Reduced synaptic depression in human neurons carrying homozygous disease-causing STXBP1 variant L446F.

MUNC18-1 is an essential protein of the regulated secretion machinery. De novo, heterozygous mutations in STXBP1, the human gene encoding this protein, lead to a severe neurodevelopmental disorder. Here, we describe the electrophysiological characteristics of a unique case of STXBP1-related disorder caused by a homozygous mutation (L446F). We engineered this mutation in induced pluripotent stem cells from a healthy donor (STXBP1LF/LF) to establish isogenic cell models. We performed morphological and electrophysiological analyses on single neurons grown on glial micro-islands. Human STXBP1LF/LF neurons displayed normal morphology and normal basal synaptic transmission but increased paired-pulse ratios and charge released, and reduced synaptic depression compared to control neurons. Immunostainings revealed normal expression levels but impaired recognition by a mutation-specific MUNC18-1 antibody. The electrophysiological gain-of-function phenotype is in line with earlier overexpression studies in Stxbp1 null mouse neurons, with some potentially human-specific features. Therefore, the present study highlights important differences between mouse and human neurons critical for the translatability of pre-clinical studies.

Neuron-specific translational control shift ensures proteostatic resilience during ER stress.

Proteostasis is essential for cellular survival and particularly important for highly specialised post-mitotic cells such as neurons. Transient reduction in protein synthesis by protein kinase R-like endoplasmic reticulum (ER) kinase (PERK)-mediated phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type-specific mechanisms that secure proteostatic stress resilience. Here, we demonstrate that PERK-deficient neurons, unlike other cell types, fully retain the capacity to control translation during ER stress. We observe rescaling of the ATF4 response, while the reduction in protein synthesis is fully retained. We identify two molecular pathways that jointly drive translational control in PERK-deficient neurons. Haem-regulated inhibitor (HRI) mediates p-eIF2α and the ATF4 response and is complemented by the tRNA cleaving RNase angiogenin (ANG) to reduce protein synthesis. Overall, our study elucidates an intricate back-up mechanism to ascertain translational control during ER stress in neurons that provides a mechanistic explanation for the thus far unresolved observation of neuronal resilience to proteostatic stress.

High-throughput assay for regulated secretion of neuropeptides in mouse and human neurons.

Neuropeptides are the largest group of chemical signals in the brain. More than 100 different neuropeptides modulate various brain functions and their dysregulation has been associated with neurological disorders. Neuropeptides are packed into dense core vesicles (DCVs), which fuse with the plasma membrane in a calcium-dependent manner. Here, we describe a novel high-throughput assay for DCV exocytosis using a chimera of Nanoluc luciferase and the DCV-cargo neuropeptide Y (NPY). The NPY-Nanoluc reporter colocalized with endogenous DCV markers in all neurons with little mislocalization to other cellular compartments. NPY-Nanoluc reported DCV exocytosis in both rodent and induced pluripotent stem cell-derived human neurons, with similar depolarization, Ca2+, RAB3, and STXBP1/MUNC18 dependence as low-throughput assays. Moreover, NPY-Nanoluc accurately reported modulation of DCV exocytosis by known modulators diacylglycerol analog and Ca2+ channel blocker and showed a higher assay sensitivity than a widely used single-cell low-throughput assay. Lastly, we showed that Nanoluc coupled to other secretory markers reports on constitutive secretion. In conclusion, the NPY-Nanoluc is a sensitive reporter of DCV exocytosis in mammalian neurons, suitable for pharmacological and genomic screening for DCV exocytosis genes and for mechanism-based treatments for central nervous system disorders.

A de novo missense mutation in synaptotagmin-1 associated with neurodevelopmental disorder desynchronizes neurotransmitter release.

