Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation

Ye Jin Chai, Emma Sierecki, Vanesa M Tomatis, Rachel S Gormal, Nichole Giles, Isabel C Morrow, Di Xia, Jürgen Götz, Robert G Parton, Brett M Collins, Yann Gambin, Frédéric A Meunier, Ye Jin Chai, Emma Sierecki, Vanesa M Tomatis, Rachel S Gormal, Nichole Giles, Isabel C Morrow, Di Xia, Jürgen Götz, Robert G Parton, Brett M Collins, Yann Gambin, Frédéric A Meunier

Abstract

Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-Syn(WT) and that of the Parkinson's disease-causing α-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the α-Syn(A30P) mutant with Munc18-1 reduced the number of α-Syn(A30P) aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.

© 2016 Chai et al.

Figures

Figure 1.
Figure 1.
Munc18-1C180Y induces the coaggregation of Munc18-1WT in vitro. (A) Structural representation of Munc18-1C180Y. (B) Principle of the coexpression assay. Munc18-1C180Y tagged with mCherry is coexpressed with a GFP-tagged Munc18-1WT. (C) Schematic representation of single-molecule fluorescence experiment in which the proteins freely diffuse in and out of the focal volume created by two lasers simultaneously exciting the GFP and mCherry fluorophores. (D) A single-molecule trace obtained for lysates coexpressing Munc18-1WT-emGFP and Munc18-1C180Y-mCherry. The number of photons detected in the green and red channels are plotted as a function of time. The trace shows a simultaneous burst in both the GFP and mCherry channels that reflects the formation of complexes containing both fluorophores. (E) Histogram of single-molecule coincidence between Munc18-1WT-emGFP and Munc18-1C180Y-mCherry coexpressed in a cell-free system. The coincidence is calculated as the ratio of intensity in the mCherry channel divided by the sum of signals in the GFP and mCherry channels. GFP-only bursts show a coincidence at 0, and mCherry-only oligomers are located at coincidence equal to 1. For the oligomers containing both fluorophores, the coincidence ratio is a measure of the stoichiometry of the assembly. More than 50 individual aggregates were analyzed for the histogram, for a total of 2,600 time points above threshold. (F) Principle of a seeding experiment. Munc18-1C180Y-mCherry is expressed in the L. tarentolae extracts, and fibrils are formed. After expression, the sample is spun down, and the aggregates are collected and sonicated to give smaller seeds visible in the mCherry channel. Those seeds are mixed into a solution of Munc18-1WT-emGFP. (G) A single-molecule trace obtained from seeding experiments. Munc18-1WT-GFP is recruited to the seeds of Munc18-1C180Y-mCherry (H) Details of individual bursts representing different aggregates diffusing through the focal volume, clearly showing codiffusion of the two fluorophores. (I) Histogram of single-molecule coincidence between Munc18-1WT-GFP and seeds of Munc18-1C180Y-mCherry. The recruitment of GFP onto the mCherry-labeled seeds was calculated from 88 individual aggregates, representing 3,789 time points.
Figure 2.
Figure 2.
Munc18-1C180Y mutation coaggregates Munc18-1WT in Munc18-1 knockout PC12 cells and hippocampal neurons. (A) Schematic diagram of the sgRNA target site. The 20-nt guide sequence comprising the 5′-end of the chimeric single guide RNA (sgRNA) is shown. This sequence pairs with the DNA target site (indicated on the bottom strand). Immediately 3′ to the target sequence is the trinucleotide protospacer adjacent motif (PAM; 5′-NGG). The CAS9 enzyme mediates a double-stranded break (DSB) ∼3 bp upstream of the PAM. (B) Cell lysates of WT (PC12 cells) and clonal Munc18-1 knockout isolates (MKO-PC12 cells) were subjected to Western blot analysis and probed with anti–Munc18-1 (red). A parallel set of lysates was probed with anti–actin antibody as a loading control (green). (C) Representative images showing coaggregates positive for Munc18-1C180Y and Munc18-1WT in MKO-PC12 cells. MKO-PC12 cells were cotransfected with Munc18-1WT-emGFP and Munc18-1WT-mCherry (top), Munc18-1C180Y-emGFP and Munc18-1C180Y-mCherry (middle), or Munc18-1WT-emGFP and Munc18-1C180Y-mCherry (bottom). Bar, 20 µm. Arrowheads indicate colocalized aggregates. (D and E) Percentage of colocalized aggregates per cell and percentage of MKO-PC12 cells containing aggregates. Data represent mean ± SEM; 10–20 cells were analyzed for each independent experiment (n = 7). (F) Representative images showing coaggregates positive for Munc18-1C180Y and Munc18-1WT in hippocampal neurons. Hippocampal neurons (8 d in vitro) were transiently cotransfected with Munc18-1WT-emGFP and Munc18-1WT-mCherry or Munc18-1C180Y-emGFP and Munc18-1C180Y-mCherry or Munc18-1WT-emGFP and Munc18-1 C180Y-mCherry. Bar, 20 µm. Percentage of colocalized aggregates per neuron (G) and percentage of hippocampal neurons containing aggregates (H). Data represent mean ± SEM; 10–20 neurons were analyzed for each independent experiment (n = 6). Arrowheads indicate the colocalized aggregates.
