ES2460665T3 - Truncated alpha synuclein fragments in Lewy body dementia - Google Patents

Abstract

An alpha-synuclein fragment, which is alpha synuclein SN1-135, with residues numbered according to SEQ ID NO: 1.

Description

BACKGROUND

[0001] Dementias with Lewy bodies (DCLs) are characterized by degeneration of the dopaminergic system, motor alterations, cognitive deficiency and Lewy body formation (CLs). (McKeith et al., Clinical and pathological diagnosis of dementia with Lewy bodies (DLB): Report of the CDLB International Workshop, Neurology (1996) 47: 1113-24). DCLs include Parkinson's disease, dementia with diffuse Lewy bodies (DCLD), variants with Lewy bodies of Alzheimer's disease (VCL) and PD and combined Alzheimer's disease (AD) and syndromes identified as multisystemic atrophy (AMS) Dementia with Lewy bodies (DCL) is a term coined to reconcile differences in the terminology of DCLs. CL disorders continue to be a common cause of movement disorders and cognitive impairment in the elderly population (Galasko et al., Clinical neuropathological correlations in Alzheimer's disease and related dementias. Arch. Neurol. (1994) 51: 888 - 95). Although its incidence continues to increase the creation of a serious public health problem, today these disorders lack approved treatments (Tanner et al., Epidemiology of Parkinson's disease and akinetic syndromes, Curr. Opin. Neurol. (2000) 13: 427-30). The cause of DCL is controversial and it is suggested that multiple factors play a role in this, including several neurotoxins and genetic susceptibility factors.

[0002] AD, PD and DCLD are the neurodegenerative disorders most commonly found in the elderly. Recent epidemiological studies have demonstrated a close clinical relationship between AD and PD, since 30% of Alzheimer's patients also have PD. Compared to the rest of the elderly population, patients with AD are more likely to develop concomitant PE. In addition, patients with PD who become demented have usually developed classical AD. Although each neurodegenerative disease seems to have a predilection for brain regions and specific cell populations, they result in different pathological characteristics, EP, AD and DCLD also share common pathological hallmarks. Patients with familial AD, Down syndrome or sporadic AD develop CLs in the tonsil, which are the classic neuropathological hallmarks of PD. In addition, each disease is associated with the degeneration of neurons, interneuronal synaptic connections and, finally, cell death, the decrease of neurotransmitters, and the abnormal accumulation of misfolded proteins, the precursors of what participates in the normal functions of the central nervous system . Biochemical studies have confirmed a relationship between EA, EP and DCL.

[0003] In recent years, a new hope has emerged to understand the pathogenesis of DCL. Specifically, several studies have shown that the synaptic alpha-synuclein protein plays a central role in the pathogenesis of PD since: (1) this protein accumulates in CLs (Spillantini et al., Nature (1997)

388: 839-40; Takeda et al., J. Pathol. (1998) 152: 367-72; Wakabayashi et al., Neurosci. Lett. (1997) 239: 45-8),

(2) mutations in the alpha-synuclein gene are harvested with rare familial forms of parkinsonism (Kruger et al., Nature Gen. (1998) 18: 106-8; Polymeropoulos, et al., Science (1997) 276 : 2045-7) and, (3) their overexpression in transgenic mice (Masliah et al., Science (2000) 287: 1265-9) and Drosophila (Feany et al., Nature (2000) 404: 394-8) mimics several pathological aspects of PD. Therefore, the fact of the accumulation of alpha-synuclein in the brain is associated with similar morphological and neurological alterations in species as diverse as humans, mice and flies suggest that this molecule contributes to the development of PD.

[0004] The neuritic plaques, which are the classic pathological hallmark of AD, consist essentially of beta-amyloid peptide (A�), a proteolytic amino acid product of the amyloid precursor protein (APP), and NAC, a proteolytic fragment of 35 amino acid alpha-synuclein. Both A� and NAC were first identified in amyloid plaques as proteolytic fragments of their respective complete proteins, for which complete cDNAs were identified and cloned. (Iwai A., Biochim. Biophys. Acta (2000) 1502: 95-109); Masliah et al., AM. J. Pathol (1996) 148: 201-10; Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90: 11282-6).

[0005] Alpha-synuclein is part of a large family of proteins, including beta- and gamma- synuclein and synoretin. Alpha-synuclein is expressed in the normal state associated with synapses and is believed to play a role in neuronal plasticine, learning and memory. Alpha-synuclein mutations have been identified in humans (h) that increase aggregation of alpha-synuclein (Ala30Pro and Ala53Thr) and are associated with rare forms of autosomal dominant forms of PD. The mechanism by which these mutations increase the propensity of alpha-synuclein to aggregate is unknown.

[0006] WO-A-2005013889 identifies new alpha-synuclein fragments in patients with Dementia with Lewy Bodies (DCL) and in transgenic animal models thereof. The fragments have a truncated C-terminal end relative to the complete alpha-synuclein. Some fragments are characterized by having a molecular weight of about 12 kDa as determined by SDS gel electrophoresis in tricine buffer and a truncation of at least ten contiguous amino acids from the C-terminal end of natural alpha-synuclein . The preferable cleavage site takes place after the residue (117) and before the residue (126) of the alpha-synuclein

natural. WO-A-2005013889 states that the identification of these new alpha-synuclein fragments has numerous applications in, for example, the discovery of pharmaceutical, diagnostic, therapeutic and transgenic animals.

Summary of the Invention

[0007] The invention features an alpha-synuclein fragment, which is alpha-synuclein SN1-135, with residues numbered according to SEQ ID NO: 1.

[0008] The invention presents screening methods for an agent with pharmacological activity useful for the treatment of Dementia with Lewy Bodies (DCL). The method includes contacting the agent with the alpha-synuclein fragment; and determining the rate or extent of aggregation of the alpha-synuclein fragment, in which a reduction in the rate or extent of aggregation with respect to the control without the agent indicates that the agent has the pharmacological activity.

[0009] Optionally, the alpha-synuclein fragment supports a mutation associated with a hereditary DCL, such as an A53T mutation. Optionally, the method includes an additional step of performing a test on a non-human animal model of DCL to determine if the agent treats or inhibits a symptom of DCL.

[0010] The invention also presents methods of screening an agent for a pharmacological activity useful in the treatment of a DCL (for example Parkinson's disease or DCLD), comprising contacting a cell expressing alpha-synuclein and processing the alpha-synuclein in the fragment with an agent. A level of the fragment in the cell is then determined with respect to the reference level in the same cell type in the absence of the agent, a reduction in the level of the fragment with respect to the reference level indicates that the agent has pharmacological activity useful in the treatment of the DCL. Optionally, the alpha-synuclein fragment contains a mutation associated with a hereditary DCL, such as an A53T mutation. The cell can be a human cell, a neuronal cell, a dopaminergic cell or a non-dopaminergic cell. Optionally, the cell is PC12 or Sy5Y cell. Optionally, the method includes a step of performing a test on a non-human animal model of DCL to determine if the agent treats or inhibits a symptom of DCL.

[0011] The invention also presents methods of screening an agent with pharmacological activity useful for treating a DCL (for example Parkinson's disease or DCLD). The method includes contacting a non-human transgenic animal that expresses the alpha-synuclein fragment and determining a level of aggregate forms of the fragment in the brain of the transgenic animal relative to the level of aggregate forms of the reference fragment in a comparable transgenic animal in absence. of the agent, a reduction in the level of the fragment of aggregate forms with respect to the reference indicates that the agent has a pharmacological activity useful in the treatment of DCL. Optionally, the alpha-synuclein fragment contains a mutation associated with a hereditary DCL, such as an A53T mutation. Optionally, the transgenic animal is a rodent. The transgenic animal can also be a Drosophila. Optionally, the method includes a step of performing a test on a non-human animal model of DCL to determine if the agent treats or inhibits a symptom of DCL.

[0012] The invention also presents methods of screening an agent with pharmacological activity useful for treating a DCL (for example Parkinson's disease or DCLD). The method includes contacting a transgenic animal that expresses the alpha-synuclein fragment and processing the alpha-synuclein in the fragment with an agent, and determining a level of the fragment in a neuronal cell relative to a reference level in the absence of the agent, a reduction in the level of fragments with respect to the reference indicates that the agent has a pharmacological activity useful in the treatment of DCL. Optionally, the alpha-synuclein fragment is 1-X, where X is 130-139. Optionally, the alpha-synuclein fragment contains a mutation associated with a hereditary DCL, such as an A53T mutation. Optionally, the transgenic animal is a rodent, a mouse or Drosophila. Optionally, the method includes a step of performing a test on a non-human animal model of DCL to determine if the agent treats or inhibits a symptom of DCL.

[0013] The invention further features a non-human transgenic animal with a genome comprising a transgene that includes a promoter operably linked to a nucleic acid segment encoding the alpha-synuclein fragment; where the expression of the fragment in the transgenic animal predisposes the animal to develop, at least, a characteristic of a DCL. Optionally, the promoter is a PDGF promoter. Optionally, at least one characteristic is a deficiency of motor function. Optionally, at least one characteristic of the transgenic animal is a deficiency of cognitive function. Optionally, the transgenic animal is a rodent, a mouse or a Drosophila.

[0014] The invention also presents methods for detecting the presence of a DCL in a patient. The methods include the detection of a level of the alpha-synuclein fragment in the cerebrospinal fluid, an increase, relative to the reference level in individuals without disease indicates the presence of DCL.

[0015] The invention furthermore presents an antibody that specifically binds to the alpha-synuclein fragment, without specifically binding to the complete alpha-synuclein. Optionally, the antibody is an antibody

Human, humanized and chimeric. Optionally, the antibody is monoclonal. Optionally, the antibody has the human isotype IgG1.

[0016] The invention also presents methods for diagnosing the presence of a DCL by functional imaging in vivo. The methods include determining a level of antibody binding in patients, where a high level of binding with respect to a reference level in individuals without disease indicates the presence of DCL.

[0017] The disclosure also presents methods for carrying out the treatment or prophylaxis of a DCL, including administration to a patient suffering from or at risk of suffering from a DCL, an effective regimen of the alpha-synuclein fragment. Optionally, the method further comprises the administration of an adjuvant that increases an immune response comprising fragment antibodies. Optionally, the fragment binds to a carrier forming a fusion protein, where the carrier increases an immune response comprising antibodies of the fragment.

[0018] The disclosure also presents methods for carrying out the treatment or prophylaxis of a DCL. The method includes the administration to a patient suffering from or at risk of suffering a DCL from an effective regimen of an antibody that specifically binds to the alpha-synuclein fragment, without binding to the intact alpha-synuclein, where the antibody performs prophylaxis. or the treatment of the disease.

BRIEF DESCRIPTION OF THE FIGURES

[0019]

Figs 1A and 1B show a Western blot of several extracts of the cortex and hippocampus of a transgenic mouse

(B) and a control matched (A) with a polyclonal antibody that binds to an epitope in SN115-122.

Fig. 2 shows a Western blot with the same antibody of Figs 1A and 1B to compare the level of the truncated form of alpha-synuclein in Triton-X100 extracts from the cortex and hippocampus of mice 3 and 12 months old .

Figs 3A and 3B show a Western blot with a different antibody called 12C1 (a monoclonal that binds to the epitope at amino acids 43-51 and 58-65) of a Triton extract from the brain of a three-month transgenic mouse (B) compared to a matched control (A).

Fig. 4 shows another Western blot using the same antibody of Fig. 3 in a Triton extract from the brain of three and twelve month old transgenic mice.

Figs 5A, 5B, 5C, 5D and 5D show Western blot with four different antibodies (B, C, D, E) and an epitope map (A) of the binding sites (SEQ ID NOS 5, 6, 7 and 8) of the antibodies to various extracts of the brains of transgenic mice.

Figs 6A, 6B and 6C show Tris extracts from the brain of a dementia patient with Lewy bodies hybridized with three different antibodies (A, B, C), subject to 2-D gel electrophoresis and Western blot. All 2D gels in this document are shown with acidic proteins on the left, and basic proteins on the right.

Figs 7A, 7B, 7C and 7D show immunoblotting of Tris extracts from the brain of a patient with dementia with Lewy bodies with four antibodies (A, B, C, D) of additional specificities.

Fig. 8 summarizes the cleavage sites with respect to epitopes (SEQ ID NO: 9) bound by antibodies used in the Western blot.

Figs 9A and 9B compare soluble Tris (A) proteins with proteins extracted from Lewy bodies (B) by 2D and Western blot electrophoresis.

Figs 10A, 10B, 10C and 10D show the immunoblotting of proteins from Lewy bodies rehybridized with several C-terminal antibodies.

Figs 11A and 11B show Western blots of various extracts of a disease-free patient and a Contursi patient with an antibody that recognizes total alpha-synuclein (A) or phospho-129 specific alpha-synuclein (B).

Fig. 12: Ion chromatogram extracted from C-terminal peptide of SN1-122.

Fig. 13: Ion chromatogram extracted from C-terminal peptide of SN1-119.

Figs 14A, 14B, 14C, 14D, 14E and 14F: 2D immunoblot with an antibody that recognizes total synuclein.

The lines mark the positions of four rows of truncated synuclein species in C. Figs 14A, 14B, 14C, 14E and 14F show different preparations from different patients and Fig. 14D is a control.

Figs 15A and 15B: 2D immunoblot comparing ELADW101 (B), which is specific for the end of SN1-119 with an antibody for total alpha-synuclein (A). Asterisks indicate spots that react with both antibodies. These spots are identified as SN1 - 119.

Figs 16A and 16B show, respectively, the labeling of Lewy bodies and neuritis with the ELADW-101 polyclonal antibody specific for the SN1-119 end.

Figs 17A and 17B are controls of a normal individual stained with ELADW-101.

Figs 18A and 18B are brain sections of a patient with DCLD stained with the 12C6 monoclonal antibody specific for the SN1-119 end.

DEFINITIONS

[0020] The term "agent" is used to describe a compound that has and may have pharmacological activity. Agents include compounds known as drugs, compounds for which a pharmacological activity has been identified but which are undergoing another pharmacological analysis, and a compound that are part of collections and libraries that are analyzed for a pharmacological activity.

