Alpha-synuclein super-mutants accelerate alpha-synuclein aggregation

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder which is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of α-synuclein. The present invention provides α-synuclein mutations which accelerate α-synuclein aggregation and can thus be utilized for transgenic animal production and generation of the first progressive PD model. Also provided is an in vitro aggregation assay which can be utilized to identify α-synuclein nucleation inhibitors for the treatment of PD.

1 . An isolated artificial α-synuclein mutant polypeptide which demonstrates accelerated aggregation as compared to naturally occurring α-synuclein mutant polypeptides.  
     
     
         2 . A polypeptide of  claim 1 , said polypeptide comprising the amino acid sequence selected from the group consisting of the amino acid sequence as set forth in at least one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.  
     
     
         3 . An in vitro aggregation assay for evaluating α-synuclein mutants which demonstrate accelerated aggregation, said assay comprising the steps of: 
 (a) generating pure α-synuclein protein;  
 (b) incubating the α-synuclein solution with defined agitation;  
 (c) centrifuging aliquots of the incubated samples at predetermined times; and  
 (4) analyzing the pellet and the supernatant of the aliquoted samples using various conventional techniques.  
 
     
     
         4 . A transgenic non-human mammal which has been genetically engineered to express the polypeptide of  claim 1  in the brain.  
     
     
         5 . An in vitro aggregation assay for high throughput screening for α-synuclein nucleation inhibitors, said assay comprising: 
 (a) generating a solution of α-synuclein or aggregation competent fragments of α-synuclein or the mutant polypeptides of claim  1 ;  
 (b) incubating said solution with a potential nucleation-affecting agent under conditions that allow α-synuclein aggregation in the absence of the agent;  
 (c) detecting the amount of aggregated α-synuclein; and  
 (d) assessing the effect of said agent on the aggregation of α-synuclein.  
 
     
     
         6 . The method of  claim 5  wherein the α-synuclein or α-synuclein fragment is labeled radioactively, enzymatically, fluorescently or antigenically.  
     
     
         7 . A method for treating, preventing, or ameliorating PD, or related disorder, comprising administering to a patient an effective amount of an α-synuclein nucleation-affecting agent identified according to claim  5 .