Figure 3.
Figure 3.
Munc18-1 EIEE4-causing mutations coaggregate Munc18-1WT in MKO-PC12 cells. (A) Positions of missense mutations (V84D, M443R, and G544D) in the Munc18-1 crystal structure are shown as a stereo representation. (B) Schematic domain diagram of EIEE4-causing Munc18-1 mutations. (C) Representative images showing coaggregates positive for Munc18-1V84D-emGFP and Munc18-1WT-mCherry (top), Munc18-1M443R-emGFP and Munc18-1WT-mCherry (middle), or Munc18-1G544D-emGFP and Munc18-1WT-mCherry (bottom) are shown. Bar, 20 µm. (D) Percentage of colocalized aggregates per cell. Data represent mean ± SEM; 15–20 cells in each independent experiment were measured; n = 5. Arrowheads indicate the colocalized aggregates.
Figure 4.
Figure 4.
Single-molecule coincidence mapping of interactions between Munc18-1C180Y and α-Syn. (A) A single-molecule trace obtained for cell-free lysates coexpressing α-SynWT-mCherry and Munc18-1C180Y-GFP. The number of photons detected in the green and red channels are plotted as a function of time. The trace shows simultaneous bursts in the GFP and mCherry channels that reflect the formation of complexes containing both fluorophores. (B) Histogram of single-molecule coincidence between α-SynWT-mCherry and Munc18-1C180Y-GFP coexpressed in a cell-free system. Note that the Munc18-1C180Y aggregates are now located on the left off the histograms, and insertion of the α-SynWT pushes the distribution to higher stoichiometry values. (C) A single-molecule trace obtained for a cell-free lysate coexpressing SNAP-25-GFP and Munc18-1C180Y-mCherry. The trace shows no simultaneous burst in the GFP and mCherry channels, suggesting that SNAP-25 does not coaggregate with Munc18-1C180Y (D) Histogram of single-molecule coincidence between SNAP-25-GFP and Munc18-1C180Y-mCherry. The diagram indicates that most mCherry-only oligomers are located at coincidence equal to 1. (E) Single-molecule coincidence between SOD1-GFP and Munc18-1C180Y-mCherry coexpressed in a cell-free lysate. (F) Histogram of single-molecule coincidence between SOD1-GFP and Munc18-1C180Y-mCherry. The trace shows that there is no coaggregation between SOD1-GFP and Munc18-1C180Y-mCherry. The data were analyzed based on >40 individual aggregates for each protein pair, representing >2,500 time points.
Figure 5.
Figure 5.
