α-synuclein multimers cluster synaptic vesicles and attenuate recycling - PubMed (original) (raw)

α-synuclein multimers cluster synaptic vesicles and attenuate recycling

Lina Wang et al. Curr Biol. 2014.

Abstract

The normal functions and pathologic facets of the small presynaptic protein α-synuclein (α-syn) are of exceptional interest. In previous studies, we found that α-syn attenuates synaptic exo/endocytosis; however, underlying mechanisms remain unknown. More recent evidence suggests that α-syn exists as metastable multimers and not solely as a natively unfolded monomer. However, conformations of α-syn at synapses--its physiologic locale--are unclear, and potential implications of such higher-order conformations to synaptic function are unknown. Exploring α-syn conformations and synaptic function in neurons, we found that α-syn promptly organizes into physiological multimers at synapses. Furthermore, our experiments indicate that α-syn multimers cluster synaptic vesicles and restrict their motility, suggesting a novel role for these higher-order structures. Supporting this, α-syn mutations that disrupt multimerization also fail to restrict synaptic vesicle motility or attenuate exo/endocytosis. We propose a model in which α-syn multimers cluster synaptic vesicles, restricting their trafficking and recycling, and consequently attenuate neurotransmitter release.

Copyright © 2014 Elsevier Ltd. All rights reserved.

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Figures

Figure 1

Figure 1. Multimeric α-syn conformations at presynaptic boutons

(A) Schematic of complementation assay (i) and molecular replacement strategy (ii). Cultured hippocampal neurons from α-syn −/− mice were transiently transfected with various VN/VC-tagged α-syn's (see “results”) and visualized after ~ 14 hours. (B)

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: Representative images of reconstituted Venus fluorescence in neurons expressing VN/VC:α-syn's (also see Supp. fig. 1A). Note these neurons are co-transfected with synaptophysin:mRFP (SyPhy:mRFP) to label boutons.

Bottom

: No fluorescence was seen in boutons expressing un-tagged VN + VC alone.

Right

: The vast majority (~85%) of SyPhy:mRFP-positive boutons also expressed VN/VC:α-syn; comparable to boutons expressing Venus:α-syn and SyPhy:mRFP (N~700 boutons for each group from two separate batches of cultures, p=0.90). (C) Overall design to compare expression-levels of transfected VN/VC:α-syn to endogenous mouse α-syn. Un-transfected cultured neurons from WT mice and VN/VC:α-syn-transfected cultured neurons from α-syn −/− mice were fixed and immunostained with an anti-α-syn antibody (guinea-pig α-syn antibody). Cell culture and immunostaining of both groups were processed in parallel. Note that while the antibody would recognize mouse α-syn in WT neurons, it would only label transfected α-syn in the VN/VC:α-syn transfected group. (D) Representative images from the two groups in (C) (left) and quantification of overall average fluorescence intensities (right; N~10 visual fields containing ~ 3000-10,000 boutons; p=0.06). Note that the number of VN/VC:α-syn transfected boutons is much lower than immunostained WT boutons (as expected with transient transfections), but the fluorescence-intensities are similar. (E) Overall design. Cultured α-syn −/− neurons were co-transfected with VN/VC:α-syn's (or Venus:α-syn) + soluble mCherry, and kinetics of initial α-syn entry and synaptic accumulation was evaluated by long-term imaging (see “results” and [21] for more details). (F) Representative frames from two time-lapse movies showing pre-synaptic accumulation of VN/VC:α-syn (top) and Venus:α-syn (bottom) over 5 hrs of imaging. (G) Quantification of average VFP intensities of boutons over 5 hrs. Note that though the kinetics of VN/VC:α-syn accumulation (black dots) is slower than Venus:α-syn (green dots) as expected, the difference is modest, suggesting that complementation is a relatively early event.

Figure 2

Figure 2. α-syn multimers cluster synaptic-vesicles

(A) Bouton-crops from neurons co-transfected with VN/VC:α-syn and mRFP:Actin (to label entire bouton-profile, see “results”). Note that reconstituted VN/VC:α-syn's only occupy a fraction of the bouton cross-sectional area. (B) Experimental design: Neurons were co-transfected with VN/VC:α-syn and markers to label the entire bouton-profile (mRFP:Actin) or synaptic-vesicles (SyPhy:mRFP); and extent of overlap was determined by custom algorithms (see “results” and “methods” for details). (C, D) Both reconstituted VN/VC:α-syn and SyPhy:GFP occupied a smaller fraction of the bouton than Venus:α-syn (~ 200 boutons analyzed for each group from two separate batches of cultures, ***p < 0.001). (D) Bouton-widths (FWHM, see methods) of VN/VC:α-syn and SyPhy:mRFP were correlated (left; r=0.36, p<0.0001), unlike VN/VC:α-syn and mRFP:Actin, further indicating associations of complemented VN/VC:α-syn's with synaptic-vesicles (N=120 boutons from two separate batches of cultures). (E)

Top

: Schematic of “synaptic-vesicle dispersion assay”. Synaptic-vesicles are labeled by SyPhy:mRFP and neurons are stimulated to disperse synaptic-vesicles (see “results”).

