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SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced readily releasable pool (RRP) size, due to increased spontaneous (miniature Excitatory Postsynaptic Current, mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y > V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.

Biallelic loss-of-function mutations in the syntaxin 3 gene have been linked to a severe retinal dystrophy in humans that presents in early childhood. In mouse models, biallelic inactivation of the syntaxin 3 gene in photoreceptors rapidly leads to their death. What is not known is whether a monoallelic syntaxin 3 loss-of-function mutation might cause photoreceptor loss with advancing age. To address this question, we compared the outer nuclear layer of older adult mice (≈ 20 months of age) that were heterozygous for syntaxin 3 with those of similarly-aged control mice. We found that the photoreceptor layer maintains its thickness in mice that are heterozygous for syntaxin 3 relative to controls and that photoreceptor somatic counts are comparable. In addition, dendritic sprouting of the rod bipolar cell dendrites into the outer nuclear layer, which occurs following the loss of functional rod targets, was similar between genotypes. Thus, syntaxin 3 appears to be haplosufficient for photoreceptor survival, even with advancing age.

Synaptic neurotransmitter release is an evolutionarily conserved process that mediates rapid information transfer between neurons as well as several peripheral tissues. Release of neurotransmitters are ensured by successive events such as synaptic vesicle docking and priming that prepare synaptic vesicles for rapid fusion. These events are orchestrated by interaction of different presynaptic proteins and are regulated by presynaptic calcium. Recent studies have identified various mutations in different components of neurotransmitter release machinery resulting in aberrant neurotransmitter release, which underlie a wide spectrum of psychiatric and neurological symptoms. Here, we review how these genetic alterations in different components of the core neurotransmitter release machinery affect the information transfer between neurons and how aberrant synaptic release affects nervous system function.

SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca 2+ -stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced readily releasable pool (RRP) size, due to increased spontaneous (miniature Excitatory Postsynaptic Current, mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y > V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion., Competing Interests: AK, JM, MØ, AM, JM, FL, WN, TS, JS No competing interests declared, (© 2023, Kádková, Murach, Østergaard et al.)

Background: STXBP 1 -related disorder ( STXBP 1 -RD) is a neurodevelopmental disorder caused by pathogenic variants in the STXBP1 gene. Its gene product MUNC18-1 organizes synaptic vesicle exocytosis and is essential for synaptic transmission. Patients present with developmental delay, intellectual disability, and/or epileptic seizures, with high clinical heterogeneity. To date, the cellular deficits of neurons of patients with STXBP 1 -RD are unknown., Methods: We combined live-cell imaging, electrophysiology, confocal microscopy, and mass spectrometry proteomics to characterize cellular phenotypes of induced pluripotent stem cell-derived neurons from 6 patients with STXBP 1 -RD, capturing shared features as well as phenotypic diversity among patients., Results: Neurons from all patients showed normal in vitro development, morphology, and synapse formation, but reduced MUNC18-1 RNA and protein levels. In addition, a proteome-wide screen identified dysregulation of proteins related to synapse function and RNA processes. Neuronal networks showed shared as well as patient-specific phenotypes in activity frequency, network irregularity, and synchronicity, especially when networks were challenged by increasing excitability. No shared effects were observed in synapse physiology of single neurons except for a few patient-specific phenotypes. Similarities between functional and proteome phenotypes suggested 2 patient clusters, not explained by gene variant type., Conclusions: Together, these data show that decreased MUNC18-1 levels, dysregulation of synaptic proteins, and altered network activity are shared cellular phenotypes of STXBP 1 -RD. The 2 patient clusters suggest distinctive pathobiology among subgroups of patients, providing a plausible explanation for the clinical heterogeneity. This phenotypic spectrum provides a framework for future validation studies and therapy design for STXBP 1 -RD., (© 2023 The Authors.)

Synaptic neurotransmitter release is an evolutionarily conserved process that mediates rapid information transfer between neurons as well as several peripheral tissues. Release of neurotransmitters are ensured by successive events such as synaptic vesicle docking and priming that prepare synaptic vesicles for rapid fusion. These events are orchestrated by interaction of different presynaptic proteins and are regulated by presynaptic calcium. Recent studies have identified various mutations in different components of neurotransmitter release machinery resulting in aberrant neurotransmitter release, which underlie a wide spectrum of psychiatric and neurological symptoms. Here, we review how these genetic alterations in different components of the core neurotransmitter release machinery affect the information transfer between neurons and how aberrant synaptic release affects nervous system function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Uzay and Kavalali.)

Biallelic loss-of-function mutations in the syntaxin 3 gene have been linked to a severe retinal dystrophy in humans that presents in early childhood. In mouse models, biallelic inactivation of the syntaxin 3 gene in photoreceptors rapidly leads to their death. What is not known is whether a monoallelic syntaxin 3 loss-of-function mutation might cause photoreceptor loss with advancing age. To address this question, we compared the outer nuclear layer of older adult mice (≈ 20 months of age) that were heterozygous for syntaxin 3 with those of similarly-aged control mice. We found that the photoreceptor layer maintains its thickness in mice that are heterozygous for syntaxin 3 relative to controls and that photoreceptor somatic counts are comparable. In addition, dendritic sprouting of the rod bipolar cell dendrites into the outer nuclear layer, which occurs following the loss of functional rod targets, was similar between genotypes. Thus, syntaxin 3 appears to be haplosufficient for photoreceptor survival, even with advancing age., Competing Interests: Conflict of Interest The authors declare that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.