Synaptic vesicle binding of α-synuclein is modulated by β- and γ-synucleins

Abstract

α-synuclein, β-synuclein, and γ-synuclein are abundantly expressed proteins in the vertebrate nervous system. α-synuclein functions in neurotransmitter release by binding to and clustering synaptic vesicles and chaperoning SNARE-complex assembly. Pathologically, aggregates originating from soluble pools of α-synuclein are deposited into Lewy bodies in Parkinson's disease and related synucleinopathies. The functions of β-synuclein and γ-synuclein in presynaptic terminals remain poorly studied. Using in vitro liposome binding studies, circular dichroism spectroscopy, immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments on isolated synaptic vesicles in combination with subcellular fractionation of brains from synuclein mouse models, we show that β-synuclein and γ-synuclein have a reduced affinity toward synaptic vesicles compared with α-synuclein, and that heteromerization of β-synuclein or γ-synuclein with α-synuclein results in reduced synaptic vesicle binding of α-synuclein in a concentration-dependent manner. Our data suggest that β-synuclein and γ-synuclein are modulators of synaptic vesicle binding of α-synuclein and thereby reduce α-synuclein's physiological activity at the neuronal synapse.

Keywords: CP: Neuroscience; Parkinson’s disease; interaction; membrane binding; multimers; neurodegeneration; synapse; synaptic vesicle; synuclein; synucleinopathy.

Figure 1.. βSyn and γSyn reveal reduced ability to bind to membranes compared with αSyn

(A) Experimental scheme of the liposome binding assay. Liposomes mixed with synuclein were floated by density gradient centrifugation. Based on the liposome distribution in the gradient, assessed by a fluorescent lipid analog, the top two fractions 1 and 2 were defined as lipid-bound fractions. (B–E) Binding of αSyn, βSyn, or γSyn to artificial small unilamellar vesicles (SUVs; composition: 70% L-α-phosphatidylcholine [PC], 30% L-α-phosphatidylserine [PS]) of 30 nm diameter (B), 100 nm diameter (D), or 200 nm diameter (E) or to synaptic vesicle mimics (30 nm diameter; composition: 36% PC, 30% L-α-phosphatidylethanolamine [PE], 12% PS, 5% L-α-phosphatidylinositol [PI], 7% sphingomyelin [SM], 10% cholesterol) (C). Binding was quantified as the sum of the top two fractions, plotted as the percentage of total synuclein in the gradient. (F and G) Same as in (B), except that different molar lipid/protein ratios were used. Data are means ± SEM (***p < 0.001 by Student’s t test in B–E and two-way ANOVA in G; n = 6–8 independent experiments). See also Figure S1. (H–J) CD spectroscopy of synucleins. Experimental scheme of the CD readouts of αSyn as unstructured or a helical (H). Secondary structure of recombinant αSyn, βSyn, or γSyn in the absence (I) or presence (J) of 30 nm charged SUVs at a molar lipid/protein ratio of 400. (K) Same as (J), except that different molar lipid/protein ratios were used, and the signal at 222 nm was plotted to highlight α helicity (***p < 0.001 by two-way ANOVA, mean of n = 3). (L and M) In vivo membrane binding of synucleins. P30 WT brain homogenates were subjected to subcellular fractionation to yield cytosolic and membrane fractions (L). Equal volumes of protein were analyzed by quantitative immunoblotting (M). Data are means ± SEM (***p < 0.001 by Student’s t test; n = 4 brains). See also Figure S2.

