Figure 4.

α-Synuclein CC dimer inhibits SV endocytosis. (A–C) Electron micrographs showing a typical control synapse, compared to synapses treated with either low or high concentrations of α-synuclein CC dimer (α-Syn-CC). L, low concentration; H, high concentration. Synapses treated with CC dimer exhibited SV recycling defects, as shown by a reduction in SVs, expanded plasma membrane (PM) evaginations (dotted lines), and greater numbers of endocytic intermediates including cisternae (C) and clathrin-coated pits (CCPs) with constricted necks (red circles). Insets show longer, branched tubules ending in clathrin coats. Arrowheads indicate their connections with the PM. Asterisks mark the complex budding structures shown in the insets. Scale bar in (A) also applies to (B,C). (D–F) 3D reconstructions reveal the extent of the endocytic defects induced by CC dimer, shown by a loss of SVs (blue), larger PM evaginations (green) and build up of cisternae (magenta), CCPs (yellow) and CCVs (white). Red slab marks the active zone. (G–K) Electron micrographs show typical CCPs at control (G) and CC dimer-treated synapses (H–K). Note the abundance of CCPs, including those with complex structures induced by CC dimer. Arrowheads indicate their connections with the PM. Scale bar in (G) applies to (H–K). (L–Q) Quantification of the SV recycling defects. The reduction in SVs (L) was compensated by increased PM evaginations, cisternae and CCPs and vesicles (M–Q). Data reported indicate the averages per synapse, as measured from single sections. Bars represent mean ± SEM from n = 26–27 synapses, 2–3 axons. Asterisks indicate statistical significance (p < 0.05) by ANOVA, as compared to controls.