Figure 8.

Cholesterol and sphingolipid depletion reduce the synaptic localization of α-synuclein. A, Hippocampal neurons transfected with GFPαsyn were treated with 250 μm mevalonic acid (MA), 4 μm mevastatin (MV), and 10 μm fumonisin B₁ (FB1) (or DMS0/methanol control) at days 12 and 15 in vitro, fixed at day 18, immunostained for SV2, and imaged by confocal microscopy. In separate cultures, nystatin (10 μg/ml, or DMSO vehicle as control) was added at day 18 for 20 min before fixation and staining for SV2. Scale bar, 5 μm. SV2 was used as synaptic marker in this case because synaptophysin has been shown to bind cholesterol (Thiele et al., 2000) and hence might be sensitive to the manipulations. B, Synaptic enrichment was quantified as the ratio of fluorescence at the bouton to fluorescence in the axon (see Fig. 7). Both chronic treatment with MA/MV/FB1 and acute treatment with nystatin substantially reduce the synaptic localization of GFPαsyn. Eight fields from two independent cultures were examined. Bars represent mean ± SD. ^*p < 0.0003 (Student's t test) between drugs and the corresponding vehicle controls.