Chaperoning SNARE Folding and Assembly.

SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.

Disease-linked mutations in Munc18-1 deplete synaptic Doc2.

Heterozygous de novo mutations in the neuronal protein Munc18-1/STXBP1 cause syndromic neurological symptoms, including severe epilepsy, intellectual disability, developmental delay, ataxia and tremor, summarized as STXBP1 encephalopathies. Although haploinsufficiency is the prevailing disease mechanism, it remains unclear how the reduction in Munc18-1 levels causes synaptic dysfunction in disease as well as how haploinsufficiency alone can account for the significant heterogeneity among patients in terms of the presence, onset and severity of different symptoms. Using biochemical and cell biological readouts on mouse brains, cultured mouse neurons and heterologous cells, we found that the synaptic Munc18-1 interactors Doc2A and Doc2B are unstable in the absence of Munc18-1 and aggregate in the presence of disease-causing Munc18-1 mutants. In haploinsufficiency-mimicking heterozygous knockout neurons, we found a reduction in Doc2A/B levels that is further aggravated by the presence of the disease-causing Munc18-1 mutation G544D as well as an impairment in Doc2A/B synaptic targeting in both genotypes. We also demonstrated that overexpression of Doc2A/B partially rescues synaptic dysfunction in heterozygous knockout neurons but not heterozygous knockout neurons expressing G544D Munc18-1. Our data demonstrate that STXBP1 encephalopathies are not only characterized by the dysfunction of Munc18-1 but also by the dysfunction of the Munc18-1 binding partners Doc2A and Doc2B, and that this dysfunction is exacerbated by the presence of a Munc18-1 missense mutant. These findings may offer a novel explanation for the significant heterogeneity in symptoms observed among STXBP1 encephalopathy patients.

KCNH6 channel promotes insulin exocytosis via interaction with Munc18-1 independent of electrophysiological processes.

Glucose-stimulated insulin secretion (GSIS) in pancreatic islet ß-cells primarily relies on electrophysiological processes. Previous research highlighted the regulatory role of KCNH6, a member of the Kv channel family, in governing GSIS through its influence on ß-cell electrophysiology. In this study, we unveil a novel facet of KCNH6's function concerning insulin granule exocytosis, independent of its conventional electrical role. Young mice with ß-cell-specific KCNH6 knockout (ßKO) exhibited impaired glucose tolerance and reduced insulin secretion, a phenomenon not explained by electrophysiological processes alone. Consistently, islets from KCNH6-ßKO mice exhibited reduced insulin secretion, conversely, the overexpression of KCNH6 in murine pancreatic islets significantly enhanced insulin release. Moreover, insulin granules lacking KCNH6 demonstrated compromised docking capabilities and a reduced fusion response upon glucose stimulation. Crucially, our investigation unveiled a significant interaction between KCNH6 and the SNARE protein regulator, Munc18-1, a key mediator of insulin granule exocytosis. These findings underscore the critical role of KCNH6 in the regulation of insulin secretion through its interaction with Munc18-1, providing a promising and novel avenue for enhancing our understanding of the Kv channel in diabetes mechanisms.

Energetics, kinetics, and pathways of SNARE assembly in membrane fusion.

Fusion of transmitter-containing vesicles with plasma membranes at the synaptic and neuromuscular junctions mediates neurotransmission and muscle contractions, respectively, thereby underlying all thoughts and actions. The fusion process is driven by the coupled folding and assembly of three synaptic SNARE proteins--syntaxin-1 and SNAP-25 on the target plasma membrane (t-SNAREs) and VAMP2 on the vesicular membrane (v-SNARE) into a four-helix bundle. Their assembly is chaperoned by Munc18-1 and many other proteins to achieve the speed and accuracy required for neurotransmission. However, the physiological pathway of SNARE assembly and its coupling to membrane fusion remains unclear. Here, we review recent progress in understanding SNARE assembly and membrane fusion, with a focus on results obtained by single-molecule manipulation approaches and electric recordings of single fusion pores. We describe two pathways of synaptic SNARE assembly, their associated intermediates, energetics, and kinetics. Assembly of the three SNAREs in vitro begins with the formation of a t-SNARE binary complex, on which VAMP2 folds in a stepwise zipper-like fashion. Munc18-1 significantly alters the SNARE assembly pathway: syntaxin-1 and VAMP2 first bind on the surface of Munc18-1 to form a template complex, with which SNAP-25 associates to conclude SNARE assembly and displace Munc18-1. During membrane fusion, multiple trans-SNARE complexes cooperate to open a dynamic fusion pore in a manner dependent upon their copy number and zippering states. Together, these results demonstrate that stepwise and cooperative SNARE assembly drive stagewise membrane fusion.

Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.

Priming of synaptic vesicles involves Munc13-catalyzed transition of the Munc18-1/syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of syntaxin-1. This interaction ensures Munc18-1 to persistently associate with syntaxin-1 during the conformational change of syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.

Reduced dynamin-1 levels in neurons lacking MUNC18-1.

MUNC18-1 (also known as syntaxin-binding protein-1, encoded by Stxbp1) binds to syntaxin-1. Together, these proteins regulate synaptic vesicle exocytosis and have a separate role in neuronal viability. In Stxbp1 null mutant neurons, syntaxin-1 protein levels are reduced by 70%. Here, we show that dynamin-1 protein levels are reduced at least to the same extent, and transcript levels of Dnm1 (which encodes dynamin-1) are reduced by 50% in Stxbp1 null mutant brain. Several, but not all, other endocytic proteins were also found to be reduced, but to a lesser extent. The reduced dynamin-1 expression was not observed in SNAP25 null mutants or in double-null mutants of MUNC13-1 and -2 (also known as Unc13a and Unc13b, respectively), in which synaptic vesicle exocytosis is also blocked. Co-immunoprecipitation experiments demonstrated that dynamin-1 and MUNC18-1 do not bind directly. Furthermore, MUNC18-1 levels were unaltered in neurons lacking all three dynamin paralogues. Finally, overexpression of dynamin-1 was not sufficient to rescue neuronal viability in Stxbp1 null mutant neurons; thus, the reduction in dynamin-1 is not the single cause of neurodegeneration of these neurons. The reduction in levels of dynamin-1 protein and mRNA, as well as of other endocytosis proteins, in Stxbp1 null mutant neurons suggests that MUNC18-1 directly or indirectly controls expression of other presynaptic genes.

Identification of residues critical for the extension of Munc18-1 domain 3a.

BACKGROUND: Neurotransmitter release depends on the fusion of synaptic vesicles with the presynaptic membrane and is mainly mediated by SNARE complex assembly. During the transition of Munc18-1/Syntaxin-1 to the SNARE complex, the opening of the Syntaxin-1 linker region catalyzed by Munc13-1 leads to the extension of the domain 3a hinge loop, which enables domain 3a to bind SNARE motifs in Synaptobrevin-2 and Syntaxin-1 and template the SNARE complex assembly. However, the exact mechanism of domain 3a extension remains elusive. RESULTS: Here, we characterized residues on the domain 3a hinge loop that are crucial for the extension of domain 3a by using biophysical and biochemical approaches and electrophysiological recordings. We showed that the mutation of residues T323/M324/R325 disrupted Munc13-1-mediated SNARE complex assembly and membrane fusion starting from Munc18-1/Syntaxin-1 in vitro and caused severe defects in the synaptic exocytosis of mouse cortex neurons in vivo. Moreover, the mutation had no effect on the binding of Synaptobrevin-2 to isolated Munc18-1 or the conformational change of the Syntaxin-1 linker region catalyzed by the Munc13-1 MUN domain. However, the extension of the domain 3a hinge loop in Munc18-1/Syntaxin-1 was completely disrupted by the mutation, leading to the failure of Synaptobrevin-2 binding to Munc18-1/Syntaxin-1. CONCLUSIONS: Together with previous results, our data further support the model that the template function of Munc18-1 in SNARE complex assembly requires the extension of domain 3a, and particular residues in the domain 3a hinge loop are crucial for the autoinhibitory release of domain 3a after the MUN domain opens the Syntaxin-1 linker region.

Munc18-1 Contributes to Hippocampal Injury in Septic Rats Through Regulation of Syntanxin1A and Synaptophysin and Glutamate Levels.

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction closely associated with mortality in the acute phase of sepsis. Abnormal neurotransmitters release, such as glutamate, plays a crucial role in the pathological mechanism of SAE. Munc18-1 is a key protein regulating neurotransmission. However, whether Munc18-1 plays a role in SAE by regulating glutamate transmission is still unclear. In this study, a septic rat model was established by the cecal ligation and perforation. We found an increase in the content of glutamate in the hippocampus of septic rat, the number of synaptic vesicles in the synaptic active area and the expression of the glutamate receptor NMDAR1. Meanwhile, it was found that the expressions of Munc18-1, Syntaxin1A and Synaptophysin increased, which are involved in neurotransmission. The expression levels of Syntaxin1A and Synaptophysin in hippocampus of septic rats decreased after interference using Munc18-1siRNA. We observed a decrease in the content of glutamate in the hippocampus of septic rats, the number of synaptic vesicles in the synaptic activity area and the expression of NMDAR1. Interestingly, it was also found that the down-regulation of Munc18-1 improved the vital signs of septic rats. This study shows that CLP induced the increased levels of glutamate in rat hippocampus, and Munc18-1 may participate in the process of hippocampal injury in septic rats by affecting the levels of glutamate via regulating Syntaxin1A and Synaptophysin. Munc18-1 may serve as a potential target for SAE therapy.

Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex.

Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.

Presynaptic Short-Term Plasticity Persists in the Absence of PKC Phosphorylation of Munc18-1.

Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1.SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.

Tyrosine phosphorylation of Munc18-1 inhibits synaptic transmission by preventing SNARE assembly.

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18-1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho-proteomics abolished the stimulatory effect of Munc18-1 on SNARE complex formation ("SNARE-templating") and membrane fusion in vitro Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18-1-null neurons expressing Munc18-1Y473D Synaptic transmission was temporarily restored by high-frequency stimulation, as well as by a Munc18-1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non-phosphorylatable Munc18-1 supported normal synaptic transmission. We propose that SFK-dependent Munc18-1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post-docking SNARE-templating role of Munc18-1, resulting in a largely abolished pool of releasable synaptic vesicles.

STXBP1 encephalopathies: Clinical spectrum, disease mechanisms, and therapeutic strategies.

Mutations in Munc18-1/STXBP1 (syntaxin-binding protein 1) are linked to various severe early epileptic encephalopathies and neurodevelopmental disorders. Heterozygous mutations in the STXBP1 gene include missense, nonsense, frameshift, and splice site mutations, as well as intragenic deletions and duplications and whole-gene deletions. No genotype-phenotype correlation has been identified so far, and patients are treated by anti-epileptic drugs because of the lack of a specific disease-modifying therapy. The molecular disease mechanisms underlying STXBP1-linked disorders are yet to be fully understood, but both haploinsufficiency and dominant-negative mechanisms have been proposed. This review focuses on the current understanding of the phenotypic spectrum of STXBP1-linked disorders, as well as discusses disease mechanisms in the context of the numerous pathways in which STXBP1 functions in the brain. We additionally evaluate the available animal models to study these disorders and highlight potential therapeutic approaches for treating these devastating diseases.

MUNC18-1 regulates the submembrane F-actin network, independently of syntaxin1 targeting, via hydrophobicity in ß-sheet 10.

MUNC18-1 (also known as STXBP1) is an essential protein for docking and fusion of secretory vesicles. Mouse chromaffin cells (MCCs) lacking MUNC18-1 show impaired secretory vesicle docking, but also mistargeting of SNARE protein syntaxin1 and an abnormally dense submembrane F-actin network. Here, we tested the contribution of both these phenomena to docking and secretion defects in MUNC18-1-deficient MCCs. We show that an abnormal F-actin network and syntaxin1 targeting defects are not observed in Snap25- or Syt1-knockout (KO) MCCs, which are also secretion deficient. We identified a MUNC18-1 mutant (V263T in ß-sheet 10) that fully restores syntaxin1 targeting but not F-actin abnormalities in Munc18-1-KO cells. MUNC18-2 and -3 (also known as STXBP2 and STXBP3, respectively), which lack the hydrophobic residue at position 263, also did not restore a normal F-actin network in Munc18-1-KO cells. However, these proteins did restore the normal F-actin network when a hydrophobic residue was introduced at the corresponding position. Munc18-1-KO MCCs expressing MUNC18-1(V263T) showed normal vesicle docking and exocytosis. These results demonstrate that MUNC18-1 regulates the F-actin network independently of syntaxin1 targeting via hydrophobicity in ß-sheet 10. The abnormally dense F-actin network in Munc18-1-deficient cells is not a rate-limiting barrier in secretory vesicle docking or fusion.This article has an associated First Person interview with the first author of the paper.

Epilepsy-causing STX1B mutations translate altered protein functions into distinct phenotypes in mouse neurons.

