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Synaptotagmin-1 (Syt-1) self-assembles into ring-like oligomers, and genetic and biochemical evidence suggest that oligomerization is needed to clamp synaptic vesicles and stabilize them for Ca 2+ -evoked release. However, oligomerization has not yet been demonstrated on lipid bilayers or studied in quantitative biophysical terms. Here we utilize single-molecule imaging methods to monitor the assembly and disassembly of oligomeric clusters of Syt-1 on lipid bilayers in real-time. Syt-1 assembled into two distinct classes of oligomers, small (5 ± 2 subunits) and large (15 ± 2 subunits). Each class assembled at a constant k on that was always proportional to its ultimate size, but both classes disassembled at the same unit rate (k off ) independent of its size. Both large and small oligomers explosively disassembled when Ca 2+ was added. The F349A mutation in the Syt-1 nearly eliminates the large class of oligomers but does not reduce the small class. Altogether, the physical-chemical properties of Syt-1 oligomers meet or exceed the physiologic requirements to function as such a clamp., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Apparently, the genomes of many organisms are pervasively transcribed, and long noncoding RNAs (lncRNAs) make up the majority of cellular transcripts. LncRNAs have been reported to play important roles in many biological processes; however, their effects on locomotion are poorly understood. Here, we presented a novel lncRNA, Locomotion Regulatory Gene (LRG), which participates in locomotion by sequestering Synaptotagmin 1 (SYT1). LRG deficiency resulted in higher locomotion speed which could be rescued by pan-neuronal overexpression but not by limited ellipsoid body, motoneuron or muscle-expression of LRG. At the molecular level, the synaptic vesicles (SVs) release and movement-related SYT1 protein was recognized as LRG-interacting protein candidate. Furthermore, LRG had no effects on SYT1 expression. Genetically, the behavioral defects in LRG mutant could be rescued by pan-neuronal knock-down of Syt1. Taken together, all the results suggested LRG exerts regulatory effects on locomotion via sequestering SYT1 thereby blocking its function without affecting its expression. Our work displays a new function of lncRNA and provides insights for revealing the pathogenesis of neurological diseases with motor disorders., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

Complexin determines magnitude and kinetics of synchronized secretion, but the underlying molecular mechanisms remained unclear. Here, we show that the hydrophobic face of the amphipathic helix at the C-terminus of Complexin II (CpxII, amino acids 115-134) binds to fusion-promoting SNARE proteins, prevents premature secretion, and allows vesicles to accumulate in a release-ready state in mouse chromaffin cells. Specifically, we demonstrate that an unrelated amphipathic helix functionally substitutes for the C-terminal domain (CTD) of CpxII and that amino acid substitutions on the hydrophobic side compromise the arrest of the pre-fusion intermediate. To facilitate synchronous vesicle fusion, the N-terminal domain (NTD) of CpxII (amino acids 1-27) specifically cooperates with synaptotagmin I (SytI), but not with synaptotagmin VII. Expression of CpxII rescues the slow release kinetics of the Ca 2+ -binding mutant Syt I R233Q, whereas the N-terminally truncated variant of CpxII further delays it. These results indicate that the CpxII NTD regulates mechanisms which are governed by the forward rate of Ca 2+ binding to Syt I. Overall, our results shed new light on key molecular properties of CpxII that hinder premature exocytosis and accelerate synchronous exocytosis., Competing Interests: MM, AP, AY, SG, MZ, WF, DB No competing interests declared, (© 2023, Makke et al.)

Background: Pathogenic missense variants in the essential synaptic vesicle protein synaptotagmin-1 (SYT1) cause a neurodevelopmental disorder characterised by motor delay and intellectual disability, hyperkinetic movement disorder, episodic agitation, and visual impairments. SYT1 is the presynaptic calcium sensor that triggers synchronous neurotransmitter release. We have previously shown that pathogenic variants around the calcium-sensing region of the critical C2B domain decrease synaptic vesicle exocytosis in neurons., Methods: Here, we have used cultured hippocampal neurons transfected with SYT1-pHluorin to examine how variants within the C2A and C2B domain of SYT1 impact evoked exocytosis., Findings: We show that recently identified variants within the facilitatory C2A domain of the protein (L159R, T196K, E209K, E219Q), as well as additional variants in the C2B domain (M303V, S309P, Y365C, G369D), share an underlying pathogenic mechanism, causing a graded and variant-dependent dominant-negative impairment in exocytosis. We establish that the extent of evoked exocytosis observed in vitro in the presence of SYT1 variants correlates with neurodevelopmental impacts of this disorder. Specifically, the severity of motor and communication impairments exhibited by individuals harbouring these variants correlates with multiple measures of exocytic efficiency., Interpretation: Together, this suggests that there is a genotype-function-phenotype relationship in SYT1-associated neurodevelopmental disorder, centring impaired evoked neurotransmitter release as a common pathogenic driver. Moreover, this points toward a direct link between control of neurotransmitter release and development of adaptive functions, providing a tractable target for therapeutic amelioration., Funding: Australian National Health and Medical Research Council, UK Medical Research Council, Great Ormond Street Hospital Children's Charity, University of Melbourne., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The Ca 2+ sensor synaptotagmin-1 (Syt1) triggers neurotransmitter release together with the neuronal sensitive factor attachment protein receptor (SNARE) complex formed by syntaxin-1, SNAP25, and synaptobrevin. Moreover, Syt1 increases synaptic vesicle (SV) priming and impairs spontaneous vesicle release. The Syt1 C 2 B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of Syt1 and the mechanism underlying Ca 2+ triggering of release are unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: Binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca 2+ -evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of Syt1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of Syt1 in vesicle priming and clamping of spontaneous release and, importantly, show that Ca 2+ triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with biophysical studies presented in [K. Jaczynska et al. , bioRxiv [Preprint] (2024). https://doi.org/10.1101/2024.06.17.599417 (Accessed 18 June 2024)], our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast SV fusion., Competing Interests: Competing interests statement:The authors declare no competing interest.