[0021] A "pharmacological" activity means that an agent shows an activity in an analysis system indicating that the agent is or may be useful in the prophylaxis or treatment of a disease. The analysis system can be in vitro, cellular, animal or human. Agents can be described as having pharmacological activity although other tests may be necessary to establish real prophylactic or therapeutic utility in the treatment of a disease.

[0022] In the context of the determination of molecular weight based on gel electrophoresis, the term "approximately" indicates the standard derivation of the expected molecular weight due to the experimental error in repetitions of the method under the same conditions. The 12 kDa molecular weight determination for certain alpha-synuclein fragments is applied to determinations using a tricine buffer.

[0023] The phrases "specifically binds" refers to a determinative binding reaction of the presence of the protein in the presence of a heterogeneous population of proteins and other biological ones. Therefore, under defined conditions, a specific ligand preferentially binds to a particular protein and does not bind in a significant amount to other proteins present in the sample. A molecule such as an antibody that specifically binds to a protein often has a constant association of at least 106 M -1 to 107 M -1, preferably 108 M -1 to 109 M -1 and, more preferably, from 1010 M -1 to 1011 M -1 or more. Numerous immunoassay formats can be used to select antibodies specifically immunoreactive to a particular protein. For example, ELISA solid phase immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine a specific immunoreactivity.

[0024] For sequential comparison, normally a sequence acts as a reference sequence, with which test sequences are compared. When a sequential comparison algorithm is used, the test and reference sequences are entered into a computer, sub-sequence coordinates are designated, if necessary, and the program parameters of the sequential algorithm are designated. The sequential comparison algorithm then calculates the percentage of sequence identity for the test sequence (s) with respect to the reference sequence, based on the designated program parameters.

[0025] The correct alignment of the sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math 2: 482 (1981), using the alignment homology algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection ( see, normally, Ausubel et al., supra).

[0026] Another example of an algorithm useful for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). The software to carry out the analyzes using BLAST is available to the public through the National Center for Biotechnology Information (http://www.nc-bi.nlm.nih.gov/). This algorithm includes, first, the identification of high-score pairs (HSPs) by identifying short words of length W in the sequence

consulted, which matches or meets a positive threshold T score when aligned with a word of the same length in the database sequence. T refers to the threshold score of the neighboring word (Altschul et al., Supra). This initial coincidence of neighboring words acts as a seed to initiate searches to find longer HSPs that contain them. Word matches extend in both directions along each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a corresponding pair of residues; always> 0) and N (penalty score for residues that do not correspond; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word matches in each direction is stopped when the cumulative alignment score decreases by the amount X of its maximum value reached; the cumulative score reaches zero or below, due to the accumulation of one or more negative score residue alignments; or the end of any sequence is reached. The BLAST W, T and X algorithm parameters determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, a cut-off value of 100, M = 5, N = - 4 and a comparison of both strands For amino acid sequences, the BLASTP program uses by default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 score matrix. The BLASTP program (using protein sequence for the nucleotide sequence) uses by default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 score matrix. (see Henikoff & Henikoff,

 Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0027] In addition, to calculate the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). A similarity measurement provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the likelihood of a coincidence between two nucleotide or amino acid sequences by chance. For example, a sequence is considered similar to another sequence if the probability of smaller sum in the comparison of the first sequence with the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0, 01, and more preferably even less than about 0.001.

[0028] For the purpose of classifying amino acid substitutions as conservative or non-conservative, the amino acids are grouped as follows: Group I (hydrophobic side chains): leu, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (the remains influence the orientation of the chain): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes with a member of another class.

[0029] The therapeutic agents of the invention are typically substantially pure of unwanted contaminants. This means that an agent is typically at least about 50% w / w (weight / weight) pure, as well as being substantially free of interfering proteins and contaminants. Sometimes the agents are at least about 80% w / w and more preferably at least 90 or about 95% w / w pure. However, using conventional protein purification techniques, homogeneous peptides of at least 99% w / w pure can be obtained.

[0030] The term "antibody" or "immunoglobulin" is used to include intact antibodies or binding fragments thereof. Normally, the fragments compete with the intact antibody from which they derive for specific binding to an antigen fragment including separate heavy chains, Fab light chains, Fab 'F (ab') 2, Fabc and Fv. The fragments are produced by recombinant DNA techniques or by chemical or enzymatic separation of intact immunoglobulins. The term "antibody" also includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. The term "antibody" also includes bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody that has two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by numerous methods, including hybridoma fusion or Fab 'fragment binding. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0031] The term "adjuvant" refers to a compound that when administered together with an antigen increases the immune response of the antigen, but when administered alone does not generate any immune response in the antigen. Adjuvants can increase an immune response through various mechanisms including lymphocyte recruitment, B and / or T cell stimulation and macrophage stimulation.

[0032] The term "patient" includes human subjects or other mammals that receive prophylactic or therapeutic treatment.

[0033] Antibody competition is determined by an assay in which the test immunoglobulin inhibits the specific binding of a reference antibody to a common antigen, such as alpha-synuclein. Be

They know numerous types of competitive binding assays, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase immunoenzymatic assay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9: 242-253 (1983)); Direct solid phase EIA with biotin-avidin (see Kirkland et al., J. Immunol. 137: 3614-3619 (1986)); direct solid phase direct test, direct solid phase direct sandwich test (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)); Direct solid phase RIA using a 1-125 label (see Morel et al., Molec. Immunol. 25 (1): 7-15 (1988)); Direct solid phase EIA with biotin-avidin (Cheung et al., Virology 176: 546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32: 77-82 (1990)). Normally, as an assay involves the use of purified antigens bound to a solid surface or cells that have it, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of marker bound to the solid surface or to the cells in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess. Antibodies identified by the competition assay (competing antibody) include antibodies that bind to the same epitope of the reference antibody and antibodies that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antibody. Usually, when a competing antibody is present in excess, it will inhibit the specific binding of a reference antibody to a common antigen in at least 50 or 75%.

[0034] Epitope coordinates are approximate (± 2 amino acids). The presence of any amino acid in an epitope is not necessary to give rise to binding.

[0035] Compositions or methods "comprising" one or more of the elements cited may include other elements that are not specifically cited. For example, a composition comprising an alpha-synuclein peptide includes both the isolated alpha-synuclein peptide and the alpha-synuclein peptide and a component with a larger polypeptide sequence.

[0036] Unless the context indicates otherwise, each embodiment, element, step or feature of the invention may be used in conjunction with any other.

DETAILED DESCRIPTION

I. General

[0037] The disclosure is argued in part in the identification of new alpha-synuclein fragments in patients with Dementia with Lewy Bodies (DCL) and transgenic animal models thereof. These diseases are characterized by aggregations of alpha-synuclein. The fragments have a truncated C-terminal end relative to the complete alpha-synuclein. Some fragments are characterized by having a molecular weight of approximately 12 kDa (corresponding to SN1 - 119), 12.5 kDa (corresponding to SN1 - 122), 13.5 kDa (whose fragment is found exclusively in patients with MCI) and 15 kDa (probably corresponding to SN1-133 or SN1-135) as determined by SDS gel electrophoresis in tricine buffer and by truncation of at least ten contiguous amino acids from the C-terminal end of alpha-synuclein natural. The cleavage site takes place, preferably, after residue 115 and before residue 136 of the natural human alpha-synuclein. Preferred cleavage sites are between residues 115 and 116, 119 and 120, between residues 122 and 123, between residues 132 and 133 and between residues 135 and 136. The identification of these new alpha-synuclein fragments has numerous applications in, for example, drug discovery, diagnostics, therapeutic and transgenic animals.

[0038] The disclosure is further based, in part, on the result that synuclein phosphorylation separates more particles (fractions enriched in Lewy bodies) related to the soluble cytosolic fraction in patients with a synucleinopathy than those related to controls Phosphorylation takes place at position 129 of the alpha-synuclein. Although knowledge of the mechanism for the practice of disclosure is not required, it is proposed that phosphorylation of alpha-synuclein leads to subsequent processing of truncated forms (i.e. cleavages between residues 132 and 133 and between residues 135 and 136) and aggregation of alpha-synuclein. The disclosure also shows that small amounts of alpha-synuclein in soluble fractions of patients with a synucleinopathy are located in lysine residues 6, 10, 12, 21, 23, 32 and 34. Ubiquitination is known to play a role in protein degradation (see, for example, Cierchanover, EMBO J. 17, 7151-7160 (1998)). Ubiquitination also makes alpha-synuclein prone to aggregate and changes its intracellular pathway from proteosomes to lysosomes. Therefore, ubiquitination can both increase alpha degradation.

- Synuclein as favoring its aggregation. The ubiquitination modulation can also be useful in the treatment of a synucleinopathy.

[0039] The disclosure presents various methods of screening agents for their appropriate activity in the treatment of DCLs. Some methods identify agents that inhibit the cleavage reaction that generates the new fragments of the disclosure. Another method identifies agents that inhibit aggregation of cleavage reaction products. Such inhibitors are practical in the treatment of DCLs. The cleavage reaction inhibitors are also useful for the purification affinity of the protease responsible for the cleavage reaction.

[0040] The disclosure also features transgenic animal models and cells that express alpha fragments

- synuclein as described above. Transgenic animal models and cells are arranged to develop features of dementia with Lewy bodies, including Lewy bodies containing fragment aggregations. Animal models and cells can be used in the screening methods described above.

[0041] The disclosure furthermore presents specific end antibodies that specifically bind to alpha-synuclein fragments without specifically binding to intact alpha-synuclein per se. These antibodies are useful in functional in vivo imaging of alpha-synuclein aggregations and also in treatment methods. The new alpha-synuclein fragments can also be used in treatment methods, optionally, together with an adjuvant.

II. Fragments of alpha-synuclein

[0042] Human alpha-synuclein is a 140 amino acid peptide that has the following amino acid sequence:

(Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90: 11282-6) .; GenBank accession number: P37840). The protein has three recognized domains, a KTKE repeat domain that covers amino acids 1-61, a NAC domain (non-amyloid component) that occurs in amino acids 60-95 and a C-terminal acid domain that occurs in amino acids. 98 to 140.

[0043] Some novel fragments of the disclosure have C-terminal truncations of at least ten contiguous amino acids, preferably at least 15 contiguous amino acids and, optionally, up to 20, 22, 23 or 25 amino acids. The fragments include all or substantially all (ie, at least 100 contiguous alpha-synuclein residues other than the deletion). Some fragments also have relatively short truncations at the N-Terminal end of up to 20 amino acids, such as deletions of residues 1-4, 1-6, 1-10 and 1-

12. Some fragments have deletions at the N-terminus of residues 1-23, 1-38 or 1-45. Preferred fragments are SN1-115, SN1-116, 1-117, SN1-118, SN1-119 , SN1-120, SN1-121, SN1-122, SN1-123, SN1-124, SN1-125, SN1-126, SN1-127, SN1-128, SN 1-129 and SN1-130. fragments SN1-115, SN1-119, SN1-120, SN1-121, SN1-122, SN1-123, SN 1-124 and SN 1-

125. Fragments SN1-115, SN1-119, SN1-122 SN1-133 and SN1-135 are especially preferable. The cleavage reaction preferably takes place at a peptide bond between amino acid residues 115 and 116, or 118 and 136, for example, particularly between waste 119 and 120 or between waste 122 and 123 or 133 and 134 or between waste 135 and 136.

[0044] C-terminal fragments of cleavage results are also included in the disclosure and can be used in the methods described below. These fragments include SN116-140, SN117-140, SN118-140, SN119-140, SN119-140, SN120-140, SN121-140, SN122-140, SN123-140, SN124-140, SN125-140, SN126-140 , SN1-127-140, SN128-140, SN129-140, SN 130-140 and SN131-140. Preferable fragments are SN116-140, SN120-140, SN123-140, SN 134-140 and SN136-140.

[0045] Other fragments of the disclosure include fragments of the N-terminal end of alpha-synuclein of 6 to 7 kDa (such as those determined by SDS electrophoresis) or 50-80 amino acids. Other fragments of the disclosure include fragments of the N-terminal end of alpha-synuclein that are free of amino acids 1-10 of the C-terminus of intact alpha-synuclein, that is, SN 1-X, where X is 130-139. Some fragments are characterized in that they have specific binding to ELADW43 antibodies (without N-terminal end) and 5C12 (111-118) and lack specific binding to 8A5 (without C-terminal end). Some fragments are characterized by specific binding to ELADW43 (without N-terminal end) and 5C12 (111-118), LB509 (115-122) and ELADW47 (118-123) and 8A5 (without C-terminal end) and lacks specific binding to ELADW43 (without N-terminal end).

[0046] Some fragments or the complete alpha-synuclein are phosphorylated at position 125 or 129 or nitrated in the tyrosine residue occupying position 125 of the alpha-synuclein. Fragments containing the amino acid serine 125 or complete alpha-synuclein may also phosphorylate at this position. The detection of enhanced phosphorylation or nitration at position 125 or phosphorylation at position 129 in a patient with respect to the mean of a population of individuals without disease is an indicator of dementia with Lewy bodies. Detection can be carried out using an antibody specific for phosphorylated or nitrated alpha-synuclein at position 125.

A level is considered improved if the standard deviation is higher than the average plus one in a population of individuals without disease.

[0047] The disclosure also features isolated peptides of up to five or ten contiguous alpha-synuclein residues with at least one of the aforementioned ubiquitination sites. These peptides can be used to compete with sites in the complete alpha-synuclein for ubiquitination or as immunogenes to generate antibodies that block the ubiquitination of the complete alpha-synuclein.

[0048] The fragments of the disclosure are different from the non-amyloid component (NAC) of the Alzheimer's disease cited above. This fragment consists of at least 28 amino acid residues (residues 60-87) and, optionally, 35 amino acid residues (residues 61-95). See et al., Biochemistry, 34: 10139-10145); Jensen et al., Biochem. J. 310 (Pt 1): 91-94 (1995); GenBank accession number S56746.

