Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

α-Synuclein promotes dilation of the exocytotic fusion pore

Nature Neuroscience volume 20, pages 681–689 (2017)Cite this article

Subjects

Abstract

The protein α-synuclein has a central role in the pathogenesis of Parkinson's disease. Like that of other proteins that accumulate in neurodegenerative disease, however, the function of α-synuclein remains unknown. Localization to the nerve terminal suggests a role in neurotransmitter release, and overexpression inhibits regulated exocytosis, but previous work has failed to identify a clear physiological defect in mice lacking all three synuclein isoforms. Using adrenal chromaffin cells and neurons, we now find that both overexpressed and endogenous synuclein accelerate the kinetics of individual exocytotic events, promoting cargo discharge and reducing pore closure ('kiss-and-run'). Thus, synuclein exerts dose-dependent effects on dilation of the exocytotic fusion pore. Remarkably, mutations that cause Parkinson's disease abrogate this property of α-synuclein without impairing its ability to inhibit exocytosis when overexpressed, indicating a selective defect in normal function.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: α-Synuclein modulates the kinetics of peptide release.
Figure 2: The analysis of VMAT2-pHluorin reveals effects of α-synuclein on the fusion pore.
Figure 3: α-Synuclein influences fusion pore closure in neurons.
Figure 4: Overexpressed and endogenous synuclein exert similar effects on peptide release.
Figure 5: Overexpressed and endogenous synuclein localizes to secretory granules in adrenal chromaffin cells.
Figure 6: Fusion pore dilation is a conserved function of the synucleins that is impaired by Parkinson's disease–associated point mutations.

Similar content being viewed by others

References

  1. Goedert, M., Spillantini, M.G., Del Tredici, K. & Braak, H. 100 years of Lewy pathology. Nat. Rev. Neurol. 9, 13–24 (2013).

    CAS  PubMed  Google Scholar 

  2. Polymeropoulos, M.H. et al. Mutation in the α-synuclein gene identified in families with Parkinson's disease. Science 276, 2045–2047 (1997).

    CAS  PubMed  Google Scholar 

  3. Krüger, R. et al. Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease. Nat. Genet. 18, 106–108 (1998).

    PubMed  Google Scholar 

  4. Zarranz, J.J. et al. The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 55, 164–173 (2004).

    CAS  PubMed  Google Scholar 

  5. Appel-Cresswell, S. et al. Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease. Mov. Disord. 28, 811–813 (2013).

    CAS  PubMed  Google Scholar 

  6. Lesage, S. et al. G51D α-synuclein mutation causes a novel parkinsonian-pyramidal syndrome. Ann. Neurol. 73, 459–471 (2013).

    CAS  PubMed  Google Scholar 

  7. Proukakis, C. et al. A novel α-synuclein missense mutation in Parkinson disease. Neurology 80, 1062–1064 (2013).

    PubMed  PubMed Central  Google Scholar 

  8. Singleton, A.B. et al. α-Synuclein locus triplication causes Parkinson's disease. Science 302, 841 (2003).

    CAS  PubMed  Google Scholar 

  9. Bendor, J.T., Logan, T.P. & Edwards, R.H. The function of α-synuclein. Neuron 79, 1044–1066 (2013).

    CAS  PubMed  Google Scholar 

  10. Larsen, K.E. et al. α-Synuclein overexpression in PC12 and chromaffin cells impairs catecholamine release by interfering with a late step in exocytosis. J. Neurosci. 26, 11915–11922 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Nemani, V.M. et al. Increased expression of α-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis. Neuron 65, 66–79 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Scott, D.A. et al. A pathologic cascade leading to synaptic dysfunction in α-synuclein-induced neurodegeneration. J. Neurosci. 30, 8083–8095 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Greten-Harrison, B. et al. αβγ-Synuclein triple knockout mice reveal age-dependent neuronal dysfunction. Proc. Natl. Acad. Sci. USA 107, 19573–19578 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Burré, J. et al. α-Synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 329, 1663–1667 (2010).

