same time, the siRNA was diluted in 150 μl of OM. siRNAs were used at a final concentration of 50 nM based on a dose- response analysis of siRNA concentrations. After 5 minutes, the siRNA and LF2000 mixtures were combined. Complexes were allowed to form for 20 minutes. During the incubation a sub confluent flask of SH-SY5Y cells was trypsinised, spun down, resuspended in OM and cells counted using a haemocytometer. Cells were diluted down to 1.33 x 10⁵ cells/ml and 1.5 mis put aside for each well to be transfected. After the siRNA:LF2000 complexes had formed, 300 μl of the complex mixture was added to 1.5 mis of cells and mixed gently. The final volume was therefore 1.8 mis per well. The cells were then allowed to stand for 10 minutes before being plated in prepared cell culture plates as described for routine differentiation. After 6 hours, 900 μl of OM supplemented with 30% FBS was added. After 18 hours, the transfection medium was removed, cells washed three times with OM and then cells covered in DMEM with 5% FBS, antibiotics, 2mM L-glutamine and 10 μM retinoic acid. Differentiation of the cells then proceeded as normal.

A similar approach was used for MESC2.10 cells. For one well of a 6 well dish, 3.9 μl of LF2000 was diluted in 150 μl of OM and incubated for 5 minutes at room temperature. As before, the siRNA was diluted in 150 μl of OM. Again, based on our titration experiments, we used siRNAs at a final concentration of 50 nM. After 5 minutes, the siRNA and LF2000 mixtures were combined and complexes allowed to form for 20 minutes. During the incubation MESC2.10 cells were trypinised, spun down, resuspended in N2 medium with no antibiotics and counted. The cells were diluted to 1.33 x 10⁵ cells/ml and 1.5 mis was put aside for each well. After the siRNA:LF2000 complexes had formed, 300 μl were added to the cells which were then allowed to stand for 10 minutes. The cells were plated in wells prepared with poly-L-lysine and fibronectin. After 5 hours the cells had adhered and the transfection medium could be removed. Cells were washed three times with N2 medium and then covered with differentiation medium. Differentiation then proceeded as described.

The siRNAs used in this study were (the sense sequence only is listed below, the antisense sequence used was complementary to the sense sequence, excluding the 3' dTdT overhang. The antisense strand may also have a 3 ' dTdT overhang) :

Scrambled (Seq ID No: 4) :5'-GCGACGUUCCUGAAACCACdtdt-3 ' ; SNCAl (Seq ID No: 1) : 5 ' -GGAAAGAC AAAAGAGGUGdtdt-3¹ ; SNC A2 (Seq ID No: 2) : 5'-GGUGUUCUCUAUGUAGGCdtdt-3^I ; SNCA3 (Seq ID No: 3) : 5'-GGAGGAAUUUUAGAAGAGGdtdt-3'

Before use the sense and antisense strands of the scrambled, SNCAl, SNCA2 and SNCA3 were annealed using techniques well known in the art.

To anneal the sense and antisense stands to form the double stranded siRNA a 20 mM siRNA duplex solution was prepared by combining: 70 μl of 2x annealing buffer (200 mM potassium acetate, 4 mM magnesium acetate, 60 mM HEPES-KOH (pH 7.4)) , sense siRNA to 20 mM final concentration, antisense siRNA to 20 mM final concentration, sterile H₂O to a final volume of 140 μl . The reaction was incubated for 1 minute at 9O⁰C, followed by 1 hour at 37⁰C. Unused siRNA duplex solution was stored frozen at -20⁰C.

Subsequent reference to the scrambled siRNA, SNCA 1, 2 or 3 refers to the double stranded form, comprising both the sense and antisense strands.

All RNA molecules were purchased from Ambion (Austin, TX) .

Immunofluorescence

Cells were plated in 24 well dishes containing glass coverslips and allowed to adhere overnight. Cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilised with IF block solution (1% fish gelatine, 0.1% Triton X-IOO, 10% normal goat serum in TBS) for 30 minutes at room temperature. Rabbit polyclonal anti-DAT (Alpha- diagnostic, San Antonio, TX) primary antibody was applied in an overnight incubation at 4° C. Cells were washed four times in IF wash solution (0.1% Triton X-100, 0.02% sodium azide in TBS) before application of AlexaFluor 488 IgG anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA). Cells were mounted using Vectashield mounting medium with propidium iodide (Vector Laboratories, Burlingame, CA) and imaged using confocal laser scanning microscopy (Carl Zeiss, Thornwood, NY) .

