α-Synuclein inhibits intersynaptic vesicle mobility and maintains recycling-pool homeostasis

Abstract

Although the presynaptic protein α-synuclein is a recognized player in neurodegeneration, its precise physiologic function(s) and/or role in human disease remains unclear. An emerging consensus from previous studies in lower-order systems is that α-synuclein interferes with vesicle-trafficking pathways; however putative neuronal correlates are unknown. Here we explore consequences of α-synuclein modulation in cultured mouse hippocampal neurons; coupling α-synuclein overexpression and knock-out model-systems with contemporary imaging paradigms. Our studies reveal an unexpected role of α-synuclein in attenuating the mobility of recycling pool (RP) vesicles between presynaptic boutons--called "superpool" trafficking--and also in maintaining the overall size of RPs at synapses. While an excess of α-synuclein led to smaller RPs and inhibited intersynaptic trafficking, an absence of α-synuclein triggered converse changes with larger RPs and enhanced intersynaptic trafficking. The data collectively suggest a model where α-synuclein maintains RP homeostasis by modulating intersynaptic vesicular dynamics, and provide a putative neuronal correlate of α-synuclein-induced impairments in vesicle-trafficking previously reported in lower-order systems.

Figure 1.

Converse effects of α-synuclein overexpression/absence on recycling pool size. A, Automated bouton sampling algorithms. Boutons were sampled by placing virtual 8 × 8 grids on a coverslip (left, large circle represents coverslip; arrows indicate sampling trajectory) and serial z-stacks were obtained, resulting in the acquisition of 64 z-stacks distributed over a 4.8 × 4.8 mm area of a 10 mm coverslip (see Materials and Methods). B, Maximum-intensity projections were computationally “stitched” into a montage (890.88 × 665.6 μm, resolution 0.16 μm/pixel) and analyzed. C, Images of WT and α-synuclein transgenic (TR) neurons loaded with FM4-64. D, Normalized average FM4-64 intensities in DIV 17–21 transgenic boutons (compared with WT) were 0.75 ± 0.01 (N ∼4000–6000 boutons were examined for each group; ***p < 0.0001. E, FM4-64-loading in α-synuclein^−/− neurons. Note increased FM-dye loading, quantified on right (relative average intensities in −/− boutons were 1.168 ± 0.0043 mean ± SEM, ***p < 0.0001; N ∼9000/7000 boutons examined in WT/−/− cultures processed in parallel). F, Left to right, Relative average FM4-64/synapsin-1/VAMP-2 intensities in −/− boutons (compared with boutons from littermate paired WT/+/− mice) were 1.12 ± 0.0010; 1.21 ± 0.0019; and 1.16 ± 0.0017 respectively (N ∼25,000–50,000 boutons/condition; ***p < 0.0001). Data from two sets of paired littermates were pooled for display, but were also independently significant.

Figure 2.

Diminished presynaptic protein levels in DIV 17–21 α-synuclein overexpressing boutons correlate with reduced recycling pools. A, B, Neurons overexpressing α-syn:GFP were fixed and stained with antibodies to endogenous mouse presynaptic proteins. Relative average fluorescence intensities of VAMP-2, amphiphysin, synapsin-1, synaptophysin and SNAP-25 in transgenic boutons (compared with controls) were 0.69 ± 0.007, 0.7 ± 0.008, 0.57 ± 0.007, 0.55 ± 0.008 and 1.12 ± 0.01 respectively, mean ± SEM, p < 0.0001, for all proteins, unpaired t test; N ∼5000–8000 boutons/group. C, Retrospective immunostaining (bouton-crops) of mouse presynaptic proteins in FM4-64-loaded boutons from WT and TR mice (see Materials and Methods). Note that boutons with low FM4-64 levels also have lower VAMP-2/synapsin-1 levels. D, FM4-64 and VAMP-2/synapsin-1 average-intensities were correlated in WT neurons (correlation coefficient r = 0.66/0.61 for VAMP-2/synapsin-1 respectively), with negative X/Y mean-shifts in TR neurons, suggesting corresponding diminutions of both presynaptic members (compare X/Y-distributions in WT-top/TR-bottom graphs). E, Representative immunostaining (bouton-crops) of WT or TR neurons with synapsin-1/VAMP-2. F, Note corresponding X/Y mean-shifts in the TR boutons stained with synapsin-1/VAMP-2 (left-panels); but lack of Y mean-shift in synapsin-1/SNAP-25 correlations (right panels; see Materials and Methods).

Figure 3.

Converse effects of α-synuclein overexpression/absence on recycling pool kinetics. A, DIV 17–21 transgenic (or WT) boutons were loaded with FM4-64, a string of boutons were selected (#1–3, “pre-bleach”), and a single bouton (#2) was selectively photobleached (“post-bleach”). Note recovery of fluorescence over time in the grayscale (left) and pseudocolor (right) images. Scaled image below shows axon continuity (arrowheads). Variations in intensities of the three boutons over time are graphically represented below. Note partial recovery of FM4-64 in the bleached bouton, reflecting exchange between boutons (see Results). B, Top, Maximum FM4-64 recovery inversely correlated with α-syn:GFP intensities in the same boutons (r = 0.56; p < 0.0001). Bottom, Kinetics of FM4-64-recovery was also significantly diminished in the upper 50^th percentile of overexpressers (see Results). N ∼40 boutons/group, ***p < 0.0001. C, Conversely, the rate of FM4-64 FRAP recovery was significantly faster in DIV 17–21 α-synuclein^−/− neurons, compared with their WT/+/− littermates (containing α-synuclein). (N ∼ 60–90 boutons/group, ***p < 0.0001). D, Pooled FM4-64-FRAP data from all experiments show an incremental diminution of FM4-64 mobility upon increasing α-synuclein dosage. ***p < 0.0001, one-way ANOVA.

Figure 4.

Direct assay of the superpool and FM4-64 exocytosis in α-syn:GFP-expressing neurons. A, Top to bottom, (1) Images of FM4-64-loaded WT/TR boutons; (2) raw kymographs showing trafficking of FM4-64-labeled vesicles (diagonal lines) between stationary boutons (vertical lines); (3) difference [DIFF (N − 1) kymographs showing inter-boutonic fluctuations of fluorescence intensities; (4) cumulative FM4-64 flux (Σ-flux, “flattened” image); (5) overlay of FM4-64/Σ-flux. B, The cumulative FM4-64 flux was significantly lower in TR neurons, compared with WT littermates [91.36 ± 9.83 arbitrary fluorescence units (AFU) and 154.3 ± 8.41 AFU, respectively; mean ± SEM, ***p < 0.0001; N = 80–120 boutons/group]. C, The cumulative FM4-64 flux was significantly lower in TR neurons, compared with WT littermates (91.36 ± 9.83 AFU and 154.3 ± 8.41 AFU, respectively; mean ± SEM, ***p < 0.0001; N = 80–120 axonal-segments/group). D, FM4-64-loading was lower in Nocodazole-treated neurons (0.88 ± 0.0044, mean ± SEM, N ∼20,000–30,000 boutons/group, ***p < 0.0001). E, WT/α-syn:GFP boutons were loaded with FM4-64 (see Materials and Methods), and stimulation-dependent (arrow) release of FM-dye from boutons was monitored (1200 action potentials at 10 Hz; 60 mA, 1 ms). Change in FM4-64-fluorescence (ΔF) was quantified as [fluorescence at each time-point (F_t) − initial fluorescence (F₀, average of all prestimulation frames)]/F₀ (Waites et al., 2011); note that not all K⁺-loaded boutons can be unloaded by these methods (see also Waites et al., 2011). Kinetics of FM4-64 decay (Materials and Methods) was slower in α-syn:GFP-expressing boutons, and was greatly attenuated in the highest α-syn:GFP expressers (Up-50, see Results). N = 1363/635 boutons, WT/TR. F, Scatter plots of α-syn:GFP intensities and the extent of FM4-64 unloading (average residual fluorescence over the last 5-frames of the decay-curves). Note that FM4-64-unloading is attenuated in high α-syn:GFP expressers. G, Bar graphs comparing FM4-64 unloading (as above) between various groups (***p < 0.0001, one-way ANOVA).