    PubMed  PubMed Central  Google Scholar 

  15. Senior, S.L. et al. Increased striatal dopamine release and hyperdopaminergic-like behaviour in mice lacking both alpha-synuclein and gamma-synuclein. Eur. J. Neurosci. 27, 947–957 (2008).

    PubMed  PubMed Central  Google Scholar 

  16. Davidson, W.S., Jonas, A., Clayton, D.F. & George, J.M. Stabilization of α-synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 273, 9443–9449 (1998).

    CAS  PubMed  Google Scholar 

  17. Pranke, I.M. et al. α-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding. J. Cell Biol. 194, 89–103 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Jensen, M.B. et al. Membrane curvature sensing by amphipathic helices: a single liposome study using α-synuclein and annexin B12. J. Biol. Chem. 286, 42603–42614 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Cooper, A.A. et al. α-Synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models. Science 313, 324–328 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Soper, J.H. et al. α-Synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae. Mol. Biol. Cell 19, 1093–1103 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Varkey, J. et al. Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins. J. Biol. Chem. 285, 32486–32493 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kamp, F. et al. Inhibition of mitochondrial fusion by α-synuclein is rescued by PINK1, Parkin and DJ-1. EMBO J. 29, 3571–3589 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Nakamura, K. et al. Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein α-synuclein. J. Biol. Chem. 286, 20710–20726 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Alabi, A.A. & Tsien, R.W. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu. Rev. Physiol. 75, 393–422 (2013).

    CAS  PubMed  Google Scholar 

  25. Taraska, J.W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S. & Almers, W. Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proc. Natl. Acad. Sci. USA 100, 2070–2075 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Fumimura, Y. et al. Analysis of the adrenal gland is useful for evaluating pathology of the peripheral autonomic nervous system in Lewy body disease. J. Neuropathol. Exp. Neurol. 66, 354–362 (2007).

    PubMed  Google Scholar 

  27. Turkka, J.T., Juujärvi, K.K., Lapinlampi, T.O. & Myllylä, V.V. Serum noradrenaline response to standing up in patients with Parkinson's disease. Eur. Neurol. 25, 355–361 (1986).

    CAS  PubMed  Google Scholar 

  28. Miesenböck, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    PubMed  Google Scholar 

  29. Dean, C. et al. Synaptotagmin-IV modulates synaptic function and long-term potentiation by regulating BDNF release. Nat. Neurosci. 12, 767–776 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Alés, E. et al. High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism. Nat. Cell Biol. 1, 40–44 (1999).

    PubMed  Google Scholar 

  31. Matsuda, N. et al. Differential activity-dependent secretion of brain-derived neurotrophic factor from axon and dendrite. J. Neurosci. 29, 14185–14198 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Holz, R.W. Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential. Proc. Natl. Acad. Sci. USA 75, 5190–5194 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Hu, G. et al. New fluorescent substrate enables quantitative and high-throughput examination of vesicular monoamine transporter 2 (VMAT2). ACS Chem. Biol. 8, 1947–1954 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Perrais, D., Kleppe, I.C., Taraska, J.W. & Almers, W. Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells. J. Physiol. (Lond.) 560, 413–428 (2004).

    CAS  Google Scholar 

  35. Fulop, T., Radabaugh, S. & Smith, C. Activity-dependent differential transmitter release in mouse adrenal chromaffin cells. J. Neurosci. 25, 7324–7332 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Onoa, B., Li, H., Gagnon-Bartsch, J.A., Elias, L.A. & Edwards, R.H. Vesicular monoamine and glutamate transporters select distinct synaptic vesicle recycling pathways. J. Neurosci. 30, 7917–7927 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Chiang, H.C. et al. Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles. Nat. Commun. 5, 3356 (2014).