Synaptotagmin-1 (Syt1) is a presynaptic calcium sensor with two calcium binding domains, C2A and C2B, that triggers action potential-induced synchronous neurotransmitter release, while suppressing asynchronous and spontaneous release. We identified a de novo missense mutation (P401L) in the C2B domain in a patient with developmental delay and autistic symptoms. Expressing the orthologous mouse mutant (P400L) in cultured Syt1 null mutant neurons revealed a reduction in dendrite outgrowth with a proportional reduction in synapses. This was not observed in single Syt1PL-rescued neurons that received normal synaptic input when cultured in a control network. Patch-clamp recordings showed that spontaneous miniature release events per synapse were increased more than 500% in Syt1PL-rescued neurons, even beyond the increased rates in Syt1 KO neurons. Furthermore, action potential-induced asynchronous release was increased more than 100%, while synchronous release was unaffected. A similar shift to more asynchronous release was observed during train stimulations. These cellular phenotypes were also observed when Syt1PL was overexpressed in wild type neurons. Our findings show that Syt1PL desynchronizes neurotransmission by increasing the readily releasable pool for asynchronous release and reducing the suppression of spontaneous and asynchronous release. Neurons respond to this by shortening their dendrites, possibly to counteract the increased synaptic input. Syt1PL acts in a dominant-negative manner supporting a causative role for the mutation in the heterozygous patient. We propose that the substitution of a rigid proline to a more flexible leucine at the bottom of the C2B domain impairs clamping of release by interfering with Syt1's primary interface with the SNARE complex. This is a novel cellular phenotype, distinct from what was previously found for other SYT1 disease variants, and points to a role for spontaneous and asynchronous release in SYT1-associated neurodevelopmental disorder.

Maximal Fusion Capacity and Efficient Replenishment of the Dense Core Vesicle Pool in Hippocampal Neurons.

Neuropeptides and neurotrophins, stored in dense core vesicles (DCVs), are together the largest currently known group of chemical signals in the brain. Exocytosis of DCVs requires high-frequency or patterned stimulation, but the determinants to reach maximal fusion capacity and for efficient replenishment of released DCVs are unknown. Here, we systematically studied fusion of DCV with single vesicle resolution on different stimulation patterns in mammalian CNS neurons. We show that tetanic stimulation trains of 50-Hz action potential (AP) bursts maximized DCV fusion, with significantly fewer fusion event during later bursts of the train. This difference was omitted by introduction of interburst intervals but did not increase total DCV fusion. Interburst intervals as short as 5 s were sufficient to restore the fusion capacity. Theta burst stimulation (TBS) triggered less DCV fusion than tetanic stimulation, but a similar fusion efficiency per AP. Prepulse stimulation did not alter this. However, low-frequency stimulation (4 Hz) intermitted with fast ripple stimulation (200 APs at 200 Hz) produced substantial DCV fusion, albeit not as much as tetanic stimulation. Finally, individual fusion events had longer durations with more intense stimulation. These data indicate that trains of 50-Hz AP stimulation patterns triggered DCV exocytosis most efficiently and more intense stimulation promotes longer DCV fusion pore openings.SIGNIFICANCE STATEMENT Neuropeptides and neurotrophins modulate multiple regulatory functions of human body like reproduction, food intake or mood. They are packed into dense core vesicles (DCVs) that undergo calcium and action potential (AP) fusion with the plasma membrane. In order to study the fusion of DCVs in vitro, techniques like perfusion with buffer containing high concentration of potassium or electric field stimulation are needed to trigger the exocytosis of DCVs. Here, we studied the relationship between DCVs fusion properties and different electric field stimulation patterns. We used six different stimulation patterns and showed that trains of 50-Hz action potential bursts triggered DCV exocytosis most efficiently and more intense stimulation promotes longer DCV fusion pore openings.

An Atypical, Staged Cell Death Pathway Induced by Depletion of SNARE-Proteins MUNC18-1 or Syntaxin-1.

