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The ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle resupply to enable fast and sustained release rates. However, the molecular mechanisms of these physiological activities remain unelucidated. Previous studies showed that the RAB‐specific GTPase‐activating protein TBC1D24 controls the endosomal trafficking of the synaptic vesicles (SVs) in Drosophila and mammalian neurons, and mutations in TBC1D24 may lead to non‐syndromic hearing loss or hearing loss associated with the DOORS syndrome in humans. In this study, we generated a knock‐in mouse model for the p. S178L mutation in TBC1D24, which leads to autosomal dominant non‐syndromic hearing loss (DFNA65). The p.S178L mutant mice show mild hearing loss and progressively declined wave I amplitude of the auditory brainstem responses. Despite the normal gross and cellular morphology of the cochlea, transmission electron microscopy reveals accumulation of endosome‐like vacuoles and a lower‐than‐normal number of SVs directly associated with the ribbons in the IHCs. Consistently, patch clamp of the IHCs shows reduced exocytosis under prolonged stimulus. ARF6, a TBC1D24‐interacting protein also involved in endosomal membrane trafficking, was underexpressed in the cochleae of the mutant mouse and has weakened in vitro interaction with the p.S178L mutant TBC1D24. Our results suggest an important role of TBC1D24 in maintaining endosomal‐mediated vesicle recycling and sustained exocytosis of hair cell ribbon synapses. [ABSTRACT FROM AUTHOR]

Synaptic-vesicle (SV) recruitment is thought to maintain reliable neurotransmitter release during high-frequency signaling. However, the mechanism underlying the SV reloading for sustained neurotransmission at central synapses remains unknown. To elucidate this, we performed direct observations of SV reloading and mobility at a single-vesicle level near the plasma membrane in cerebellar mossy fiber terminals using total internal reflection fluorescence microscopy, together with simultaneous recordings of membrane fusion by capacitance measurements. We found that actin disruption abolished the rapid SV recruitment and reduced sustained release. In contrast, induction of actin polymerization and stabilization did not affect vesicle recruitment and release, suggesting that the presence of actin filaments, rather than actin dynamics, was required for the rapid recruitment. Single-particle tracking experiments of quantum dot-labeled vesicles, which allows nanoscale resolution of vesicle mobility, revealed that actin disruption caused vesicles to diffuse more rapidly. Hidden Markov modeling with Bayesian inference revealed that SVs had two diffusion states under normal conditions: free-diffusing and trapped. After disruption of the actin filament, vesicles tended to have only the free-diffusing state. F-actin staining showed that actin filaments were localized outside the active zones (AZs) and surrounded some SV trajectories. Perturbation of SV mobility, possibly through interference with biomolecular condensates, also suggested that the restricted diffusion state determined the rate of SV recruitment. We propose that actin filaments confined SVs near the AZ to achieve rapid and efficient recruitment followed by priming and sustained synaptic transmission. [ABSTRACT FROM AUTHOR]

Monoamine neurotransmitters generated by de novo synthesis are rapidly transported and stored into synaptic vesicles at axon terminals. This transport is essential both for sustaining synaptic transmission and for limiting the toxic effects of monoamines. Here, synthesis of the monoamine histamine by histidine decarboxylase (HDC) and subsequent loading of histamine into synaptic vesicles are shown to be physically and functionally coupled within Drosophila photoreceptor terminals. This process requires HDC anchoring to synaptic vesicles via interactions with N-ethylmaleimide-sensitive fusion protein 1 (NSF1). Disassociating HDC from synaptic vesicles disrupts visual synaptic transmission and causes somatic accumulation of histamine, which leads to retinal degeneration. We further identified a proteasome degradation system mediated by the E3 ubiquitin ligase, purity of essence (POE), which clears mislocalized HDC from the soma, thus eliminating the cytotoxic effects of histamine. Taken together, our results reveal a dual mechanism for translocation and degradation of HDC that ensures restriction of histamine synthesis to axonal terminals and at the same time rapid loading into synaptic vesicles. This is crucial for sustaining neurotransmission and protecting against cytotoxic monoamines. Synopsis: Neurotransmitters are key for synaptic transmission at axon terminals, but their transport modalities remain debated. This study demonstrates that coupling of histamine synthesis with its loading into synaptic vesicles by anchoring histidine decarboxylase (HDC) to synaptic vesicles is critical to maintaining neurotransmission and preventing cytotoxic side effects. Coupling histamine synthesis with synaptic vesicle loading is critical for neurotransmitter homeostasis and synaptic transmission. HDC is co-transported with synaptic vesicles to axonal terminals through binding with vesicle-fusing ATPase NSF1. HDC remaining in soma is degraded by the E3 ubiquitin ligase POE via the proteasome. Aberrant accumulation of histamine in the soma is cytotoxic and causes retinal degeneration. Anchoring of histidine decarboxylase to synaptic vesicles is critical to maintaining neurotransmission and preventing cytotoxic side effects. [ABSTRACT FROM AUTHOR]