Munc18-1C180Y mutation induces the aggregation of α-Syn in PC12 cells and hippocampal neurons. (A) Representative images showing coaggregates positive for α-Syn and Munc18-1WT (top) and α-Syn and Munc18-1C180Y (bottom) in PC12 cells. Bars, 20 µm. Arrowhead indicates the colocalized aggregate. PC12 cells were cotransfected with either α-Syn-mCherry and Munc18-1WT-emGFP or α-Syn-mCherry and Munc18-1C180Y-emGFP. (B) Percentage of colocalized aggregates. (C) Area occupied by α-Syn aggregates. A mean of 8–20 cells were analyzed for each independent experiment (n = 4; ***, P < 0.001, unpaired Student’s t test). (D) Representative images showing coaggregates positive for α-Syn and Munc18-1WT (top) and α-Syn and Munc18-1C180Y (bottom) in hippocampal neurons. Hippocampal neurons were cotransfected either α-Syn-mCherry and Munc18-1WT-emGFP or α-Syn-mCherry and Munc18-1C180Y-emGFP at 8 d in vitro. Bars, 20 µm. Arrowhead indicates the colocalized aggregates. (E) Percentage of colocalized aggregates. (F) Area occupied by α-Syn aggregates. 10–15 neurons in each independent experiment were analyzed (n = 5; ***, P < 0.001, unpaired Student’s t test). (G) PC12 cells were either cotransfected with Munc18-1WT-emGFP and α-Syn-mCherry or Munc18-1C180Y-emGFP and α-Syn-mCherry. They were lysed, solubilized, and immunoprecipitated with anti-GFP antibodies. Western blot analysis was performed using anti–α-Syn and anti–Munc18-1 antibodies. (H) The interaction between α-Syn and Munc18-1 was quantified after normalizing against the amount of immunoprecipitated Munc18-1. Data represent mean ± SEM of band intensities normalized to control values of Munc18-1WT (n = 3; ***, P < 0.001, unpaired Student’s t test).
Figure 6.
Figure 6.
Munc18-1 EIEE4-causing mutations recruit endogenous α-Syn in MKO-PC12 cells. (A) MKO-PC12 cells were transfected with EIEE4-causing Munc18-1 mutants, fixed, and immunolabeled with anti–α-Syn antibody. Representative images of MKO-PC12 cells expressing EIEE4-causing Munc18-1 mutants and probed with endogenous α-Syn. Bar, 20 µm. Arrowheads indicate colocalized aggregates. (B) Percentage of colocalized aggregates per MKO-PC12 cell. (C) Percentage of MKO-PC12 cells containing aggregates. Data represent mean ± SEM. 10–20 cells were analyzed for each independent experiment (n = 3).
Figure 7.
Figure 7.
Munc18-1C180Y mutation recruits endogenous α-Syn aggregation and forms Lewy body–like structures. Hippocampal neurons were transfected with Munc18-1WT-emGFP and Munc18-1C180Y-emGFP and immunolabeled with anti–α-Syn antibody. (A) Representative images of hippocampal neurons expressing Munc18-1C180Y and probed for endogenous α-Syn. Bars: 20 µm; (magnified images) 5 µm. Arrowhead indicates colocalized aggregates. 11 neurons were analyzed per experiment (n = 3 independent experiment, so a total of 33 neurons were examined). (B) Relative frequency distribution of the size of Lewy body–like structures. (C) Representative correlative fluorescence electron micrograph of a Munc18-1C180Y–positive aggregate in MKO-PC12 cells. Bars: (left) 5 µm; (center) 2 µm; (right) 500 nm. Arrowheads indicate cellular aggregates. (D) Representative images of hippocampal neurons transfected with either Munc18-1WT-emGFP or Munc18-1C180Y-emGFP. Nuclei were stained with DAPI. Bars, 10 µm. Arrowhead indicates a spheroid and arrow indicates pyknotic nucleus. (E) Percentage of pyknotic nuclei analyzed from Munc18-1WT-emGFP (control) and Munc18-1C180Y-emGFP transfected neurons. Data represent mean ± SEM (n = 6 regions of interest containing 7–12 transfected neurons each; ***, P < 0.001, unpaired Student’s t test).
Figure 8.
Figure 8.
PD-linked α-Syn mutants coaggregate Munc18-1WT and endogenous Munc18-1. (A) MKO-PC12 cells were cotransfected with either α-SynWT-GFP or indicated PD-linked α-Syn mutants and Munc18-1WT-mCherry. Representative images showing coaggregates positive for PD-linked α-Syn mutants and Munc18-1WT-mCherry in MKO-PC12 cells. Bars, 20 µm. (B) Percentage of colocalized aggregates. (C) Percentage of MKO-PC12 cells containing aggregates. 10–20 cells in each independent experiment were quantified. Data represent mean ± SEM; n = 5. (D) α-SynWT-GFP or PD-linked α-Syn mutants were transfected in PC12 cells and endogenous Munc18-1 was immunolabeled with anti–Munc18-1 antibody. Representative images showing coaggregates positive for GFP-tagged PD-linked α-Syn mutants and endogenous Munc18-1 in PC12 cells. Bars, 20 µm. Arrowheads indicate the colocalized aggregates. (E) Percentage of colocalized aggregates. (F) Percentage of PC12 cells containing aggregates. Data represent mean ± SEM. 10–20 cells were quantified in each independent experiment (n = 3).