[0049] Unless the context indicates otherwise, reference to alpha-synuclein or its fragments includes the natural human amino acid sequence indicated above, or fragments thereof, as well as analogs that include allelic, species and induced variants ( for example, E83Q, A90V, A76T). Analog amino acids are assigned the same numbers as corresponding amino acids in the natural human sequence when the analog and human sequence are aligned to the maximum. The analogs normally differ from natural peptides in one, in two or in few positions, often by virtue of conservative substitutions. Some natural allelic variants are genetically associated with the associated DCL. The term "allelic variant" is used to refer to variations between genes or different individuals of the same species and the corresponding variations in the proteins encoded by the genes. Allelic variants include E46K, A30P and A53T (the first letter indicates the amino acid in SEQ ID No. 1, the number is the codon position in SEQ ID No. 1 and the second letter is the amino acid in the allelic variant). Analogs can include any combination of allelic variants. The A53T variation is associated with increased levels of phosphorylation at position 129 of alpha-synuclein in an individual with the mutation relative to the phosphorylation norm in disease-free individuals lacking the mutation. The analogs show a sequence identity of at least 80 or 90% with natural peptides. Some analogs also include unnatural amino acids or modifications of amino acids N or C

- Terminal in one, in two or in few positions. For example, the glutamic acid residue can be replaced with iso-aspartic acid. Some examples of unnatural amino acids are amino acids D, alpha, alpha-disubstituted, N-alkyl amino acids, lactic acid, 4-hydroxyproline, gamma-carboxyglutamate, epsilon-N, N, N-trimethillisin, epsilon-N-acetylillin, O - phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, omega-N-methylaginine, - alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, gamma-amino butyric acid, homoserine, citrulline and acid isoaspartic The analogs usually bind specifically to a population of polyclonal antibodies generated against natural human alpha-synuclein, and each end of an analog of a specific fragment of a natural human alpha-synuclein also specifically binds to a monoclonal antibody that is specific to the end of the respective end of the natural fragment. The disclosure also features D-peptides, whose D amino acids can be substituted for the corresponding natural L-amino acids of alpha-synuclein in most or all positions. A fragment designated in the Snx-y form refers to an alpha-synuclein fragment that begins at amino acid X and ends at amino acid Y, and contains each amino acid between X and Y. This fragment can (but does not need) to bind to a heterologous polypeptide but not to other human alpha-synuclein amino acids so that the fragment begins before X or ends after Y. The residues in a fragment are listed according to SEQ ID No. 1, where the fragment is aligned to the maximum with SEQ ID No. 1 as described above using default parameters.

[0050] Alpha-synuclein, its fragments and analogs can be synthesized by solid phase peptide synthesis or by recombinant expression, or can be obtained from natural sources. Automatic peptide synthesizers are commercially available from numerous suppliers, such as Applied Biosystems, Foster City, California. Recombinant expression can be performed in bacteria, as in E. coli, yeasts, insect cells or mammalian cells. The procedures for recombinant expression are described in Sambrok et al., Molecular Cloning: A laboratory Manual (C. S. H. P. Press, NY, 2nd Ed. 1989).

III. Dementias with Lewy bodies

[0051] Dementias with Lewy bodies (DCLs) are characterized by degeneration of the dopaminergic system, motor disorders, cognitive deficiency and Lewy body formation (Cls). (McKeith et al., Clinical and pathological diagnosis of dementia with Lewy bodies (DLB): Report of the CDLB International Workshop, Neurology (1996) 47: 1113-24). Lewy bodies are spherical deposits of protein found in nerve cells. Its presence in the brain affects its normal function, disrupting the action of chemical messengers, including acetylcholine and dopamine. Dementias with Lewy bodies include Parkinson's disease (including idiopathic Parkinson's disease (PD)), dementia with diffuse Lewy bodies (DCLD), also known as dementia with Lewy bodies. Combined Alzheimer's disease (AD) and Parkinson's and multisystemic atrophy (AMS). DCLD shares symptoms with both Alzheimer's disease and Parkinson's disease. DCLD differs from Parkinson's disease, mainly by the location of Lewy bodies. Other dementias with bodies

of Lewy include pure autonomic failure, dysphagia with Lewy bodies, indicental DCL, inherited DCL (for example mutations of the alpha-synuclein gene, PARK3 and PARK4) and multisystemic atrophy (for example olivopontocerebellar atrophy, striatonigral degeneration and Shy syndrome - Drager).

IV. GMO cells and animals

[0052] The disclosure features transgenic animals that have a genome comprising a transgene with a nucleic acid segment encoding a truncated form of C-Terminal alpha-synuclein described above. The transgene is preferably present in all, or substantially all, of the somatic and germ cells of the transgenic animal. The nucleic acid segment encoding the truncated form of C-terminal of alpha-synuclein is operably linked to one or more regulatory segments that allow the truncated form of alpha-synuclein to be expressed in neuronal cells of the animal. Promoters such as the rat neuron-specific enolase promoter, the human beta-actin promoter, the human platelet-derived growth factor B chain (PDGF-B) promoter, the sodium channel promoter can be used. rat, the mouse myelin basic protein promoter, the human copper and zinc superoxide dismutase promoter and the mammalian POU domain regulatory promoter. The PDGF promoter is particularly appropriate. Optionally, an inducible promoter is used. Also suitable is the mouse metallothionin promoter, which can be regulated by adding heavy metals such as zinc to the water or to the mouse diet. These transgenic animals can be produced by the same general approaches described in (Masliah et al., Am. J. Pathol. (1996) 148:

 201-10 and Feany et al., Nature (2000) 404: 394-8)) for transgenic animals with complete alpha-synuclein or in US Patent 5,811,633 (for transgenic animals with a mutant form of APP). Optionally, transgenic animals that carry a transgene that expresses a truncated alpha-synuclein protein can intersect with other transgenic models of neurogenic disease, such as models with Alzheimer's disease. For example, transgenic animals that carry a transgene that expresses a truncated alpha-synuclein protein may cross with transgenic animals that carry an APP transgene expressed with an FAD mutation as described, for example, in Games et al., Nature 373, 523 (1995) McConlogue et al., US 5,612,486, Hsiao et al., Science 274, 99 (1996); Staufenbiel et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Sturchler - Pierrat et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Borchelt et al., Neuron 19, 939-945 (1997) .The procedure for carrying out this crossing is described for example, in Masliah et al., PNAS USA 98: 12245-12250 (2001), which describes a crossing among transgenic mice expressing complete alpha-synuclein with PDAPP mice as described by Games et al. Transgenic animals are preferably rodents, such as mice or rats, or insects, such as Drosophila. Transgenic animals can be produced by the introduction of a transgene in the germinal phase, in which case all, or substantially all (except for the rare losses of somatic mutation), the cells of the transgenic animal include the transgene integrated into the genome. Transgenes can be introduced by microinjection, nuclear transfer or viral infection in cells or animals. Lentiviruses are particularly suitable for the latter. Alternatively, transgenes can be introduced by viral infection in the animal's brain. These transgenes are not part of the germ cells of recipient animals, but can target regions of the brain responsible for the disease (for example, the black substance). These animal models incorporate an alpha-synuclein in the brain cell genome and are arranged to develop at least one characteristic of a synucleinopathy. Lentiviruses are an appropriate vehicle for introducing an alpha-synuclein transgene into the brain (see Brain Pathology 13, 364 372 (2003); Bjorklund, Trends Neurosci. 26, 386-92 (2003), Lotharius et al., J. Biol. Chem. 277, 38884-94 (2002), Zhou et al., Brain Research 866, 33-43 (2000)).

[0053] The expression of truncated forms of alpha-synuclein in animal models causes animals willing to develop at least one characteristic of a dementia with Lewy bodies. These characteristics include increased levels of intracellular deposits of alpha-synuclein, increased formation of Lewy bodies, and decreased motor and cognitive functions compared to normal non-transgenic animals of the same species. These transgenic animals are useful for analyzing agents for their pharmacological activity in the treatment of dementia with Lewy bodies.

[0054] The disclosure also features cells transformed with truncated alpha-synuclein, which forms inclusion bodies with added truncated alpha-synuclein. Transformed cells are preferably neuronal cells, such as GT1-7 neuronal cells (Hsue et al. Am. J. Pathol. 157: 401-410 (2000)), PC12 cells

or SY5Y neuroblastoma cells. PEAK cells can also be used. The cells are preferably human cells. A vector comprising a segment encoding a truncated form of alpha-synuclein operably linked to one or more regulatory sequences that ensure expression of the truncated expression is transfected into the cells. Transfected cells can be used to analyze agents for their activity by eliminating alpha-synuclein inclusions.

V.

 Screening methods

[0055] The disclosure presents several screening methods to identify agents that have a useful pharmacological activity in the treatment of DCL. The methods include analyzes that can be carried out in vitro, in transgenic cells or animals, and that test numerous parameters as activity indicators. Agents that have activity in these analyzes can be retested in secondary analyzes of animal models of

DCL or in clinical trials to determine activity against behavioral symptoms or other symptoms of these diseases.

1. In vitro

[0056] In vitro assays can be carried out to test the ability of an agent to inhibit the aggregation of truncated forms of alpha-synuclein, particularly SN1-115, SN1-119, SN1-122, SN1-133 and SN1-

135. The base format for analyzing in vivo aggregation of alpha-synuclein, although only in the context of complete alpha-synuclein, is described in (Wood, J. Biol. Chem. 274, 19509-19512 (1999)). Truncated fragments can be phosphorylated to carry out the assay. The assay can also be carried out with phosphorylated complete alpha-synuclein. Phosphorylation is preferably at position 129. Synuclein can be phosphorylated in vitro using a serine kinase. In the present methods, the assay is carried out in the presence of an agent being tested. The rate or extent of aggregation of alpha-synuclein in the presence of an agent is determined and compared with the rate or extent of alpha-synuclein in a contemporary control.

or historical in which the agent was omitted. A reduction in the rate or extent of aggregation in the presence of the agent relative to the control indicates that the agent has activity inhibiting the aggregation of truncated forms of alpha-synuclein. This activity is potentially useful in the treatment or prevention of dementias with Lewy bodies.

2. Cellular assays

[0057] Some cell assays are performed on cells transfected with nucleic acids encoding truncated forms of alpha-synuclein as described above (particularly SN1-115, SN1-119, SN1-122, SN1-133 and SN1-135) , optionally, with a hereditary variation, such as Ala30Pro or Ala53Thr. Cells may also carry mutations in other genes associated with Parkinson's disease, such as a repeat kinase rich in leucine PARK8. These cells are in contact with a test agent, and the rate or extent of aggregation of the alpha-synuclein is measured. The rate or extent of aggregation of the alpha-synuclein is then compared to that of similarly transfected control cells in the absence of the agent. Aggregation can be controlled by immunohistochemical analysis, optimal microscopes, sedimentation or by gel analysis. Gel analysis can detect the formation of larger dimers, trimers or oligomers, as well as the inability of synuclein to enter gels due to high levels of oligomerization. A reduction in the rate or extent of aggregation in the presence of the test agent with respect to the control indicates that the agent has pharmacological activity by inhibiting the aggregation of truncated forms of alpha-synuclein. This activity is potentially useful in the treatment or prevention of dementias with Lewy bodies.

[0058] Other cell assays are performed on cells transfected with nucleic acids encoding complete alpha-synuclein, optionally with an inherited variation, such as Ala30Pro or Ala53Thr. Cells may also have mutations in other genes associated with Parkinson's disease, such as leucine-rich repeat kinase, PARK8. Similar assays can also be carried out on cells that naturally express alpha-synuclein. Said cells are contacted with an agent under test conditions and the rate or extent of formation of truncated forms of alpha-synuclein and / or phosphorylated or nitrated forms of synuclein are measured. The presence of these forms can be detected by Western blotting using one or more antibodies in alpha-synuclein. End-specific antibodies (ie, antibodies that bind to a truncated form without binding to the complete alpha-synuclein) are particularly useful for this analysis. Collections of antibodies with different epitope specificities can also be used. For example, the presence of truncated forms of alpha-synuclein can be shown by the presence of bands when they are immunoblotted with antibodies that recognize an N-terminal epitope of an amino acid segment defined approximately by amino acids 115-125 or 118-135 (particularly SN1 - 115, SN1-119, SN1-122, SN1-133 and SN1-135) of intact alpha-synuclein, and lacking bands when immunoblotted with an antibody that recognizes a C-terminal epitope of this region. The rate or extent of formation of truncated forms of alpha-synuclein and / or phosphorylated or nitrated forms in the presence of the agent is compared with the control cells in the absence of the agent. A reduction in the rate or extent of formation of truncated forms of alpha-synuclein in the presence of the test agent with respect to the control indicates that the agent has an activity that inhibits the processing of alpha-synuclein in its truncated forms. This activity is useful for trying

or prevent DCL.

3. Testing in transgenic animals

[0059] Transgenic animals have a transgene that expresses a truncated form of alpha-synuclein as described above (particularly SN1-115, SN1-119, SN1-122, SN1-133 or SN1-135), optionally, with a variation hereditary, such as Ala30Pro or Ala53Thr. Transgenic animals can also carry mutations in other genes associated with Parkinson's disease, such as a repeat kinase rich in PARK8 leucine. This animal is contacted with a test agent and the rate and extent of aggregation of the truncated form of alpha-synuclein is measured compared to that of a contemporary or historical control. The control is normally a similar transgenic animal of the same species that has not been exposed to the agent. The

Aggregation of alpha-synuclein in a transgenic animal can be analyzed by Western blotting or immunohistochemistry as described in the examples. Alternatively or additionally, the activity of the agent in these transgenic animals can be determined from behavioral characteristics, such as motor or cognitive characteristics, as described in the Examples. In these tests, the activity of the agent is shown from the improved motor or cognitive characteristics (ie, decreased deficiency of such characteristics) with respect to a comparable transgenic control animal not exposed to the agent.