Two recent papers by Mehta et al. and Zhu et al. in this issue (https://doi.org/10.1083/jcb.202311191) discover that synaptotagmin-1, the primary calcium sensor at the synapse, forms biomolecular condensates, identifying a new layer of regulation in calcium-triggered synaptic vesicle exocytosis., (© 2024 Tromm and Milovanovic.)

Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion., (© 2024 Zhu et al.)

Fusion pore opening is a transient intermediate state of synaptic vesicle exocytosis, which is highly dynamic and precisely regulated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and synaptotagmin-1 (Syt1). Yet, the regulatory mechanism is not fully understood. In this work, using single-channel membrane fusion electrophysiology, we determined that SNAREpins are important for driving fusion pore opening and dilation but incapable of regulating the dynamics. When Syt1 was added, the closing frequency of fusion pores significantly increased, while the radius of fusion pores mildly decreased. In response to Ca2+, SNARE/Syt1 greatly increased the radius of fusion pores and reduced their closing frequency. Moreover, the residue F349 in the C2B domain of Syt1, which mediates Syt1 oligomerization, was required for clamping fusion pore opening in the absence of Ca2+, probably by extending the distance between the two membranes. Finally, in Ca2+-triggered fusion, the primary interface between SNARE and Syt1 plays a critical role in stabilizing and dilating the fusion pore, while the polybasic region of Syt1 C2B domain has a mild effect on increasing the radius of the fusion pore. In summary, our results suggest that Syt1, SNARE, and the anionic membrane synergically orchestrate the dynamics of fusion pore opening in synaptic vesicle exocytosis., (© The Author(s) (2024). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been proposed to act as a 'pre-assembly' mechanism for endocytosis that ensures high-fidelity retrieval of SV cargo. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1 K326A/K328A ) and SV2A knockdown. SV2A co-clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2A T84A ). Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin-1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Acute kidney injury (AKI) is common and an independent risk factor for mortality in patients with paraquat (PQ) poisoning. Currently, no specific antidote is available. Synaptotagmin-1 (SYT1) has been identified as a key protein that facilitates PQ efflux in PQ-resistant A549 cells, thereby preventing PQ-induced lung injury. However, the protective effect of STY1 on PQ-induced AKI remains to be elucidated. This study exposed human kidney 2 (HK-2) cells overexpressing SYT1 to PQ. These cells exhibited significantly lower levels of growth inhibition, reactive oxygen species production, early apoptosis, and PQ accumulation compared to the parent HK-2 cells. Transcriptomic screening and Western blot analysis revealed that SYT1 overexpression significantly promoted the expression of glucose transporter 2 (GLUT2). Inhibition of GLUT2 completely abolished the protective effects of SYT1 overexpression in HK-2 cells and restored intracellular PQ concentrations. Further immunoprecipitation-shotgun and RNA interference experiments revealed that SYT1 binds to and stabilizes the protein SERPINE1 mRNA-binding protein 1 (SERBP1), enhancing the stability of GLUT2 mRNA and its protein levels. In summary, SYT1 antagonizes PQ intracellular accumulation and prevents nephrocyte toxicity by up-regulating SERBP1/GLUT2 expression. This study identifies a potential target for the treatment of PQ-induced AKI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Migrasomes, organelles crucial for cell communication, undergo distinct stages of nucleation, maturation, and expansion. The regulatory mechanisms of migrasome formation, particularly through biological cues, remain largely unexplored. This study reveals that calcium is essential for migrasome formation. Furthermore, we identify that Synaptotagmin-1 (Syt1), a well-known calcium sensor, is not only enriched in migrasomes but also indispensable for their formation. The calcium-binding ability of Syt1 is key to initiating migrasome formation. The recruitment of Syt1 to migrasome formation sites (MFS) triggers the swelling of MFS into unstable precursors, which are subsequently stabilized through the sequential recruitment of tetraspanins. Our findings reveal how calcium regulates migrasome formation and propose a sequential interaction model involving Syt1 and Tetraspanins in the formation and stabilization of migrasomes., (© 2024 Han and Yu.)

We examined the role of protein tyrosine phosphatase receptor sigma (PTPRS) in the context of Alzheimer's disease and synaptic integrity. Publicly available datasets (BRAINEAC, ROSMAP, ADC1) and a cohort of asymptomatic but "at risk" individuals (PREVENT-AD) were used to explore the relationship between PTPRS and various Alzheimer's disease biomarkers. We identified that PTPRS rs10415488 variant C shows features of neuroprotection against early Tau pathology and synaptic degeneration in Alzheimer's disease. This single nucleotide polymorphism correlated with higher PTPRS transcript abundance and lower p(181)Tau and GAP-43 levels in the CSF. In the brain, PTPRS protein abundance was significantly correlated with the quantity of two markers of synaptic integrity: SNAP25 and SYT-1. We also found the presence of sexual dimorphism for PTPRS, with higher CSF concentrations in males than females. Male carriers for variant C were found to have a 10-month delay in the onset of AD. We thus conclude that PTPRS acts as a neuroprotective receptor in Alzheimer's disease. Its protective effect is most important in males, in whom it postpones the age of onset of the disease., (© 2024. The Author(s).)

