Alpha-synuclein accumulates in the brains of sporadic Parkinson's disease 
patients as a major component of Lewy bodies, and mutations in alpha-synuclein 
are associated with familial forms of Parkinson's disease. The pathogenic 
mechanisms that precede and promote the aggregation of alpha-synuclein into Lewy 
bodies in neurons remain to be determined. Here, we constructed a series of 
alpha-synuclein-enhanced green fluorescent protein (alpha-synucleinEGFP, 
SynEGFP) fusion proteins to address whether the Parkinson's disease-associated 
mutations alter the subcellular distribution of alpha-synuclein, and to use as a 
tool for experimental manipulations to induce aggregate formation. When 
transfected into mouse cultured primary neurons, the 49-kDa alpha-synucleinEGFP 
fusion proteins are partially truncated to a approximately 27-kDa form. This 
non-fluorescent carboxy-terminally modified fusion protein spontaneously forms 
inclusions in the neuronal cytoplasm. A marked increase in the accumulation of 
inclusions is detected following treatment with each of three proteasome 
inhibitors, n-acetyl-leu-leu-norleucinal, lactacystin and MG132. Interestingly, 
Ala30Pro alpha-synucleinEGFP does not form the cytoplasmic inclusions that are 
characteristic of wild-type and Ala53Thr alpha-synucleinEGFP, supporting the 
idea that the Ala30Pro alpha-synuclein protein conformation differs from 
wild-type alpha-synuclein. Similar inclusions are formed if alpha-synuclein 
carboxy-terminus is modified by the addition of a V5/6xHistidine epitope tag. By 
contrast, overexpression of unmodified alpha-synuclein does not lead to 
aggregate formation. Furthermore, synphilin-1, an alpha-synuclein interacting 
protein also found in Lewy bodies, colocalizes with the carboxy-terminally 
truncated alpha-synuclein fusion protein in discrete cytoplasmic inclusions.Our 
finding that manipulations of the carboxy-terminus of alpha-synuclein lead to 
inclusion formation may provide a model for studies of the pathogenic mechanisms 
of alpha-synuclein aggregation in Lewy bodies.