[0060] Other tests are carried out on transgenic animals with a transgene that expresses a complete form of alpha-synuclein, optionally with an inherited variation, such as Ala30Pro or Ala53Thr or mutations in other genes associated with Parkinson's disease, such as a kinase. of repetition rich in leucine PARK8. Similar assays can be carried out in non-transgenic animals expressing endogenous alpha-synuclein. These animals are contacted with a test agent, and the speed or extent of the appearance of truncated forms of alpha-synuclein is detected (particularly SN1-115, SN1-119, SN1-122, SN1-133 and SN1-135 ), optionally, with a hereditary variation, such as Ala30Pro or Ala53Thr. These forms can be detected using Western blotting or immunohistochemical analysis using the appropriate anti-alpha-synuclein antibodies (as described for cell assays). The speed or extent of the appearance of truncated forms of alpha-synuclein and / or phosphorylated or nitrated forms is compared with the speed or extent of appearance of such forms in a contemporary or historical control that constitutes a transgenic animal that has not been exposed to the agent. A reduction in the speed or extent of the appearance of the truncated forms of alpha-synuclein in the animal exposed to the test agent with respect to the control indicates that the agent has activity inhibiting the processing of complete alpha-synucleia in truncated forms.

4. Agents to be screened

[0061] Agents to be screened include alpha-synuclein antibodies, alpha-synuclein peptides, drugs known or suspected of having activity in the treatment of DCL, natural products and combinatorial libraries. Preferred alpha-synuclein peptides are relatively short peptides of 30, 25, 20, 10, 5 or less amino acids including amino acids 114-117, 117-126, 118-125, 117-120, 120-124, 130-136 , 132-138, 131-135, 132-134, 133-137, 134-136 alpha-synuclein. Optionally, an amino acid immediately at the N-terminal end of the cleavage site that generates truncated forms of C-terminal alpha-synuclein is replaced with a transition state analog amino acid that forms a non-hydrolyzable bond between the two amino acids that flank the cleavage site, for example, between waste 115 - 116,119 - 120, 122

-

 123, 133-134 and 135-136 of alpha-synuclein. Some examples of analogs are transition state analogs which are statin, hydroxyethylene, hydroxyetelamine, AHPPA, ACHPA and derivatives thereof. One or more amino acids of a natural alpha-synuclein sequence can also be substituted with other natural amino acids.

[0062] Natural products to be screened can also be obtained from the National Cancer Institute's Natural Product Repository, Bethesda, MD. Random libraries of peptides or other compounds can also be screened for suitability. Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step design. These compounds include polypeptides, beta spin mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, N-substituted oligomeric glycines and oligocarbamates. Large combinatorial libraries of the compounds can be constructed from the method of encoded synthetic libraries (ESL) described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO 95 / 30642. Peptide libraries can also be generated by phage expression methods. See, for example, Devlin, WO 91 / 18980. Combinatorial libraries can be screened initially for their suitability by determining their ability to bind to alpha-synuclein.

IV. Toxicity tests

[0063] Strategies analogous to those described in screening assays can be used to determine whether existing drugs, foods, environmental toxins and other compounds exert toxic effects by promoting the processing, phosphorylation or aggregation of alpha-synuclein. These tests are carried out in the same manner as the screening tests. The toxic activity is indicated by the opposite result to the pharmacological activity in the screening tests.

VII. Protease isolation

[0064] The processing of complete alpha-synuclein in the truncated forms of the disclosure is carried out by a protease. The protease can be purified using an inhibitor identified by the screening methods mentioned above. A preferred inhibitor is an alpha-synuclein peptide of, for example, up to 20 amino acids adjacent to SEQ ID NO: including residues 114-117, 111-126, 113-126, 113-119, 117 121 or 120-125 , or 130-136, 132-138, 131-135, 133-134, 133-137, or 135-136, in which an N-Terminal residue from the cleavage site (eg, between residues 115-116, 119-120, 122-123, 133-134 and 135-136) have been replaced by a transition state analog. This inhibitor is used as a reagent of

affinity purification to purify the protease from brain cell extracts. These cells can be obtained from a corpse of a normal individual or one that has suffered a DCL. Protease levels may rise in the latter. The protease can be evaluated by presenting it with an alpha-synuclein substrate and controlling the formation of cleavage products. The end-specific antibodies described above are practical for the detection of cleavage products. The substrate can be, for example, the natural human form of alpha-synuclein described above, a fragment thereof with residues flanking both sides of the cleavage site or a mutant form thereof in which the mutation is associated with a form hereditary of the DCL. Optionally, the C-Terminal end of the substrate can be immobilized in the solid phase, and the N-terminal end in a marker. The cleavage of the substrate releases the label in the liquid phase. The liquid phase can be easily separated from the solid phase, and the amount of labeling quantified as a measure of proteolytic activity.

VIII. Endpoint Specific Antibodies

[0065] The invention features endpoint specific antibodies. These antibodies specifically bind to the truncated form of alpha-synuclein (at the C-terminal end), without specifically binding to the complete alpha-synuclein. These antibodies are useful for functional in vivo imaging of alpha-synuclein deposits, as therapeutic agents (see below), and for detecting cleavage products resulting from proteolytic cleavage of alpha-synuclein in the screening methods described above. End-specific antibodies are also described in their corresponding C-terminal fragments, for example, 116-140, 117-140, 118-140, 119-140, 120-140, 121-140, 122-140, 123-140 , 124 - 140, 125 - 140, 126 - 140, 134 - 140 and 136 -

140. Preferable fragments are 116-140, 120-140, 123-140, 134-140 and 136-140. End-specific antibodies recognize the N-terminal ends of these fragments so that they specifically bind to the fragment without specifically bind complete alpha-synuclein.

[0066] Preferred end-specific antibodies are ELADW-101 (polyclonal) and 12C6 (monoclonal) specific for the C-terminal ends of SN1-119 and ELADW-105 (polyclonal) and 7G8 (monoclonal) specific for C-ends. terminal of SN1-122. Monoclonal are mouse monoclonal expressed by hybridomas produced by conventional methods.

[0067] These antibodies can be generated by immunizing a laboratory animal with alpha-synuclein or a fragment thereof to induce the antibodies and screen the resulting antibodies to identify those with the desired binding specificity. Optionally, immunization can be carried out with relatively short peptides of less than 20 amino acids, typically 7 or 8 amino acids that include the C-terminal end of the truncated fragments of the disclosure (eg, SN 99-118, SN106-115, SN 110-119, SN-113-122, SN126-133, SN128-135). Optionally, these short peptides bind to a carrier that helps to obtain the immune response. For example, the CGGDMPVD peptide (SEQ ID No. 10) corresponding to amino acids SN 115-119 with a CGG linkage is useful for generating antibodies such as ELADW-101 and 12C6, and the CGGVDPDN peptide (SEQ ID No. 10) corresponding to amino acids 118-122 with a CGG bond is useful for generating ELADW-105 and 7G8 antibodies.

[0068] Optionally, specific binding to a labeled or immobilized truncated fragment can be carried out together with unlabeled complete alpha-synuclein. Optionally, large antibody libraries can be analyzed simultaneously using the phage expression technique.

[0069] The production of non-human monoclonal antibodies, for example murides, guinea pigs, primates, rabbits or rats can be carried out as described in Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988). The complete Freund's adjuvant followed by an incomplete adjuvant for the immunization of laboratory animals is preferable. Rabbits or guinea pigs are normally used to create polyclonal antibodies. Mice are normally used to create monoclonal antibodies. Binding can be done, for example, by Western blotting or ELISA. The smallest fragment that shows specific binding to the antibody defines the epitope of the antibody. Alternatively, the epitope specificity can be determined by a competition assay in which a test and reference antibody compete for alpha-synuclein binding. If the test and reference antibodies compete, then they bind to the same epitope or epitopes close enough that the binding of one antibody interferes with the binding of the other.

[0070] Chimeric and humanized antibodies have specificity and affinity similar or equal to those of mouse

or to that of another non-human antibody that provides the starting material for the construction of a chimeric or humanized antibody. Humanized antibodies are antibodies whose light and heavy chains have been constructed, normally, by genetic engineering, from immunoglobulin gene segments belonging to different species. For example, the variable segments (V) of the genes of a mouse monoclonal antibody can bind to human constant segments (C), such as IgG1 and IgG4. The human isotype IgG1 is preferable. In some methods, the antibody isotype is human IgG1. IgM antibodies can also be used in some methods. A typical chimeric antibody is therefore a hybrid protein consisting of the V domain or antigen-binding domain of a mouse and in the C or effector domain of a human antibody.

[0071] Humanized antibodies have substantially variable region frame residues of a human antibody (called the receptor antibody) and complementarity by determining regions of substantially a murine antibody, (called donor immunoglobulin). See Queen et al., Proc. Natl. Acad. Sci. USA

86: 10029-10033 (1989), WO 90/07861, US 5,693,762, US 5,693,761, US 5,585,089, US 5,530,101, and Winter, US 5,225,539. The constant region (s), if present, are also substantially or completely human immunoglobulin. The human variable domains are normally chosen from human antibodies whose sequence structures show a high degree of sequence identity with the domains of the murine variable region from which the CDRs are derived. The residues of the heavy and light chain variable framework region can be derived from the same or different human antibody sequences. The sequence of human antibodies may be sequences of natural human antibodies or they may be sets of sequences of different human antibodies. See Carter et al., WO 92 / 22653. Some amino acids from the residues of the structure of the human variable region are selected for substitution based on their possible influence on the conformation and / or binding of CDR to the antigen. The investigation of these possible influences is carried out by designing, examining the characteristics of amino acids in particular locations or empirically observing the effects of substitution or mutagenesis of particular amino acids.

[0072] Human antibodies with alpha-synuclein are provided by numerous techniques described below. Some human antibodies are selected from competitive binding experiments or, otherwise, to have the same epitope specificity as a particular mouse antibody. Techniques for producing human antibodies include the triome methodology of Oestberg et al., Hybridoma 2: 361-367 (1983); Oestberg, US Patent No. 4,634,664; and Engleman et al., US Patent 4,634,666 on the use of transgenic non-human mammals with transgenes encoding at least one segment of the human immunoglobulin locus as described in, for example, Lonberg et al., WO93 / 1222, US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati , WO 91/10741 and phage expression methods, see, for example, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743 and US 5,565,332.

[0073] The variable regions of the heavy and light chain of chimeric, humanized or human antibodies can bind to at least a part of a human constant region. The choice of the constant region depends, in part, on whether the antibody-dependent complement and / or cell mediated toxicity is desired. For example, the IgG1 and IgG3 isotypes have complement activity and the IgG2 and IgG4 isotypes do not. The choice of the isotype can also affect the passage of the antibody to the brain. The human isotype IgG1 is preferable. The constant light chain regions can be lambda or kappa. Antibodies can be expressed as tetramers with two light and two heavy chains, as separate heavy chains, light chains such as Fab, Fab 'F (ab') 2, and Fv or as single chain antibodies in which the light and heavy chain variable domains They are joined by a separator.

[0074] In another disclosure, monoclonal antibodies specifically bind an epitope at residues 109-120 or 115-122 of alpha-synuclein, or a discontinuous epitope is also provided at residues 43-51 and 58-65 or one specific for the C-terminal end of alpha-synuclein, including humanized chimeric and human forms thereof. An alpha-synuclein-specific C-terminal end antibody can be recognized by the ability to specifically bind alpha-synuclein as a free protein without specifically binding to alpha-synuclein as a component of a fusion protein when the C-terminal end of alpha-synuclein binds to a second peptide. These antibodies can be screened for their therapeutic activity, and if positive results are obtained, they can be used in therapeutic methods. Antibodies can also be employed by detecting alpha-synuclein fragments as described above.

IX. Diagnostics

[0075] The invention features functional imaging methods of CLs in vivo in a patient as defined in the claims. These methods are useful for diagnosing or confirming a diagnosis of dementia with Lewy or EP bodies or susceptibility to them. For example, the methods can be used in a patient with symptoms of dementia. If the patient has CLs, the patient is likely to suffer from dementia with Lewy bodies. The methods can also be used in patients without symptoms. The presence of abnormal amylode deposits indicates susceptibility to future symptomatic disease. The methods are also useful for controlling the progression of the disease and / or the response to treatment in patients who have previously been diagnosed with dementia with Lewy bodies.

[0076] The methods work by administering an end-specific antibody as described above that binds to the alpha-synuclein in the patient and then detecting the antibody after binding. If desired, an elimination response can be avoided using antibody fragments that lack a complete constant region, such as Fabs. In some methods, the same antibody can serve as both a diagnostic and a treatment reagent.

[0077] Diagnostic scanning can also be performed using an antibody specific for alpha

- phosphorylated synuclein, such as monoclonal 11A5 (phosphorus specific) or 5C12 (binds to phosphorylated and non-phosphorylated forms of alpha-synuclein) described in the pending application 10/984, 192. The presence of phosphorylated alpha-synuclein associated with Alpha-synuclein deposits is an indicator of a synucleinopathy or susceptibility to it. The presence of ubiquitinated alpha-synuclein is also an indicator of disease. This can be detected using a two-step assay in which the alpha-synuclein is precipitated with a first alpha-synuclein antibody, and the amount of ubiquitinated alpha-synuclein is detected using a ubiquitin antibody.

[0078] Diagnostic reagents can be administered by intravenous injection into the patient's body, or directly into the brain by intracranial injection or by drilling the skull. The reagent dose should be in the same range as the treatment methods. Normally, the reagent is labeled, although in some methods, the primary reagent with affinity for alpha-synuclein is not labeled and a secondary labeling agent is used to bind the primary reagent. The choice of the marker depends on the detection means. For example, a fluorescent marker is appropriate for optical detection. The use of paramagnetic markers is appropriate for tomographic detection without surgical intervention. Radioactive markers can also be detected using PET or SPECT.

[0079] The diagnosis is carried out by comparing the number, size and / or intensity of the labeled loci to the corresponding reference values. Reference values may represent the average levels in a population of individuals without disease. The reference values can also represent previous levels determined in the same patient. For example, the reference values can be determined in a patient before starting treatment and then compared with the measured values. A decrease in the values with respect to the reference indicates a positive response to the treatment.

[0080] End-specific antibodies are also useful for determining whether truncated forms of alpha

-

 Synuclein are present in the cerebrospinal fluid or in other body fluids or tissues. The presence of these forms at significantly different levels, usually higher, (ie, higher or lower than average plus one of the standard deviation) in a patient with respect to the normal level in a population of individuals without disease is indicative of the presence or susceptibility of a DCL.

X.