Immunofluorescence of differentiated cells

Cells were differentiated in 24 well dishes containing glass cover slips coated as described. After differentiation, cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilised with IF block solution (1% fish gelatine, 0.1% Triton X-100, 10% normal goat serum in TBS) for 30 minutes at room temperature. The following primary antibodies and dilutions were used: mouse monoclonal anti-α-synuclein (1:500) (Abeam, Cambridge, UK) , mouse monoclonal antibody SMI-31 (1 :500) (Sternberger Moleuclar Inc, Baltimore, MD) , rabbit monoclonal anti-MAP2 antibody (1:1000) (Chemicon, Temecula, CA) . The primary antibody was applied in an overnight incubation at 4⁰C. Cells were washed four times in IF wash solution (0.1% Triton X-100, 0.02% sodium azide in TBS) before application of the appropriate AlexaFluor 488 IgG secondary antibody (Invitrogen) . Cells were mounted using Vectashield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) and imaged using confocal laser scanning microscopy (Carl Zeiss) . Measurement of cell viability

Cells were grown in 24 well dishes and, once differentiation was complete, they were washed with Dulbecco's PBS (D-PBS) supplemented with calcium and magnesium. Cell viability was assessed using vital stains. D-PBS with 4 μM Calciein AM and 4 μM Ethidium homodimer-1 (Invitrogen, Carlsbad, CA) was added to the cells and incubated at 37°C for 30 minutes. Cells were then immediately imaged using a 2Ox objective on an inverted fluorescent microscope (Nikon) and pictures taken with a CCD camera (Hamamatsu Photonics, Shizuoka, Japan) . Images from six randomly selected fields of view were captured in each well and live cells were counted blind using Volocity software (Improvision, Lexington, MA) . Results were expressed as the mean number of cells per field of view from three separate experiments.

Cell viability was also determined in 24 well plates using the MTT assay (Sigma, St. Louis, MO) as described by Brown et al (2006 J Neurochem 98, 495-505) . Absorbance was measured at 570 nm in 96 well plates using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale, CA) . Background absorbance was subtracted at 690 nm. To measure the effect of MPP + toxicity, cells were exposed to 5mM MPP + in media without serum or antibiotics for 12 hours. Control cells were treated with media without serum or antibiotics for the same duration. Experiments were performed in triplicate and repeated three times.

³H Dopamine uptake measurements and data analysis

³H Dopamine (³HDA) uptake measurements were performed on cells in 6 well dishes. The cells were washed three times in Dulbecco's Phosphate Buffered Saline with calcium and magnesium (D-PBS) (Invitrogen, Carlsbad, CA) . ³HDA was resupended in D-PBS to the desired concentration with or without 10 μM Mazindol. For measuring the time course of uptake, 20 nM ³HDA was applied for periods of up to 10 minutes. For kinetic analysis, up to 30 nM ³HDA was added along with non-radioactive dopamine to produce final concentrations of 15 nM - 1 μM. Uptake was measured over two minutes. For each experimental well, 1 ml of ³HDA mixture was added for the required time before being aspirated and cells washed twice with ice cold PBS. At the end of the experiment the cells were dissolved in 1 ml IN NaOH solution. 500 μl of cell lysis solution was added to 5 ml of OptiPhase Supermix (PerkinElmer, Wellesley, MA) and scintillation quantified using a scintillation counter (Beckman Coulter, Fullerton, CA) . Results were normalised to protein content measured in a sample of the cell lysate using the BCA method as described.

Kinetic parameters of ³HDA uptake were determined using linear regression of Eadie-Hofstee plots from five separate experiments and confirmed using non-linear regression analysis fitting the data to the Michaelis-Menten formula with Poisson weighting (GraphPad Prism, GraphPad Software, San Diego, CA) . There was good agreement between the two methods. ANOVA of this data was performed using the extra sum-of-squares F test.

RESULTS

SH-SY5Y cells are a suitable dopaminergic model

The human neuroblastoma cell line SH-SY5Y has been widely used as a dopaminergic neuronal cell culture model (Takahashi et al. , 1994 J

Neural Transm Gen Sect 98:107-118; Fang et al. , 1995

Psychopharmacology (Berl) 121:373-378) . To confirm that these cells were suitable for this invention endogenous expression of the dopamine related genes tyrosine hydroxylase (TH) , vesicular monoamine transporter 2 (VMAT2) , DAT and α-synuclein was demonstrated at the

RNA level by reverse-transcription PCR (RT-PCR) (Figure IA) . Endogenous expression levels of the α-synuclein and DAT proteins as well as the neuronal marker protein Tau was demonstrated by Western blot analysis (Figure IB) .

