alpha-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons

alpha -Synuclein accumulates in the brains of sporadic Parkinsons 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 a-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 transiected into mouse cultured primary neurons, the 49-kDa alpha -synucleinEGFP fusion proteins are partially truncated to a similar to 27-kDa form. This nonfluorescent 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 a-synucleinEGFP does not form the cytoplasmic inclusions that are characteristic of wild-type and AlaS3Thr alpha -synucleinEGFP. supporting the idea that the Ala30Pro alpha -syyncceen 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 -synnuclein 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. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.