    PubMed  Google Scholar 

  38. van de Bospoort, R. et al. Munc13 controls the location and efficiency of dense-core vesicle release in neurons. J. Cell Biol. 199, 883–891 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Asensio, C.S. et al. Self-assembly of VPS41 promotes sorting required for biogenesis of the regulated secretory pathway. Dev. Cell 27, 425–437 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Fortin, D.L. et al. Neural activity controls the synaptic accumulation of α-synuclein. J. Neurosci. 25, 10913–10921 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Unni, V.K. et al. In vivo imaging of α-synuclein in mouse cortex demonstrates stable expression and differential subcellular compartment mobility. PLoS One 5, e10589 (2010).

    PubMed  PubMed Central  Google Scholar 

  42. George, J.M., Jin, H., Woods, W.S. & Clayton, D.F. Characterization of a novel protein regulated during the critical period for song learning in the zebra finch. Neuron 15, 361–372 (1995).

    CAS  PubMed  Google Scholar 

  43. Bartels, T., Choi, J.G. & Selkoe, D.J. α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature 477, 107–110 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Burré, J., Sharma, M. & Südhof, T.C. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc. Natl. Acad. Sci. USA 111, E4274–E4283 (2014).

    PubMed  PubMed Central  Google Scholar 

  45. Wang, L. et al. α-synuclein multimers cluster synaptic vesicles and attenuate recycling. Curr. Biol. 24, 2319–2326 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Fortin, D.L. et al. Lipid rafts mediate the synaptic localization of α-synuclein. J. Neurosci. 24, 6715–6723 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Shi, L. et al. SNARE proteins: one to fuse and three to keep the nascent fusion pore open. Science 335, 1355–1359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Braun, A.R. & Sachs, J.N. α-Synuclein reduces tension and increases undulations in simulations of small unilamellar vesicles. Biophys. J. 108, 1848–1851 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Nuscher, B. et al. α-Synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study. J. Biol. Chem. 279, 21966–21975 (2004).

    CAS  PubMed  Google Scholar 

  50. DeWitt, D.C. & Rhoades, E. α-Synuclein can inhibit SNARE-mediated vesicle fusion through direct interactions with lipid bilayers. Biochemistry 52, 2385–2387 (2013).

    CAS  PubMed  Google Scholar 

  51. Ninkina, N. et al. Neurons expressing the highest levels of gamma-synuclein are unaffected by targeted inactivation of the gene. Mol. Cell. Biol. 23, 8233–8245 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Fellner, L. et al. Toll-like receptor 4 is required for α-synuclein dependent activation of microglia and astroglia. Glia 61, 349–360 (2013).

    PubMed  PubMed Central  Google Scholar 

  53. Baksi, S., Tripathi, A.K. & Singh, N. Alpha-synuclein modulates retinal iron homeostasis by facilitating the uptake of transferrin-bound iron: implications for visual manifestations of Parkinson's disease. Free Radic. Biol. Med. 97, 292–306 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Langone, F. et al. Metformin protects skeletal muscle from cardiotoxin induced degeneration. PLoS One 9, e114018 (2014).

    PubMed  PubMed Central  Google Scholar 

  55. Turner, T.N. et al. Loss of δ-catenin function in severe autism. Nature 520, 51–56 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Hao, Z. et al. Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells. J. Cell Sci. 128, 1365–1374 (2015).

    CAS  PubMed  Google Scholar 

  57. Zhang, S., Wang, P., Ren, L., Hu, C. & Bi, J. Protective effect of melatonin on soluble Aβ1–42-induced memory impairment, astrogliosis, and synaptic dysfunction via the Musashi1/Notch1/Hes1 signaling pathway in the rat hippocampus. Alzheimers Res. Ther. 8, 40 (2016).

