Truncated Fragments of Alpha-Synuclein in Lewy Body Disease

Abstract

The application identifies novel fragments of alpha-synuclein in patients with Lewy Body Disease (LBD) and transgenic animal models thereof. These diseases are characterized by aggregations of alpha-synuclein. The fragments have a truncated C-terminus relative to full-length alpha-synuclein. Some fragments are characterized by 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-terminus of natural alpha-synuclein. The site of cleavage preferably occurs after residue 117 and before residue 126 of natural alpha-synuclein. The identification of these novel fragments of alpha-synuclein has a number of application in for example, drug discovery, diagnostics, therapeutics, and transgenic animals.

1 . A method of screening for an agent having a pharmacological activity useful for treating a Lewy Body Disease (LBD) comprising:
 contacting the agent with phosphorylated alpha-synuclein or a phosphorylated fragment thereof, wherein the fragment is characterized by presence of at least 100 contiguous amino acids of intact alpha-synuclein and a deletion of 1-11 contiguous amino acids from the C-terminus of intact alpha-synuclein;   determining the rate or extent of aggregation of the alpha synuclein or fragment of alpha-synuclein, wherein a reduction in the rate or extent of aggregation relative to a control lacking the agent indicates the agent has the pharmacological activity.   
     
     
         2 . The method of  claim 1 , wherein the agent is contacted with intact alpha synuclein or SN1-133 or SN1-135 numbered according to SEQ ID NO:1. 
     
     
         3 . The method of  claim 1 , wherein the fragment of alpha-synuclein bears a mutation associated with a hereditary LBD. 
     
     
         4 . The method of  claim 3 , wherein the mutation is an A53T mutation. 
     
     
         5 . The method of  claim 1 , further comprising conducting a trial in a human having a LBD or an animal model of LBD to determine whether the agent treats or inhibits a symptom of the LBD. 
     
     
         6 . A method of screening for an agent having a pharmacological activity useful for treating a Lewy Body Disease (LBD) comprising:
 contacting the agent with a fragment of alpha synuclein, which is SN1-115, SN1-133 or SN1-135, with residues numbered according to SEQ ID NO:1;   determining the rate or extent of aggregation of the alpha synuclein or fragment of alpha-synuclein, wherein a reduction in the rate or extent of aggregation relative to a control lacking the agent indicates the agent has the pharmacological activity.   
     
     
         7 . The method of  claim 6 , wherein the fragment of alpha-synuclein bears a mutation associated with a hereditary LBD. 
     
     
         8 . The method of  claim 37  wherein the mutation is an A53T mutation. 
     
     
         9 . The method of  claim 6 , further comprising conducting a trial in a human having a LBD or an animal model of LBD to determine whether the agent treats or inhibits a symptom of the LBD. 
     
     
         10 . A method of screening an agent for a pharmacological activity useful in treating a LBD, comprising
 contacting a cell expressing alpha-synuclein and processing the alpha-synuclein into a fragment with an agent, wherein the fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO:1;   determining a level of the fragment in the cell relative to a baseline level in the same cell type in the absence of the agent, a reduction in the level of the fragment relative to the baseline indicating the agent has the pharmacological activity useful in treating a LBD.   
     
     
         11 . The method of  claim 10 , wherein the fragment of alpha-synuclein bears a mutation associated with a hereditary LBD. 
     
     
         12 . The method of  claim 11 , wherein the mutation is an A53T mutation. 
     
     
         13 . The method of  claim 10 , further comprising conducting a trial in a human having a LBD or an animal model of LBD to determine whether the agent treats or inhibits a symptom of the LBD. 
     
     
         14 . The method of  claim 13 , wherein the Lewy Body disease is Parkinson's disease or Diffuse Lewy Body Disease (DLBD). 
     
     
         15 . A method of screening for an agent having a pharmacological activity useful for treating a LBD, comprising:
 contacting a transgenic animal having a transgene expressing a fragment of alpha-synuclein, wherein the fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO:1;   determining a level of aggregated forms of the fragment in the brain of the transgenic animal relative to a baseline level of aggregated forms of the fragment in a comparable transgenic animal in the absence of the agent, a reduction in the level of the aggregated forms fragment relative to the baseline indicating the agent has a pharmacological activity useful in treating a LBD.   
     
     
         16 . The method of  claim 15 , wherein the fragment of alpha-synuclein bears a mutation associated with a hereditary LBD. 
     
     
         17 . The method of  claim 16 , wherein the mutation is an A53T mutation. 
     
     
         18 . The method of  claim 15 , wherein the transgenic animal is a mouse. 
     
     
         19 . The method of  claim 15 , wherein the transgenic animal is a  Drosophila.    
     
     
         20 . The method of  claim 15 , further comprising conducting a trial in a human having a LBD or an animal model of LBD to determine whether the agent treats or inhibits a symptom of the LBD. 
     
     
         21 . The method of  claim 20 , wherein the LBD is Parkinson's disease or DLBD. 
     