Treatment methods

[0081] The disclosure presents various methods to prevent or treat dementia with Lewy bodies in patients suffering from or at risk of suffering from said disease. Therapeutic agents include the truncated form of alpha-synuclein described above, and end-specific antibodies such as those described above. Optionally, the fragments are particularly phosphorylated at position 129. Other agents described include complete phosphorylated alpha-synuclein, preferably at position 129, agents that inhibit phosphorylation of alpha-synuclein, or that favor the elimination of phosphorylated alpha-synuclein by ubiquitination. or similar, or that favors or inhibits ubiquitination. The general approaches to administering agents to patients suffering from or at risk of suffering from DCL are described in the pending applications USSN 60 / 423,012 filed on November 1, 2002, and PCT US00 / 15239 filed on June 1, 2000, and PCT / US03 / 34527, filed October 31, 2003.

[0082] Patients willing for treatment include individuals at risk of having a DCL but who do not show symptoms, as well as patients who do show symptoms. In addition, the present methods can be administered prophylactically to individuals who have a known genetic risk of suffering from a DCL. These individuals include those with family members who have suffered from the disease, and those who are at risk of having it as determined by genetic tests or biochemical markers. The genetic risk markers of PD include mutations in the alpha-synuclein or in the Parkin, UCHLI and CYP2D6 genes; particularly mutations at positions 30 and 53 of the alpha-synuclein gene. Individuals who already had Parkinson's disease can be recognized for their clinical manifestations, including resting tremor, muscle stiffness, bradykinesia and postural instability.

[0083] In some methods, the patient has no clinical symptoms or risk factors for amyloidogenic disease other than that characterized by Lewy bodies. In some methods, the patient has no clinical symptoms or disease risk factors characterized by extracellular amyloid deposits. In some methods, the patient has no disease characterized by amyloid deposits of peptide A. In some methods, the patient has no clinical symptoms or risk factor for Alzheimer's disease. In some methods, the patient suffers from concurrent Alzheimer's disease and a disease characterized by Lewy bodies. In some methods, the patient has concurrent Alzheimer's and Parkinson's diseases.

[0084] In asymptomatic patients, treatment can begin at any age (for example 10, 20, 30). Normally, however, it is not necessary to begin treatment until the patient reaches 40, 50, 60 or 70 years. Treatment usually involves multiple doses over a period of time. The treatment can be controlled by test antibody or activated T cell or B cell responses to a therapeutic agent (eg, a truncated form of an alpha-synuclein peptide) over time. If the answer drops, a booster dose is recommended.

[0085] In prophylactic applications, the pharmaceutical compositions or medicaments are administered to a patient susceptible to, or at risk of, a DCL in a regimen comprising a quantity and frequency of administration of the composition or medicament sufficient to eliminate or reduce the risk, reduce the severity or delay the onset of the disease, including physiological, biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes present during the development of the disease. In therapeutic applications, compositions or treatments are administered to a patient susceptible to suffering from such disease, in a regimen comprising a quantity and frequency of administration of the composition sufficient to cure, or at least partially stop, the symptoms of disease (physiological, biochemical, histological and / or behavioral), including its complications and intermediate pathological phenotypes present during the development of the disease. An amount suitable to achieve a therapeutic or prophylactic treatment is defined as an effective dose therapeutically or prophylactically. A combination of quantity and frequency of dose suitable for achieving a therapeutic or prophylactic treatment is defined as an effective therapeutic or prophylactic regimen. In both prophylactic and therapeutic regimens, agents are usually administered in several doses until sufficient immune response is achieved. Normally, the immune response is controlled and repeated doses are administered if the immune response begins to decline.

[0086] In some methods, administration of an agent results in the reduction of intracellular levels of the added alpha-synuclein. In some methods, the administration of the agents results in a reduction in the levels of the truncated C-terminal forms of the alpha-synuclein. In some methods, the administration of an agent results in an improvement of a clinical symptom of a DCL, such as motor function or cognitive function in the case of Parkinson's disease. In some methods, the reduction in intracellular levels of the added alpha-synuclein or the improvement in a clinical symptom of the disease is monitored at intervals after the administration of an agent.

[0087] The effective doses of the compositions of the present disclosure for the treatment of the conditions described above vary depending on various factors, including means of administration, target site, physiological state of the patient, whether the patient is a human being or an animal. , other treatments administered, and if the treatment is prophylactic or therapeutic. Generally, the patient is a human being, although non-human mammals including transgenic mammals can also be treated. Treatment doses should be assessed to improve safety and efficacy.

[0088] In some methods, the agent is a truncated alpha-synuclein fragment or a fragment capable of inducing antibodies in the alpha-synuclein. The amount of said fragment depends on whether adjuvant is also administered with high dosages required in the absence of the adjuvant. The amount of a fragment for administration sometimes varies between 1 - 500 μg per patient and more usually between 5 - 500 μg per injection for administration in humans. Occasionally, a high dose of 1-2 mg per injection is used. It is generally used for each injection in humans 10, 20, 50 or 100 μg. The mass of the fragment also depends on the mass index of the immunogenic epitope in the fragment for the mass of the fragment as a whole. Between 10-3 to 10-5 micromoles of immunogenic epitope per microgram of fragment are used. The period of injections can vary significantly from once a day, once a year or once every ten years. On any day that the immunogen dosage is provided, the dosage is greater than 1 μg / patient and usually greater than 10 μg / patient if the adjuvant is also administered, and greater than 10 μg / patient and generally greater than 100 μg / patient in the absence of adjuvant. A typical regimen consists of an immunization followed by booster injections at time intervals, such as 6 week intervals. Another regimen consists of an immunization followed by booster injections 1, 2 and 12 months later. Another regimen involves an injection every two months for life. Alternatively, booster injections may be irregularly based as indicated in the control of the immune response.

[0089] Truncated alpha-synuclein fragments can also be administered in the form of nucleic acids encoding fragments operably linked to one or more regulatory elements to ensure expression of the truncated alpha-synuclein fragment. The doses for nucleic acids encoding immunogens range between 10 ng and 1 g, 100 ng and 100 mg, 1 μg and 10 mg, or 30 - 300 μg of DNA per patient. The doses for infectious viral vectors range from 10-100, or more, virions per dose.

[0090] Some methods involve passive immunization with a specific end antibody. In such methods, the dosage ranges between 0.0001 and 100 mg / kg, and more generally between 0.01 and 5 mg / kg of the host's body weight. For example, the dosages may be 1 mg / kg body weight or 10 mg / kg body weight or range between 1-10 mg / kg or, in other words, 70 mg or 700 mg, or range between 70-700 mg, respectively, for a 70 kg patient. An example of the treatment regimen involves administration once every two weeks, or once a month, or once every 3-6 months. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the ranges indicated. The antibody is generally administered multiple times. The intervals between the single doses can be weekly, monthly or annual. The intervals may also be irregular as indicated by measuring the blood levels of a

antibody in alpha-synuclein in a patient. In some methods, the dosage is adjusted to reach a concentration of plasma antibodies of 1 - 1000 μg / ml and in some methods of 25 - 300 μg / ml. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show a longer half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The dosage and frequency may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage is sometimes required in relatively short intervals until the disease is reduced or terminated, and preferably until the patient shows partial or complete improvement of the disease symptoms. From there, a prophylactic regimen can be administered to the patient.

[0091] The therapeutic agents can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intracranially, intrathecally, intraperitoneally, intranasally, or intramuscularly for prophylactic and / or therapeutic treatment. The most common route of administration of an immunogenic agent is the subcutaneous route, although other routes may be equally effective. The next most common route is intramuscular injection. This type of injection is usually done in the muscles of the arm or legs. In some methods, agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection. Intramuscular injection or intravenous infusion is preferred for administration of an antibody. In some methods, particular therapeutic antibodies are injected directly into the skull. In some methods, the antibodies are administered as sustained release compositions or devices, such as a MedipadTM device. Small molecules that act by inhibiting the processing of alpha-synuclein protease can be administered intravenously if small molecules pass through the blood brain barrier for therapeutic or prophylactic efficacy, or directly in the skull.

[0092] The agents of the disclosure may optionally be administered in combination with other agents that are partially at least effective in the treatment of DCL. The agents of the disclosure can also be administered in conjunction with other agents that increase the passage of the agents of the disclosure through the blood-brain barrier.

[0093] Immunogenic agents are sometimes administered in combination with an adjuvant. Various adjuvants can be used in combination with a peptide, such as alpha-suniclein, to induce an immune response. Preferred adjuvants increase the intrinsic response in an immunogen without causing conformational changes in the immunogen that affects the qualitative form of the response. Preferred adjuvants include aluminum hydroxide and aluminum phosphate, 3 monophosphoryl lipid A Des-O-acylated (MPLTM) (see GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa)). Stimulon TM QS - 21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al., In Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); and US Patent No. 5,057,540), (Aquila BioPharmaceuticals, Framingham, MA). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killer mycobacteria. Another adjuvant is CpG (WO 98/40100).

[0094] Alternatively, the alpha-synuclein can be coupled to an adjuvant. However, such coupling should not substantially change the conformation of alpha-synuclein to affect the nature of the immune response thereof. The adjuvants can be administered as a component of a therapeutic composition with an active agent or they can be administered separately, before, concurrently with, or after administration of the therapeutic agent.

[0095] A preferred class of adjuvants are aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulatory agents such as MPL or 3-DMP, QS-21, polymeric or monomeric amino acids such as polyglutamic acid or polylysine. Another class of adjuvants are oil-in-water emulsion formulations. Such adjuvants may be used with or without other specific immunostimulatory agents such as muramyl peptides (eg, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2 - (1'-2 'dipalmitoyl-sn-glycerol-3-hydroxy-phosphoryloxy) -ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-AI-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP) teramide TM), or other components of the bacterial cell wall. Oil-in-water emulsions include (a) MF59 (WO 90/14837), which contains 5% squalene, 0.4% Tween 80, 0.5% Span 85 (optionally containing various amounts of MTP - PE) formulated into particles smaller than one micron using a microfluidizer such as the Model 110 Y microfluidizer (Microfluidics, Newton MA), (b) SAF, containing 10% squalene, 0.4% Tween 80, L121 pluronic polymer 5% blocked, and thr-MPD, either microfluidized in a submicron emulsion or vortexed to generate an emulsion of size

major particle, and (c) RibiTM adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% squalene, 0.2% Tween 80, and one or more components of the bacterial cell wall of the formed group by monophosphorylipid A (MPL), trehalose dimicolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM).

[0096] Another class of preferred adjuvants are saponin adjuvants, such as Stimulon ™ (Qs-21, Aquila, Framingham, MA) or particles generated therefrom such as ISCOMs (immunostimulatory complexes) and ISCOMATRIX. Other adjuvants include RC-529, GM-CSF, Freund's complete adjuvant (ACF) and incomplete Freund's adjuvant (AIF). Other adjuvants include cytokines, such as interleukins (eg, IL-1, IL-2, IL-4, IL-6, IL-12, IL-13, and IL-15), macrophage colony stimulating factor (M - CSF), granulocyte and macrophage colony stimulating factor (GM - CSF), and tumor necrosis factor (FNT). Another class of adjuvants are glycolipid analogs that include N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in a sugar residue by an amino acid, as immunomodulators or adjuvants (see US Patent No. 4,855 .283). Heat shock proteins, for example, HSP70 and HSP90, can also be used as adjuvants.

[0097] An adjuvant with an alpha-synuclein fragment can be administered as a single composition, or it can be administered before, simultaneously with, or after administration of the alpha-synuclein fragment. The alpha-synuclein fragment and the adjuvant can be packaged and supplied in the same vial or can be packaged in separate vials and mixed before use. The alpha fragment and adjuvant

- Synuclein are packaged with a label indicating the intended therapeutic application. If the alpha-synuclein fragment and adjuvant are packaged separately, the package includes instructions for mixing before use. The choice of an adjuvant and / or carrier depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosage plan, the effectiveness of the adjuvant for the species being vaccinated, and, in humans, A pharmaceutically acceptable adjuvant is one that has been approved or is being approved for administration in humans by the relevant regulatory bodies. For example, Freund's complete adjuvant is not suitable for administration in humans. Alum, MPL and QS-21 is preferred. Optionally, two or more adjuvants can be used simultaneously. Preferred combinations include alum with MPL, alum with QS-21, MPL or RC-529 with GM-CSF, and alum, QS-21 and MPL together. Likewise, Freund's incomplete adjuvant (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998)) can be used, optionally in combination with any alum, QS-21, MPL and combinations thereof.

[0098] The agents of the disclosure are often administered as pharmaceutical compositions comprising an active therapeutic agent, ie; various pharmaceutically acceptable components. See Remington’s Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980). The preferred form depends on the mode of administration and intended therapeutic application. The compositions may also include, depending on the desired formulation, non-toxic pharmaceutically acceptable carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for administration in humans or animals. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological buffered saline, Ringer's solutions, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

[0099] Pharmaceutical compositions may also include slowly metabolized large macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (Sepharose (TM) functionalized with latex, agarose, cellulose and the like), polymeric amino acids, copolymers of amino acids, and lipid aggregates (such as oil drops or liposomes). In addition, these carriers can function as immunostimulatory agents (ie, adjuvants).

[0100] For parenteral administration, the agents of the disclosure can be administered as injectable doses of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as oils in water, saline solution. , glycerol or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffered substances and the like can be presented in the compositions. Other components of the pharmaceutical compositions are those with petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. The antibodies can be administered in the form of a depot injection or implant preparation, which can be formulated in such a way as to allow sustained release of the active ingredient. An exemplary composition comprises a 5 mg / mL monoclonal antibody, formulated in an aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCI. Compositions for parenteral administration are substantially sterile, substantially isotonic and manufactured under GMP conditions of the FDA or similar organisms.

[0101] Generally, the compositions are prepared as injectables, as solutions or liquid suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles before the injection can be prepared. The preparation can also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymer to enhance the effect of the adjuvant, as discussed above (see Langer, Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997) The agents can be administered in the form of a reservoir injection or implant preparation that can be formulated to allow sustained or pulsatile release of the active ingredient.