Syntaxin 1B (STX1B) is a core component of the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that is critical for the exocytosis of synaptic vesicles in the presynapse. SNARE-mediated vesicle fusion is assisted by Munc18-1, which recruits STX1B in the auto-inhibited conformation, while Munc13 catalyses the fast and efficient pairing of helices during SNARE complex formation. Mutations within the STX1B gene are associated with epilepsy. Here we analysed three STX1B mutations by biochemical and electrophysiological means. These three paradigmatic mutations cause epilepsy syndromes of different severity, from benign fever-associated seizures in childhood to severe epileptic encephalopathies. An insertion/deletion (K45/RMCIE, L46M) mutation (STX1BInDel), causing mild epilepsy and located in the early helical Habc domain, leads to an unfolded protein unable to sustain neurotransmission. STX1BG226R, causing epileptic encephalopathies, strongly compromises the interaction with Munc18-1 and reduces expression of both proteins, the size of the readily releasable pool of vesicles, and Ca2+-triggered neurotransmitter release when expressed in STX1-null neurons. The mutation STX1BV216E, also causing epileptic encephalopathies, only slightly diminishes Munc18-1 and Munc13 interactions, but leads to enhanced fusogenicity and increased vesicular release probability, also in STX1-null neurons. Even though the synaptic output remained unchanged in excitatory hippocampal STX1B+/- neurons exogenously expressing STX1B mutants, the manifestation of clear and distinct molecular disease mechanisms by these mutants suggest that certain forms of epilepsies can be conceptualized by assigning mutations to structurally sensitive regions of the STX1B-Munc18-1 interface, translating into distinct neurophysiological phenotypes.

Munc18-1 haploinsufficiency impairs learning and memory by reduced synaptic vesicular release in a model of Ohtahara syndrome.

Ohtahara syndrome, also known as type 4 of Early Infantile Epileptic Encephalopathy with suppression bursts (EIEE-4) is currently an untreatable disorder that presents with seizures and impaired cognition. EIEE-4 patients have mutations most frequently in the STXBP1 gene encoding a Sec protein, munc18-1. The exact molecular mechanism of how these munc18-1 mutations cause impaired cognition, remains elusive. The leading haploinsufficiency hypothesis posits that mutations in munc18-1 render the protein unstable leading to its degradation. Expression driven by the healthy allele is not sufficient to maintain the physiological function resulting in haploinsufficiency. The aim of this study has been to understand how munc18-1 haploinsufficiency causes cognitive impairment seen in EIEE-4. Here we present results from behavioral to cellular effects from a mouse model of munc18-1 haploinsufficiency. Munc18-1 heterozygous knock-out mice showed impaired spatial learning and memory in behavior tests as well as reduced synaptic plasticity in hippocampal CA1 long-term potentiation. Cultured munc18-1 heterozygous hippocampal neurons had significantly slower rate of synaptic vesicle release and decreased readily releasable vesicle pool compared to wild-type control neurons in fluorescent FM dye assays. These results demonstrate that reduced munc18-1 levels are sufficient to impair learning and memory by reducing neurotransmitter release. Therefore, our study implicates munc18-1 haploinsufficiency as a primary cause of cognitive impairment seen in EIEE-4 patients.

SNARE Complex-Associated Proteins in the Lateral Amygdala of Macaca mulatta Following Long-Term Ethanol Drinking.

BACKGROUND: Recent work with long-term ethanol (EtOH) self-administration in nonhuman primate models has revealed a complex array of behavioral and physiological effects that closely mimic human alcohol abuse. Detailed neurophysiological analysis in these models suggests a myriad of pre- and postsynaptic neurobiological effects that may contribute to the behavioral manifestations of long-term EtOH drinking. The molecular mechanisms regulating presynaptic effects of this chronic EtOH exposure are largely unknown. To this end, we analyzed the effects of long-term EtOH self-administration on the levels of presynaptic SNARE complex proteins in Macaca mulatta basolateral amygdala, a brain region known to regulate both aversive and reward-seeking behaviors. METHODS: Basolateral amygdala samples from control and EtOH-drinking male and female monkeys were processed. Total basolateral amygdala protein was analyzed by Western blotting using antibodies directed against both core SNARE and SNARE-associated proteins. We also performed correlational analyses between protein expression levels and a number of EtOH drinking parameters, including lifetime grams of EtOH consumed, preference, and blood alcohol concentration. RESULTS: Significant interactions or main effects of sex/drinking were seen for a number of SNARE core and SNARE-associated proteins. Across the range of EtOH-drinking phenotypes, SNAP25 and Munc13-1 proteins levels were significantly different between males and females, and Munc13-2 levels were significantly lower in animals with a history of EtOH drinking. A separate analysis of very heavy-drinking individuals revealed significant decreases in Rab3c (females) and complexin 2 (males). CONCLUSIONS: Protein expression analysis of basolateral amygdala total protein from controls and animals following long-term EtOH self-administration suggests a number of alterations in core SNARE or SNARE-associated components that could dramatically alter presynaptic function. A number of proteins or multiprotein components were also correlated with EtOH drinking behavior, which suggest a potentially heritable role for presynaptic SNARE proteins.