Communication within the nervous system relies on the calcium‐triggered release of neurotransmitter molecules by exocytosis of synaptic vesicles (SVs) at defined active zone release sites. While decades of research have provided detailed insight into the molecular machinery for SV fusion, much less is known about the mechanisms that form functional SVs during the development of synapses and that control local SV reformation following exocytosis in the mature nervous system. Here we review the current state of knowledge in the field, focusing on the pathways implicated in the formation and axonal transport of SV precursor organelles and the mechanisms involved in the local reformation of SVs within nerve terminals in mature neurons. We discuss open questions and outline perspectives for future research. [ABSTRACT FROM AUTHOR]

Ribbon synapses of inner hair cells (IHCs) are uniquely designed for ultrafast and indefatigable neurotransmission of the sound. The molecular machinery ensuring the efficient, compensatory recycling of the synaptic vesicles (SVs), however, remains elusive. This study showed that hair cell knock-out of murine Dmxl2, whose human homolog is responsible for nonsyndromic sensorineural hearing loss DFNA71, resulted in auditory synaptopathy by impairing synaptic endocytosis and recycling. The mutant mice in the C57BL/6J background of either sex had mild hearing loss with severely diminished wave I amplitude of the auditory brainstem response. Membrane capacitance measurements of the IHCs revealed deficiency in sustained synaptic exocytosis and endocytic membrane retrieval. Consistent with the electrophysiological findings, 3D electron microscopy reconstruction showed reduced reserve pool of SVs and endocytic compartments, while the membrane-proximal and ribbon-associated vesicles remain intact. Our results propose an important role of DMXL2 in hair cell endocytosis and recycling of the SVs. [ABSTRACT FROM AUTHOR]

The presynapse performs an essential role in brain communication via the activity‐dependent release of neurotransmitters. However, the sequence of events through which a presynapse acquires functionality is relatively poorly understood, which is surprising, since mutations in genes essential for its operation are heavily implicated in neurodevelopmental disorders. We addressed this gap in knowledge by determining the developmental trajectory of synaptic vesicle (SV) recycling pathways in primary cultures of rat hippocampal neurons. Exploiting a series of optical and morphological assays, we revealed that the majority of nerve terminals displayed activity‐dependent calcium influx from 3 days in vitro (DIV), immediately followed by functional evoked exocytosis and endocytosis, although the number of responsive nerve terminals continued to increase until the second week in vitro. However, the most intriguing discovery was that activity‐dependent bulk endocytosis (ADBE) was only observed from DIV 14 onwards. Importantly, optimal ADBE recruitment was delayed until DIV 21 in Fmr1 knockout neurons, which model Fragile X Syndrome (FXS). This implicates the delayed recruitment of ADBE as a potential contributing factor in the development of circuit dysfunction in FXS, and potentially other neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Age-related impairment of the diaphragm causes respiratory complications. Neuromuscular junction (NMJ) dysfunction can be one of the triggering events in diaphragm weaknesses in old age. Prominent structural and functional alterations in diaphragm NMJs were described in elderly rodents, but NMJ changes in middle age remain unclear. Here, we compared diaphragm muscles from young adult (3 months) and middle-aged (12 months) BALB/c mice. Microelectrode recordings, immunofluorescent staining, electron microscopy, myography, and whole-body plethysmography were used. We revealed presynaptic (i) and postsynaptic (ii) changes. The former (i) included an increase in both action potential propagation velocity and neurotransmitter release evoked by low-, moderate-, and high-frequency activity but a decrease in immunoexpression of synapsin 1 and synaptic vesicle clustering. The latter (ii) consisted of a decrease in currents via nicotinic acetylcholine receptors and the area of their distribution. These NMJ changes correlated with increased contractile responses to moderate- to high-frequency nerve activation. Additionally, we found alterations in the pattern of respiration (an increase in peak inspiratory flow and a tendency of elevation of the tidal volume), which imply increased diaphragm activity in middle-aged mice. We conclude that enhancement of neuromuscular communication (due to presynaptic mechanism) accompanied by improved contractile responses occurs in the diaphragm in early aging. [ABSTRACT FROM AUTHOR]

