α-Synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons

P. J. McLean, H. Kawamata, B. T. Hyman

Research output: Contribution to journalArticle

166 Scopus citations

Abstract

α-Synuclein accumulates in the brains of sporadic Parkinson's disease patients as a major component of Lewy bodies, and mutations in α-synuclein are associated with familial forms of Parkinson's disease. The pathogenic mechanisms that precede and promote the aggregation of α-synuclein into Lewy bodies in neurons remain to be determined. Here, we constructed a series of α-synuclein-enhanced green fluorescent protein (α-synucleinEGFP, SynEGFP) fusion proteins to address whether the Parkinson's disease-associated mutations alter the subcellular distribution of α-synuclein, and to use as a tool for experimental manipulations to induce aggregate formation. When transfected into mouse cultured primary neurons, the 49-kDa α-synucleinEGFP fusion proteins are partially truncated to a ∼ 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 α-synucleinEGFP does not form the cytoplasmic inclusions that are characteristic of wild-type and Ala53Thr α-synucleinEGFP, supporting the idea that the Ala30Pro α-synuclein protein conformation differs from wild-type α-synuclein. Similar inclusions are formed if α-synuclein carboxy-terminus is modified by the addition of a V5/6 × Histidine epitope tag. By contrast, overexpression of unmodified α-synuclein does not lead to aggregate formation. Furthermore, synphilin-1, an α-synuclein interacting protein also found in Lewy bodies, colocalizes with the carboxy-terminally truncated α-synuclein fusion protein in discrete cytoplasmic inclusions. Our finding that manipulations of the carboxy-terminus of α-synuclein lead to inclusion formation may provide a model for studies of the pathogenic mechanisms of α-synuclein aggregation in Lewy bodies.

Original languageEnglish (US)
Pages (from-to)901-912
Number of pages12
JournalNeuroscience
Volume104
Issue number3
DOIs
StatePublished - Jun 14 2001

Keywords

  • Aggregation
  • Lewy body
  • Parkinson's disease
  • Synphilin-1
  • α-Synuclein

ASJC Scopus subject areas

  • Neuroscience(all)

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