The presynaptic proteins MUNC18-1, syntaxin-1, and SNAP25 drive SNARE-mediated synaptic vesicle fusion and are also required for neuronal viability. Their absence triggers rapid, cell-autonomous, neuron-specific degeneration, unrelated to synaptic vesicle deficits. The underlying cell death pathways remain poorly understood. Here, we show that hippocampi of munc18-1 null mice (unknown sex) express apoptosis hallmarks cleaved caspase 3 (CC-3) and phosphorylated p53, and have condensed nuclei. However, side-by-side in vitro comparison with classical apoptosis induced by camptothecin uncovered striking differences to syntaxin-1 and MUNC18-1 depleted neurons. First, live-cell imaging revealed consecutive neurite retraction hours before cell death in MUNC18-1 or syntaxin-1 depleted neurons, whereas all neurites retracted at once, directly before cell death in classical apoptosis. Second, CC-3 activation was observed only after loss of all neurites and cellular breakdown, whereas CC-3 is activated before any neurite loss in classical apoptosis. Third, a pan-caspase inhibitor and a p53 inhibitor both arrested classical apoptosis, as expected, but not cell death in MUNC18-1 or syntaxin-1 depleted neurons. Neuron-specific cell death, consecutive neurite retraction, and late CC-3 activation were conserved in syntaxin-1 depleted human neurons. Finally, no indications were observed for involvement of other established cell death pathways, including necroptosis, Wallerian degeneration, autophagic cell death, and pyroptosis. Together, these data show that depletion of presynaptic proteins MUNC18-1 or syntaxin-1 triggers an atypical, staged cell death pathway characterized by consecutive neurite retraction, ultimately leading to, but not driven by, apoptosis.SIGNIFICANCE STATEMENT Neuronal cell death can occur via a multitude of pathways and plays an important role in the developing nervous system as well as neurodegenerative diseases. One poorly understood pathway to neuronal cell death takes place on depletion of presynaptic SNARE proteins syntaxin-1, SNAP25, or MUNC18-1. The current study demonstrates that MUNC18-1 or syntaxin-1 depleted neurons show a new, atypical, staged cell death that does not resemble any of the established cell death pathways in neurons. Cell death on MUNC18-1 or syntaxin-1 depletion is characterized by consecutive neurite retraction, ultimately involving, but not driven by, classical apoptosis.

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.

Power and optimal study design in iPSC-based brain disease modelling.

Studies using induced pluripotent stem cells (iPSCs) are gaining momentum in brain disorder modelling, but optimal study designs are poorly defined. Here, we compare commonly used designs and statistical analysis for different research aims. Furthermore, we generated immunocytochemical, electrophysiological, and proteomic data from iPSC-derived neurons of five healthy subjects, analysed data variation and conducted power simulations. These analyses show that published case-control iPSC studies are generally underpowered. Designs using isogenic iPSC lines typically have higher power than case-control designs, but generalization of conclusions is limited. We show that, for the realistic settings used in this study, a multiple isogenic pair design increases absolute power up to 60% or requires up to 5-fold fewer lines. A free web tool is presented to explore the power of different study designs, using any (pilot) data.

Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes.

Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. Here, using adrenal chromaffin cells, we identify the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, we show that the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, lead us to propose that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes.

Neuromodulator release in neurons requires two functionally redundant calcium sensors.

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

SALM1 controls synapse development by promoting F-actin/PIP2-dependent Neurexin clustering.

Synapse development requires spatiotemporally regulated recruitment of synaptic proteins. In this study, we describe a novel presynaptic mechanism of cis-regulated oligomerization of adhesion molecules that controls synaptogenesis. We identified synaptic adhesion-like molecule 1 (SALM1) as a constituent of the proposed presynaptic Munc18/CASK/Mint1/Lin7b organizer complex. SALM1 preferentially localized to presynaptic compartments of excitatory hippocampal neurons. SALM1 depletion in excitatory hippocampal primary neurons impaired Neurexin1ß- and Neuroligin1-mediated excitatory synaptogenesis and reduced synaptic vesicle clustering, synaptic transmission, and synaptic vesicle release. SALM1 promoted Neurexin1ß clustering in an F-actin- and PIP2-dependent manner. Two basic residues in SALM1's juxtamembrane polybasic domain are essential for this clustering. Together, these data show that SALM1 is a presynaptic organizer of synapse development by promoting F-actin/PIP2-dependent clustering of Neurexin.

CaMKII controls neuromodulation via neuropeptide gene expression and axonal targeting of neuropeptide vesicles.

Ca2+/calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α- and ßCaMKII-deficient (αßCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αßCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αßCaMKII DKO neurons was restored by active ßCaMKII but not inactive ßCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to ßCaMKII- and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.

Munc18-1 is essential for neuropeptide secretion in neurons.