    PubMed  PubMed Central  Google Scholar 

  58. Sirkis, D.W., Edwards, R.H. & Asensio, C.S. Widespread dysregulation of peptide hormone release in mice lacking adaptor protein AP-3. PLoS Genet. 9, e1003812 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Hua, Z. et al. v-SNARE composition distinguishes synaptic vesicle pools. Neuron 71, 474–487 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Jullié, D., Choquet, D. & Perrais, D. Recycling endosomes undergo rapid closure of a fusion pore on exocytosis in neuronal dendrites. J. Neurosci. 34, 11106–11118 (2014).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank members of the Edwards laboratory for discussion, D. Jullié for help with the pH oscillation experiment, A. Bertholet (UCSF) for the TOM20 antibody and B. Calagui and S. Batarni for technical assistance. We also thank K. Bohannon, M. Bittner and R. Holz for sharing data and providing suggestions. This work was supported by grants from NINDS (NS062715), NIDA (DA10154) and the Weill Institute for Neurosciences (to R.H.E.), the John and Helen Cahill Family Endowment for Research on Parkinson's Disease (to R.H.E.), a fellowship from NINDS (to T.L.) and a fellowship from the A.P. Giannini Foundation (to J.B.).

Author information

Author notes

  1. Todd Logan and Jacob Bendor: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Neurology and Physiology, UCSF School of Medicine, University of California, San Francisco, San Francisco, California, USA

    Todd Logan, Jacob Bendor, Chantal Toupin & Robert H Edwards

  2. Graduate Program in Biomedical Sciences, UCSF School of Medicine, University of California, San Francisco, San Francisco, California, USA

    Todd Logan & Robert H Edwards

  3. Department of Biochemistry & Biophysics, UCSF School of Medicine, University of California, San Francisco, San Francisco, California, USA

    Kurt Thorn

Authors
  1. Todd Logan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jacob Bendor
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chantal Toupin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kurt Thorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Robert H Edwards
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.H.E., T.L. and J.B. designed the research and wrote the manuscript. T.L. and J.B. performed the experiments and analyzed the data, with assistance from C.T. K.T. provided essential technical assistance with the chromaffin cell imaging experiments.

Corresponding author

Correspondence to Robert H Edwards.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Endogenous and lentiviral expression of α-synuclein in adrenal chromaffin cells.

(a) Chromaffin cells from wild type (wt) or synuclein triple knockout (TKO) mice were transduced with either lentivirus encoding human α-synuclein (SYN) or empty vector, cultured for 72 h and immunostained for the over-expressed protein using the human-specific α-synuclein antibody 15G7 (left) as well as the dense core vesicle protein secretogranin II (SgII) to identify chromaffin cells (right).

(b) Chromaffin cells from wt and synuclein TKO mice were immunostained using the α-synuclein-specific antibody syn-1, which detects both rodent and human isoforms. Comparison to the TKO shows that wt cells express low levels of endogenous α-synuclein in a diffuse cytosolic distribution. Scale bar, 5 μm. (c) Quantification of whole cell α-synuclein immunofluorescence detected with syn-1 antibody in (b) indicates ~5-fold overexpression after lentiviral infection. Values indicate mean ± SEM, n = 10 cells for each group. a.u., arbitrary units

Supplementary Figure 2 Synuclein does not influence calcium influx during stimulation.

(a) Chromaffin cells from wild type and synuclein TKO mice were transduced with lentivirus encoding wild type human α-synuclein (SYN) or empty vector (wt and TKO) 3-5 days before imaging, incubated in Fluo-5F AM (6 μM) for 15 min and imaged by TIRF microscopy during the addition of 45 mM K+. Values represent mean ± SEM. (b) Peak Fluo-5F fluorescence observed during stimulation is unaffected by synuclein (p = 0.4571 by one-way ANOVA; F(2, 32) = 0.8023). wt, n = 13 cells; SYN, n = 12 cells; TKO, n = 10 cells from two independent cultures.

Supplementary Figure 3 BDNF-pHluorin events reflect fusion pore opening and peptide release.