     
         22 . A method of screening an agent for a pharmacological activity useful for treating a LBD, comprising:
 contacting a transgenic animal having a transgene expressing alpha-synuclein and processing the alpha-synuclein into a fragment with an agent, wherein 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 relative to a baseline level in the absence of the agent, a reduction in the level of the fragments relative to the baseline indicating the agent has the pharmacological activity useful for treating the LBD.   
     
     
         23 . The method of  claim 22 , wherein the fragment of alpha-synuclein bears a mutation associated with a hereditary LBD. 
     
     
         24 . The method of  claim 23 , wherein the mutation is an A53T mutation. 
     
     
         25 . The method of  claim 22 , wherein the transgenic animal is a mouse. 
     
     
         26 . The method of  claim 22 , wherein the transgenic animal is a  Drosophila.    
     
     
         27 . The method of  claim 22 , further comprising conducting a trial in a human having a LBD or an animal model of LBD to determine whether the agent treats or inhibits a symptom of the LBD. 
     
     
         28 . The method of  claim 27 , wherein the LBD is Parkinson's disease or DLBD. 
     
     
         29 . A transgenic animal having a genome comprising a transgene comprising a promoter operably linked to a nucleic acid segment encoding a fragment of alpha-synuclein wherein the fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO:1;
 wherein expression of the fragment in the transgenic animal disposes the animal to develop at least one characteristic of a LBD.   Screening any animal or cell   
     
     
         30 . The transgenic animal of  claim 29 , wherein the promoter is a PDGF promoter. 
     
     
         31 . The transgenic animal of  claim 29 , wherein the at least one characteristic is an impairment of motor function. 
     
     
         32 . The transgenic animal of  claim 29 , wherein the at least one characteristic is an impairment of cognitive function. 
     
     
         33 . The transgenic animal of  claim 29  that is a mouse. 
     
     
         34 . The transgenic animal of  claim 29  that is a  Drosophila.    
     
     
         35 . A method of detecting presence or susceptibility to an LBD in a patient, comprising
 detecting a level of a fragment of alpha-synuclein in body fluid, wherein the fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO:1;   a level greater than a baseline level in undiseased individuals indicating presence or susceptibility to LBD.   
     
     
         36 . An antibody that specifically binds to a fragment of alpha-synuclein, wherein 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 full-length alpha synuclein. 
     
     
         37 . The antibody of  claim 36  that is a human antibody. 
     
     
         38 . The antibody of  claim 36  that is a humanized antibody. 
     
     
         39 . The antibody of  claim 36  that is a monoclonal antibody. 
     
     
         40 . The antibody of  claim 36  having human isotype IgG1. 
     
     
         41 . The antibody of  claim 36  designated 12C6 or 7G8. 
     
     
         42 . A method of diagnosing presence of or susceptibility to LBD, comprising
 administering to a patient an antibody that specifically binds to SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO:1 without specifically binding to full length alpha-synuclein;   determining a level of binding of the antibody in the patients, wherein a higher level of binding relative to a base line level in undiseased individuals indicates presence or susceptibility to the LBD.   
     
     
         43 . A method of effecting treatment or prophylaxis of a LBD, comprising administering to a patient suffering from or at risk of a LBD, an effective regime of phosphorylated alpha synuclein or a phosphorylated fragment of alpha-synuclein, wherein the fragment is characterized by presence of at least 100 contiguous amino acids of intact alpha-synuclein and a deletion of 1-11 contiguous amino acids from the C-terminus of intact alpha-synuclein; and thereby effecting treatment or prophylaxis of the LBD. 
     
     
         44 . The method of  claim 43 , wherein the fragment is SN1-133 or SN1-135. 
     
     
         45 . The method of  claim 43 , further comprises administering an adjuvant that augments an immune response comprising antibodies to the fragment. 
     
     
         46 . The method of  claim 43 , wherein the fragment is linked to a carrier forming a fusion protein, wherein the carrier augments an immune response comprising antibodies to the fragment. 
     
     
         47 . A method of effecting treatment or prophylaxis of a LBD, comprising administering to a patient suffering from or at risk of a LBD, an effective regime of a fragment of alpha-synuclein, wherein the fragment is SN1-115, SN1-133 or SN1-135 numbered according to SEQ ID NO:1; or a subfragment of any of these including at least five continuous residues starting from the C-terminus of one of the fragments, and thereby effecting treatment or prophylaxis of the LBD. 
     
     
         48 . A method of effecting treatment or prophylaxis of a LBD comprising administering to a patient suffering from or at risk of a LBD an effective regime of an antibody that specifically binds to a fragment of alpha-synuclein, wherein 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 effects prophylaxis or treatment of the disease. 
     
     
         49 . A method of purifying a protease that cleaves intact alpha-synuclein to form a fragment, wherein the fragment is SN1-115, SN1-133 or SN1-135 with residues numbered according to SEQ ID NO:1; comprising
 identifying an inhibitor of the protease;   contacting the inhibitor with a cellular or tissue extract containing the protease, whereby the protease binds to the inhibitor; and   releasing the protease from the inhibitor.   
     
     
         50 . The method of  claim 49 , wherein the inhibitor is a peptide of alpha-synuclein comprising a contiguous segment of at least 5 residues of intact alpha-synuclein between positions 129-139, or 111-119. 
     
     
         51 . The method of  claim 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.