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VAMP2 chaperones α-synuclein in synaptic vesicle co-condensates

Nature Cell Biology volume 26, pages 1287–1295 (2024)Cite this article

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Abstract

α-Synuclein (α-Syn) aggregation is closely associated with Parkinson’s disease neuropathology. Physiologically, α-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of α-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)—a component of the SNARE complex that resides on SVs—directly interacts with the carboxy-terminal region of α-Syn through charged residues to regulate α-Syn’s function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, α-Syn and other components. Moreover, VAMP2 binding protects α-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains α-Syn’s function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson’s disease.

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Fig. 1: SVs and α-Syn co-condense.
Fig. 2: The juxtamembrane region of VAMP2 interacts with the C-terminal region of α-Syn.
Fig. 3: Disruption of the α-Syn–VAMP2 interface abolishes co-condensation.
Fig. 4: Disruption of the α-Syn–VAMP2 interface impairs the α-Syn function of SV trafficking.
Fig. 5: VAMP2 prevents α-Syn aggregation and cytotoxicity.

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Data availability

All of the data are available within the paper or its supplementary materials. The XL-MS raw data and pLink searched data have been uploaded to iProX with the accession number IPX0006924001. Any data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

C.L. and D.L. thank the Major State Basic Research Development Program (2019YFE0120600 to C.L.), National Natural Science Foundation of China (82188101 and 32171236 to C.L. and 92353302 and 32170683 to D.L.), Science and Technology Commission of Shanghai Municipality (grant number 22JC1410400 to C.L.), Shanghai Pilot Program for Basic Research—Chinese Academy of Sciences Shanghai Branch (grant number JCYJ-SHFY-2022-005 to C.L.), Shanghai Basic Research Pioneer Project and Chinese Academy of Sciences Project for Young Scientists in Basic Research (grant number YSBR-095 to C.L.) for support. This work was supported by the National Institutes of Health (R01NS102181, R01NS113960, 1RF1NS126342 and R21NS127939 to J.B. and R01NS121077 to J.D. and J.B.), Selma Schottenstein Harris Lab for Research in Parkinson’s and Michael J. Fox Foundation (16661 to J.D. and K.B.S. and 16164 to J.B.). We thank L. Xu for help with the cell culture experiments and Life Science Editors for editing services. Grammarly was used to check the grammar and style of the manuscript.

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Authors and Affiliations

  1. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    Chuchu Wang, Kai Zhang, Jiaojiao Hu, Chunyu Zhao, Jiali Qiang, Shouqiao Hou, Zhenying Liu, Jinge Gu, Yaoyang Zhang, Yanshan Fang & Cong Liu

  2. Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA

    Chuchu Wang & Axel T. Brunger

  3. Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Chuchu Wang & Axel T. Brunger

  4. University of Chinese Academy of Sciences, Beijing, China

    Chuchu Wang, Kai Zhang, Jiaojiao Hu, Chunyu Zhao, Jiali Qiang, Shouqiao Hou, Zhenying Liu & Jinge Gu

  5. Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Bin Cai, Zhiqi Tian, Xiao Hu, Daniel Hall & Jiajie Diao

  6. Helen and Robert Appel Alzheimer’s Disease Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA

    Jillian E. Haller, Kathryn E. Carnazza & Jacqueline Burré

  7. State Key Laboratory of Chemical Biology, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    Yaoyang Zhang, Yanshan Fang & Cong Liu

  8. Department of Neurology, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Kim B. Seroogy

  9. Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, China

    Dan Li

  10. Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai, China

    Dan Li

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Contributions

C.L., A.T.B., D.L. and J.D. designed and directed the study. C.W., J.H., C.Z., Z.T., D.H., J.Q., S.H., Z.L., J.G. and Y.Z. prepared the proteins, performed the cellular and cross-linking experiments and carried out the ThT, circular dichroism, electron microscopy, NMR and mass spectrometry measurements. C.W., K.Z. and Y.F. conducted the cultured neuron experiments. B.C., Z.T. and X.H. performed the single-vesicle experiments. J.E.H., K.E.C. and J.B. analysed SNARE complex assembly and synaptic transmission. C.W., K.B.S., J.B., C.L., A.T.B., D.L. and J.D. analysed the data, discussed the results and wrote the manuscript.

Corresponding authors

Correspondence to Dan Li or Jiajie Diao.

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Extended data

Extended Data Fig. 1 α-Syn, VAMP2, and SVs form a multi-component condensed phase.