Neuropeptide secretion from dense-core vesicles (DCVs) controls many brain functions. Several components of the DCV exocytosis machinery have recently been identified, but the participation of a SEC1/MUNC18 (SM) protein has remained elusive. Here, we tested the ability of the three exocytic SM proteins expressed in the mammalian brain, MUNC18-1/2/3, to support neuropeptide secretion. We quantified DCV exocytosis at a single vesicle resolution upon action potential train-stimulation in mouse CNS neurons (of unknown sex) using pHluorin- and/or mCherry-tagged Neuropeptide-Y (NPY) or Brain-Derived Neurotrophic Factor (BDNF). Conditional inactivation of Munc18-1 abolished all DCV exocytosis. Expression of MUNC18-1, but not MUNC18-2 or MUNC18-3, supported DCV exocytosis in Munc18-1 null neurons. Heterozygous (HZ) inactivation of Munc18-1, as a model for reduced MUNC18-1 expression, impaired DCV exocytosis, especially during the initial phase of train-stimulation, when the release was maximal. These data show that neurons critically and selectively depend on MUNC18-1 for neuropeptide secretion. Impaired neuropeptide secretion may explain aspects of the behavioral and neurodevelopmental phenotypes that were observed in Munc18-1 HZ mice.SIGNIFICANCE STATEMENT:Neuropeptide secretion from dense-core vesicles (DCVs) modulates synaptic transmission, sleep, appetite, cognition and mood. However, the mechanisms of DCV exocytosis are poorly characterized. Here, we identify MUNC18-1 as an essential component for neuropeptide secretion from DCVs. Paralogs MUNC18-2 or -3 cannot compensate for MUNC18-1. MUNC18-1 is the first protein identified to be essential for both neuropeptide secretion and synaptic transmission. In heterozygous Munc18-1 neurons, that have a 50% reduced MUNC18-1 expression and model the human STXBP1 syndrome, DCV exocytosis is impaired, especially during the initial phase of train-stimulation, when the release is maximal. These data show that MUNC18-1 is essential for neuropeptide secretion and that impaired neuropeptide secretion upon reduced MUNC18-1 expression may contribute to the symptoms of STXBP1 syndrome.

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.

Pool size estimations for dense-core vesicles in mammalian CNS neurons.

Neuropeptides are essential signaling molecules transported and secreted by dense-core vesicles (DCVs), but the number of DCVs available for secretion, their subcellular distribution, and release probability are unknown. Here, we quantified DCV pool sizes in three types of mammalian CNS neurons in vitro and in vivo Super-resolution and electron microscopy reveal a total pool of 1,400-18,000 DCVs, correlating with neurite length. Excitatory hippocampal and inhibitory striatal neurons in vitro have a similar DCV density, and thalamo-cortical axons in vivo have a slightly higher density. Synapses contain on average two to three DCVs, at the periphery of synaptic vesicle clusters. DCVs distribute equally in axons and dendrites, but the vast majority (80%) of DCV fusion events occur at axons. The release probability of DCVs is 1-6%, depending on the stimulation. Thus, mammalian CNS neurons contain a large pool of DCVs of which only a small fraction can fuse, preferentially at axons.

Single-case experimental designs for bumetanide across neurodevelopmental disorders: BUDDI protocol.

BACKGROUND: Bumetanide is a selective NKCC1 chloride importer antagonist which is being repurposed as a mechanism-based treatment for neurodevelopmental disorders (NDDs). Due to their specific actions, these kinds of interventions will only be effective in particular subsets of patients. To anticipate stratified application, we recently completed three bumetanide trials each focusing on different stratification strategies with the additional objective of deriving the most optimal endpoints. Here we publish the protocol of the post-trial access combined cohort study to confirm previous effects and stratification strategies in the trial cohorts and in new participants. METHOD/DESIGN: Participants of the three previous cohorts and a new cohort will be subjected to 6 months bumetanide treatment using multiple baseline Single Case Experimental Designs. The primary outcome is the change, relative to baseline, in a set of patient reported outcome measures focused on direct and indirect effects of sensory processing difficulties. Secondary outcome measures include the conventional questionnaires 'social responsiveness scale', 'repetitive behavior scale', 'sensory profile' and 'aberrant behavior scale'. Resting-state EEG measurements will be performed at several time-points including at Tmax after the first administration. Assessment of cognitive endpoints will be conducted using the novel Emma Tool box, an in-house designed battery of computerized tests to measure neurocognitive functions in children. DISCUSSION: This study aims to replicate previously shown effects of bumetanide in NDD subpopulations, validate a recently proposed treatment prediction effect methodology and refine endpoint measurements. TRIAL REGISTRATION: EudraCT: 2020-002196-35, registered 16 November 2020, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-002196-35/NL.

