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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.