[0102] Additional formulations suitable for other modes of administration include oral, intranasal, pulmonary formulations, suppositories, and transdermal applications. For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; said suppositories can be formed from mixtures containing the active ingredient in a range between 0.5% and 10%, preferably 1% -2%. Oral formulations include excipients, such as the pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose and magnesium carbonate. These compositions are presented in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% -95% of the active ingredient, preferably 25% -70%.

[0103] Topical application may result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with a cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins (see Glenn et al., Nature 391, 851 (1998)). Co-administration can be achieved by using the components as a mixture or as bound molecules obtained by chemical cross-linking or expression as a fusion protein. Alternatively, transdermal delivery can be achieved using a cutaneous route or using transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).

Examples

1. Detection of truncated forms of alpha-synuclein in a transgenic animal

[0104] Transgenic mice that have a nucleic acid encoding intact alpha-synuclein operably linked to the PDGF promoter were analyzed with 6 weeks, 3 months and 12 months. The animals were sacrificed and the cortex and hippocampal tissue of four mice (2 males / 2 females) were grouped. The tissue was homogenized in TBS (250 mM NaCl), and centrifuged at 150,000 x g for 15 minutes. The pellet was then extracted with 1% Triton-X 100 for 30 minutes at 4 degrees and centrifuged as before. The resulting pellet was then extracted with 1% SDS for 30 minutes at 25 degrees and centrifuged as before. Finally, the pellet was extracted with 8 M urea / 1% SDS. This procedure resulted in four extracts that will be referred to below in the description as Tris, Triton, SDS and urea extracts.

[0105] Figures 1A and 1B show a Western blot of extracts from a transgenic mouse and a control paired with an ELADW-47 antibody. This antibody is a polyclonal antibody that binds to an epitope in SN115-122 (although not necessarily requires each amino acid for some binding to occur). The antibody preferentially binds to the human form of alpha-synuclein although it also binds to a lesser extent to the mouse form. Figures 1A and 1B show a 14 kDa alpha-synuclein band for both the control mouse and the transgenic mouse. The band is more powerful for the transgenic mouse than for the control mouse. For the different extracts, the band is more intense in the Triton extract. This extract solubilizes the alpha-synuclein-bound membrane and possibly type inclusions with Lewy bodies. Tris and in particular the Triton extractions of the transgenic mouse (although not the control one) show a 12 kDa band in a tricine buffer. This is a truncated form of alpha-synuclein. The molecular weight of the band corresponds to a length of 115-120 amino acids.

[0106] Figure 2 shows a Western blot with the same antibody as Figures 1A and 1B to compare the level of the truncated form of alpha-synuclein in 3 and 12 month old mice. The figure shows that the truncated form appears more strongly in 3-month-old mice. Again, the truncated band does not appear in control mice. The more intense appearance of the truncated early form of alpha-synuclein in the development of transgenic mice indicates that the truncated form of alpha-synuclein plays a role in the pathogenesis of dementia with Lewy bodies.

[0107] Figures 3A and B show a Western blot with a different antibody called 12C1 (binds to the epitope at amino acids 43-51, 58-65, and in the monoclonal antibody IgG1k). This antibody binds similarly to the mouse and human forms of alpha-synuclein in an epitope that includes amino acids 43-51 and 58-65. Figure 3 shows the truncated band of 12 kDa in the Triton extract of mice transgenic The same band appears more weakly in the Triton extract of control mice. Therefore, the processing of alpha-synuclein for a truncated form occurs in both transgenic and normal mice, although it is stronger in the latter. The greater scope of processing in transgenic mice may be due to processing directly from human alpha-synuclein, or it may be due to the presence of alpha-synuclein that targets alpha-synuclein by a route used to a lesser extent in non-transgenic mice. .

[0108] Figure 4 also shows a Western blot using the same antibody as Figure 3. This gel shows two additional bands of molecular weights of 6 or 7 kDa. The 7 kDa band appears more strongly in transgenic mice than in control mice. The 6 kDa band appears only in the transgenic mouse, and then only in the 3 month sample. The 6 or 7 kDa bands are indicative of shorter N-terminal fragments of the 50-80 amino acid alpha-synuclein.

[0109] Figures 5A, B, C, D, and E show Western blots with four different antibodies and epitope maps of antibody binding sites. ELADW-44 is a polyclonal antibody that binds only to the human form of alpha-synuclein (i.e., not in the mouse form). It binds to the epitope at amino acids 103-105. ELADW-47 is a polyclonal antibody that preferentially binds to the human form but also binds to the mouse form. It binds to an epitope at amino acids 115-122. ELADW-48 is a polyclonal antibody that binds in the same way to mouse and human forms. It binds to an epitope at amino acids 131 and 140. 8A5 is a monoclonal antibody that binds in the same way to mouse and human forms. It joins the C-terminal of the alpha

- synuclein. Figures 5A-E show that of these four antibodies, only ELADW-47 generated a 12 kDa band indicative of a truncated form of alpha-synuclein. The result that ELADW-48 did not originate this band is useful in mapping the cleavage site. Because ELADW-47 joined and ELADW-48 did not join, the cleavage site is limited by the N-Terminal end of the ELADW-47 epitope and the C-Terminal amino acid of the ELADW-48 epitope. In addition, due to that some amino acids of the ELADW-47 epitope must be present to allow binding, and of the ELADW-48 epitope must be present to prevent binding, the cleavage site is further limited in a region in amino acids 118-135. When this data considered with the size of the truncated fragment (approximately 115-120 amino acids), the probable cleavage site is around amino acids 118-121. The lack of binding by the C-terminal 8A5 antibody is consistent with this cleavage site. The lack of binding of the ELADW-44 antibody, however, requires further comments. The lack of cleavage can be explained if a truncated form of the alpha-synuclein results in cleavage that adapts to a different conformation for an intact alpha-synuclein avoiding ELADW-44 binding. Alternatively, the truncated form of the alpha- Synuclein occurs in transgenic mice to a greater extent than in normal mice that are represented in the form of mouse alpha-synuclein. In this case, the greater amount of truncated form in transgenic mice would be due to the presence of the human alpha-synuclein that is more directed to the mouse alpha-synuclein by a processing path leading to an alpha-synuclein in relation to The situation in a control mouse.

2. Detection of truncated forms of alpha-synuclein in the brain of a patient with DCLD

[0110] This example compares the alpha-synuclein species in CL for the remaining particle and soluble protein fractions of a brain with DCLD. CLs and soluble protein were prepared from the cortex of a single patient with DCLD (see Jensen et al., J. Biol. Chem. 275 21500-21507 (2000)). The tissue was homogenized in Tris / sucrose (0.32 mM) / EDTA (5 mM) and in buffer with protease inhibitors. The homogenate was centrifuged at 1,000 x g. The supernatant was further centrifuged at 150,000 x g. The supernatant of this centrifugation was used to prepare a soluble Tris protein fraction. The 1,000 x g pellet was resuspended and used to prepare a fraction with Lewy bodies. Lewy bodies were purified by immunoprecipitation on magnetic beads containing antisinuclein antibodies. The precipitate was extracted with 7 M urea / 2 M thiourea / 4% CHAPS. Un extracted material was reextracted with urea / thiourea / CHAPS. Extracts from this step and the previous extraction were pooled and analyzed by 2D PAGE and immunoblot. The non-extracted residue was subjected to an additional extraction with 90% formic acid. This extract was stored diluted in 9% formic acid. The extract was analyzed by SDS PAGE and RP-HPLC. Little or no synuclein was found in this last extract, and the present result resembled the material extracted by urea / thiourea / CHAPS, indicating that urea / thiourea / CHAPS provided complete extraction.

[0111] The synuclein species were resolved in 2D gels and detected by Western blots. All Western 2D blots were shown with more acidic proteins on the left, more basic on the right. Multiple species of alpha-synuclein, including phosphorylated and truncated species, occurred both in the CL and in the soluble fraction of the brain. The predominant truncations were present in the C-terminal region of the alpha-synuclein at amino acids 118-125. A larger additional clivated fragment near the C-terminal was observed. A beta or gamma-synuclein was not detected in the CL preparation despite being found in the soluble protein fraction. The alpha-synuclein in the CL preparation differed in that the soluble fraction had additional cleavages in C-terminal, and in general the truncated alpha-synuclein species were enriched in the CLs relative to the soluble protein fraction. In addition, the multiple species of the alpha

- Synuclein of greater molecular mass, 25-35 kDa, was detected only in the CL preparation. This includes ubiquitinated species, identified by us and others (Tofaris et al. J. Biol. Chem. 278 (45): 44405-44411, 2003). The truncated C-terminal fragments are the same size as those observed in the transgenic mouse model of Example 1 indicating a role in the pathogenesis of the disease.

[0112] Figures 6A, B, C show Tris extracts hybridized with different antibodies, subjected to 2D gel electrophoresis and subjected to Western blotting. Dark spots appear to the left of the graphs that represent the complete alpha-synuclein. The most notable feature is the four spots on

the Sin - 1 graph that are absent in the 8A5 graph. These four spots represent truncated forms of alpha

- synuclein unable to bind to antibody 8A5 due to the lack of the C-terminal amino acid. These truncations correspond to SN forms between 1-118 and 1-125. Numerous additional spots and adjacent to the complete alpha-synuclein spots have been seen at the bottom. The spots on the bottom of the whole spots represent minor truncations of the C-terminal (i.e., 1-X synuclein, where X is 130-139), since they react with antibodies in phosphorylated S129 but not with 8A5. The adjacent spot of the complete spots, except the right, represents a minor deletion of the N-terminal (due to the lack of this spot in the immunoblot with ELADW-43).

[0113] Figures 7A, b, C, D show immunoblotting with additional antibodies. The four spots are present with 5C12 (111-118). Two of the spots are present with ELADW-47 (115-122) and the spots are absent with LB509 8115-123). The spots may differ from each other both in molecular weight and in the presence or absence of post-translational modification, such as nitration or phosphorylation. The change of these fragments towards a basic pH, in relation to complete synuclein, is consistent with the removal of a part of the C-terminal acid sequence. These results set the cleavage sites at amino acids 120-125 of the alpha-synuclein. Also noteworthy are various spots made under (lower molecular weight) or to the left (lower pH) of the unmodified synuclein spots. This may represent forms of synuclein that have undergone a small extension of the N-terminal truncation and / or a different post-translational modification with respect to the main spots. Note that some spots visualized by ELADW-43 and 8A5 on immunoblotting of the soluble protein are beta-synucleins, particularly the prominent spot on the left and slightly above the complete alpha.

[0114] Figure 8 summarizes the cleavage sites relative to epitopes bound by antibodies used in Western blotting.

[0115] Figures 9A, B compare soluble Tris proteins with proteins extracted from Lewy bodies by 2D electrophoresis and Western blotting. Immunoblotting of Tris on the left shows four spots in a lower molecular weight representing truncated forms of alpha-synuclein (probably in the range of amino acids 1-120 to 1-125). These are of relatively low intensity compared to the representative spots of the complete alpha-synuclein. The immunoblot of proteins from Lewy bodies shows more representative spots of the truncated forms of the alpha-synuclein in the range of 1-120 to 1-125. However, these are of greater intensity in relation to the representative spots of the alpha - complete synuclein. Also, two spots that move faster than complete alpha-synuclein but slower than the collection of spots at the bottom of the immunoblot are evident. These spots probably represent truncations in the 1-X range, where X is amino acid 130-139. As described above, these spots react with antibodies in phosphorylated S129 but not with 8A5.

[0116] Figures 10A, B, C, and D show immunoblotting of Lewy body proteins re-hybridized with several C-terminal antibodies. All spots appeared with Sin - 1 (91 - 96) and 5C12 (111 - 118). With ELADW - 47, the stain is performed with a higher speed and in the most basic position where the Sin - 1 and 5C12 immunoblotting are not found. In the immunoblot of LB509, the 12 kDa spots corresponding to those of the soluble Tris samples disappear or are barely visible, although the row of spots just above them ("level 3") still reacts. The absence or reduced intensity of some spots on ELADW-47 and LB509 immunoblotting indicates that these spots represent truncated forms of alpha-synuclein and are consistent with cleavage that occurs between amino acids 120 and 125.

3. Detection of alpha-synuclein added in a transgenic animal

[0117] Transgenic animals are sacrificed and brains are removed for neurochemical and neuropathological analyzes. Briefly, the right hemicerebro is frozen and homogenized during the immunoreactivity determinations of human alpha-synuclein added and not aggregated by Western blotting (Masliah et al., Science (2000) 287: 1265). The left hemicerebro is fixed in 4% paraformaldehyde, serially sectioned in the vibratom for immunocytochemistry and ultrastructural analysis.

[0118] Sections of the brain were immunostained with a rabbit polyclonal antibody against human alpha-synuclein (1: 500). After an overnight incubation at 4 ° C, the sections were incubated with a biotinylated anti-rabbit secondary antibody followed by the horseradish D-peroxidase complex (HRP) (1: 200, ABC Elite, Vector).

[0119] The reaction was visualized with 0.1% of 3, 3, - diaminobenzidine tetrahydrochloride (DAB) in 50 nM Tris

- HCI (pH 7.4) with 0.01% H2O2 and the sections were mounted on slides under Entellan. Immunoreactivity levels are semiquantitatively evaluated by optical densitometry using Quantimet 570C. These sections were also studied by an image analysis to determine the number of immunoreactive inclusions of alpha-synuclein and this reliable measure of aggregation of alpha-synuclein acts as a valuable index of antiplatelet effects (Masliah et al. Science (2000 ) 287: 1265).

[0120] Analyzes of neurodegeneration patterns are achieved by analyzing synaptic and dendritic densities in the hippocampus, frontal cortex, temporal cortex and basal ganglia using sections of double immunostained vibratoms for synaptophysin and microtubule-associated protein 2 (MAP2) and were visualized with MCBL. Additional neurodegeneration analyzes are achieved by determining the immunoreactivity of tyrosine hydroxylase (TH) in caudoputamen and in the black substance (SN) as described previously (Masliah, et al. (2000)). Sections will be captured with the MCBL and each individual image is interactively evaluated so that the immunoreactive terminals of the TH that show pixel intensity are included in a linear range. A scale is adjusted to determine the pixel ratio to μm. This information is then used to calculate the percentage of the area of the neuropil covered by immunoreactive terminals of the TH. These same sections are also used to evaluate the number of TH neurons in the SN.