(a) Representative dual color imaging traces of BDNF-pHluorin exocytotic events with fast (left) and slow (right) rise times. The loss of FFN206 fluorescence indicates that slow as well as fast rise times can reflect exocytosis, and the lag between onset of FFN loss and onset of BDNF-pHluorin unquenching for the slow event illustrates the effect of the fusion pore on behavior of the two reporters. 30/35 pHluorin events with rise time >150 ms and 32/48 with a shorter rise time exhibit an associated FFN release event. The high proportion (~30%) of new arrivals with extremely short duration (in many cases <10 ms) (data not shown) makes it impossible to detect all FFN release events with the alternating 30 ms illumination required for dual imaging of the two fluorophores. Size bar indicates 1 μm.

(b) Scatterplot of the time constant for FFN206 loss as a function of BDNF-pHluorin rise time. Linear regression demonstrates a correlation (R2 = 0.12, p = 0.008), accounting for the higher proportion of fast pHluorin events with undetectable FFN release. n = 83 events from 2 cells (c) Representative traces of a decaying chromaffin cell BDNF-pHluorin event quenched by low pH buffer (MES) (left), a stable event quenched by MES (center), and a stable event protected from quenching by MES (right). afu, arbitrary fluorescence units (d) All events in the process of decay were quenched by low pH MES buffer, eliminating the possibility that decay of BDNF-pHluorin fluorescence results from vesicle movement out of the TIRF plane. For stable events, MES application did not always result in quenching, indicating that the fusion pore can limit BDNF-pHluorin release but retain continuity with the external solution. In addition, only stable events have the potential for complete pore closure. (e) Experiments performed in the absence and presence of bafilomycin (0.6 μM) showed no difference in the kinetics of BDNF-pHluorin fluorescence decay, excluding a role for vesicle reacidification in the rate of release. wt, n = 213 / 124 events; SYN, n = 117 / 60 events; TKO, n = 178 / 190 events (+ / - bafilomycin)

Supplementary Figure 4 Effect of synuclein on BDNF-pHluorin release.

(a) Time course of a single exocytotic event with slow rise time and interrupted release. a.u., arbitrary units (b) The time to 90% peak BDNF-pHluorin fluorescence was determined as in Figure 1b, and represented here for wild type, α-synuclein overexpression and synuclein TKO as mean ± SEM (****, p < 0.0001 by Kruskal-Wallis one-way ANOVA; H = 84.20). (c) The mean time to peak per cell plotted as a function of event number per cell shows that single cells do not distort the analysis of individual exocytotic events. (d) The peak fluorescence was normalized and the mean time course of fluorescence decay determined for all exocytotic events on a per cell basis. For all comparisons, p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test (F(2, 842) = 181.0). n = 19 cells per group (c,d) (e) Representative kymographs depicting the BDNF-pHluorin event classes presented in Figure 1. (f) Cumulative frequency distribution of the data shown in Figure 1d.

Supplementary Figure 5 Synuclein expression does not affect the number of docked chromaffin granules or their intravesicular pH.

Chromaffin cells from wt or TKO mice were transduced with lentivirus encoding BDNF-pHluorin, cultured for 5 days and imaged by TIRF microscopy. BDNF-pHluorin-expressing LDCVs were identified using 50 mM NH4Cl to alkalinize acidic compartments, the fluorescent punctae subjected to automated filtering and analyzed in ImageJ. (a) Synuclein loss (TKO) or over-expression (SYN) do not affect the number of docked vesicles per cell. (b) Synuclein expression does not affect lumenal pH. The fold change in fluorescence due to 50 mM NH4Cl was determined for individual LDCVs and the averages per cell plotted along with mean ± SEM. n.s., not significant by one-way ANOVA (p = 0.806 for (a) (F(2, 30) = 0.2173) and p = 0.772 for (b) (F(2, 30) = 0.261)); wt, n = 12 cells, 162 vesicles; TKO, n = 11 cells, 154 vesicles; SYN, n = 10 cells, 151 vesicles

Supplementary Figure 6 Stimulated release of BDNF-pHluorin at 2 mM external Ca2+.