Differentiating neurons express increasing levels of neuronal markers and α-synuclein

Several different protocols have been developed to differentiate SH-SY5Y cells based on sequential treatment with retinoic acid followed by BDNF (Garcia-Perez et al 1998 J Neurosci Res 52, 445-452; Encinas et al. J Neurochem 75, 991-10032000; Gimenez-Cassina et al 2006 J Neurosci Res 84, 755-767) . For this study, a slightly modified protocol from that described by Gimenez-Cassina et al (2006) was used (see Methods for details) .

MESC2.10 cells, are derived from 1st trimester human mesencephalic cells (Lotharius et al 2002 J Biol Chem 277, 38884-38894) . MESC2.10 cells are immortalised by a v-myc oncogene under the control of a tetracycline sensitive transactivator. This allows the cells to replicate indefinitely in culture. Upon the addition of tetracycline, v-myc expression is abolished and, in cooperation with GDNF and dcAMP, cells become post-mitotic and adopt a neuronal phenotype.

By monitoring changes in cellular morphology of SH-SY5Y cells and MESC2.10 cells differentiation of the two cell types was studied (Figure 1) . The development of neurite-like extensions, which form a complex network in both cell types (Figure IA) , was observed using light microscopy. Immunofluorescence studies were undertaken using two markers of cytoskeletal polarity, SMI-31 and microtubule associated protein 2 (MAP2) (Figure IB) . The SMI-31 antibody recognises axon- specific phosphoepitopes of cytoskeletal proteins, including MAPlB and high-molecular weight filament protein (HMW-NFP) (Ulloa et al 1993 Embo J 12, 1633-1640; Garcia-Perez et al 1998 J Neurosci Res 52, 445- 452) . The MAP2 antibody preferentially recognises heavy isoforms of MAP2 associated with mature neurons (Diez-Guerra & Avila 1995 Eur J Biochem 227, 68-77) . Both differentiated cell types showed expression patterns consistent with the cytoskeletal morphology found in primary neurons .

To confirm the neuronal phenotype, expression levels of the neuronal maker tau was measured on immunoblots from protein lysate harvested from cells grown under differentiation conditions for different lengths of time (Figure 2) . At baseline, undifferentiated SH-SY5Y cells expressed moderate levels of tau and this was markedly increased by the end of differentiation. Undifferentiated MESC2.10 cells expressed no detectable tau, however high expression levels of the protein were observed after differentiation .

It has been suggested that α-synuclein may have a role in neuronal differentiation (Sidhu et al 2004 Ann N Y Acad Sci 1035, 250-270) . In keeping with this, α-synuclein is up-regulated during differentiation of MESC2.10 (Lotharius et al 2002 J Biol Chem 277, 38884-38894) and human embryonic carcinoma line NTERA-2 (NT2) cells (Satoh & Kuroda, 2001 Parkinsonism Relat Disord 8, 7-17) . Proliferating MESC2.10 cells exhibit minimal levels of α-synuclein but, by the end of differentiation they express relatively high levels (Figure 2) . The same was true for SHSY-5Y cells which expressed a moderate amount of α-synuclein at baseline which increased more than three-fold by the end of the 10 day differentiation period. Both neuronal models therefore displayed increased α-synuclein expression during differentiation. RNAi can suppress α-synuclein expression

Small interfering RNA (siRNA) molecules were shown to suppress endogenous α-synuclein levels in SH-SY5Y and MESC2.10 cells.

To first confirm successful delivery of the siRNA molecules, labelled scrambled siRNAs were transfected, according to the aforementioned protocol, and intracellular localisation confirmed by immunofluorescence studies (Figure 4A) .

Three different siRNA (double stranded) molecules designed to specifically target α-synuclein (SNCAl, 2 and 3) , and a scrambled control (double stranded) siRNA moleucle, were then transfected into SH-SY5Y cells and expression of α-synuclein protein was assayed 72 hours later (Figure 4B) . Quantification of the protein levels showed a highly significant effect of siRNA treatment on α-synuclein protein levels (ANOVA, p < 0.001) (Figure 4C) . SNCAl and 2 both produced 80-90% reduction in α-synuclein compared to untransfected cells and SNCA3 produced 67% reduction (p < 0.001 for each) . SNCAl and SNCA2 were selected for further studies. The scrambled control or carrier agent only control did not produce any significant down regulation of α-synuclein indicating that down regulation was specific to the targeted sequences. None of the siRNAs produced down regulation of actin, again confirming the specificity of down regulation.

