Reduction of Immunoreactivity Against the C-Terminal Region of the Intracellular α-Synuclein by Exogenous α-Synuclein Aggregates: Possibility of Conformational Changes

Background: The formation of intracellular aggregates containing alpha-synuclein (alpha-syn) is a main pathological feature of Parkinson disease. The propagation of alpha-syn aggregation via cell-to-cell transmission has been implicated in the progression of Parkinson disease. Objective: Our aim is to clarify the molecular mechanisms underlying the formation of intracellular aggregation by extracellular alpha-syn. Methods: We investigated the effects of exogenous alpha-syn aggregates on intracellular alpha-syn immunoreactivity in alpha-syn-overexpressing SH-SY5Y cells using two antibodies to distinct epitopes of alpha-syn. To obtain alpha-syn aggregates, alpha-syn solution was aged with continuous agitation. Results: Immunoreactivity against the acidic C-terminal domain of the intracellular alpha-syn was reduced by exposure to aged alpha-syn, whereas that against the hydrophobic non-amyloid component region was not changed. The reduction in immunoreactivity was not suppressed by protease inhibitors but was mimicked by neutralization of the negative charges on the C-terminal of the intracellular alpha-syn induced by spermine or extracellular acidification. Conclusions: These results suggest that the reduction in immunoreactivity is attributed not to proteolytic cleavage but to a conformational change at the C-terminus of the intracellular alpha-syn. The conformational change at the C-terminus of the intracellular alpha-syn might be involved in an initial step of fibril formation by exogenous alpha-syn aggregates.