Figure 9.
Figure 9.
Munc18-1 controls endogenous α-Syn propensity to aggregate in vitro and in gene-edited neurosecretory cells. (A) Seeds of Munc18-1C180Y do not recruit α-Syn in vitro. A single-molecule trace obtained from seed experiments. α-Syn-GFP is not recruited to the seeds of Munc18-1C180Y-mCherry. (B) Histogram of single-molecule coincidence between α-Syn-GFP and seeds of Munc18-1C180Y-mCherry. (C) A single-molecule trace obtained for cell-free lysates expressing α-SynA30P-GFP alone. (D) A single-molecule trace obtained for cell-free lysates coexpressing α-SynA30P-GFP and Munc18-1WT-mCherry (E) Detailed analysis of the distribution of fluorescence bursts from C and D, showing a reduction in bright events caused by the chaperone activity of Munc18-1. (F) Munc18-1WT-emGFP was transfected in MKO-PC12 cells, and FACS was applied to separate low-expressing from high-expressing Munc18-1 WT-emGFP transfected MKO-PC12 cells. MKO-PC12 cells together with control PC12 cells were platted for 4 h, fixed, and immunolabeled for α-Syn. Representative image of endogenous α-Syn from indicated cells. Bars, 20 µm. (G) FACS forward scatterplot showing gates used to select cells with half and full expression levels of Munc18-1WT-emGFP (H) The area occupied by endogenous α-Syn was quantified and the frequency distribution plotted. (I) The number of aggregates per cell and the percentage of cells containing aggregates were measured in PC12 cells and Munc18-1WT-emGFP low- to higher-expressing MKO-PC12 cells. Data represent mean ± SEM. 10–20 cells for each independent experiment were quantified (n = 3). One-way ANOVA was performed (**, P < 0.01; *** P < 0.001; n.s, not significant).
Figure 10.
Figure 10.
Munc18-1WT reexpression in MKO-PC12 cells rescues the propensity α-SynWT and two PD-linked α-Syn mutants to aggregate. α-SynWT-GFP or PD-linked α-Syn mutants were transfected in PC12 cells or MKO-PC12 cells (top and middle). α-SynWT-GFP and Munc18-1WT–mCherry or PD-linked α-Syn mutants and Munc18-1WT-mCherry were cotransfected in MKO-PC12 cells to rescue the Munc18-1WT expression (bottom). (A) Representative images of aggregates positive for α-SynWT and PD-linked α-Syn mutants in PC12 cells (top) or in MKO-PC12 cells (middle), and MKO-PC12 cells expressing Munc18-1WT-mCherry (bottom). Bar, 20 µm. Arrowheads indicate the colocalized aggregates. The number of aggregates per cell (B) and the percentage of cells containing aggregates (C) were quantified. Data represent mean ± SEM. 10–20 cells for each independent experiment were quantified (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001; one way ANOVA).