Bottom

: A time-series showing dispersion of synaptic-vesicles from a bouton (elapsed time in seconds on lower left, asterisk marks the start of stimulation). (F) Quantification of synaptic-vesicle dispersion using above assay. While Venus:α-syn diminishes dispersion-kinetics (compared to vector), the dispersion is further attenuated by VN/VC:α-syn (note that error bars are too small to be seen). Extent of dispersion quantified in inset (19.5%, 13.6% and 9% of total synaptic-vesicles were dispersed in vector, Venus:α-syn and VN/VC:α-syn groups respectively; ***p < 0.001, unpaired t test).

Figure 3

Figure 3. Biochemical analyses of α-syn multimers

(A) VN/VC:α-syn's were introduced into HEK293T cells or neurons (by viruses), expressed for the times indicated, and cell-lysates were analyzed by Native/SDS-PAGE. (B) Native-PAGE show α-syn higher-order multimers immunoblotted with two α-syn antibodies and an anti-GFP antibody that also recognizes YFP (note disruption upon boiling). The red arrow marks the position where bands are typically seen, black arrow marks putative monomeric α-syn in neurons. An SDS-PAGE immunoblotted with anti-GFP marks the VFP-fragments. Each experiment was repeated 3-5 times with similar results. (C) In-vitro reconstitution assay. Purified synaptic-vesicles and cytosol from α-syn −/− mouse brains were mixed with WT-α-syn purified from bacteria with/without a chemical cross linker (DSG). Vesicle membrane bound and unbound fractions were separated by centrifugation and analyzed by SDS-PAGE. (D) Both monomeric and cross-linked α-syn multimers bound to synaptic-vesicles (a synaptophysin stain confirms that all synaptic-vesicles are in the bound fraction). Red and black arrows mark positions of putative tetramers and monomers. Experiment was repeated twice with similar results.

Figure 4

Figure 4. Mechanistic links between α-syn multimerization and synaptic function

(A) Schematic of the α-syn helices (shaded) and position of the six mutations. (B) Neurons from α-syn −/− mice were transfected with VN/VC:WT or VN/VC:TsixK α-syn's and fluorescence was quantified in boutons. There were clear diminutions in the TsixK datasets as shown in the representative images and quantification below. (C) “Synaptic-vesicle dispersion” assay: Neurons were co-transfected with SyPhy:mRFP (to label synaptic-vesicles) and untagged WT or TsixK α-syn (or vector alone). Boutons were stimulated and decay of RFP fluorescence from boutons was quantified (see “results”). Note that while WT α-syn attenuates activity-induced synaptic-vesicle dispersion, the TsixK mutant has no effect on vesicle-trafficking (N=number of boutons). (D) Synaptic recycling evaluated by vGlut-pHluorin assays. Cultured neurons were co transfected with vGlut-pHluorin and either untagged WT α-syn or TsixK α-syn. Fluorescence-change of the pH-sensitive vGlut-pHluorin probe reflects synaptic-vesicle recycling in this assay (see “results” and “methods”). Representative panels show fluorescence intensity change of vGlut-pHluorin upon 600 AP stimulation and NH4Cl perfusion. Note that NH4Cl alkalinizes all vesicles, revealing the total (recycling + resting) pool in these neurons. (E, F) Representative ensemble average of vGlut-pHluorin traces from empty vector, WT α-syn or TsixK α-syn transfected neurons (N=number of boutons). Note that while WT α-syn nattenuates neurotransmitter release and decreases mean recycling-pools compared to vector-controls, TsixK α-syn fails to show this effect; quantified in (F) (all data normalized to total pools). Recycling/total pool for vector=43±2.17 %; WT α-syn =28±2.38%; TsixK α-syn =39±2.29% (~ 160 boutons on 7-9 coverslips were analyzed for each group from three separate batches of cultures; ***p < 0.001 compared to vector by one-way ANOVA followed by Dunnet's post hoc test). Total (alkalinized) pools of vector, WT-α-syn and TsixK-α-syn groups were 317.1 ± 16 AFU, 317.5 ± 11 AFU and 376 ± 18 AFU (mean ± SEM) respectively).

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