Figure 2.. Reduced presynaptic localization of γSyn but not βSyn compared with αSyn

(A–C) Synaptosome isolation. Experimental scheme (A). Enrichment analysis of proteins in synaptosomal preparations (B, C). Brains of P40 WT mice were homogenized and subjected to subcellular fractionation to yield synaptosomes. Twenty micrograms of brain homogenate and synaptosomes was analyzed by quantitative immunoblotting (Syb2, synaptobrevin-2; αTub, α-tubulin; NF165, neurofilament of 165 kDa). Data are means ± SEM (****p < 0.0001 by Student’s t test; n = 4 brains). (D–K) Synaptic targeting of synucleins. Cultured cortical WT mouse neurons were analyzed at 27 days in vitro for co-localization with the indicated proteins (D–G). Synapsin-promoter-driven expression of TdTomato (D) was achieved via lentiviral transduction. Co-localization was quantitated using Pearson’s coefficient (H–K). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Student’s t test; n = 6 independent cultures). Scale bar, 10 μm. See also Figure S2.

Figure 3.. Synucleins interact with one another in a specific conformation

(A) Synuclein labeling scheme for FRET experiments. Single-cysteine substitutions were introduced into synucleins at positions 8 and 96 for αSyn and γSyn and at positions 8 and 85 for βSyn for modification with Alexa 488- or Alexa 546-maleimide. (B) SDS-PAGE analysis of 5 mg purified Alexa 488- or Alexa 546-labeled recombinant αSyn. (C–E) Experimental scheme for the FRET experiments in the presence of 30 nm diameter charged (C), 100 nm diameter charged (D), or 30 nm diameter neutral liposomes (E), with expected outcomes. (F and G) Emission spectra in Figures S5 and S6 were used for calculation of FRET signals. Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Student’s t test; n = 4–9 independent experiments). See also Figures S3 and S4.

Figure 4.. βSyn and γSyn reduce synaptic targeting of αSyn in a dose-dependent manner

(A) Experimental scheme of the βSyn and γSyn titration experiments. ICC, immunocytochemistry; IB, immunoblot. (B–E) Analysis of synuclein levels. αβγ-Syn triple-knockout neurons were transduced with lentiviral vectors expressing αSyn only (A; 5 μL of 40× lentiviral particles) or constant αSyn levels (5 μL of 40× lentiviral particles) with increasing amounts of βSyn or γSyn (B–E; 0.2, 0.5, 1, 2, or 5 μL of 40× lentiviral particles). Data are means ± SEM (**p < 0.01, ***p < 0.001 by Student’s t test; n = 11–12 independent cultures). All synucleins were myc-tagged, enabling direct comparison of their levels on the same blot using an antibody to myc. (F–I) Synaptic targeting of αSyn. αβγ-Syn triple-knockout neurons were transduced as in (B)–(E). Synaptic targeting was quantified by co-localization with synapsin and Pearson’s coefficient (I). Data are means ± SEM (*p < 0.05 by Student’s t test; n = 3 cultures). Scale bar, 10 μm. See also Figures S5A–S5D. (J–M) Same as in (F)–(I), except that co-localization of αSyn was assessed with SV2 in the presence of αSyn only (J, M; 5 μL of 40× lentiviral particles) or constant αSyn levels (5 μL of 40× lentiviral particles) with increasing amounts of βSyn or γSyn (K–M; 1 or 5 μL of 40× lentiviral particles). Data are means ± SEM (*p < 0.05, **p < 0.01 by Student’s t test; n = 3 cultures). Scale bar, 10 μm. See also Figure S5. (N–Q) αSyn enrichment in synaptosomes from mice of different genotypes. Synaptosomes were isolated from mouse brain homogenates of mice lacking αSyn, βSyn, or γSyn via subcellular fractionation (N; see Figures 2B and 2C for data obtained from WT mice), and 20 μg of homogenate and synaptosomes was analyzed by quantitative immunoblotting (Syb2, synaptobrevin-2; aTub, α-tubulin; NF165, neurofilament of 165 kDa; O–Q). Data are means ± SEM (*p < 0.05, **p < 0.01, ****p < 0.0001 by Student’s t test; n.s., not significant; n = 6–8 mice). See also Figure S7.