References

    1. Alvarez-Barón E., Bien C.G., Schramm J., Elger C.E., Becker A.J., and Schoch S.. 2008. Autoantibodies to Munc18, cerebral plasma cells and B-lymphocytes in Rasmussen encephalitis. Epilepsy Res. 80:93–97. 10.1016/j.eplepsyres.2008.03.007
    1. Auluck P.K., Chan H.Y., Trojanowski J.Q., Lee V.M., and Bonini N.M.. 2002. Chaperone suppression of α-synuclein toxicity in a Drosophila model for Parkinson’s disease. Science. 295:865–868. 10.1126/science.1067389
    1. Barcia G., Chemaly N., Gobin S., Milh M., Van Bogaert P., Barnerias C., Kaminska A., Dulac O., Desguerre I., Cormier V., et al. . 2014. Early epileptic encephalopathies associated with STXBP1 mutations: could we better delineate the phenotype? Eur. J. Med. Genet. 57:15–20. 10.1016/j.ejmg.2013.10.006
    1. Burré J., Sharma M., and Südhof T.C.. 2014. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc. Natl. Acad. Sci. USA. 111:E4274–E4283. 10.1073/pnas.1416598111
    1. Cantuti-Castelvetri I., Klucken J., Ingelsson M., Ramasamy K., McLean P.J., Frosch M.P., Hyman B.T., and Standaert D.G.. 2005. Alpha-synuclein and chaperones in dementia with Lewy bodies. J. Neuropathol. Exp. Neurol. 64:1058–1066. 10.1097/01.jnen.0000190063.90440.69
    1. Deprez L., Weckhuysen S., Holmgren P., Suls A., Van Dyck T., Goossens D., Del-Favero J., Jansen A., Verhaert K., Lagae L., et al. . 2010. Clinical spectrum of early-onset epileptic encephalopathies associated with STXBP1 mutations. Neurology. 75:1159–1165. 10.1212/WNL.0b013e3181f4d7bf
    1. Donovan L.E., Higginbotham L., Dammer E.B., Gearing M., Rees H.D., Xia Q., Duong D.M., Seyfried N.T., Lah J.J., and Levey A.I.. 2012. Analysis of a membrane-enriched proteome from postmortem human brain tissue in Alzheimer’s disease. Proteomics Clin. Appl. 6:201–211. 10.1002/prca.201100068
    1. Gagoski D., Mureev S., Giles N., Johnston W., Dahmer-Heath M., Škalamera D., Gonda T.J., and Alexandrov K.. 2015. Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems. J. Biotechnol. 195:1–7. 10.1016/j.jbiotec.2014.12.006
    1. Galvin J.E., Uryu K., Lee V.M., and Trojanowski J.Q.. 1999. Axon pathology in Parkinson’s disease and Lewy body dementia hippocampus contains alpha-, beta-, and gamma-synuclein. Proc. Natl. Acad. Sci. USA. 96:13450–13455. 10.1073/pnas.96.23.13450
    1. Gambin Y., Schug A., Lemke E.A., Lavinder J.J., Ferreon A.C., Magliery T.J., Onuchic J.N., and Deniz A.A.. 2009. Direct single-molecule observation of a protein living in two opposed native structures. Proc. Natl. Acad. Sci. USA. 106:10153–10158. 10.1073/pnas.0904461106
    1. Gambin Y., VanDelinder V., Ferreon A.C., Lemke E.A., Groisman A., and Deniz A.A.. 2011. Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing. Nat. Methods. 8:239–241. 10.1038/nmeth.1568
    1. Hamdan F.F., Piton A., Gauthier J., Lortie A., Dubeau F., Dobrzeniecka S., Spiegelman D., Noreau A., Pellerin S., Côté M., et al. . 2009. De novo STXBP1 mutations in mental retardation and nonsyndromic epilepsy. Ann. Neurol. 65:748–753. 10.1002/ana.21625
    1. Hamdan F.F., Gauthier J., Dobrzeniecka S., Lortie A., Mottron L., Vanasse M., D’Anjou G., Lacaille J.C., Rouleau G.A., and Michaud J.L.. 2011. Intellectual disability without epilepsy associated with STXBP1 disruption. Eur. J. Hum. Genet. 19:607–609. 10.1038/ejhg.2010.183