4. Analysis of alpha-synuclein in patients with DCL

[0121] To determine which a-synuclein species are enriched in, or only of the disease tissue, brain samples have been examined from patients with multisystemic atrophy (AMS), and a mutation of familial Parkinson's disease ( A53T; Contursi family). Brain particle fractions with AMS and Contursi were prepared by homogenizing brain tissue in 50 mM Tris, 140 mM NaCI and 1% Triton, respectively matched by age, control patients ("normal") were prepared in a manner identical for brain disease. The samples were analyzed by Western blots of 1D gels and by ELISA as described below, also in 2D gels. Part of the fraction of the particles was analyzed. The rest was centrifuged. The supernatant was also analyzed. The pellet was extracted in 7 M urea. The supernatant of this extraction was analyzed. The pellet was further extracted in 7 M urea / 1% SDS. The supernatant was analyzed. Western blots using an antibody to detect total alpha-synuclein or specifically phosphorylated alpha-synuclein at position 129 are shown in Figures 11 and A.

[0122] Synuclein was fractionated differently from the particle fraction of the Contursi brain versus the control brain. Most of the synuclein in the normal brain particle fraction was soluble after homogenization in the Tris-sucrose buffer, but almost all of the Contursi brain synuclein required urea plus SDS for solubilization that suggests a huge amount of Lewy bodies in this patient The synuclein in the Contursi patient was markedly different from that of the control patient in the amount of phosphorylation being 129. Only a small part of the phosphorylated α-synuclein was detected in the control patient (groups on the left), while the Contursi patient (right groups) had an extremely large amount of phospho-synuclein compared to Western blots. Therefore, the insolubility of the synuclein in the Contursi brain was associated with a large increase in the phosphorylation of the synuclein in being 129. The a-synuclein in the Contursi patient also differed from the normal brain in the distribution of truncations of C- terminal. C-terminal truncated a-synucleia was observed in both the normal brain particle fractions and in the Contursi. However, all detectable truncations were highly insoluble (urea / SDS extract) in the Contursi patient, while those in the control brain were soluble (Tris-sucrose buffered extract). Enrichment of the C-terminal truncated synuclein in the CL-enriched fraction of a Contursi patient obtained the same results as our finding of the C-terminal truncated synuclein enriched in the DCLD. The brain with AMS was also enriched in phospho (ser 129) -a-synuclein revealed in C-terminal truncation and abundant phosphorylation and other acid modifications in CL were observed. High levels of phospho (being 129) were also observed in the brain of a patient with DCLD in relation to disease-free control.

5. Identification of truncated synuclein species of the brain with DCLD by LC-MS / MS

[0123] To generate an en-synclein-enriched group, a solubilized fraction of urea / thiourea / CHAPS particles from a brain with DCLD was first purified by anion exchange chromatography. The resulting fractions were analyzed by Western Blot and separated into crude groups enriched in truncated, complete or phospho-synuclein. The truncated group was further purified by affinity chromatography (5C12 antibody conjugated to Sepharose). Next, the fractions were concentrated separately and separated by capillary HPLC.

[0124] The three largest peaks of a fraction (designated C6) were subsequently digested with trypsin and analyzed by LC-MS / MS to determine the identity and composition of the protein present in the sample. Peak 1 was analyzed and a-synclein was identified with the sequence coverage that encompasses amino acids 1-97. Since the sequence coverage ends at the C-terminal with a lysine residue, which is a cleavage site of trypsin, additional peptides may be presented downstream.

[0125] Peak 2 of fraction C6 was analyzed and found to contain an a-synuclein sequence at positions 11-97. Again, because the tryptic fragments at terminals N and C are absent; The exact composition of the species is not able to be determined. In addition, most of the protein present in this

The fraction is not synuclein, but a form of myelin basic protein that is copurified with this synuclein group.

[0126] The analysis of peak 3 identified two different species of synuclein, both starting at amino acid 1, with one ending at position 119D and the other at position 122N. These two truncated forms were identified using a search in the database designated to detect truncated peptides truncated in C-terminal. The list of search results in the database of the truncated peptide fragments in C-terminal is shown below (Table 1) together with their respective extracted ion chromatograms (Figures 12 and 13) illustrating the intensity of the precursor (not fragmented) of the ion signals for these peptides when compared to the reference level.

Table 1: Results of the search in the database for peak 3 of fraction C6

#2

Reference scanner (s) Alpha_synuclein (C122) 2690-2694 (SEQ ID No. 2) 2344-2679 (SEQ ID No. 3) DQLGKNEEGAPQEGILEDMPVDPDN sequence. KDQLGKNEEGAPQEGILEDMPVDPDN MH + 2710.22 2838.31 Load 2 3 XCorr 4.53 2.38

.

#3

 Alpha_sinuclein (C119) 2675 (SEQ ID NO: 4) KDQLGKNEEGAPQEGILEDMPVD. 2512.19 2 1.94

[0127] The search identified 122N truncation twice, finding two different triptych sequences for 122N species with a peptide resulting from a lost triptych cleavage of the N-terminal lysine residue. Also, the Xcorr, or the cross-correlation function, for the first listed sequence of the 122N variant is very high. The three additional truncated forms of SN1-115, SN1-133 and SN1-35 were also found in brains with DCLD. The truncations were identified by MS / MS matching the spectra of the peptide fragmentation of the C-terminal truncated a-sicnuclein peptides against a theoretical spectrum generated by the TurboSequest Mass spectrum analysis software (ThermoElectron license, Inc). The two forms were phosphorylated in ser129. The higher the Xcorr value, the greater the security in the match that lends greater support to the data.

[0128] Figures 14 A-F show immunoblotting of 2D gels from extracts of Lewy body preparations of patients P48 and P52 with DCL and a P2 patient with AMS (strictly speaking, the AMS preparation contains glial cortical inclusions) prepared as It has been done before compared to a control. The probe is an antibody of total alpha-synuclein. Identification numbers (A / 1, U / 1, etc.) refer to different preparations of Lewy bodies, made on different days and used in different cortical regions. The different preparations of sick patients are shown in Figures 14A, B, C, E and F. The control is shown in Figure 14D. There are no synuclein modifications that were consistently present or absent in the AMS preparation compared to those with DCL, so the two pathologies will be analyzed together. Most preparations that have the same adduct groups move above the complete synuclein monomer although the relative amount of each group varies. The truncated C-synuclein between 16 and 12 kDa can be grouped into four levels, ranging from full length in the first level to the 12 kD fragment in the fourth. The relative amounts of different truncation vary. P52U / 2 and P28U / 1 have no specific level 3 in detectable DCL. P48 has very little truncated C material; Level 3 spots are barely visible (although present), and Level 4 is not much more intense, if at all, than soluble proteins. Figure 15B shows the results of the rehybridization of a 2D immunoblot of the preparation of Lewy P52U / 2 bodies with ELADW-101. An anterior probe of the same immunoblot with the Sin-1 monoclonal antibody is shown on the left for comparison (Figure 15A). Three asterisks mark the spots that overlap on the two probes. The P28U / 1 immunoblot was re-hybridized with identical results. The ELADW-101 polyclonal antibody, directed against the termination of Asp 119, reacts specifically with the 3 most prominent spots in the fourth 12 kD row of truncated species. Multiple spots suggest different forms of SN1-119 that differ by load modifications, for example, phosphorylation, of the remaining sequence.

[0129] The non-floating cortical brain sections of a normal patient and with DCLD were hybridized with a new epitope-specific ELADW-101 antibody. The results are shown in Figures 16A and B (diseased brain) and in Figures 17A and B (controls). The regions in boxes marked A and B are shown augmented in the panels on the right. A typical LB and LN are marked with arrows. Only weak synaptic staining is seen in the normal brain. The same experiment was performed with the new epitope-specific 12C6 antibody. The results are shown in Figures 18A and B. The boxed regions marked A and B are shown augmented in the panels on the right. Typical LB and LN are marked with arrows. In panel B, an arrow indicates a granular cytoplasmic staining by 12C6. This staining is not seen in the normal brain. These results show that the truncated alpha-synuclein fragment of SN1-119 is enriched in patients with dementia with Lewy bodies.

6. Behavioral analysis in a transgenic animal

[0130] For locomotive activity, mice were analyzed for 2 days on the rotating roller (San Diego) Instruments, San Diego, CA)), as previously described (Masliah, et al. (2000)). On the first day, the mice are trained in 5 trials: the first at 10 rpm, the second at 20 rpm and the third at the fifth at 40 rpm. On the second day, mice are tested in 7 trials at 40 rpm each. Mice are placed individually in the cylinder and the rotation speed is increased from 0 to 40 rpm for 240 seconds. The duration of the mice in the roller (latency drop) is recorded and used as a measure of motor function.

[0131] Mice test cognitive ability in the Morris water maze (Morris, Learn Motivat. 12; 239-260 (1981)). In this procedure, the animal is placed in a circular pool with water, with an escape platform submerged beneath the surface of the water. A visible marker is placed on the platform so that the animal can find it by heading towards a nearby visual signal. Alternatively, the animals will be provided with a more complex form of the test in which there are no formal signs to mark the location of the platform. In this way, the animal must learn the location of the platform with respect to the distal visual cues. The duration of the animal in water is inversely related to its cognitive ability.

7. Analysis of aggregated alpha-synuclein fragments in a cell line

[0132] GT1-7 neuronal cells (Hsue et al. Am. J. Pathol. 157: 401-410 (2000)) are transfected with an expression vector pCR3.1-T (Invitrogen, Carlsbad, CA) that expresses a truncated alpha-synuclein fragment as described above in the murine alpha-synuclein and compared with cells transfected only with the expression vector. Cells transfected only with the vector have a fibroblastic appearance while cells transfected with alpha-synuclein are rounded, with inclusion bodies on the visible cell surface through a light and confocal scanning microscope. Transfected GT1-7 cells can be used to screen agents for activity in the elimination of synuclein inclusions.

[0133] The disclosure comprises at least the following numbered embodiments:

one.

A screening method for an agent that has a pharmacological activity useful for the treatment of dementia with Lewy bodies (DCL) comprising:

contacting the agent with a phosphorylated alpha-synuclein or a phosphorylated fragment of the same, in which the fragment is characterized by the presence of at least 100 amino acids contours of intact alpha-synuclein and a deletion of 1-11 contiguous amino acids of C - intact alpha-synuclein terminal; determine the rate or extent of aggregation of the alpha-synuclein or alpha-fragment synuclein, in which a reduction in the rate or extent of aggregation with respect to a control lacking agent, indicates that the agent has a pharmacological activity.

2.

The method of embodiment 1, wherein the agent is contacted with intact alpha-synuclein or with SN1-133 or SN1-135 numbered according to SEQ ID NO. 1.

3.

The method of embodiment 1, wherein the alpha-synuclein fragment contains a mutation associated with hereditary DCL.

Four.

The method of embodiment 3, wherein the mutation is an A53T mutation.

5.

The method of embodiment 1, further comprising performing an assay on a human being having a DCL or an animal model of DCL to determine whether the agent treats or inhibits a symptom of DCL.

6.

A screening method for an agent that has a pharmacological activity useful for the treatment of dementia with Lewy bodies (DCL) comprising:

contacting the agent with an alpha-synuclein fragment, which is SN1-115, SN1-133

or SN1-135, with numbered residues according to SEQ ID NO: 1; determine the rate or extent of aggregation of the alpha-synuclein or alpha-fragment synuclein, in which a reduction in the rate or extent of aggregation with respect to a control lacking agent, indicates that the agent has a pharmacological activity.

7.

The method of embodiment 6, wherein the alpha-synuclein fragment contains a mutation associated with hereditary DCL.

8.

The method of embodiment 37, wherein the mutation is an A53T mutation.

9.

The method of embodiment 6, further comprising performing an assay on a human being having a DCL or an animal model of DCL to determine whether the agent treats or inhibits a symptom of DCL.

10. A method of screening an agent for a pharmacological activity useful in the treatment of DCL, which comprises contacting a cell that expresses alpha-synuclein, and processes the alpha-synuclein in a fragment with an agent, in which the fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO: 1;

10 determine a level of the fragment in the cell in relation to the reference level in the same cell type in the absence of an agent, a reduction in the level of the fragment in relation to the reference indicating that the agent has a pharmacological activity useful in the DCL treatment.

11. The method of embodiment 10, wherein the alpha-synuclein fragment contains a mutation associated with a hereditary DCL.

12.

The method of embodiment 11, wherein the mutation is an A53T mutation.

13.

The method of embodiment 10, further comprising performing a test on a human being who

20 has a DCL or an animal model of DCL to determine if the agent treats or inhibits a symptom of DCL.

14. The method of embodiment 13, in which dementia with Lewy bodies is Parkinson's disease

or dementia with diffuse Lewy bodies (DCLD). 25

15. A method of screening an agent for a pharmacological activity useful in the treatment of DCL, comprising:

contacting a transgenic animal that has a transgene that expresses an alpha-fragment

Synuclein, wherein the fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO: 1; determine a level of aggregate forms of the fragment in the brain of a transgenic animal in relation to the reference level of aggregate forms comparable to a transgenic animal in the absence of the agent, a reduction in the level of aggregate forms of the fragment in relation to the

A reference indicating that the agent has a useful pharmacological activity in the treatment of DCL.

16. The method of embodiment 15, wherein the alpha-synuclein fragment contains a mutation associated with a hereditary DCL.

The method of embodiment 16, wherein the mutation is an A53T mutation.

18. The method of embodiment 15, wherein the transgenic animal is a mouse.

19. The method of embodiment 15, wherein the transgenic animal is a Drosophila. Four. Five

20. The method of embodiment 15, further comprising performing an assay on a human being having a DCL or an animal model of DCL to determine whether the agent treats or inhibits a symptom of DCL.

50 21. The method of embodiment 20, wherein the DCL is a Parkinson's disease or a DCLD.