Members of the synaptophysin and synaptogyrin family are vesicle proteins with four transmembrane domains. In spite of their abundance in synaptic vesicle (SV) membranes, their role remains elusive and only mild defects at the cellular and organismal level are observed in mice lacking one or more family members. Here, we show that coexpression with synapsin in fibroblasts of each of the four brain-enriched members of this family--synaptophysin, synaptoporin, synaptogyrin 1, and synaptogyrin 3--is sufficient to generate clusters of small vesicles in the same size range of SVs. Moreover, mice lacking all these four proteins have larger SVs. We conclude that synaptophysin and synaptogyrin family proteins play an overlapping function in the biogenesis of SVs and in determining their small size. [ABSTRACT FROM AUTHOR]

Neurons are highly polarized cells that are composed of a single axon and multiple dendrites. Axon-dendrite polarity is essential for proper tissue formation and brain functions. Intracellular protein transport plays an important role in the establishment of neuronal polarity. However, the regulatory mechanism of polarized transport remains unclear. Here, we show that Rab6, a small GTPase that acts on the regulation of intracellular vesicular trafficking, plays key roles in neuronal polarization and brain development. Central nervous system-specific Rab6a/b double knock-out (Rab6 DKO) mice of both sexes exhibit severe dysplasia of the neocortex and the cerebellum. In the Rab6 DKO neocortex, impaired axonal extension of neurons results in hypoplasia of the intermediate zone. In vitro, deletion of Rab6a and Rab6b in cultured neurons from both sexes causes the abnormal accumulation of synaptic vesicle precursors (SVPs) adjacent to the Golgi apparatus, which leads to defects in axonal extension and the loss of axon-dendrite polarity. Moreover, Rab6 DKO causes significant expansion of lysosomes in the soma in neurons. Overall, our results reveal that Rab6- mediated polarized transport of SVPs is crucial for neuronal polarization and subsequent brain formation. [ABSTRACT FROM AUTHOR]

