Formation of intracellular aggregates of α-synuclein.

(A) Confocal images of double immunostaining for phosphorylated α-synuclein and GAD after treatment with preformed fibrils of α-synuclein. The region marked by a white square in A is magnified in B. Immunoreactivity of phosphorylated α-synuclein was observed as intracellular fibrous aggregates or inclusion bodies. GAD-positive neurons indicated by # were free of α-synuclein aggregate formation. GAD signals, including GAD-positive neurites, were not colocalized with phosphorylated α-synuclein. (C) In the absence of fibril treatment, exogenous human α-synuclein was diffusely distributed in the cell body of GAD neurons (Control). After fibril treatment, intracellular inclusions positive for α-synuclein were induced in the GAD-positive cells expressing exogenous α-synuclein. Cell bodies are shown surrounded by white dotted lines. ‘N’ indicates the location of the nucleus. Three independent cultures were performed and in all cases confirmed that intracellular inclusions were predominantly formed in GAD-negative neurons. Exogenous expression of human α-synuclein enhanced the aggregate formation in GAD-positive cells. These results were quantified and are described in Table 1. Scale bars: 10 µm.