Figure 4. α–Synuclein reduces the recycling pool of synaptic vesicles.

(A) Hippocampal neurons cotransfected with VGLUT1-pHluorin and either human αsyn or empty vector were stimulated at 30 Hz for 3 s to activate specifically the readily releasable pool of synaptic vesicles. n=3 coverslips, 60 boutons per condition. Inset, peak ΔF/F₀ values normalized to the response in the vector control shows a substantial reduction in the cells over-expressing human αsyn. n=6 coverslips, 120 boutons per condition from 2 independent transfections. p<0.0001 by two-tailed, unpaired t-test. (B) Neurons expressing VGLUT1-pHluorin were stimulated with Tyrode’s solution containing 500 mM sucrose in the presence of 1μM bafilomycin to prevent reacidification of the internalized vesicles, and imaged in the absence of sucrose (to avoid distortion by changes in refractive index) both before and after stimulation. The change in ΔF/F₀ normalized to vector control shows a reduction in neurons over-expressing human αsyn. n=9 coverslips, 180 boutons per condition from 3 independent transfections. p<0.05 by two-tailed, unpaired t-test. (C) Hippocampal neurons transfected with either αsyn-IRES2-GFP or IRES2-GFP were loaded with 15 μM FM 4-64 by 10 Hz stimulation for 60 s, maintained in the dye for 60 additional seconds to allow full endocytosis, and washed extensively before destaining at 10 Hz for 120 seconds. n = 3 coverslips, 71 boutons for αsyn-transfected, 73 nerve terminals for vector. αsyn over-expression reduces the amount of releasable dye uptake (inset). p < 0.05, n = 6 coverslips, 153 boutons for αsyn-over-expressing cells, and 208 boutons for vector control from 2 independent transfections. (D) Hippocampal neurons transfected with VGLUT1-pHluorin and either human αsyn or empty vector were stimulated at 10 Hz for 150 s in the presence of 1 μM bafilomycin to reveal the full recycling pool, followed by treatment with NH₄Cl (arrow) to reveal the total synaptic vesicle pool. n = 60 boutons from 3 coverslips for each condition (E) Left panel - cultures were transfected and stimulated as in C but with controls stimulated in the presence of 1 mM as well as 2 mM Ca²⁺. n = 60 boutons from 3 coverslips per condition. Right panel - peak ΔF/F₀ before addition of NH₄Cl (normalized to 2 mM Ca²⁺ control) shows a reduction in the presence of over-expressed αsyn but no significant reduction of control boutons stimulated in 1 mM Ca²⁺. p<0.01 by one-way ANOVA with Tukey’s post hoc tests, p>0.05 for vector in 2 mM Ca²⁺ versus vector in 1 mM Ca²⁺, p<0.01 for vector in 2 mM Ca²⁺ versus αsyn in 2 mM Ca²⁺, and p<0.05 for vector in 1 mM Ca²⁺ versus αsyn in 2 mM Ca²⁺. n=120 boutons from 6 coverslips per condition and 2 independent transfections. Values represent mean ± SEM.