    1. Han G.A., Malintan N.T., Saw N.M., Li L., Han L., Meunier F.A., Collins B.M., and Sugita S.. 2011. Munc18-1 domain-1 controls vesicle docking and secretion by interacting with syntaxin-1 and chaperoning it to the plasma membrane. Mol. Biol. Cell. 22:4134–4149. 10.1091/mbc.E11-02-0135
    1. Han L., Jiang T., Han G.A., Malintan N.T., Xie L., Wang L., Tse F.W., Gaisano H.Y., Collins B.M., Meunier F.A., and Sugita S.. 2009. Rescue of Munc18-1 and -2 double knockdown reveals the essential functions of interaction between Munc18 and closed syntaxin in PC12 cells. Mol. Biol. Cell. 20:4962–4975. 10.1091/mbc.E09-08-0712
    1. Jacobs E.H., Williams R.J., and Francis P.T.. 2006. Cyclin-dependent kinase 5, Munc18a and Munc18-interacting protein 1/X11alpha protein up-regulation in Alzheimer’s disease. Neuroscience. 138:511–522. 10.1016/j.neuroscience.2005.11.017
    1. Johnston W.A., and Alexandrov K.. 2014. Production of eukaryotic cell-free lysate from Leishmania tarentolae. Methods Mol. Biol. 1118:1–15. 10.1007/978-1-62703-782-2_1
    1. Kim Y.E., Hipp M.S., Bracher A., Hayer-Hartl M., and Hartl F.U.. 2013. Molecular chaperone functions in protein folding and proteostasis. Annu. Rev. Biochem. 82:323–355. 10.1146/annurev-biochem-060208-092442
    1. Kovtun O., Mureev S., Jung W., Kubala M.H., Johnston W., and Alexandrov K.. 2011. Leishmania cell-free protein expression system. Methods. 55:58–64. 10.1016/j.ymeth.2011.06.006
    1. Lang L., Zetterström P., Brännström T., Marklund S.L., Danielsson J., and Oliveberg M.. 2015. SOD1 aggregation in ALS mice shows simplistic test tube behavior. Proc. Natl. Acad. Sci. USA. 112:9878–9883. 10.1073/pnas.1503328112
    1. Law C., Schaan Profes M., Levesque M., Kaltschmidt J.A., Verhage M., and Kania A.. 2016. Normal molecular specification and neurodegenerative disease-like death of spinal neurons lacking the SNARE-associated synaptic protein Munc18-1. J. Neurosci. 36:561–576. 10.1523/JNEUROSCI.1964-15.2016
    1. Malintan N.T., Nguyen T.H., Han L., Latham C.F., Osborne S.L., Wen P.J., Lim S.J., Sugita S., Collins B.M., and Meunier F.A.. 2009. Abrogating Munc18-1-SNARE complex interaction has limited impact on exocytosis in PC12 cells. J. Biol. Chem. 284:21637–21646. 10.1074/jbc.M109.013508
    1. Maries E., Dass B., Collier T.J., Kordower J.H., and Steece-Collier K.. 2003. The role of alpha-synuclein in Parkinson’s disease: insights from animal models. Nat. Rev. Neurosci. 4:727–738. 10.1038/nrn1199
    1. Martin S., Tomatis V.M., Papadopulos A., Christie M.P., Malintan N.T., Gormal R.S., Sugita S., Martin J.L., Collins B.M., and Meunier F.A.. 2013. The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane. J. Cell Sci. 126:2353–2360. 10.1242/jcs.126813
    1. Martin S., Papadopulos A., Tomatis V.M., Sierecki E., Malintan N.T., Gormal R.S., Giles N., Johnston W.A., Alexandrov K., Gambin Y., et al. . 2014. Increased polyubiquitination and proteasomal degradation of a Munc18-1 disease-linked mutant causes temperature-sensitive defect in exocytosis. Cell Reports. 9:206–218. 10.1016/j.celrep.2014.08.059