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 Syt1 PL -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 Syt1 PL -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 Syt1 PL was overexpressed in wild type neurons. Our findings show that Syt1 PL 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. Syt1 PL 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., (© 2024. The Author(s).)

Synaptic dysfunction is a core component of the pathophysiology of schizophrenia. However, the genetic risk factors and molecular mechanisms related to synaptic dysfunction are still not fully understood. The Stonin 2 (STON2) gene encodes a major adaptor for clathrin-mediated endocytosis (CME) of synaptic vesicles. In this study, we showed that the C-C (307Pro-851Ala) haplotype of STON2 increases the susceptibility to schizophrenia and examined whether STON2 variations cause schizophrenia-like behaviors through the regulation of CME. We found that schizophrenia-related STON2 variations led to protein dephosphorylation, which affected its interaction with synaptotagmin 1 (Syt1), a calcium sensor protein located in the presynaptic membrane that is critical for CME. STON2 307Pro851Ala knockin mice exhibited deficits in synaptic transmission, short-term plasticity, and schizophrenia-like behaviors. Moreover, among seven antipsychotic drugs, patients with the C-C (307Pro-851Ala) haplotype responded better to haloperidol than did the T-A (307Ser-851Ser) carriers. The recovery of deficits in Syt1 sorting and synaptic transmission by acute administration of haloperidol effectively improved schizophrenia-like behaviors in STON2 307Pro851Ala knockin mice. Our findings demonstrated the effect of schizophrenia-related STON2 variations on synaptic dysfunction through the regulation of CME, which might be attractive therapeutic targets for treating schizophrenia-like phenotypes., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

GPR133 (ADGRD1) is an adhesion G-protein-coupled receptor that signals through Gαs/cyclic AMP (cAMP) and is required for the growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca 2+ -dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca 2+ -sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca 2+ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM slows tumor growth, suggesting tumorigenic functions of ESYT1. Our findings demonstrate a mechanism for the modulation of GPR133 signaling by increased cytosolic Ca 2+ , which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels., Competing Interests: Declaration of interests D.G.P. and NYU Grossman School of Medicine own an EU and Hong Kong patent titled “Method for treating high-grade gliomas” on the use of GPR133 as a treatment target in glioma. D.G.P. and collaborators at NYU Grossman School of Medicine have filed a patent application titled “Anti-CD97 antibodies and antibody-drug conjugates”. D.G.P. has received consultant fees from Tocagen, Synaptive Medical, Monteris, Robeaute, Advantis, and Servier Pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sociability stands as a crucial factor in the evolutionary success of all mammalian species. Notably, enriched environment (EE) housing has been shown to enhance sociability in mice. However, the precise underlying molecular mechanism remains elusive. In this study, we established an EE paradigm, housing mice for a 14-day period. Both enhanced sociability and an increased spine density in the medial prefrontal cortex (mPFC) of mice subjected to EE were detected. To elucidate the potential molecular pathway, we conducted high-performance liquid chromatography tandem mass spectrometry (HPLC-MS) analysis of the entire mPFC from both EE and home-caged (HC) housed mice. Our analysis identified 16 upregulated and 20 downregulated proteins in the EE group. Among them, Extended Synaptotagmin 1 (ESyt1), an activity-dependent endoplasmic reticulum (ER)-plasma membrane (PM) tethering protein associated with synaptic function and growth, emerged as a potentially key player in the increased synapse formation and enhanced sociability observed in EE-housed mice. Further investigation, involving the knockdown of ESyt1 expression via sh ESyt1 lentivirus in the mPFC, revealed that ESyt1 is crucial for increased spine density of mPFC and enhanced sociability of mice in an enriched environment but not in normal condition. Overall, our findings uncover a novel mechanistic insight into the positive influence of environmental enrichment on social behavior via ESyt1-mediated pathways., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We report the case of a Spanish pediatric patient with developmental delay, hypotonia, feeding difficulties, visual problems, and hyperkinetic movements. Whole-exome sequencing uncovered a new heterozygous de novo Synaptotagmin 1 (SYT1) missense variant, NM_005639.3:c.930T>A (p.Asp310Glu), in a female proband. This gene encodes the synaptotagmin-1 (SYT1) protein, which is a component of a protein complex involved in the fusion of synaptic vesicles with the presynaptic membrane. Pathogenic SYT1 variants have been associated with Baker-Gordon syndrome (BAGOS), an autosomal dominant neurodevelopmental disorder. Although up to 30 cases have been identified worldwide, to the best of our knowledge, this is the first patient described with mitochondrial respiratory chain deficiencies and rod-cone dysfunction. In conclusion, our data expand both the genetic and phenotypic spectrum associated with SYT1 variants.