(a) Dynamic light scattering measurement of the size distribution of SVM in solution. (b) Molecular weight and concentration of PEG pairs, (c) SVM lipid, and (d) NaCl concentrations scoring positive (blue dots) or negative (red dots) for the appearance of droplets. 100 μM VAMP21-96, and 100 μM α-Syn were used for all the titrations. Additionally, 1 mM SVMs, 5% (w/v) PEG 3350, or 1 mM SVMs and 5% (w/v) PEG 3350 were used for the titration in (b), (c), or (d), respectively. Fluorescence images of (e, top) 100 μM α-Syn (containing 1% Alexa555-α-Syn), (e, middle) 100 μM VAMP21-96 (containing 1% Alexa647-V2), (e, bottom) 100 μM α-Syn (containing 1% Alexa555-α-Syn) and 100 μM VAMP21-96 (containing 1% Alexa647-V2), (f) 100 μM α-Syn (containing 1% α-Syn-Alexa488) and 1 mM SVM with 100 μM other synaptic proteins (90 μM SNAP-25, 5 μM Munc18, and 5 μM Munc13), or (g) 100 μM α-Syn (containing 1% Alexa555-α-Syn) and 1 mM carboxyfluorescein-green-labeled SVM with 100 μM VAMP21-96 (containing 1% Alexa647-V2) in the buffer A (10 mM HEPES-Na pH 7.4, 5 mM Zn2+, 5% PEG 3350), respectively. DIC: differential interference contrast. (h) The final SV product was fractionated into three fractions by gradient centrifugation. The SVs in the bottom layer were used for experiments. (i) Western blot of fractions collected from each step of SV purification. The fraction containing SV is highlighted in red boxes. Other fractions are labeled in abbreviations (see details in Methods). The fractions were immunoblotted for tubulin (cytosol marker), synaptophysin (SV marker), Rpt4 (proteasome marker), VDAC (mitochondria marker), and Sec61B (endoplasmic reticulum marker), respectively. (j) Representative negative staining TEM image of endogenous SVs (bottom layer) purified from mouse brains. Uncropped blots are available in the Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 The C-terminal region of α-Syn electrically interacts with the juxtamembrane region of VAMP2.

(a) Top: residue-resolved relative NMR cross-peak intensity ratios (I/I0) of 100 μM N15α-Syn in 200 μM soluble VAMP2 (VAMP21-96) to that in solution. Bottom: residue-resolved relative NMR cross-peak intensity ratios (I/I0) of 100 μM soluble N15VAMP2 in 150 μM α-Syn to that in solution. (b) The C-terminal region of α-Syn is important for interacting with VAMP2. The 2D 1H-15N HSQC spectrum of 15N-labled soluble VAMP2 (black) and in the presence of α-Syn1-100 alone (red) are almost identical, which validates that the C-terminal end of α-Syn is responsible for the interaction between α-Syn and VAMP2. (c) Circular dichroism spectroscopy of α-Syn1-100 alone (black), in the presence of DOPS (red) or DOPC (blue) liposomes, shows that the C-terminal truncation does not influence membrane binding of α-Syn. The molar ratios of VAMP2 to α-Syn are indicated. (d) The turbidity was measured for several mixtures: 1 mM SVM alone, SVM mixed with α-Syn only, SVM with VAMP21-96 and α-Syn, SVM with VAMP21-96 and α-Syn1-100, and SVM with VAMP21-78 and α-Syn. The wavelength was 600 nm, and the molar ratio of liposome:VAMP2:α-Syn was 10:1:1. Data are means ± SD; n = 3 replicates. (e) Comparison of expression levels of α-Syn-GFP (Fig. 1e) and α-Syn1-100-GFP (Fig. 3b) in cultured cortical neurons by immunoblotting. (f) 2D 1H-15N HSQC of 15N-labled soluble VAMP2 only (black) and in the presence of α-Syn5A (E105A, D119A, E126A, E130A, E137A) alone (red) are nearly identical. (g) Per residue chemical shift changes of soluble VAMP2 signals upon α-Syn5A titration. Source numerical data and uncropped blots are available in the Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 The negatively charged phosphatidylserine (PS) is important in α-Syn-induced clustering.