(a) Overexpression of α-syn (SYN) reduces the frequency of BDNF-pHluorin exocytotic events (****, p < 0.0001 by one-way ANOVA with Tukey’s post hoc comparison; F(2, 52) = 10.98). wt, n = 16 cells; SYN, n = 18 cells; TKO, n = 21 cells from three independent cultures (b) As described in figure S4, the release of BDNF-pHluorin was determined across all events by normalizing the peak fluorescence and presenting the mean loss of fluorescent cargo per cell. p < 0.0001 for all comparisons by one-way ANOVA with Tukey’s post hoc comparison (F(2, 253) = 65.65). wt, n = 11 cells; SYN, n = 9 cells; TKO, n = 11 cells (c) BDNF-pHluorin event classes (defined in Figure 1C) showed a similar distribution to events recorded at 5 mM Ca++. Synuclein overexpression reduced the fraction of events with pore closure relative to the synuclein TKO (***, p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison (F(2, 28) = 5.334; q = 4.602, TKO vs SYN)). n = 11 (wt), 9 (SYN) and 11 (TKO)

(d) Overexpression of α-synuclein reduces and loss of all synucleins increases the time constant of BDNF-pHluorin fluorescence decay. For all events that decayed completely to baseline fluorescence values, the time constants of decay were determined by fitting single exponentials to the decay component of the trace. The distributions differ with p < 0.01 for wt-SYN comparison, p < 0.05 for wt-TKO and p < 0.001 for SYN-TKO (Kolmogorov-Smirnov). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by Kruskal-Wallis one-way ANOVA with Dunn’s post hoc test (H = 43.76). wt, n = 213 events; SYN, n = 117 events; TKO, n = 178 events

Supplementary Figure 7 Distribution of VMAT2-pHluorin event kinetics per cell.

The mean kinetic parameters for each cell (obtained from Fig. 2) are plotted as a function of event number. The effects of over-expressed wild type human α-synuclein on latency to decay (a) and time constant of decay (b) do not reflect the distribution of event numbers per cell. Data points represent individual cells.

Supplementary Figure 8 Quantification of α-synuclein overexpressed in rat hippocampal neurons.

Primary, dissociated rat hippocampal neurons were co-electroporated with BDNF-pHluorin, synaptophysin-mCherry and either α-synuclein or empty vector as described in Figure 3. After 14 days in vitro, the neurons were harvested, and α-synuclein levels from four independent cultures (1-4) quantified by fluorescent western analysis (a). (b) α-Synuclein immunoreactivity was normalized to that of actin and expressed as fold-overexpression relative to cells transfected with empty vector. mean ± SEM, 4.6 ± 0.5

Supplementary Figure 9 Quenching of BDNF-pHluorin by low pH in wild-type and synuclein TKO neurons.

Wild type (wt) and synuclein TKO hippocampal neurons were transfected with BDNF-pHluorin and imaged 14-20 days later, stimulating at 50 Hz for 5 s followed by transient quenching of the cell surface fluorescence at pH 5.5 as in Figure 3a-d. (a,b) Loss of synuclein has no effect on the frequency of exocytotic events per coverslip (p = 0.436 by unpaired t-test; t(17) = 0.798) (a) or the proportion of slowly decaying events in each coverslip quenchable with acidic buffer (p = 0.258 by unpaired t-test; t(17) = 1.170) (b). Data indicate mean ± SEM. (c) Events were classified as already decayed at the time of acid application or for those that were not, either quenched or unquenched by the low pH (p = 0.16 by Chi-square test). n = 397 events from three cultures for both wt (11 coverslips) and TKO (8 coverslips).

Supplementary Figure 10 Role of reacidification in the decay of NPY-pHluorin fluorescence.