MicroRNAs (miRNAs) are important post-transcriptional factors involved in the regulation of gene expression and play crucial roles in biological processes related to milk fat metabolism. Our previous study revealed that miR-19a expression was significantly higher in the mammary epithelial cells of high-milk fat cows than in those of low-milk fat cows. However, the precise molecular mechanisms underlying these differences remain unclear. In this study, we found a high expression of miR-19a in the mammary tissues of dairy cows. The regulatory effects of miR-19a on bovine mammary epithelial cells (BMECs) were analyzed using cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays, which demonstrated that miR-19a significantly inhibited BMEC proliferation. Transfection of the miR-19a mimic into BMECs significantly upregulated the expression of milk fat marker genes LPL, SCAP, and SREBP1, promoting triglyceride (TG) synthesis and lipid droplet formation, whereas the miR-19a inhibitor exhibited the opposite function. TargetScan and miRWalk predictions revealed that synaptotagmin 1 (SYT1) is a target gene of miR-19a. A dual luciferase reporter gene assay, RT-qPCR, and western blot analyses revealed that miR-19a directly targets the 3'-untranslated region (UTR) of SYT1 and negatively regulates SYT1 expression. Functional validation revealed that overexpression of SYT1 in BMECs significantly downregulated the expression of LPL, SCAP, and SREBP1, and inhibited TG synthesis and lipid droplet formation. Conversely, the knockdown of SYT1 had the opposite effect. Altogether, miR-19a plays a crucial role in regulating the proliferation and differentiation of BMECs and regulates biological processes related to TG synthesis and lipid droplet formation by suppressing SYT1 expression. These findings provide a strong foundation for further research on the functional mechanisms underlying milk fat metabolism in dairy cows., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca 2+ -triggered exocytosis of synaptic vesicles (SVs). In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P 2 -triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin mined from the egg transcriptome of spider L. tredecimguttatus, was previously found to promote the release of dopamine from PC12 cells. However, the relevant molecular mechanism has not been fully clear. Here LETX-VI was demonstrated to rapidly penetrate the plasma membrane of PC12 cells via the vesicle exocytosis/endocytosis cycle, during which vesicular transmembrane protein synaptotagmin 1 (Syt1) functions as a receptor, with its vesicle luminal domain interacting with the C-terminal region of LETX-VI. The C-terminal sequence of LETX-VI is the functional region for both entering cells and promoting dopamine release. After gaining entry into the PC12 cells, LETX-VI down-regulated the phosphorylation levels of Syt1 at T201 and T195, thereby facilitating vesicle fusion with plasma membrane and thus promoting dopamine release. The relevant mechanism analysis indicated that LETX-VI has a protein phosphatase 2A (PP2A) activator activity. The present work has not only probed into the Syt1-mediated action mechanism of LETX-VI, but also revealed the structure-function relationship of the toxin, thus suggesting its potential applications in the drug transmembrane delivery and treatment of the diseases related to dopamine release and PP2A activity deficiency., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1 K189-192A and Syt1 K326-327A ) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release. SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes., (Copyright © 2022 the authors.)

The molecular mechanisms underlying somatodendritic dopamine (DA) release remain unresolved, despite the passing of decades since its discovery. Our previous work showed robust release of somatodendritic DA in submillimolar extracellular Ca 2+ concentration ([Ca 2+ ] o ). Here we tested the hypothesis that the high-affinity Ca 2+ sensor synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its Ca 2+ dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices from male and female mice to monitor evoked DA-dependent D2 receptor-mediated inhibitory currents (D2ICs). Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased pulse train-evoked D2ICs, revealing a functional role for Syt7. The assessment of the Ca 2+ dependence of pulse train-evoked D2ICs confirmed robust DA release in submillimolar [Ca 2+ ] o in wild-type (WT) neurons, but loss of this sensitivity with intracellular Syt7 Ab or in Syt7 knock-out (KO) mice. In millimolar [Ca 2+ ] o , pulse train-evoked D2ICs in Syt7 KOs showed a greater reduction in decreased [Ca 2+ ] o than seen in WT mice; the effect on single pulse-evoked DA release, however, did not differ between genotypes. Single-cell application of a Syt1 Ab had no effect on train-evoked D2ICs in WT SNc DA neurons, but did cause a decrease in D2IC amplitude in Syt7 KOs, indicating a functional substitution of Syt1 for Syt7. In addition, Syt1 Ab decreased single pulse-evoked D2ICs in WT cells, indicating the involvement of Syt1 in tonic DA release. Thus, Syt7 and Syt1 play complementary roles in somatodendritic DA release from SNc DA neurons. SIGNIFICANCE STATEMENT The respective Ca 2+ dependence of somatodendritic and axonal dopamine (DA) release differs, resulting in the persistence of somatodendritic DA release in submillimolar Ca 2+ concentrations too low to support axonal release. We demonstrate that synaptotagmin7 (Syt7), a high-affinity Ca 2+ sensor, underlies phasic somatodendritic DA release and its Ca 2+ sensitivity in the substantia nigra pars compacta. In contrast, we found that synaptotagmin 1 (Syt1), the Ca 2+ sensor underlying axonal DA release, plays a role in tonic, but not phasic, somatodendritic DA release in wild-type mice. However, Syt1 can facilitate phasic DA release after Syt7 deletion. Thus, we show that both Syt1 and Syt7 act as Ca 2+ sensors subserving different aspects of somatodendritic DA release processes., (Copyright © 2022 the authors.)