Frontotemporal dementia and amyotrophic lateral sclerosis are common forms of neurodegenerative disease that share overlapping genetics and pathologies. Crucially, no significantly disease-modifying treatments are available for either disease. Identifying the earliest changes that initiate neuronal dysfunction is important for designing effective intervention therapeutics. The genes mutated in genetic forms of frontotemporal dementia and amyotrophic lateral sclerosis have diverse cellular functions, and multiple disease mechanisms have been proposed for both. Identification of a convergent disease mechanism in frontotemporal dementia and amyotrophic lateral sclerosis would focus research for a targetable pathway, which could potentially effectively treat all forms of frontotemporal dementia and amyotrophic lateral sclerosis (both familial and sporadic). Synaptopathies are diseases resulting from physiological dysfunction of synapses, and define the earliest stages in multiple neuronal diseases, with synapse loss a key feature in dementia. At the presynapse, the process of synaptic vesicle recruitment, fusion and recycling is necessary for activity-dependent neurotransmitter release. The unique distal location of the presynaptic terminal means the tight spatio-temporal control of presynaptic homeostasis is dependent on efficient local protein translation and degradation. Recently, numerous publications have shown that mutations associated with frontotemporal dementia and amyotrophic lateral sclerosis present with synaptopathy characterized by presynaptic dysfunction. This review will describe the complex local signalling and membrane trafficking events that occur at the presynapse to facilitate neurotransmission and will summarize recent publications linking frontotemporal dementia/amyotrophic lateral sclerosis genetic mutations to presynaptic function. This evidence indicates that presynaptic synaptopathy is an early and convergent event in frontotemporal dementia and amyotrophic lateral sclerosis and illustrates the need for further research in this area, to identify potential therapeutic targets with the ability to impact this convergent pathomechanism. [ABSTRACT FROM AUTHOR]

Rotenone is a pesticide used in research for its ability to induce changes similar, in vivo and in vitro, to those observed in Parkinson's disease (PD). This includes a selective death of dopaminergic neurons in the substantia nigra. Nonetheless, the precise mechanism through which rotenone modifies structure and function of neurons remains unclear. The PC12 cells closely resemble dopamine terminal neurons. This makes it a preferred model for studying the morphology of central dopamine neurons and predicting neurotoxicity. In this paper, we investigated the effects of 0.5 µM rotenone for 24–48 h on PC12 cell viability and ultrastructure (TEM), trying to identify primary and more evident alterations that can be related to neuronal damages similar to that seen in animal PD models. Cell viability decreased after 24 h rotenone treatment, with a further decrease after 48 h. Ultrastructural changes included vacuolar degeneration, mitochondrial mild swelling, decrease in the number of neuropeptide granules, and the loss of cell-to-cell adhesion. These findings are in agreement with previous research suggesting that rotenone, by inhibiting energy production and increasing ROS generation, is responsible for significant alterations of the ultrastructure and cell death of PC12 cells. Our data confirm the link between rotenone exposure, neuronal damage, and changes in dopamine metabolism, suggesting its role in the pathogenesis of PD. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is an age‐related chronic neurological disorder, mainly characterized by the pathological feature of α‐synuclein (α‐syn) aggregation, with the exact disease pathogenesis unclear. During the onset and progression of PD, synaptic dysfunction, including dysregulation of axonal transport, impaired exocytosis, and endocytosis are identified as crucial events of PD pathogenesis. It has been reported that over‐expression of α‐syn impairs clathrin‐mediated endocytosis (CME) in the synapses. However, the underlying mechanisms still needs to be explored. In this study, we investigated the molecular events underlying the synaptic dysfunction caused by over‐expression of wild‐type human α‐syn and its mutant form, involving series of proteins participating in CME. We found that excessive human α‐syn causes impaired fission and uncoating of clathrin‐coated vesicles during synaptic vesicle recycling, leading to reduced clustering of synaptic vesicles near the active zone and increased size of plasma membrane and number of endocytic intermediates. Furthermore, over‐expressed human α‐syn induced changes of CME‐associated proteins, among which synaptojanin1 (SYNJ1) showed significant reduction in various brain regions. Over‐expression of SYNJ1 in primary hippocampal neurons from α‐syn transgenic mice recovered the synaptic vesicle density, clustering and endocytosis. Using fluorescence‐conjugated transferrin, we demonstrated that SYNJ1 re‐boosted the CME activity by restoring the phosphatidylinositol‐4,5‐bisphosphate homeostasis. Our data suggested that over‐expression of α‐syn disrupts synaptic function through interfering with vesicle recycling, which could be alleviated by re‐availing of SYNJ1. Our study unrevealed a molecular mechanism of the synaptic dysfunction in PD pathogenesis and provided a potential therapeutic target for treating PD. [ABSTRACT FROM AUTHOR]

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function. [ABSTRACT FROM AUTHOR]