In a similar study in MESC2.10 cells, SNCAl produced a 90% reduction in α-synuclein compared to untransfected cells and SNCA2 produced a 50% reduction (Figure 5) . Again the scrambled control had no effect. To summarise, by using RNAi an 80% reduction in endogenous α-synuclein levels was achieved in SH-SY5Y and a 90% reduction in MESC2.10 cells.

Cells differentiate normally with α-synuclein down regulation

The increase in α-synuclein expression during differentiation may indicate the protein is involved in cell differentiation. To test this, differentiation in cells transfected with SNCAl was compared to control cells treated with the scrambled SNCAl siRNA. The morphological and gene expression characteristics of the cells were determined using antibodies for α-synuclein and the neuronal marker antibody SMI-31 (Figure 6) . Consistent with the expression analysis from western blot analysis, SNCAl treated SH-SY5Y and MESC2.10 cells showed minimal α-synuclein immunoreactivity compared to scrambled control treated cells. By contrast, the level of immunoreactivity to the SMI-31 was similar in both groups. Furthermore no differences in the morphological characteristics of cells with or without α-synuclein suppression were observed. Cells treated with SNCAl or scrambled control siRNA both developed an extensive network identical to that seen in untransfected cells indicating that differentiation had proceeded normally in cells lacking α-synuclein.

To be certain that differentiation was unaffected by α-synuclein down regulation the expression levels of the neuronal marker tau was analysed by immunoblotting (Figure 7) . Tau levels increase with neuronal differentiation and may therefore be used as a marker of differentiation. Levels of tau were the same in all groups confirming that both SH-SY5Y and MESC2.10 cells were fully differentiated despite α-synuclein down regulation. Down regulation of α-synuclein does not alter baseline cell viability

It has been suggested that α-synuclein has a neuroprotective function (da Costa et al. , 2000 J Biol Chem 275:24065-24069; Hashimoto et al. , 2002 J Biol Chem 277: 11465-11472; Seo et al. , 2002 Faseb J 16:1826-1828) . Consequently, the effect of α-synuclein down regulation on baseline cell viability was assessed. Cell growth was measured every 24 hours for five days after transfection of siRNAs (Figure 8A). During this time, α- synuclein protein levels were also evaluated by Western blot and showed suppression/down regulation of α-synuclein persisted for the duration of the experiment (Figure 8B) . There was no difference in cell viability in cells treated with SNCAl , SNCA2 and scrambled siRNAs.

For experimental control, in this and other experiments, each experiment was performed in triplicate and results are expressed as the mean ± SEM. Normality of data and homogeneity of variance was confirmed using the KS statistic and Levene's test respectively. One or two-way independent ANOVA was performed. Post hoc comparisons were carried out using the Bonferroni adjustment with p < 0.05 as *, p < 0.01 as ** and p < 0.001 as ***. The introduction of double stranded RNA into cells can induce a non-specific interferon response which may confound results (Sledz et al. , 2003 Nat Cell Biol 5 :834-839) . To control for this, a scrambled siRNA control was included. Furthermore, two siRNAs with different sequences to target α-synuclein were used. The results obtained with these two molecules were consistent, confirming that the effects observed were due to α-synuclein down regulation and not specific to a particular siRNA molecule.

The results demonstrate that reducing α-synuclein levels below normal is not harmful to human cells. It was demonstrated that SNCAl and SCNA2- mediated down regulation of α-synuclein lasted for up to five days (Figure 8B) and did not affect cell viability (Figure 8A) .

Studies were also undertaken to demonstrate that down regulation of α-synuclein also did not affect cell viability in MESC2.10 cells.

In this study to determine if α-synuclein reduction adversely affected cell viability, cell survival was measured in two ways: (i) counting of live cells and (ii) the MTT assay, a biochemical test based on mitochondrial function (Figure 9) . To count living cells cultures were treated with calcein AM, a compound that is cleaved by esterases in living cells to form a green fluorescent molecule. Dead cells were stained red with ethidium homodimer 1. In this experiment, we found no difference in the number of cells between control and α-synuclein down regulated groups for differentiation SH-SY5Y cells (unpaired Student's t-test, p = 0.60) or MESC2.10 cells (unpaired Student's t-test, p = 0.67) . To confirm this result the MTT assay was used which relies on the formation of MTT formazan by mitochondrial reduction of MTT. Again, it was found that MTT formazan formation was the same with or without α-synuclein down regulation for SH-SY5Y (unpaired Student's t-test, p = 0.81) and MESC2.10 cells (unpaired Student's t-test, p = 0.80) . Therefore, using two independent methods, it was demonstrated that α-synuclein reduction does not significantly affect cellular survival in normal differentiation conditions in either of the two types of human neurons tested.