Purpose: Synaptotagmin-1 (SYT1) is a critical mediator of neurotransmitter release in the central nervous system. Previously reported missense SYT1 variants in the C2B domain are associated with severe intellectual disability, movement disorders, behavioral disturbances, and electroencephalogram abnormalities. In this study, we expand the genotypes and phenotypes and identify discriminating features of this disorder., Methods: We describe 22 individuals with 15 de novo missense SYT1 variants. The evidence for pathogenicity is discussed, including the American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria, known structure-function relationships, and molecular dynamics simulations. Quantitative behavioral data for 14 cases were compared with other monogenic neurodevelopmental disorders., Results: Four variants were located in the C2A domain with the remainder in the C2B domain. We classified 6 variants as pathogenic, 4 as likely pathogenic, and 5 as variants of uncertain significance. Prevalent clinical phenotypes included delayed developmental milestones, abnormal eye physiology, movement disorders, and sleep disturbances. Discriminating behavioral characteristics were severity of motor and communication impairment, presence of motor stereotypies, and mood instability., Conclusion: Neurodevelopmental disorder-associated SYT1 variants extend beyond previously reported regions, and the phenotypic spectrum encompasses a broader range of severities than initially reported. This study guides the diagnosis and molecular understanding of this rare neurodevelopmental disorder and highlights a key role for SYT1 function in emotional regulation, motor control, and emergent cognitive function., Competing Interests: Conflict of Interest The authors declare that there are no commercial associations that might pose or create the appearance of a conflict of interest with the information presented in this manuscript., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Synaptotagmins belong to a large family of proteins. Although various synaptotagmins have been implicated as Ca 2+ sensors for vesicle replenishment and release at conventional synapses, their roles at retinal ribbon synapses remain incompletely understood. Zebrafish is a widely used experimental model for retinal research. We therefore investigated the homology between human, rat, mouse, and zebrafish synaptotagmins 1-10 using a bioinformatics approach. We also characterized the expression and distribution of various synaptotagmin (syt) genes in the zebrafish retina using RT-PCR, qPCR, and in situhybridization, focusing on the family members whose products likely underlie Ca 2+ -dependent exocytosis in the central nervous system (synaptotagmins 1, 2, 5, and 7). Most zebrafish synaptotagmins are well conserved and can be grouped in the same classes as mammalian synaptotagmins, based on crucial amino acid residues needed for coordinating Ca 2+ binding and determining phospholipid binding affinity. The only exception is synaptotagmin 1b, which lacks 34 amino acid residues in the C2B domain and is therefore unlikely to bind Ca 2+ there. Additionally, the products of zebrafish syt5a and syt5b genes share identity with mammalian class 1 and 5 synaptotagmins. Zebrafish syt1, syt2, syt5, and syt7 paralogues are found in the zebrafish brain, eye, and retina, excepting syt1b, which is only present in the brain. The complementary expression pattern of the remaining paralogues in the retina suggests that syt1a and syt5a may underlie synchronous release and syt7a and syt7b may mediate asynchronous release or other Ca 2+ -dependent processes in different retinal neurons., (© 2021 Wiley Periodicals LLC.)

Background: miR-34c has been found to be implicated in the pathological process of Alzheimer's disease, diabetes, and its complications., Objective: To investigate the underlying mechanisms of miR-34c in the pathogenesis of diabetic encephalopathy (DE)., Methods: Diabetes mellitus rats were developed by incorporating a high-fat diet and streptozotocin injection. Morris water maze test and novel object recognition test were used to assess the cognitive function of rats. Expression of miR-34c were detected by fluorescence in situ hybridization and qRT-PCR. Immunofluorescence and western blot were used to evaluate synaptotagmin 1 (SYT1) and AdipoR2 or other proteins. Golgi staining was performed to investigate dendritic spine density., Results: The increased miR-34c induced by advanced glycation end-products (AGEs) was mediated by ROS-JNK-p53 pathway, but not ROS-Rb-E2F1 pathway, in hippocampus of DE rats or in HT-22 cells. miR-34c negatively regulated the expression of SYT1, but not AdipoR2, in hippocampal neurons. miR-34c inhibitor rescued the AGE-induced decrease in the density of dendritic spines in primary hippocampal neurons. Administration of AM34c by the intranasal delivery increased the hippocampus levels of SYT1 and ameliorated the cognitive function in DE rats. The serum levels of miR-34c were increased in patients with DE comparing with normal controls., Conclusion: These results demonstrated that AGE-induced oxidative stress mediated increase of miR-34c through ROS-JNK-p53 pathway, resulting in synaptic deficits and cognitive decline by targeting SYT1 in DE, and the miR-34c/SYT1 axis could be considered as a novel therapeutic target for DE patients.

Alzheimer's disease (AD) is characterized by progressive synaptic dysfunction, deterioration of neuronal transmission, and consequently neuronal death. Although there is no treatment for AD, exposure to enriched environment (EE) in mice, as well as physical and mental activity in human subjects have been shown to have a protective effect by slowing the disease's progression and reducing AD-like cognitive impairment. However, the molecular mechanism of this mitigating effect is still not understood. One of the mechanisms that has recently been shown to be involved in neuronal degeneration is microRNAs (miRNAs) regulation, which act as a post-transcriptional regulators of gene expression. miR-128 has been shown to be significantly altered in individuals with AD and in mice following exposure to EE. Here, we focused on elucidating the possible role of miR-128 in AD pathology and found that miR-128 regulates the expression of two proteins essential for synaptic transmission, SNAP-25, and synaptotagmin1 (Syt1). Clinically relevant, in 5xFAD mouse model for AD, this miRNA's expression was found as downregulated, resembling the alteration found in the hippocampi of individuals with AD. Interestingly, exposing WT mice to EE also resulted in downregulation of miR-128 expression levels, although EE and AD conditions demonstrate opposing effects on neuronal functioning and synaptic plasticity. We also found that miR-128 expression downregulation in primary hippocampal cultures from 5xFAD mice results in increased neuronal network activity and neuronal excitability. Altogether, our findings place miR-128 as a synaptic player that may contribute to synaptic functioning and plasticity through regulation of synaptic protein expression and function., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER-PM communication in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca 2+ -dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER., (© 2021 Benavente et al.)