(a) Wild type mouse hippocampal neurons transfected with NPY-pHluorin were stimulated at 50 Hz for 5 s in the absence (con) or presence of bafilomycin (0.6 μM, +BafA). Individual traces were fit to a plateau followed by single exponential decay. Events that failed to show fluorescence decay were scored as no decay, and those that did show decay were classified as fast if τ < 5 s or slow if τ > 5 s (p < 0.01 by Chi-square test). n=198 events / 3 coverslips for both conditions. (b) Cumulative frequency distribution of τdecay values, including events that did not decay. p = 0.25 by Kolmogorov-Smirnov.

Supplementary Figure 11 Distribution of neuronal event kinetics per coverslip.

The mean kinetic parameters for each coverslip are plotted as a function of event number, using the data shown in Figure 4. The effects of wild type human α-synuclein over-expression on latency to decay (a) and time constant of decay (b) do not reflect event number. Similarly, the effects of the synuclein TKO on latency to decay (c) and time constant of decay (d) do not vary systematically with event number per coverslip.

Supplementary Figure 12 Synuclein has a primary effect on fusion pore dilation.

(a,b) NPY-pHluorin events from rat hippocampal neurons over-expressing human α-synuclein (syn) and controls were separated into those with time constants of fluorescent decay more than 5 s (a) and less than 1 s (b), and the latency to decay plotted by cumulative frequency (p = 0.97 for a and < 0.0001 for b by Kolmogorov-Smirnov). n(a) = 65 events / 5 coverslips for control and 23 events / 8 coverslips for syn; n(b) = 197 events for control and 210 for syn (c,d) NPY-pHluorin events from the hippocampal neurons of wt and TKO mice were separated into similar groups as in (a) and (b) and displayed by cumulative frequency. Synuclein affects the latency to decay only for more rapidly decaying events (p = 0.054 for c and < 0.0001 for d by Kolmogorov-Smirnov). n(c) = 99 events / 10 coverslips for wt and 185 events / 11 coverslips for TKO; n(d) = 750 events for wt and 474 events for TKO. Insets indicate mean values ± SEM. n.s., not significant (p = 0.853 for a and 0.0753 for c); ****, p < 0.0001 by Mann-Whitney; U = 727.5 (a), 13982 (b), 7984 (c) and 121060 (d)

Supplementary Figure 13 Endogenous synuclein concentrates at presynaptic terminals in cultured neurons.

Primary hippocampal neurons from wt or synuclein TKO mice were cultured for 18 days, fixed, immunostained for α/β-synuclein using the H3C antibody (green), and for the vesicular glutamate transporter 1 (VGLUT1, red) and imaged by confocal microscopy. Size bar, 5 μm.

Supplementary Figure 14 Synuclein does not colocalize with mitochondria in adrenal chromaffin cells.

Chromaffin cells from wt mice transduced with lentivirus encoding human α-synuclein were immunostained for synuclein using the H3C antibody and for mitochondria using an antibody to the outer membrane protein TOM20. The images were obtained by structured illumination and are shown here as reconstructions of a 120 nm-thick slice located within 0.5 μm of the cell-coverglass interface. Size bar, 2.5 μm.

Supplementary Figure 15 Mutations associated with Parkinson’s disease do not perturb the localization of human α-synuclein to LDCVs in synuclein TKO mice.

Chromaffin cells from synuclein TKO mice were isolated, transduced with either empty vector or lentivirus encoding one of PD-associated α-synuclein mutants and immunostained 5 days later for α-synuclein (H3C, green) as well as the dense core vesicle protein secretogranin II (SgII, red). Representative TIRF images show that both mutants localize to secretory vesicles even in the absence of endogenous synuclein. Size bar, 2.5 μm.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Logan, T., Bendor, J., Toupin, C. et al. α-Synuclein promotes dilation of the exocytotic fusion pore. Nat Neurosci 20, 681–689 (2017). https://doi.org/10.1038/nn.4529

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.4529

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing