Figure 1.
Low expression of α-synuclein in inhibitory neurons.
Cells expressing the inhibitory neuronal marker proteins GAD (A), parvalbumin (C), and somatostatin (D) (indicated by #) showed low expression of α-synuclein. Cells with high expression of α-synuclein are labeled with asterisks. The region marked by a white square in A is magnified in B. Immunoreactivity of GAD was not colocalized with that of α-synuclein (B). Three independent cultures were performed and the differential expression pattern of α-synuclein was reproducibly confirmed. These images were further subjected to the quantitative colocalization analyses shown in Fig. S2. Scale bars: 10 µm in A, C, and D; 5 µm in B.
Figure 2.
Differential expression of α-synuclein during cell maturation.
(A) Expression of α-synuclein was examined at the indicated times after cell dissemination. Immunoreactivity of α-synuclein increased during cell maturation. By 30 h after cell dissemination, some NeuN-positive cells expressed α-synuclein (asterisks), but other NeuN-positive cells did not. (B) Expression of GAD at 30 h was not sufficient to distinguish inhibitory neurons from other types of neurons. Three independent cultures were performed. Scale bars: 10 µm.
Figure 3.
Presynaptic localization of α-synuclein in excitatory neurons.
(A, B) Confocal images of double immunostaining for α-synuclein and synaptotagmin. The region marked by a white square in A is magnified in B. Arrowhead in B indicates the presynapse, expressing both α-synuclein and synaptotagmin. However, there are some synaptotagmin-positive synapses lacking α-synuclein (arrow). (C, D) Confocal images of double immunostaining for α-synuclein and vGluT-1, an excitatory neuronal marker protein. The region marked by a white square in C is magnified in D. α-Synuclein is colocalized with vGluT-1 in D (arrowheads). Three independent cultures were performed and colocalization between α-synuclein and vGluT-1 was reproducibly confirmed. Panel C was subjected to the quantitative colocalization analysis shown in Fig. S2. Scale bars: 10 µm in A and C; 5 µm in B and D.
Figure 4.
Colocalization of synapsin and α-synuclein during presynaptic membrane recycling.
Synapsin immunoreactivity was observed as punctate signals before treatment (Control). After membrane depolarization by applying a medium containing high potassium, the punctate signals of synapsin changed shape and fused to the presynaptic plasma membrane (High K). The change in localization was reversed by treatment with a low-potassium medium (High K-Recovery). α-Synuclein was colocalized with synapsin during presynaptic membrane recycling (arrowheads). However, there are also instances of synapsin immunoreactivity without α-synuclein expression (arrows). Three independent cultures were performed, and these staining patterns were observed in all cultures. Scale bar: 5 µm.
Figure 5.
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.
Table 1.
Figure 6.
Localization of α-synuclein in the hippocampal CA1 region.
(A, B) Confocal images of hippocampal neurons double immunostained for vGluT-1 and α-synuclein. The region marked by a white square in A is magnified in B. α-Synuclein is colocalized with vGluT-1 in B (arrowheads). (C, D) Confocal images double immunostained for α-synuclein and GAD. The region marked by a white square in C is magnified in D. As indicated by arrows in D, colocalization of α-synuclein and GAD was not detected. Mouse brains from two littermates were used for the immunohistochemical study. This experiment was repeated three times, and the differential expression pattern of α-synuclein was reproducibly confirmed. Panels A and C were used for the quantitative colocalization analyses shown in Fig. S2. Scale bars: 10 µm. indicates stratum pyramidale.