All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered release of neurotransmitters and hormones. Expansion of this small pore to release cargo is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here, we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins. Pore dilation relied on calcium-induced insertion of the tandem C2 domain hydrophobic loops of Syt1 into the membrane, previously shown to reorient the C2 domain. Mathematical modelling suggests that C2B reorientation rotates a bound SNARE complex so that it exerts force on the membranes in a mechanical lever action that increases the height of the fusion pore, provoking pore dilation to offset the bending energy penalty. We conclude that Syt1 exerts novel non-local calcium-dependent mechanical forces on fusion pores that dilate pores and assist neurotransmitter and hormone release., Competing Interests: ZW, ND, ZM, ST, BO, EK No competing interests declared, (© 2021, Wu et al.)

Background: Migraine is a multifactorial disorder that is more frequent (two to four times) in women than in men. In recent years, our research group has focused on the role of neurotransmitter release and its regulation. Neurexin (NRXN2) is one of the components of the synaptic vesicle machinery, responsible for connecting intracellular fusion proteins and synaptic vesicles. Our aim was to continue exploring the role and interaction of proteins involved in the control and promotion of neurotransmission in migraine susceptibility., Methods: A case-control study was performed comprising 183 migraineurs (148 females and 35 males) and 265 migraine-free controls (202 females and 63 males). Tagging single nucleotide polymorphisms of NRXN2 were genotyped to assess the association between NRXN2 and migraine susceptibility. The χ 2 test was used to compare allele frequencies in cases and controls and odds ratios were estimated with 95% confidence intervals. Haplotype frequencies were compared between groups. Gene-gene interactions were analysed using the Multifactor Dimensionality Reduction v2.0., Results: We found a statistically significant interaction model (p = 0.009) in the female group between the genotypes CG of rs477138 (NRXN2) and CT of rs1158605 (GABRE). This interaction was validated by logistic regression, showing a significant risk effect [OR = 4.78 (95%CI: 1.76-12.97)] after a Bonferroni correction. Our data also supports a statistically significant interaction model (p = 0.011) in the female group between the GG of rs477138 in NRXN2 and, the rs2244325's GG genotype and rs2998250's CC genotype of CASK. This interaction was also validated by logistic regression, with a protective effect [OR = 0.08 (95%CI: 0.01-0.75)]. A weak interaction model was found between NRXN2-SYT1. We have not found any statistically significant allelic or haplotypic associations between NRXN2 and migraine susceptibility., Conclusions: This study unravels, for the first time, the gene-gene interactions between NRXN2, GABRE - a GABA A -receptor - and CASK, importantly it shows the synergetic effect between those genes and its relation with migraine susceptibility. These gene interactions, which may be a part of a larger network, can potentially help us in better understanding migraine aetiology and in development of new therapeutic approaches.

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., Competing Interests: The authors declare no competing interest.

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca 2+ and a number of Ca 2+ sensors including synaptotagmins (Syts) and Double C 2 domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C 2 domain architecture, but are functionally diverse in their subcellular location, Ca 2+ -binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca 2+ -sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca 2+ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca 2+ sensors that may be implicated in the spontaneous release of glutamate., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Epilepsy is accompanied by abnormal neurotransmission, and microRNAs, as versatile players in the modulation of gene expression, are important in epilepsy pathology. Here, we found that miR-128 expression was elevated in the acute seizure phase and decreased during the recurrent seizure phase after status epilepticus in mice. Both SNAP-25 and SYT1 are regulated by miR-128 in vitro and in vivo. Overexpressing miR-128 in cultured neurons decreased neurotransmitter released by suppressing SNAP-25 and SYT1 expression. Anti-miR-128 injection before kainic acid (KA) injection increased the sensitivity of mice to KA-induced seizures, while overexpressing miR-128 at the latent and recurrent phases had a neuroprotective effect in KA-induced seizures. Our study shows for the first time that miR-128, a key regulator of neurotransmission, plays an important role in epilepsy pathology and that miR-128 might be a potential candidate molecular target for epilepsy therapy., Competing Interests: Declaration of competing interest We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is a progressive neurodegenerative disease affecting the ventral midbrain dopaminergic neurons, resulting in motor defects mainly tremor, rigidity, and bradykinesia along with a wide array of non-motor symptoms. The current study is focused on determining the potential druggable targets of PD by consolidating gene expression profiling and network methodology. Initially, the differentially expressed genes were established from which the central network was constructed by assimilating the interacting partners. Investigating the topological parameters of the network, the genes SYT1, CXCR4, CDC42, KIT, RET, DRD2, NTN1, PRKACB, KDR, NR4A2, SLC18A2, CCK, TH, KCNJ6, and TAC1 were identified as the hub genes and can be explored as potential candidate genes for PD therapeutics. Gene ontology and cluster analysis of the hub genes has provided further insights about the pathophysiology of the disease. Among the hub genes, PRKACB is observed in relatively all the enriched pathways which are modulated by G protein-coupled receptors through protein kinases. Further, we noticed SYT1 as a novel biomarker for PD. Moreover, the regulatory network was constructed with the hub genes as seed nodes with associated transcription factors (TFs) and microRNA (miRNAs). In this analysis, we identified MYC as the major TF and the miRNAs miR-21, miR-155, miR-7, and miR26A1 have a significant role in modulating the hub genes. Briefly, these significant hub genes and their enriched pathways, TFs, and miRNAs have aided in the better understanding of molecular mechanisms underlying PD and its potential core target genes.