Normal Molecular Specification and Neurodegenerative Disease-Like Death of Spinal Neurons Lacking the SNARE-Associated Synaptic Protein Munc18-1.

The role of synaptic activity during early formation of neural circuits is a topic of some debate; genetic ablation of neurotransmitter release by deletion of the Munc18-1 gene provides an excellent model to answer the question of whether such activity is required for early circuit formation. Previous analysis of Munc18-1(-/-) mouse mutants documented their grossly normal nervous system, but its molecular differentiation has not been assessed. Munc18-1 deletion in mice also results in widespread neurodegeneration that remains poorly characterized. In this study, we demonstrate that the early stages of spinal motor circuit formation, including motor neuron specification, axon growth and pathfinding, and mRNA expression, are unaffected in Munc18-1(-/-) mice, demonstrating that synaptic activity is dispensable for early nervous system development. Furthermore, we show that the neurodegeneration caused by Munc18-1 loss is cell autonomous, consistent with apparently normal expression of several neurotrophic factors and normal GDNF signaling. Consistent with cell-autonomous degeneration, we demonstrate defects in the trafficking of the synaptic proteins Syntaxin1a and PSD-95 and the TrkB and DCC receptors in Munc18-1(-/-) neurons; these defects do not appear to cause ER stress, suggesting other mechanisms for degeneration. Finally, we demonstrate pathological similarities to Alzheimer's disease, such as altered Tau phosphorylation, neurofibrillary tangles, and accumulation of insoluble protein plaques. Together, our results shed new light upon the neurodegeneration observed in Munc18-1(-/-) mice and argue that this phenomenon shares parallels with neurodegenerative diseases. SIGNIFICANCE STATEMENT: In this work, we demonstrate the absence of a requirement for regulated neurotransmitter release in the assembly of early neuronal circuits by assaying transcriptional identity, axon growth and guidance, and mRNA expression in Munc18-1-null mice. Furthermore, we characterize the neurodegeneration observed in Munc18-1 mutants and demonstrate that this cell-autonomous process does not appear to be a result of defects in growth factor signaling or ER stress caused by protein trafficking defects. However, we find the presence of various pathological hallmarks of Alzheimer's disease that suggest parallels between the degeneration in these mutants and neurodegenerative conditions.

Extension of Helix 12 in Munc18-1 Induces Vesicle Priming.

UNLABELLED: Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324-339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil-coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324-339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca(2+) dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming. SIGNIFICANCE STATEMENT: The essential postdocking role of Munc18-1 in vesicular exocytosis has remained elusive, but recent data led to the hypothesis that the extension of helix 12 in Munc18 within domain 3a leads to synaptobrevin-2/VAMP2 interaction and SNARE complex formation. Using both lack-of-function and gain-of-function mutants, we here report that the conformation of helix 12 predicts vesicle priming and secretory amplitude in living chromaffin cells. The effects of mutants on secretion could not be explained by differences in syntaxin-1 chaperoning/localization or vesicle docking, and the fusion kinetics and calcium dependence were unchanged, indicating that the effect of helix 12 extension is specific for the vesicle-priming step. We conclude that a conformational change within helix 12 is responsible for the essential postdocking role of Munc18-1 in neurosecretion.

Regulation of dendrite growth and maintenance by exocytosis.

Dendrites lengthen by several orders of magnitude during neuronal development, but how membrane is allocated in dendrites to facilitate this growth remains unclear. Here, we report that Ras opposite (Rop), the Drosophila ortholog of the key exocytosis regulator Munc18-1 (also known as STXBP1), is an essential factor mediating dendrite growth. Neurons with depleted Rop function exhibit reduced terminal dendrite outgrowth followed by primary dendrite degeneration, suggestive of differential requirements for exocytosis in the growth and maintenance of different dendritic compartments. Rop promotes dendrite growth together with the exocyst, an octameric protein complex involved in tethering vesicles to the plasma membrane, with Rop-exocyst complexes and exocytosis predominating in primary dendrites over terminal dendrites. By contrast, membrane-associated proteins readily diffuse from primary dendrites into terminals, but not in the reverse direction, suggesting that diffusion, rather than targeted exocytosis, supplies membranous material for terminal dendritic growth, revealing key differences in the distribution of materials to these expanding dendritic compartments.

The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane.

Munc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a 'closed', fusion-incompetent to an 'open', fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1(Δ317-333)) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1(Δ317-333) to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1(Δ317-333) binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1(Δ317-333) could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.