Doc2 Proteins Are Not Required for the Increased Spontaneous Release Rate in Synaptotagmin-1-Deficient Neurons.

Regulated secretion is controlled by Ca2+ sensors with different affinities and subcellular distributions. Inactivation of Syt1 (synaptotagmin-1), the main Ca2+ sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca2+ affinities and/or lower cooperativities. Such sensors could include Doc2a and Doc2b, which have been implicated in spontaneous and asynchronous neurotransmitter release and compete with Syt1 for binding SNARE complexes. Here, we tested this hypothesis using triple-knock-out mice. Inactivation of Doc2a and Doc2b in Syt1-deficient neurons did not reduce the high spontaneous release rate. Overexpression of Doc2b variants in triple-knock-out neurons reduced spontaneous release but did not rescue synchronous release. A chimeric construct in which the C2AB domain of Syt1 was substituted by that of Doc2b did not support synchronous release either. Conversely, the soluble C2AB domain of Syt1 did not affect spontaneous release. We conclude that the high spontaneous release rate in synaptotagmin-deficient neurons does not involve the binding of Doc2 proteins to Syt1 binding sites in the SNARE complex. Instead, our results suggest that the C2AB domains of Syt1 and Doc2b specifically support synchronous and spontaneous release by separate mechanisms. (Both male and female neurons were studied without sex determination.)SIGNIFICANCE STATEMENT Neurotransmission in the brain is regulated by presynaptic Ca2+ concentrations. Multiple Ca2+ sensor proteins contribute to synchronous (Syt1, Syt2), asynchronous (Syt7), and spontaneous (Doc2a/Doc2b) phases of neurotransmitter release. Genetic ablation of synchronous release was previously shown to affect other release phases, suggesting that multiple sensors may compete for similar release sites, together encoding stimulus-secretion coupling over a large range of synaptic Ca2+ concentrations. Here, we investigated the extent of functional overlap between Syt1, Doc2a, and Doc2b by reintroducing wild-type and mutant proteins in triple-knock-out neurons, and conclude that the sensors are highly specialized for different phases of release.

Loss of MUNC18-1 leads to retrograde transport defects in neurons.

Loss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its target-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether loss of MUNC18-1 causes defects in intracellular membrane transport pathways in primary murine neurons that may explain neurodegeneration. Electron, confocal and super resolution microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall, ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. By synchronizing protein trafficking by conditional cargo retention in the endoplasmic reticulum using selective hooks (RUSH) and immunocytochemistry, we show that anterograde Endoplasmic Reticulum-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin B-subunit transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking in an antibody uptake assay. We conclude that MUNC18-1 deficient neurons have normal anterograde but reduced retrograde transport to the Golgi. The impairments in retrograde pathways suggest a role of MUNC18-1 in endosomal SNARE-dependent fusion and provide a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons.

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.

Homozygous STXBP1 variant causes encephalopathy and gain-of-function in synaptic transmission.

Heterozygous mutations in the STXBP1 gene encoding the presynaptic protein MUNC18-1 cause STXBP1 encephalopathy, characterized by developmental delay, intellectual disability and epilepsy. Impaired mutant protein stability leading to reduced synaptic transmission is considered the main underlying pathogenetic mechanism. Here, we report the first two cases carrying a homozygous STXBP1 mutation, where their heterozygous siblings and mother are asymptomatic. Both cases were diagnosed with Lennox-Gastaut syndrome. In Munc18-1 null mouse neurons, protein stability of the disease variant (L446F) is less dramatically affected than previously observed for heterozygous disease mutants. Neurons expressing Munc18L446F showed minor changes in morphology and synapse density. However, patch clamp recordings demonstrated that L446F causes a 2-fold increase in evoked synaptic transmission. Conversely, paired pulse plasticity was reduced and recovery after stimulus trains also. Spontaneous release frequency and amplitude, the readily releasable vesicle pool and the kinetics of short-term plasticity were all normal. Hence, the homozygous L446F mutation causes a gain-of-function phenotype regarding release probability and synaptic transmission while having less impact on protein levels than previously reported (heterozygous) mutations. These data show that STXBP1 mutations produce divergent cellular effects, resulting in different clinical features, while sharing the overarching encephalopathic phenotype (developmental delay, intellectual disability and epilepsy).