Background: Alcohol addiction is characterized by persistent neuroadaptations in brain structures involved in motivation, emotion, and decision making, including the medial prefrontal cortex, the nucleus accumbens, and the amygdala. We previously reported that induction of alcohol dependence was associated with long-term changes in the expression of genes involved in neurotransmitter release. Specifically, Syt1, which plays a key role in neurotransmitter release and neuronal functions, was downregulated. Here, we therefore examined the role of Syt1 in alcohol-associated behaviors in rats., Methods: We evaluated the effect of Syt1 downregulation using an adeno-associated virus (AAV) containing a short hairpin RNA against Syt1. Cre-dependent Syt1 was also used in combination with an rAAV2 retro-Cre virus to assess circuit-specific effects of Syt1 knockdown (KD)., Results: Alcohol-induced downregulation of Syt1 is specific to the prelimbic cortex (PL), and KD of Syt1 in the PL resulted in escalated alcohol consumption, increased motivation to consume alcohol, and increased alcohol drinking despite negative consequences ("compulsivity"). Syt1 KD in the PL altered the excitation/inhibition balance in the basolateral amygdala, while the nucleus accumbens core was unaffected. Accordingly, a projection-specific Syt1 KD in the PL-basolateral amygdala projection was sufficient to increase compulsive alcohol drinking, while a KD of Syt1 restricted to PL-nucleus accumbens core projecting neurons had no effect on tested alcohol-related behaviors., Conclusions: Together, these data suggest that dysregulation of Syt1 is an important mechanism in long-term neuroadaptations observed after a history of alcohol dependence, and that Syt1 regulates alcohol-related behaviors in part by affecting a PL-basolateral amygdala brain circuit., (Copyright © 2020 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca 2+ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca 2+ -dependent liposome fusion between synaptobrevin liposomes and syntaxin-1-SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca 2+ binding to the two C 2 domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca 2+ -dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca 2+ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons., Competing Interests: The authors declare no competing interest.

Background and Aims: The molecular mechanisms by which the liver develops steatotic disease still remain unclear. Previous studies using nutritional and genetic models of hepatic steatosis in mice showed that liver synaptotagmin 1 (Syt1) expression was associated with lipid droplet area. Hepatic Syt1 overexpression was used as a tool to explore its effect on hepatic and plasma lipids., Methods and Results: To find out a cause-effect, hepatic mouse Syt1 mRNA was cloned into a vector driving hepatocyte-specific expression and administered by hydrodynamic injection to male Apoe-deficient mice fed on a Western diet, the latter as a model of rapid spontaneous steatosis development. Hepatic microsomal, large vesicle, lysosomal and plasma membrane fractions were enriched in SYT1 protein following gene overexpression. In these conditions, very low density lipoprotein esterified cholesterol increased. Likewise, the transgene caused an alteration in lipid droplet surface and a positive correlation between Syt1 expression and hepatic total cholesterol content. A lipidomic approach evidenced a decrease in lysophosphatidylcholine, phosphatidylcholine and triglycerides in isolated plasma membrane fraction. Expressions of genes involved in biosynthesis of bile acids, fatty acid metabolism, lipoprotein dynamics and vesicular transport were modified by the increased SYT1 expression., Conclusions: These results indicate that this protein is involved in hepatic management of lipids and in the regulation of genes involved in lipid metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

At neuronal synapses, synaptotagmin-1 (syt1) acts as a Ca 2+ sensor that synchronizes neurotransmitter release with Ca 2+ influx during action potential firing. Heterozygous missense mutations in syt1 have recently been associated with a severe but heterogeneous developmental syndrome, termed syt1-associated neurodevelopmental disorder. Well-defined pathogenic mechanisms, and the basis for phenotypic heterogeneity in this disorder, remain unknown. Here, we report the clinical, physiological, and biophysical characterization of three syt1 mutations from human patients. Synaptic transmission was impaired in neurons expressing mutant variants, which demonstrated potent, graded dominant-negative effects. Biophysical interrogation of the mutant variants revealed novel mechanistic features concerning the cooperative action, and functional specialization, of the tandem Ca 2+ -sensing domains of syt1. These mechanistic studies led to the discovery that a clinically approved K + channel antagonist is able to rescue the dominant-negative heterozygous phenotype. Our results establish a molecular cause, basis for phenotypic heterogeneity, and potential treatment approach for syt1-associated neurodevelopmental disorder., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: The quest for reliable fluid biomarkers tracking synaptic disruption is supported by the evidence of a tight association between synaptic density and cognitive performance in neurodegenerative diseases (NDD), especially Alzheimer's disease (AD)., Areas Covered: Neurogranin (Ng) is a post-synaptic protein largely expressed in neurons involved in the memory networks. Currently, Ng measured in CSF is the most promising synaptic biomarker. Several studies show Ng elevated in AD dementia with a hippocampal phenotype as well as in MCI individuals who progress to AD. Ng concentrations are also increased in Creutzfeldt Jacob Disease where widespread and massive synaptic disintegration takes place. Ng does not discriminate Parkinson's disease from atypical parkinsonisms, nor is it altered in Huntington disease. CSF synaptosomal-associated protein 25 (SNAP-25) and synaptotagmin-1 (SYT-1) are emerging candidates., Expert Opinion: CSF Ng revealed a role as a diagnostic and prognostic biomarker in NDD. Ng increase seems to be very specific for typical AD phenotype, probably for a prevalent hippocampal involvement. Synaptic biomarkers may serve different context-of-use in AD and other NDD including prognosis, diagnosis, and tracking synaptic damage - a critical pathophysiological mechanism in NDD - thus representing reliable tools for a precision medicine-oriented approach to NDD.

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca 2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1., Competing Interests: JV, EC No competing interests declared, (© 2020, Vevea and Chapman.)

The epilepsy-linked gene SV2A , has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity. SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an individual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity., (Copyright © 2020 the authors.)

