Analysis of alpha-synuclein aggregation and associated pathologies in a cellular model of multiple system atrophy

Abstract

Intracellular aggregation of alpha-synuclein is a neuropathological hallmark of a variety of neurodegenerative diseases, including Parkinson's disease and multiple system atrophy (MSA). The deposition of the normally neuronal alpha-synuclein protein within (oligodendro)glial cytoplasmic inclusions (GCIs) and its relationship with oligodendrocyte death in MSA have not been elucidated. We developed a cellular model that recapitulates features of the degenerative process that occurs in MSA. Ectopic human alpha-synuclein expression in primary oligodendrocytes was achieved by deriving cultures from transgenic mice, which express human alpha-synuclein under the control of the myelin proteolipid protein promoter. High level of alpha-synuclein expression in oligodendrocytes induced alpha-synuclein accumulation, proteasome dysfunction and alters their sensitivity to proteasome inhibition. The large perinuclear inclusions formed, displayed biochemical and histological features of GCIs including the presence of phosphorylated and oxidized forms of alpha-synuclein as well as components of the ubiquitin-proteasome system and molecular chaperones. The fibrillar (amyloid) nature of alpha-synuclein inclusions was indicated by thioflavin S staining and proven by immunoelectron microscopy. Under these conditions, alpha-synuclein accumulation was accompanied by oligodendrocyte apoptosis, as evidenced by caspase-3 activation and chromatin condensation. These effects were not due to secondary effects of proteasome inhibitors, because high-level over-expression by lentiviral delivery of alpha-synuclein alone, but not beta-synuclein, caused protein aggregation and cell death. Moreover, lenti-beta-synuclein prevented aggregation in alpha-synuclein transgenic oligodendrocytes challenged with proteasome inhibitor. Thus, induction of alpha-synuclein and pathological impairment of the ubiquitin-proteasome system may synergistically contribute to GCI formation and ultimately cause oligodendrocyte dysfunction and cell death in MSA.