22. A method of screening an agent for a pharmacological activity useful in the treatment of DCL, comprising:

55 contacting a transgenic animal that has a transgene that expresses alpha-synuclein and processes the alpha-synuclein in a fragment with an agent, in which the fragment is SN1-115, SN1

- 133 or SN1-135 numbered according to SEQ ID NO: 1; determining a level of the fragment in a neuronal cell in relation to the reference level in the absence of the agent, a reduction in the level of the fragments in relation to the reference indicates that

60 the agent has a useful pharmacological activity in the treatment of DCL.

23. The method of embodiment 22, wherein the alpha-synuclein fragment contains a mutation associated with a hereditary DCL.

65 24. The method of embodiment 23, wherein the mutation is an A53T mutation.

25.

The method of embodiment 22, wherein the transgenic animal is a mouse.

26.

The method of embodiment 22, wherein the transgenic animal is a Drosophila.

27.

The method of embodiment 22, further comprising performing an assay on a human being having a DCL or an animal model of DCL to determine whether the agent treats or inhibits a symptom of DCL.

28.

The method of embodiment 27, wherein the DCL is a Parkinson's disease or a DCLD.

29.

A transgenic animal having a genome comprising a transgene comprising a promoter operably linked to a nucleic acid segment encoding an alpha-synuclein fragment, wherein the fragment is numbered SN1-115, SN1-133 or SN1-135 according to SEQ ID No. 1; in which the expression of the fragment in the transgenic animal predisposes the animal to develop at least one characteristic of the DCL.

30

The transgenic animal of embodiment 29, wherein the promoter is a PDGF promoter.

31.

The transgenic animal of embodiment 29, wherein at least one characteristic is a deficiency of motor function.

32

The transgenic animal of embodiment 29, in which at least one characteristic is a deficiency of cognitive function.

33.

The transgenic animal of embodiment 29 is a mouse.

3. 4.

The transgenic animal of embodiment 29 is a Drosophila.

35

A method of detecting the presence or susceptibility of DCL in a patient, which comprises detecting a level of an alpha-synuclein fragment in a body fluid, in which the fragment is SN1

- 115, SN1-133 or SN1-135 numbered according to SEQ ID NO: 1; a level higher than the reference level in individuals without disease that indicates the presence or susceptibility in the DCL.

36.

An antibody that specifically binds to an alpha-synuclein fragment, in which the fragment is SN1-115, SN116-140, SN1-133, SN1-135, SN134-140 or SN136-140 numbered according to SEQ ID NO: 1 ; without specifically binding to complete alpha-synuclein.

37.

The antibody of embodiment 36 is a human antibody.

38.

The antibody of embodiment 36 is a humanized antibody.

39.

The antibody of embodiment 36 is a monoclonal antibody.

40

The antibody of embodiment 36 having a human IgG1 isotype.

41.

The antibody of embodiment 36 designated 12C6 or 7G8.

42

A method for detecting the presence or susceptibility of DCL in a patient, comprising administering to an patient an antibody that specifically binds SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO: 1; without specifically binding to complete alpha-synuclein; determining a level of antibody binding in patients, in which a higher level of binding with respect to the reference level in individuals without disease, indicates the presence or susceptibility in the DCL.

43

A method for effecting the treatment or prophylaxis of a DCL, which comprises administering to a patient suffering from, or at risk of a DCL, an effective regimen of phosphorylated alpha-synuclein or a phosphorylated fragment of alpha-synuclein, in which the fragment is characterized by the presence of at least 100 contiguous amino acids of intact alpha-synuclein and a deletion of 1-11 contiguous C-terminal amino acids of intact alpha-synuclein; and thus, performs the treatment or prophylaxis of the DCL.

44.

The method of embodiment 43, wherein the fragment is SN1-133 or SN1-135.

Four. Five.

The method of embodiment 43, further comprising the administration of an adjuvant that increases an immune response comprising antibodies in the fragment.

46.

The method of embodiment 43, wherein the fragment is linked to a carrier that forms a fusion protein, wherein the carrier increases an immune response comprising antibodies in the fragment.

47

A method for effecting the treatment or prophylaxis of a DCL, which comprises administering to a patient suffering from, or at risk of a DCL, an effective regimen of an alpha-synuclein fragment, in which the fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO: 1; or a subfragment of any of these that includes at least five contiguous residues that start at C-terminal of one of the fragments, and thereby, carry out the treatment or prophylaxis of the DCL.

48.

A method for effecting the treatment or prophylaxis of a DCL, which comprises administering to a patient suffering from, or at risk of a DCL, an effective regimen of an antibody that specifically binds to an alpha-synuclein fragment, in which the fragment is SN1-115, SN1-133 or SN1-135; with residues numbered according to SEQ ID No. 1 without binding to intact alpha-synuclein, whereby the antibody performs the prophylaxis or treatment of the disease.

49.

A method of purification of a protease that clones intact alpha-synuclein to form a fragment, in which the fragment is SN1-115, SN1-133 or SN1-135; with waste numbered according to SEQ ID No. 1; which comprises identifying a protease inhibitor; contacting the inhibitor with a cell or tissue extract containing the protease, whereby the protease binds to the inhibitor; and release the inhibitor protease.

fifty.

The method of embodiment 49, wherein the inhibitor is an alpha-synuclein peptide comprising a contiguous segment of at least 5 intact alpha-synuclein residues between positions 129-139, or 111-119.

51.

The method of embodiment 49, wherein at least one of the residues is a transition state analog.

52

A monoclonal antibody designated 12C6 or 7G8 or a chimeric or humanized form thereof.

53.

An alpha-synuclein fragment, which is alpha-synuclein SN1-133 or SN1-135, with residues numbered according to SEQ ID NO: 1.

54

The fragment of embodiment 53, wherein the alpha-synuclein fragment contains a mutation associated with dementia with hereditary Lewy bodies (DCL), for example, an A53T mutation.

55.

An alpha-synuclein fragment, which is alpha-synuclein SN134-140 or SN136-140, with numbered residues according to SEQ ID NO: 1.

56.

A screening method for an agent that has a pharmacological activity useful for the treatment of dementia with Lewy bodies (DCL) comprising:

contacting the agent with an alpha-synuclein fragment, defined in the claims. 53 or 54; determine the rate or extent of aggregation of the alpha-synuclein or alpha-fragment synuclein, in which a reduction in the rate or extent of aggregation with respect to a control lacking agent, indicates that the agent has a pharmacological activity.

57.

A method according to embodiment 56, wherein the alpha-synuclein fragment is phosphorylated.

58.

A screening method for an agent that has a pharmacological activity useful for the treatment of dementia with Lewy bodies (DCL) comprising:

contacting a cell expressing alpha-synuclein and processing the alpha-synuclein in a fragment with an agent, in which the agent is defined in embodiment 53 or 54; determining a level of the fragment in the cell in relation to the reference level in the same cell type in the absence of the agent, a reduction in the level of the fragment in relation to the reference indicates that the agent has a pharmacological activity useful in the treatment of the DCL

59.

A method of screening an agent for a pharmacological activity useful in the treatment of DCL, comprising:

contacting a transgenic animal having a transgene expressing an alpha-synuclein fragment, as defined in claims 53 or 54, with an agent; determine a level of aggregate forms of the fragment in the brain of a transgenic animal relative to the reference level of the aggregate forms of the fragment comparable to a transgenic animal in the absence of the agent, a reduction in the level of aggregate forms of the fragment in relation to the reference indicates that the agent has a useful pharmacological activity in the treatment

5 of the DCL, in which the transgenic animal is preferably a mouse or a Drosophila.

60. A method of screening an agent for a pharmacological activity useful in the treatment of DCL, comprising:

10 contacting a transgenic animal that has a transgene that expresses the alpha-synuclein and processes the alpha-synuclein in a fragment defined in embodiments 53 or 54 with an agent, determining a level of the fragment in a neuronal cell relative to the level reference in the absence of the agent, a reduction in the level of the fragments in relation to the reference indicates that the agent has a pharmacological activity useful in the treatment of DCL, in which the animal

Transgenic is preferably a mouse or a Drosophila.

61. The method of embodiments 56-60, further comprising conducting a test on a human being having a DCL or an animal model of DCL to determine whether the agent treats or inhibits a symptom of DCL, in which DCL is preferably Parkinson's disease or a DCLD.

62. A transgenic animal, preferably a mouse or a Drosophila, that has a genome comprising a transgene comprising a promoter operably linked to a nucleic acid segment encoding an alpha-synculein fragment as defined in embodiment 53 ; in which the expression of the fragment in the transgenic animal predisposes the animal to develop at least

A characteristic of DCL, in which the promoter is preferably a PDGF promoter, or in which at least one characteristic is preferably a deficiency of motor function or a deficiency of cognitive function.

63. A method of detecting the presence or susceptibility of DCL in a patient, comprising:

30 detecting a level of an alpha-synuclein fragment as defined in embodiment 53 in a body fluid; a higher level of union with respect to the reference level in individuals without disease that indicates the presence or susceptibility in the DCL.

64. An antibody that specifically binds to an alpha-synuclein fragment, as defined in claims 53 or 55, without specifically binding to a complete alpha-synuclein, wherein the antibody is preferably a human or humanized antibody. , more preferably a human isotype igG1.

65. A method for diagnosing the presence of or susceptibility in DCL, which comprises:

determining a level of binding of an antibody according to embodiment 64 when it depends on embodiment 53 in a patient, in which a higher level of binding relative to the reference level in 45 individuals without disease indicates the presence or susceptibility in the DCL.

66. An alpha-synuclein fragment, in which the fragment is defined in embodiments 53 or 54, or a subfragment that includes at least five contiguous residues beginning at C-terminal of one of the fragments, or an antibody in the alpha-synuclein as defined in embodiment 64 when

50 depends on embodiment 53, for use in a method of treatment or prophylaxis of dementia with Lewy bodies.

67. A method for purifying a protease that clones intact alpha-synuclein to form a fragment, as defined in embodiment 53, comprising:

55 identify a protease inhibitor; contacting the inhibitor with a cell or tissue extract containing the protease, whereby the protease binds to the inhibitor; Y; release the inhibitor protease, in which the inhibitor is optionally an alpha-peptide

60 synuclein comprising a contiguous segment of at least 5 intact alpha-synuclein residues between positions 129-139, or 111-119, preferably in which at least one of the residues is a transition state analog.

SEQUENCE LIST

55 15

Claims (13)

 Claims

one.

An alpha-synuclein fragment, which is alpha synuclein SN1-135, with residues numbered according to SEQ ID NO: 1.

2.

A fragment of claim 1, wherein the alpha-synuclein fragment contains a mutation associated with dementia with hereditary Lewy bodies (DCL), preferably an A53T mutation.

3.

A method of screening an agent that has a pharmacological activity useful for the treatment of dementia with Lewy bodies (DCL) comprising:

contacting the agent with an alpha-synuclein fragment, as defined in the claims 1 or 2; Y determine the rate or extent of aggregation of alpha-synuclein or alpha-fragment synuclein, in which a reduction in the rate or extent of aggregation with respect to a control lacking agent, indicates that the agent has a pharmacological activity.

Four.

A method according to claim 3, wherein the alpha-synuclein fragment is phosphorylated.

5.

A method of screening an agent that has a pharmacological activity useful for the treatment of (DCL) comprising:

contacting a cell expressing alpha-synuclein and processing the alpha-synuclein in a fragment with an agent, wherein the agent is defined in embodiments 1 or 2; and determining a level of the fragment in the cell in relation to the reference level in the same cell type in the absence of the agent, a reduction in the level of the fragment in relation to the reference indicates that the agent has a pharmacological activity useful in the treatment of the DCL.

6.

A method of screening an agent for a pharmacological activity useful in the treatment of DCL, comprising:

contacting a non-human transgenic animal having a transgene that expresses an alpha-synuclein fragment, as defined in claims 1 or 2, with an agent; and determining a level of aggregate forms of the fragment in the brain of a non-human transgenic animal relative to the reference level of aggregate forms comparable to a non-human transgenic animal in the absence of the agent, a reduction in the level of aggregate forms of the The reference fragment indicates that the agent has a pharmacological activity useful in the treatment of DCL, in which the non-human transgenic animal is preferably a mouse or a Drosophila.

7.

A method of screening an agent for a pharmacological activity useful in the treatment of DCL, comprising:

contacting a non-human transgenic animal that has a transgene that expresses alpha-synuclein and processes alpha-synuclein in a fragment, as defined in claims 1 or 2, with an agent; and determining a level of the fragment in a neuronal cell of a non-human transgenic animal in relation to the line level in the absence of the agent, a reduction in the level of the fragments in relation to the reference indicates that the agent has a useful pharmacological activity in the treatment of DCL, in which the non-human transgenic animal is preferably a mouse or a Drosophila.

8.

The method of claims 3-7, further comprising conducting an assay in a non-human animal model of DCL to determine whether the agent treats or inhibits a symptom of DCL, wherein the DCL is preferably Parkinson's disease. or the DCLD.

9.

A non-human transgenic animal, preferably a mouse or a Drosophila, which has a genome comprising a transgene comprising a promoter operably linked to a nucleic acid segment encoding an alpha-synculein fragment as defined in claims 1 or 2; wherein the expression of the fragment in the non-human transgenic animal predisposes the non-human animal to develop at least one characteristic of DCL, in which the promoter is preferably a PDGF promoter, or in which at least one characteristic is preferably a deficiency of motor function or a deficiency of cognitive function.

10.

A method to detect the presence of DCL in a patient, comprising:

detecting the level of an alpha-synuclein fragment as defined in claim 1 or in claim 2 in the cerebrospinal fluid; a higher level than the reference level in individuals without disease that indicates the presence of the DCL.

eleven.

An antibody that specifically binds to an alpha-synuclein fragment, as defined in claims 1 or 2, without specifically binding to a complete alpha-synuclein, wherein the antibody is preferably a human or humanized antibody, more preferably a human isotype igG1.

12.

A method of diagnosing the presence of DCL by functional image in vivo, comprising:

determining a level of binding of an antibody according to claim 11 in the brain of a patient, wherein a higher level of binding relative to the reference level in individuals without disease indicates the presence of DCL. 13. An alpha-synuclein fragment, which is defined in claim 1 or claim 2, or an alpha-synuclein antibody as defined in claim 11, for use in a method of treatment or prophylaxis of dementia with Lewy bodies.