Dopamine powerfully controls neural circuits through neuromodulation. In the vertebrate striatum, dopamine adjusts cellular functions to regulate behaviors across broad time scales, but how the dopamine secretory system is built to support fast and slow neuromodulation is not known. Here, we set out to identify Ca 2+ -triggering mechanisms for dopamine release. We find that synchronous dopamine secretion is abolished in acute brain slices of conditional knockout mice in which Synaptotagmin-1 is removed from dopamine neurons. This indicates that Synaptotagmin-1 is the Ca 2+ sensor for fast dopamine release. Remarkably, dopamine release induced by strong depolarization and asynchronous release during stimulus trains are unaffected by Synaptotagmin-1 knockout. Microdialysis further reveals that these modes and action potential-independent release provide significant amounts of extracellular dopamine in vivo. We propose that the molecular machinery for dopamine secretion has evolved to support fast and slow signaling modes, with fast release requiring the Ca 2+ sensor Synaptotagmin-1., Competing Interests: AB, JL, PN, CL, PK No competing interests declared, (© 2020, Banerjee et al.)

Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo., Competing Interests: The authors have declared that no competing interests exist.

Calcium (Ca 2+ )-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca 2+ -influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca 2+ -regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of 'central' SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free 'peripheral' SNAREpins to produce a stably clamped vesicle. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca 2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles., Competing Interests: SR, MB, JC, JR, SK No competing interests declared, (© 2020, Ramakrishnan et al.)

Synaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.

Regulated secretion is controlled by Ca 2+ sensors with different affinities and subcellular distributions. Inactivation of Syt1 (synaptotagmin-1), the main Ca 2+ sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca 2+ 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 Ca 2+ concentrations. Multiple Ca 2+ 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 Ca 2+ 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., (Copyright © 2020 the authors.)

Synaptotagmin 1 (Syt1) synchronizes neurotransmitter release to action potentials (APs) acting as the fast Ca 2+ release sensor and as the inhibitor (clamp) of spontaneous and delayed asynchronous release. While the Syt1 Ca 2+ activation mechanism has been well-characterized, how Syt1 clamps transmitter release remains enigmatic. Here we show that C2B domain-dependent oligomerization provides the molecular basis for the Syt1 clamping function. This follows from the investigation of a designed mutation (F349A), which selectively destabilizes Syt1 oligomerization. Using a combination of fluorescence imaging and electrophysiology in neocortical synapses, we show that Syt1 F349A is more efficient than wild-type Syt1 (Syt1 WT ) in triggering synchronous transmitter release but fails to clamp spontaneous and synaptotagmin 7 (Syt7)-mediated asynchronous release components both in rescue (Syt1 -/- knockout background) and dominant-interference (Syt1 +/+ background) conditions. Thus, we conclude that Ca 2+ -sensitive Syt1 oligomers, acting as an exocytosis clamp, are critical for maintaining the balance among the different modes of neurotransmitter release., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Numerous studies have verified that electroacupuncture (EA) can relieve neuropathic pain through a variety of mechanisms. Synaptotagmin 1 (Syt-1), a synaptic vesicle protein for regulating exocytosis of neurotransmitters, was found to be affected by EA stimulation. However, the roles of Syt-1 in neuropathic pain and EA-induced analgesic effect remain unclear. Here, the effect of Syt-1 on nociception was assessed through an antibody blockade, siRNA silencing, and lentivirus-mediated overexpression of spinal Syt-1 in rats with spared nerve injury (SNI). EA was used for stimulating bilateral "Sanjinjiao" and "Zusanli" acupoints of the SNI rats to evaluate its effect on nociceptive thresholds and spinal Syt-1 expression. The mechanically and thermally nociceptive behaviors were assessed with paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different temperatures, respectively, at day 0, 7, 8, 14, and 20. Syt-1 mRNA and protein levels were determined with qRT-PCR and Western blot, respectively, and its distribution was observed with the immunohistochemistry method. The results demonstrated Syt-1 antibody blockade and siRNA silencing increased ipsilateral PWTs and PWLs of SNI rats, while Syt-1 overexpression decreased ipsilateral PWTs and PWLs of rats. EA significantly attenuated nociceptive behaviors and down-regulated spinal Syt-1 protein levels (especially in laminae I-II), which were reversed by Syt-1 overexpression. Our findings firstly indicate that Syt-1 is involved in the development of neuropathic pain and that EA attenuates neuropathic pain, probably through suppressing Syt-1 protein expression in the spinal cord.

The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca 2+ -sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex 'stuttering' mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.

Phosphatidylserine (PS), a negatively charged phospholipid present predominantly at the inner leaflet of the plasma membrane, has been widely implicated in many cellular processes including membrane trafficking. Along this line, PS has been demonstrated to be important for endocytosis, however, the involved mechanisms remain uncertain. By monitoring clathrin-mediated endocytosis (CME) of single vesicles in mouse chromaffin cells using cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate in the present study that the fission-pore duration is reduced by PS addition, indicating a stimulatory role of PS in regulating the dynamics of vesicle fission during CME. Furthermore, our results show that the PS-mediated effect on the fission-pore duration is Ca 2+ -dependent and abolished in the absence of synaptotagmin 1 (Syt1), implying that Syt1 is necessary for the stimulatory role of PS in vesicle fission during CME. Consistently, a Syt1 mutant with a defective PS-Syt1 interaction increases the fission-pore duration. Taken together, our study suggests that PS-Syt1 interaction may be critical in regulating fission dynamics during CME., (© 2019 International Society for Neurochemistry.)