(a) Experimental scheme of single-vesicle assay for monitoring vesicle clustering. A saturated layer of DiI-labeled SVM was immobilized on an imaging surface via biotin–NeutrAvidin interactions. After free-floating DiD-labeled SVM was injected into the sample chamber, clustering in the presence or absence of α-Syn was determined by counting the number of spots arising from the fluorescence emission of DiD upon excitation at 633 nm. (b) The numbers of interacting DiD-labeled, protein-free SVMs—with or without PS—interacting on the imaging surface was assessed both in the presence of α-Syn or not. (c) The numbers of interacting DiD-labeled, full-length VAMP2 reconstituted SVMs—with or without PS—interacting on the imaging surface was assessed both in the presence of α-Syn or not. (b-c) Bar graphs: quantification of interacting vesicles; data are means ± SD; unpaired two-tailed t-test; n (left to right) = 8, 20, 25, 12, or 16, 16, 16, 16 for (b) or (c), respectively; n refers to the number of random imaging locations in the same channel. (d) Residue-resolved relative NMR signal intensity ratios (I/I0) of α-Syn titrated by DOPC liposome at indicated protein/lipid molar ratios. Dashed lines highlight the residue positions 30 and 95. Source numerical data is available in the Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Disrupting the interface between α-Syn and VAMP2 impairs the catalysis role of α-Syn in SNARE complex assembly.

Expression levels of three SNARE proteins in HEK293T cells co-transfected with plasmids expressing (a, top; c, top; and d, middle) syntaxin-1, (a, middle; c, middle; and d, right) SNAP-25, and (a, bottom; c, bottom; and d, left) VAMP2 at a 1:1:1 ratio, together (a,b) with an increasing amount (0-3-fold) of α-Syn variant plasmids (WT and α-Syn100), and (c,d) with 4-fold expression plasmids of GFP, α-SynWT and α-Syn5K. Cell lysates were immunoblotted for the indicated SNAREs, followed by quantitation for representative immunoblots (see Fig. 4e). Total expression levels of individual SNARE proteins or balancing pCMV5-emerald in transfected HEK293T cells were analyzed by melting SNARE-complexes in SDS sample buffer (100 °C for 20 min), and quantitated by immunoblotting, normalized to α-tubulin (α-Tub) or heat shock cognate 71 kDa protein. HEK293T cell lysates (e) (n = 6 independent replicates) or primary neuron lysates (f) (n = 5 independent replicates) were immunoblotted for SNARE complexes and α-Syn followed by quantification and normalization to heat shock cognate 71 kDa protein (Hsc70) or β-tubulin-III (Tuj1) levels. Data shown are means ± SD; unpaired two-tailed t-test; n = 3 (a,b) or 6 (d) independent cultures. Source numerical data and uncropped blots are available in the Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 Disrupting the interface between α-Syn and VAMP2 impairs the role of α-Syn in synaptic transmission.

(a) Graphic annotation of the multi-electrode array (MEA) assay on cultured primary neurons. (b-d) Representative images of conducting MEA assay in the α-Syn knockout neurons (b) or knockout neurons expressing wildtype α-Syn (c) or α-Syn5K (d) mutant. (e-g) MEA data from α-Syn knockout neurons or knockout neurons expressing wildtype α-Syn or α-Syn5K mutant were subjected to analysis of mean firing rate (e), burst frequency (f), and burst percentage (g). In the Violin plots, the lower dashed line represents the first quartile, the middle dashed line represents the median, and the upper dashed line represents the third quartile; unpaired two-tailed t-test; n (left to right) = 88, 88, 88, or 82, 73, 80 or 84, 80, 86 firing events for (e) or (f) or (g), respectively. Source numerical data is available in the Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 VAMP2 prevents α-Syn aggregation in vitro and in neurons.

(a) ThT spectra and (b) negative staining TEM images show α-Syn (50 μM) amyloid aggregation in the presence of PS liposomes with VAMP25A (S80A, K83A, K85A, R86A, K87A) mutant or VAMP2. Data are means ± SD; error bars represent the SD of three replicates. (c) Immunofluorescence images of hippocampal neurons transfected with the same amount of plasmids of full-length and C-terminally-truncated α-Syn-GFP, respectively, for 12 days with anti-P-S129-α-Syn. This antibody stains the human hippocampus of Parkinson’s disease brains but shows no staining in normal brains. Source numerical data is available in the Source Data Extended Data Fig. 6.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

List of identified inter-cross-linked peptides between α-Syn and VAMP21–96 and vesicles.

Supplementary Table 2

List of identified intra-α-Syn-linked peptides in α-Syn and VAMP21–96 and vesicles.

Supplementary Table 3

List of identified intra-VAMP2-linked peptides in α-Syn and VAMP21–96 and vesicles.

Source data

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Uncropped western blots.

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Wang, C., Zhang, K., Cai, B. et al. VAMP2 chaperones α-synuclein in synaptic vesicle co-condensates. Nat Cell Biol 26, 1287–1295 (2024). https://doi.org/10.1038/s41556-024-01456-1

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