Suppressing α-synuclein results in decreased dopamine uptake velocity via DAT

DAT is a plasma membrane protein that is selectively expressed in dopaminergic neurons of the substantia nigra and the ventral tegmental area of the brain. It mediates internalisation of dopamine from the extracellular space and is critical in regulating both intra and extra- cellular dopamine levels and limiting dopamine toxicity. Immunofluorescence studies show that endogenous DAT expression in SH-S Y5 Y cells is primarily cytoplasmic, in a distribution consistent with association to internal cellular membranes (Figure 10A) .

The function of endogenous DAT was confirmed by incubating cells with ³HDA for different durations and measuring uptake of ³HDA by scintillation counting (Figure 10B) . There was substantial uptake of dopamine by SH-S Y5 Y cells. This was linear for the first three minutes but dopamine continued to accumulate in the cells for at least 10 minutes. Subsequent experiments were conducted using a two minute incubation time to ensure the reaction was measured during the linear part of the curve. By adding mazindol, a specific DAT antagonist, it was shown that dopamine uptake can be blocked (Figure 10B) . The inhibition of ³HDA uptake by the DAT antagonist mazindol confirmed that uptake in SH-S Y5 Y cells was specific to DAT.

The kinetic parameters of DAT were also determined using enzyme saturation analysis (Figure 11 A) . The K_m (a measure of the affinity of DAT for dopamine) and V_max (the maximum velocity of dopamine transport) were found to be 155 nM and 287 pmol/mg/min respectively, values consistent with other studies (Bennett et al. , 1998; John et al. , 2006) .

The effect of suppressing α-synuclein expression on the MT kinetics was studied. Dopamine uptake velocity was measured after no treatment or 72 hours after treatment with SNCAl, SNCA2 or scrambled siRNAs (Figure HA) . Non-specific uptake determined in the presence of 10 μM mazindol was the same in all conditions (data not - shown) . The resulting V_11111x and K_n, values are summarised in Table 1. Treatment with siRNAs had a significant effect on uptake velocity (ANOVA, p < 0.001) . Scrambled control siRNA treated cells had a V_max of 332 pmol/mg/min, not significantly different to untransfected cells. The V_max in cells treated with SNCAl or SNCA2 was significantly lower at 166 pmol/mg/min (p < 0.01) and 206 pmol/mg/min (P < 0.05), respectively.

-*^-m * max

(nM) (pmol/mg/min)

SNCAl 139.4 ± 17.3 166.4 ± 12.4

SNCA2 154.8 ± 44.0 206.6 ± 32.4

Scrambled 183.7 ± 37.5 332.0 ± 40.9

Untransfected 155.0 ± 23.9 287.3 ± 24.3

Table 1. Kinetic parameters of DAT. Eadie-Hofstee transformation of the data from ³HDA saturation analysis was used to determine the kinetic parameters of DAT in cells treated with SNCAl, SNCA2 and scrambled siRNA as well as untransfected cells. Data is shown ± SE.

It was also demonstrated that siRNA-mediated down regulation of α- synuclein had no effect on total levels of DAT protein expression (Figure HB) .

The introduction of double stranded RNA into cells can induce a nonspecific interferon response which may confound results (Sledz et al. , 2003 Nat Cell Biol 5:834-839) . To control for this, a scrambled siRNA control was included in the experiments. Furthermore, two siRNAs with different sequences to target α-synuclein were used. The results obtained with these two molecules were consistent, confirming that the effects observed were due to α-synuclein down regulation and not specific to a particular siRNA molecule. Overall, the results show that suppressing/down regulating α-synuclein, by up to 80%, results in up to a 50% decrease in dopamine uptake velocity via DAT (RNAi mediated α-synuclein knock-down reduced the V_raax by up to 50%) . Importantly, the difference in uptake velocity is not due to a change in enzyme affinity for dopamine with treatment having no effect on K_n,. These data suggest that α-synuclein down regulation leads to reduced V_raax of dopamine uptake via DAT through a reduction in functional DAT on the cell surface.

This data shows that suppressing/down regulation of normal human α- synuclein expression reduces the rate of dopamine accumulation within cells.