    1. McKeith I. 2004. Dementia with Lewy bodies. Dialogues Clin. Neurosci. 6:333–341.
    1. Mignot C., Moutard M.L., Trouillard O., Gourfinkel-An I., Jacquette A., Arveiler B., Morice-Picard F., Lacombe D., Chiron C., Ville D., et al. . 2011. STXBP1-related encephalopathy presenting as infantile spasms and generalized tremor in three patients. Epilepsia. 52:1820–1827. 10.1111/j.1528-1167.2011.03163.x
    1. Misura K.M., Scheller R.H., and Weis W.I.. 2000. Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex. Nature. 404:355–362. 10.1038/35006120
    1. Mureev S., Kovtun O., Nguyen U.T., and Alexandrov K.. 2009. Species-independent translational leaders facilitate cell-free expression. Nat. Biotechnol. 27:747–752. 10.1038/nbt.1556
    1. Orimo S., Uchihara T., Nakamura A., Mori F., Kakita A., Wakabayashi K., and Takahashi H.. 2008. Axonal α-synuclein aggregates herald centripetal degeneration of cardiac sympathetic nerve in Parkinson’s disease. Brain. 131:642–650. 10.1093/brain/awm302
    1. Otsuka M., Oguni H., Liang J.S., Ikeda H., Imai K., Hirasawa K., Imai K., Tachikawa E., Shimojima K., Osawa M., and Yamamoto T.. 2010. STXBP1 mutations cause not only Ohtahara syndrome but also West syndrome--result of Japanese cohort study. Epilepsia. 51:2449–2452. 10.1111/j.1528-1167.2010.02767.x
    1. Papadopulos A., Martin S., Tomatis V.M., Gormal R.S., and Meunier F.A.. 2013. Secretagogue stimulation of neurosecretory cells elicits filopodial extensions uncovering new functional release sites. J. Neurosci. 33:19143–19153. 10.1523/JNEUROSCI.2634-13.2013
    1. Pevsner J., Hsu S.C., and Scheller R.H.. 1994. n-Sec1: a neural-specific syntaxin-binding protein. Proc. Natl. Acad. Sci. USA. 91:1445–1449. 10.1073/pnas.91.4.1445
    1. Polymeropoulos M.H., Lavedan C., Leroy E., Ide S.E., Dehejia A., Dutra A., Pike B., Root H., Rubenstein J., Boyer R., et al. . 1997. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science. 276:2045–2047. 10.1126/science.276.5321.2045
    1. Prusiner S.B., Woerman A.L., Mordes D.A., Watts J.C., Rampersaud R., Berry D.B., Patel S., Oehler A., Lowe J.K., Kravitz S.N., et al. . 2015. Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism. Proc. Natl. Acad. Sci. USA. 112:E5308–E5317. 10.1073/pnas.1514475112
    1. Ran F.A., Hsu P.D., Wright J., Agarwala V., Scott D.A., and Zhang F.. 2013. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8:2281–2308. 10.1038/nprot.2013.143
    1. Saibil H. 2013. Chaperone machines for protein folding, unfolding and disaggregation. Nat. Rev. Mol. Cell Biol. 14:630–642. 10.1038/nrm3658
    1. Saitsu H., Kato M., Mizuguchi T., Hamada K., Osaka H., Tohyama J., Uruno K., Kumada S., Nishiyama K., Nishimura A., et al. . 2008. De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy. Nat. Genet. 40:782–788. 10.1038/ng.150
    1. Saitsu H., Kato M., Okada I., Orii K.E., Higuchi T., Hoshino H., Kubota M., Arai H., Tagawa T., Kimura S., et al. . 2010. STXBP1 mutations in early infantile epileptic encephalopathy with suppression-burst pattern. Epilepsia. 51:2397–2405. 10.1111/j.1528-1167.2010.02728.x
    1. Spillantini M.G., Schmidt M.L., Lee V.M., Trojanowski J.Q., Jakes R., and Goedert M.. 1997. α-synuclein in Lewy bodies. Nature. 388:839–840. 10.1038/42166
    1. Tavyev Asher Y.J., and Scaglia F.. 2012. Molecular bases and clinical spectrum of early infantile epileptic encephalopathies. Eur. J. Med. Genet. 55:299–306. 10.1016/j.ejmg.2012.04.002
    1. Tomatis V.M., Papadopulos A., Malintan N.T., Martin S., Wallis T., Gormal R.S., Kendrick-Jones J., Buss F., and Meunier F.A.. 2013. Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin. J. Cell Biol. 200:301–320. 10.1083/jcb.201204092
    1. Verhage M., Maia A.S., Plomp J.J., Brussaard A.B., Heeroma J.H., Vermeer H., Toonen R.F., Hammer R.E., van den Berg T.K., Missler M., et al. . 2000. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science. 287:864–869. 10.1126/science.287.5454.864
    1. Walhout A.J.M., Temple G.F., Brasch M.A., Hartley J.L., Lorson M.A., van den Heuvel S., and Vidal M.. 2000. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328:575–592. 10.1016/S0076-6879(00)28419-X
    1. Wang J., Farr G.W., Hall D.H., Li F., Furtak K., Dreier L., and Horwich A.L.. 2009. An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans. PLoS Genet. 5:e1000350 10.1371/journal.pgen.1000350

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