Article Figures & Data
Figures
- Figure 1.
WT monomeric α-synuclein inhibits slow and fast vesicle endocytosis at calyces of Held in slices from posthearing rats, but its mutants do not. A, Native PAGE (from left to right); monomeric human WT α-synuclein (purified by M.H.), that obtained from Sigma-Aldrich (His tag), and monomeric A30P and A53T α-synuclein mutants from Sigma-Aldrich, all dissolved in pipette solution. B1, Exo–endocytic Cm changes of calyceal presynaptic terminals evoked by ICa (induced by a 20 ms pulse stepping from −80 mV to +10 mV). Left, Intraterminal loading of monomeric WT α-synucleins (3.6 μm) from different sources similarly slowed endocytic Cm changes without affecting exocytic Cm changes or ICa (red and yellow traces superimposed with control black traces, each averaged from records in seven to eight terminals). Right, A30P or A53T α-synuclein mutants (3.6 μm), loaded into presynaptic terminals, had no significant effect (superimposed blue or green traces, averaged from records in four to six terminals). B2, Bar graphs showing the effects of WT α-synucleins, His tag WT α-synuclein, and A30P and A53T mutants on the 50% decay time of endocytic Cm (left), the magnitudes of exocytic Cm jump (middle), and presynaptic Ca2+ current charge (QCa, right). C, Intraterminal loading of α-synuclein (His tag) at different concentrations (0.7–22 μm) slowed vesicle endocytosis in a concentration-dependent manner. Top, Cm traces; data from six to seven calyces were averaged, normalized at the peak, and superimposed. Bottom, Half endocytic decay time at different α-synuclein concentrations (n = 6–7 for each data point). Asterisks indicate a significant difference from the control (with no α-synuclein). D, Top, Cm changes induced by 20 ms depolarizing pulses (arrows indicate stimulations) repeated at 1 Hz for 20 s at calyceal terminals loaded with α-synuclein (His tag, red), A30P (blue), A53T mutant (green), or without loading (black, control). Traces represent average Cm changes from four to seven calyces. Bottom left, Endocytic rate during the train (fF/s, ordinate) estimated from a linear regression line fit to the Cm decay 0.45–1 s after each pulse versus stimulus number (abscissa) in calyceal terminals with α-synuclein (red, n = 5), A30P (blue, n = 6), A53T (green, n = 4), or without them (control: black, n = 7). The endocytic Cm decay after each pulse (initial rate: 56.1 ± 12 fF/s) became gradually faster and reached a maximal rate of 183 ± 14 fF/s at around the 10th pulse (mean value of 10th–20th events, n = 7). α-Synuclein decreased the maximal rate to 96.8 ± 17 fF/s (n = 5, p < 0.05). Bottom center, Bar graphs indicating the maximal endocytic rate in the presence of α-synuclein, A30P, A53T mutants, or in their absence. Bottom right, Cumulative ΔCm during the 20 s train (ordinate) versus stimulus number (abscissa) in calyceal terminals loaded with WT α-synuclein (red, n = 5), A30P (blue, n = 6), A53T (green, n = 4), or without loading (control, black, n = 7). WT α-synuclein significantly reduced the cumulative ΔCm (p < 0.05 from 11th stimulus), whereas A30P mutant significantly increased the cumulative ΔCm (p < 0.05 from sixth stimulus) compared with controls. A53T mutant had no effect.
- Figure 2.
The tubulin depolymerizers nocodazole and photostatin-1 can rescue the inhibitory effects of α-synuclein on vesicle endocytosis. A, B, Coloading of PIP2 (50 μm, n = 5, red in A) or latrunculin A (25 μm, n = 6, red in B) together with WT α-synuclein had no significant rescuing effect (n.s. in A, p = 0.20) for the inhibitory effect of α-synuclein on vesicle endocytosis. Yellow trace in A indicates the endocytic inhibitory effect of WT α-synuclein for comparison. Bar graphs indicate endocytic 50% decay time, averaged from data in five to seven calyces. Latrunculin A loaded alone in calyceal terminals had no effect on vesicle endocytosis (n = 5, blue trace in B). C, Intraterminal loading of nocodazole (10 μm) together with WT α-synuclein with (red) or without (orange) His tag (3.6 μm) rescued the inhibitory effect of α-synuclein on vesicle endocytosis induced by a single 20 ms pulse. Nocodazole alone (blue trace) had no effect. Bar graphs indicate endocytic 50% decay time averaged from data in five to seven calyces. D, Nocodazole coloading (10 μm) rescued the inhibitory effects of α-synuclein (His tag, 3.6 μm) on accelerating fast endocytosis induced by a train of 20 ms pulse at 1 Hz. Left, Traces of control (black), nocodazole alone (blue), and WT α-synuclein coloaded with nocodazole (red, superimposed). Center and right, Endocytic rates were similar in control (black), nocodazole alone (blue), and α-synuclein + nocodazole (red), as shown in time plots and bar graphs (n = 5–7). E, PST-1 reversibly rescued endocytic inhibitory effects of α-synuclein. Intraterminal loading of PST-1 (10 μm) alone had no effect under 390 nm (black trace) or 510 nm (green trace) illumination (left sample traces), but, when loaded with α-synuclein (His tag, 3.6 μm, right traces), rescued its inhibitory effect on vesicle endocytosis under 390 nm illumination (black trace), but not under 510 nm light (right sample traces). In sample traces, ΔCm amplitude was normalized at the peak to compare endocytic time courses. Time plots (middle panel) indicate endocytic 50% decay time under 390 or 510 nm illumination in the presence of PST-1 and α-synuclein (filled circles) and PST-1 alone (open circles). In the presence of PST-1 and α-synuclein, the endocytic rate was slowed when PST-1 was inactivated by 510 nm light, but became faster when returned to 390 nm illumination. Right panel summarizes the mean half endocytic decay time from five terminals at three epochs in the time plot showing significant and reversible rescue by PST-1 from α-synuclein-induced endocytic inhibition under 390 nm illumination. Asterisks indicate a significant difference (*p < 0.05).
- Figure 3.
α-Synuclein A53T mutant inhibits vesicle endocytosis at calyces of Held in prehearing rats in a MT-independent manner. A, In rat calyces of Held from prehearing rats (P7–P9), both WT α-synuclein (His tag, 3.6 μm, n = 5, red) and A53T (3.6 μm, n = 5, blue) inhibited endocytic capacitance change elicited by a 20 ms depolarizing pulse (p < 0.05), whereas the A30P mutant (3.6 μm, n = 5, green) had no effect (p = 0.42). Bar graphs indicate mean endocytic recovery (%) from exocytic ΔCm 50 s after stimulation. B, Endocytic inhibition by WT α-synuclein (His tag) rescued by nocodazole (10 μm, n = 4–5, no significant difference between nocodazole alone and WT α-synuclein + nocodazole, p = 0.89). C, Endocytic inhibition by the A53T mutant could not be rescued by nocodazole (10 μm, n = 4–5, significant difference between nocodazole alone and A53T + nocodazole, p < 0.05). Asterisks indicate a significant difference (*p < 0.05).
- Figure 4.
Toxic effects of WT α-synuclein on high-frequency neurotransmission. A, WΤ α-synuclein (His tag) has no effect on basal transmission at 0.03 Hz. EPSCs (bottom traces) evoked by presynaptic action potentials (top traces) at 0.03 Hz in the presence (red) or absence (black) of WT α-synuclein (3.6 μm, superimposed). In bar graphs, the mean amplitude of EPSCs was 6.8 ± 1.3 nA (n = 6) and 6.1 ± 1.4 nA (n = 6) in the absence and presence of α-synuclein, respectively (p = 0.72, Student's t test). The decay time constant of EPSCs was 0.96 ± 0.09 ms (n = 6) and 1.1 ± 0.1 ms (n = 6) in the presence and absence of α-synuclein, respectively (p = 0.29). B, Left, EPSCs evoked by a train of 30 stimuli at 100 Hz in the presence (red) or absence (control, black) of α-synuclein (His tag, 3.6 μm) in calyceal terminals. Right, Cumulative amplitudes of EPSCs during a 100 Hz train with (red, n = 6) or without (black, n = 6) WT α-synuclein. WT α-synuclein had no effect on cumulative amplitude of EPSCs (p = 0.91). C, Recovery of EPSCs from STD in the presence (red) and absence (black) of α-synuclein (His tag, 3.6 μm) in calyceal terminals. The 100 Hz stimulation was followed by test stimuli at different interstimulus intervals (0.02–20 s, protocol on the top panel). Sample traces in insets indicate EPSCs at different intervals after STD (superimposed). Right bar graphs indicate mean and SEMs of fast and slow recovery time constants (τ) from five to six synapses showing a significant prolongation (*) by α-synuclein of the slow recovery time constant in double exponential fits (solid lines in time plots); 6.2 ± 0.8 s in control, 12 ± 2 s with α-synuclein. There was no significant difference in fast recovery time constants. D, Nocodazole (10 μm) rescued the inhibitory effect of α-synuclein (His tag) on recovery of EPSCs from STD (n = 7). Recovery in the presence of α-synuclein alone (Fig. 4B) is indicated by a dashed line. Loading nocodazole alone (10 μm, blue, n = 6) had a similar recovery time to control (black). Bar graphs in the right panel indicate no significant effect of α-synuclein on the recovery time after STD in the presence of nocodazole. E, Left panels show postsynaptic APs (output) in response to presynaptic APs (input) elicited at 100 Hz in the control (left) in the presence of α-synuclein (middle) and α-synuclein + nocodazole (right) at different time periods during sustained stimulation. Center panels indicate the inhibitory effect of α-synuclein on synaptic fidelity (in output/input ratio; filled red circles, n = 4; left) compared with control (open black circles, n = 5). Right panel shows rescuing effect of coloaded nocodazole (10 μm, right panel, open red circles, n = 4). Nocodazole alone (10 μm) had no effect (blue-filled circles, n = 4). The dashed line indicates impaired synaptic fidelity by α-synuclein for comparison.