Dopamine is toxic to cells because it is readily metabolised into reactive oxygen species and maintaining intracellular levels within acceptable parameters is essential. DAT is a vital component in this process and it has been shown that increasing DAT activity correlates to cell death in Parkinson's disease (Storch et al. , 2004 Journal of Neural Transmission 111:1267) . The results presented here suggest that α-synuclein's function is to promote dopamine accumulation in cells by increasing DAT function. Therefore, removing or reducing α-synuclein can reduce dopamine accumulation and, consequently, reduce oxidative stress within cells and increase their viability.

Down regulation of α-synuclein makes neurons resistant to Parkinson's disease (PD) related compounds

To evaluate the effect of α-synuclein down regulation on cell survival in models of PD, cultures were treated with MPP+ , rotenone and dopamine. These three compounds are known to recapitulate many of the features of PD and have been used extensively in studies related to PD pathogenesis (summarised in Figure 12) .

Both MESC2.10 and differentiated SH-SY5Y cells were rendered resistant to MPP+ by α-synuclein down regulation. There was a 50% decrease in

MESC cell viability with treatment of 1-5 mM of MPP+ . This was totally prevented by α-synuclein down regulation (two-way ANOVA, p <

0.001) . A similar degree of neuroprotection was observed in differentiated SH-SY5Y cells with α-synuclein down regulation (two-way ANOVA, p < 0.001) .

Studies of rotenone toxicity also showed benefits to α-synuclein down regulation. Control MESC and differentiated SH-SY5Y cells were very sensitive to rotenone toxicity, down regulation of α-synuclein rendered MESC cells resistant to rotenone (two-way ANOVA, p < 0.001) . The same trend was also noted for differentiated SH-SY5Y cells.

Dopamine, a neurotransmitter synthesised by dopaminergic neurons in the midbrain, is known to be toxic due to its breakdown to free radical species (Graham et al. , 1978) . A major hypothesis for PD pathogenesis is that dopaminergic neurons are particularly susceptible to oxidative stress because they contain dopamine (Xu et al. , 2002) . In this study it was shown that 5 μM of dopamine was sufficient to significantly reduce both SH-SY5Y and MESC viability in control cells, α-synuclein down regulation prevented this (two-way ANOVA, p < 0.05 for MESC cells, p < 0.001 for SH-SY5Y) . Regulation of α-synuclein protects cells from MPP + toxicity

To further demonstrate the effect of α-synuclein down regulation on cell survival in models of PD, the following study further demonstrates that α-synuclein down regulation protects cells from MPP+ toxicitiy.

To determine if the decrease in dopamine uptake observed with α-synuclein down regulation was associated with changes in the cellular response to neurotoxins, the effect of down regulation of α-synuclein on MPP + toxicity was studied. MPP + is known to cause Parkinson's disease. l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) , and its active metabolite MPP + , have long been established as one of the best models of Parkinson's disease (Watanabe et al. , 2005 Med Sci Monit ll :RA17-23) . MPP + causes selective degeneration of nigrostriatal dopaminergic neurones by inducing oxidative stress through inhibiting complex I of the mitochondrial electron transport chain. Like dopamine, MPP + requires the presence of DAT to enter cells and it is known that SH-SY5Y cells are susceptible to MPP+ toxicity. To evaluate the effect of α-synuclein suppression/down regulation on MPP + toxicity, cells were treated with siRNAs and 48 hours later exposed to either 5 mM of MPP + or control media for 12 hours (Figure 13) . Cell viability was then measured. In cells not exposed to MPP + , there was no effect of siRNA treatment on cell viability. Treatment with MPP + resulted in a highly significant reduction in cellular viability by 40% (ANOVA, p < 0.001) . This could be almost completely prevented by treatment with either SNCAl or SNCA2 (p < 0.001 for each) . This data shows that RNAi-mediated down regulation of α-synuclein protects human cells from MPP+ toxicity. This work shows that suppressing normal human α-synuclein expression protects against neurotoxic insult in a model of Parkinson's disease.

This result demonstrates that treatments to reduce normal physiological α-synuclein expression has a therapeutic potential, this could be applied to all forms of Parkinson's disease. In particular it could be applied to sporadic Parkinson's disease where there is no increase in α-synuclein expression levels. Indeed, in most cases of Parkinson's disease there is no increase in α-synuclein level observed. Indeed, fewer than 10 cases of increased α-synuclein levels due to gene multiplication mutations in the world have been identified to far, so it is fair to say that very nearly all ( > 99.9%) of Parkinson's disease patients have normal physiological levels of α-synuclein. A recent study has shown no increased α-synuclein expression in 190 patients with sporadic Parkinson's disease (Gispert et al. , 2005 Arch Neurol 62:96-98) .

RNAi-mediated down regulation of α-synuclein expression reduces the uptake velocity of the dopamine transporter DAT and protects neuronal cells from MPP + toxicity. Taken together, these data are consistent with a mechanism in which α-synuclein down regulation protects cells from MPP + toxicity by reducing the activity of DAT. MPP + is a widely-used model of dopamine toxicity, and this work demonstrates that α-synuclein down regulation is a potentially powerful therapy for Parkinson's disease.

Cell Survival after Dopamine Treatment

SH-SY5Y cells were treated with either a scrambled control siRNA or SNCAl designed to down regulate α-synuclein. 48 hours later the cells were washed twice with Dulbecco's modified Eagle's medium (DMEM) and exposed to 200 μM of dopamine in DMEM + 2 mM L-glutamine for 15 hours. Survival of cells was then measured using the MTT assay as previously described and illustrated in Figure 14. The experiment was performed in triplicate. Overall, treatment to reduce α-synuclein levels resulted in a 60% increase in cell survival compared to control cells without α-synuclein down regulation (p < 0.01) .

One of the leading hypotheses in Parkinson's disease research is that a major factor leading to the death of dopaminergic cells is the dysregulation of dopamine metabolism (Perez and Hastings, 2004 J

Neurochem 89: 1318-1324) . Dopamine is known to be toxic to neurons because it is rapidly metabolised into a variety of highly reactive oxygen species that cause oxidative damage to cellular components (Graham et al. , 1978 MoI Pharmacol 14: 644-653) . In this experiment, it is shown that down regulating α-synuclein expression can protect cells from toxicity caused by dopamine, and, therefore, (knocking down) down regulating α-synuclein below normal physiological levels the pathways integral to the development of Parkinson's disease maybe inhibited.

Down regulation of α-synuclein prevents α-synuclein aggregation

A prevailing hypothesis in the field of PD research is that cell death may be related to aggregation of α-synuclein (Lansbury & Lashuel, 2006 Nature 443:774-779) . Intermediate products of aggregation called protofibrils are known to be toxic due to a number of mechanisms (Voiles & Lansbury, 2002 Biochemistry 41 :4595-4602) . (Figure 5) . The production of triton soluble α-synuclein aggregates in cells treated with MPP+ was evaluated. Treatment with MPP + caused a significant aggregation of α-synuclein into higher molecular weight oligomers and multimers. Down regulation of α-synuclein prevented this. This is likely to be part of the mechanism by which α-synuclein down regulation prevents neuronal death in models of PD.

Down regulation of α-synuclein prevents MPP + induced NOS activation

It is emerging that nitrosative stress is also very important in PD pathogenesis and other forms of neurodegeneration (Jenner 2003; Sacksteder et al. 2006) . Reactive nitrogen species are formed from NO, generated by the conversion of L-arginine to L-citrulline by the enzyme nitric oxide synthase (NOS) (Guix et al. 2005). In pathological situations NO can be toxic, rapidly reacting with superoxide to form peroxynitrite (Halliwell 2006) . Peroxynitrite has a half life in physiological conditions of about 1 second and, while being deleterious on its own, also degrades to form toxic hydroxyl radicals (Beckman et al. 1990) . In brains from patients with PD there is increased expression of iNOS and nNOS in the substantia nigra and basal ganglia compared to controls (Eve et al. 1998; Gatto et al. 2000; Hunot et al. 1996) . Lewy bodies stain heavily with antibodies directed against 3-nitrotyrosine, an indicator of peroxynitrite- mediated protein modification (Duda et al. 2000; Giasson et al. 2000; Good et al. 1998) . Moreover, animal studies using NOS inhibitors or NOS knockout animals have confirmed a vital role for NOS derived species in PD pathogenesis. Inhibitors of NOS protect mice and baboons from MPTP induced dopaminergic cell loss (Hantraye et al. 1996; Przedborski et al. 1996; Schulz et al. 1995) . Animals lacking NOS are also resistant to MPTP toxicity (Dehmer et al. 2000; Liberatore et al. 1999; Matthews et al. 1997; Przedborski et al. 1996) .

This study illustrates that MPP + causes a significant increase in NOS activity as assayed by conversion of arginine to citruline (Figure 16) . Down regulation of α-synuclein prevents this occurring (two-way ANOVA, p < 0.05) . This is therefore also likely to be part of the mechanism by which α-synuclein down regulation protects cells. By preventing NOS activation, cells will be exposed to less reactive nitrogen species, less oxidative stress and therefore their survival increased.

Claims

1. Use of an agent that down regulates α-synuclein to down regulate α-synuclein in a subject with normal physiological levels of α-synuclein expression.

2. The use of claim 1 to treat one or more neurodegenerative diseases.

3. Use of an agent that down regulates α-synuclein in the preparation of a medicament for the treatment of a neurodegenerative disease in a subject with normal physiological levels of α-synuclein.

4. The use of claim 2 or 3 wherein the neurodegenerative disease is sporadic Parkinson's disease.

5. Use of an agent that down regulates α-synuclein in the preparation of a medicament for the treatment of sporadic Parkinson's disease.

6. A method of treating a neurodegenerative disease in a subject with normal physiological levels of α-synuclein comprising down regulating α- synuclein in the subject.

7. A method of treating sporadic Parkinson's disease comprising down regulating α-synuclein.

8. The use or method of any preceding claim wherein the level and/or activity of α-synuclein protein in a cell or population of cells is reduced by between about 40% and about 90%.

9. The use or method of claim 8 wherein the level and/or activity of α-synuclein is reduced in cells of the midbrain.

10. The use or method of any preceding claim for prophylactic or therapeutic treatment.

11. The use or method of any preceding claim wherein α-synuclein is down regulated using an agent selected from the group comprising: an RNAi agent, such as a siRNA molecule; an antisense RNA molecule; an antisense DNA molecule; a ribozyme; an antibody; and any other small molecule that down regulates α-synuclein expression or a combination thereof.

12. The use or method of claim 11 wherein α-synuclein is down regulated using siRNA comprising an antisense strand of RNA which is complementary to a part of the mRNA transcribed from the SNCA gene, or is sufficiently complementary to a part of the SNCA mRNA to reduce α-synuclein expression.

13. The use or method of claim 12 wherein the siRNA is between about 15 to about 30 nucleotide base pairs.

14. The use or method of claim 12 or 13 wherein the siRNA comprises the sequence of Sequence ID No: 1 , 2 or 3 or a variant thereof.

15. The use or method of claim 14 wherein the siRNA comprises a sequence with at least 95% sequence identity to the sequence of Sequence ID No: 1, 2 or 3.

16. A siRNA comprising a nucleotide sequence identical to the sequence of Sequence ID No: 1, 2, or 3, or a variant thereof.

17. The siRNA of claim 16 wherein the siRNA comprises a sequence with at least 95% sequence identity to the sequence of Sequence ID No: 1, 2 or 3.

18. A vector comprising a promoter, a nucleotide sequence of sequence ID No 1, 2 or 3 or a variant thereof operatively linked to said promoter.

19. A method of treating a neurodegenerative disorder in a subject with normal physiological levels of α-synuclein comprising administering to the subject a therapeutically or prophylactically effective amount of an agent for down regulating α-synuclein.

20. A method of treating sporadic Parkinson's disease comprising administering to a subject a therapeutically or prophylactically effective amount of an agent for down regulating α-synuclein.

21. A method of treating a neurodegenerative disorder in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of an siRNA comprising the sequence of Sequence ID No: 1, 2 or 3 or a variant thereof with at least 95% identity to the sequence of Sequence ID No: 1, 2 or 3.

22. A method of reducing α-synuclein in a cell with normal physiological levels of α-synuclein comprising introducing into the cell an effective amount of an agent to down regulate expression of the α- synuclein gene.

23. A pharmaceutical composition for down regulating α-synuclein in a subject with normal physiological levels of α-synuclein comprising one or more agents capable of down regulating α-synuclein and a pharmaceutically acceptable carrier.

24. A pharmaceutical composition for down regulating α-synuclein in a subject with sporadic Parkinson's disease comprising one or more agents capable of down regulating α-synuclein and a pharmaceutically acceptable carrier.

25. A method for treating sporadic Parkinson's disease, comprising determining whether a subject has sporadic Parkinson's disease then administering to the subject a therapeutically effective amount of an agent to down regulate α-synuclein.

26. The method of claim 19, 20, 22 or 25 or the composition of claim 23 or 24 wherein the agent capable of down regulating α-synuclein is selected from the group comprising an RNAi agent, a ribozyme, an antisense molecule, an antibody, a naturally occurring or synthetic polypeptide, and a small molecule.

27. The method or composition of claim 26 wherein the agent targets SNCA RNA or the protein product thereof.

28. A method for protecting human neuronal cells with normal physiological levels of α-synuclein from neurotoxic insult by down regulating α-synuclein.

29. The method of claim 28 wherein α-synuclein is down regulated using RNA interference (RNAi) .