Figure 5.. Synucleins directly modulate one another’s ability to associate with membranes

(A) Experimental scheme of the liposome binding assay. (B) Liposome binding of αSyn, quantified as the sum of the top two fractions as a percentage of total αSyn in the gradient, was analyzed in the absence or presence of equal amounts of βSyn or γSyn. Data are means ± SEM (*p < 0.05, **p < 0.01 by Student’s t test; n = 6–15 independent experiments). Experiments with twice the amount of αSyn in αSyn-only flotations revealed results identical to the ones shown (data not shown). (C and D) Liposome binding of βSyn (C) or γSyn (D) was analyzed in the absence or presence of equal amounts of αSyn by a flotation assay as in (B). Data are means ± SEM (*p < 0.05 by Student’s t test; n = 6–15 independent experiments). Experiments with twice the amount of βSyn (C) or γSyn (D) in βSyn- or γSyn-only flotations revealed results identical to the ones shown above (data not shown).

Figure 6.. Heteromerization of βSyn or γSyn with αSyn on synaptic vesicles reduces binding of αSyn

(A) Experimental scheme of the synaptic vesicle isolation procedure. (B) Synaptic vesicles separate into two distinct populations on the sucrose gradient: a free synaptic vesicle (SV) pool devoid of plasma membrane markers and other organelles, and a pool that is docked to the plasma membrane and part of the active zone. The dotted lines indicate where blots were merged (note that fractions 23 and 25 were loaded on both gels to enable cross-membrane comparison). (C–H) FRET experiments on synaptic vesicles. Synaptic vesicles were immunoisolated using magnetic beads and an antibody to the vesicle protein SV2 in the presence of Alexa-labeled recombinant synucleins, with beads lacking vesicles as controls (C). Upon washing, fluorescence spectra were recorded for the indicated FRET pairs (D–F; Don, donor; Acc, acceptor), and FRET was calculated in the presence (G) or absence (H) of synaptic vesicles. Data are means ± SEM (*p < 0.05, **p < 0.01 by Student’s t test; n = 5–6 independent experiments). See also Figure S7. (I–N) Immunoprecipitation experiments on synaptic vesicles. Synaptic vesicles were immunoisolated as in (C) in the presence of recombinant purified synucleins, with beads lacking synaptic vesicles as controls (I). Upon washing, the amount of bound αSyn, βSyn, or γSyn when incubated alone (J, K) or when 1- or 4-fold molar amounts of βSyn or γSyn were added (L, M) was quantified (N). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test; n = 6 independent experiments).

Figure 7.. Summary of data and proposed model of the effect of βSyn or γSyn on αSyn

(A) Summary of our findings. Synucleins exist in a dynamic equilibrium between an α-helical, multimeric synaptic-vesicle-bound state and a natively unstructured cytosolic state. Different binding affinities of the synucleins for synaptic vesicles shift this equilibrium more toward synaptic vesicles or the cytosol, resulting in robust membrane binding of αSyn (red) and less robust binding of βSyn (green) and γSyn (blue), also indicated by arrow thicknesses. The binding of αSyn/βSyn or αSyn/γSyn heterodimers depends on the dose of βSyn and γSyn and shifts the equilibrium for αSyn more toward the cytosolic pool. (B) Model of cellular effects of βSyn and γSyn on αSyn function and dysfunction. In the presynaptic terminal, αSyn cycles between a cytosolic and a synaptic-vesicle-bound pool (1). Binding to synaptic vesicles leads to synaptic vesicle clustering (2), which restricts synaptic vesicle mobility and thereby provides a reserve pool for long-term functioning of the nerve terminal. Via this process, αSyn promotes SNARE-complex assembly at the presynaptic plasma membrane and thereby affects neurotransmitter release (3). Via heteromultimerization, βSyn and γSyn reduce synaptic-vesicle-bound αSyn (1), which leads to a reduction in αSyn’s physiological activities (2, 3). Rendering αSyn less membrane bound through the presence of βSyn and γSyn may increase the aggregation-prone cytosolic pool of αSyn (4) or, alternatively, binding of αSyn to βSyn or to γSyn may shield the aggregation-prone residues in αSyn, thereby also modifying αSyn-mediated pathology (4).