Munc13-1 is essential for vesicle docking and fusion at the active zone of synapses. Here, we report that Munc13-1 self-assembles into molecular clusters within diacylglycerol-rich microdomains present in phospholipid bilayers. Although the copy number of Munc13-1 molecules in these clusters has a broad distribution, a systematic Poisson analysis shows that this is most likely the result of two molecular species: monomers and mainly hexameric oligomers. Each oligomer is able to capture one vesicle independently. Hexamers have also been observed in crystals of Munc13-1 that form between opposed phospholipid bilayers [K. Grushin, R. V. Kalyana Sundaram, C. V. Sindelar, J. E. Rothman, Proc. Natl. Acad. Sci. U.S.A. 119, e2121259119 (2022)]. Mutations targeting the contacts stabilizing the crystallographic hexagons also disrupt the isolated hexamers, suggesting they are identical. Additionally, these mutations also convert vesicle binding from a cooperative to progressive mode. Our study provides an independent approach showing that Munc13-1 can form mainly hexamers on lipid bilayers each capable of vesicle capture. [ABSTRACT FROM AUTHOR]

Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca21-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca2+-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both in vitro and in vivo. Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals. [ABSTRACT FROM AUTHOR]

The multidomain adaptor protein amphiphysin‐1 (Amph1) is an important coordinator of clathrin‐mediated endocytosis in non‐neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid‐binding N‐BAR (Bin/Amphiphysin/Rvs) domain, central proline‐rich (PRD) and clathrin/AP2 (CLAP) domains, and a C‐terminal SH3 domain. Amph1 interacts with both lipids and proteins, with all of these interactions required for SV endocytosis, with the exception of the Amph1 PRD. The Amph1 PRD associates with the endocytosis protein endophilin A1, however, the role of this interaction in SV endocytosis has not been investigated. In this study, we set out to determine whether the Amph1 PRD and its interaction with endophilin A1 was essential for efficient SV endocytosis at typical small central synapses. To achieve this, domain‐specific interactions of Amph1 were validated using in vitro GST pull‐down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions of Amph1 in the control of SV endocytosis. Importantly, we identified the interaction site for endophilin A1 within the Amph1 PRD and exploited specific binding mutants to reveal a key role for this interaction in SV endocytosis. Finally, we determined that the formation of the Amph1‐endophilin A1 complex is dependent on the phosphorylation status of Amph1‐S293 within the PRD and that the phosphorylation status of this residue is essential for efficient SV regeneration. This work, therefore, reveals a key role for the dephosphorylation‐dependent Amph1‐endophilin A1 interaction in efficient SV endocytosis. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is manifested as skeletal muscle denervation, loss of motor neurons and finally severe respiratory failure. Mutations of RNA-binding protein FUS are one of the common genetic reasons of ALS accompanied by a 'dying back' type of degeneration. Using fluorescent approaches and microelectrode recordings, the early structural and functional alterations in diaphragm neuromuscular junctions (NMJs) were studied in mutant FUS mice at the pre-onset stage. Lipid peroxidation and decreased staining with a lipid raft marker were found in the mutant mice. Despite the preservation of the end-plate structure, immunolabeling revealed an increase in levels of presynaptic proteins, SNAP-25 and synapsin 1. The latter can restrain Ca2+-dependent synaptic vesicle mobilization. Indeed, neurotransmitter release upon intense nerve stimulation and its recovery after tetanus and compensatory synaptic vesicle endocytosis were markedly depressed in FUS mice. There was a trend to attenuation of axonal [Ca2+]in increase upon nerve stimulation at 20 Hz. However, no changes in neurotransmitter release and the intraterminal Ca2+ transient in response to low frequency stimulation or in quantal content and the synchrony of neurotransmitter release at low levels of external Ca2+ were detected. At a later stage, shrinking and fragmentation of end plates together with a decrease in presynaptic protein expression and disturbance of the neurotransmitter release timing occurred. Overall, suppression of synaptic vesicle exo–endocytosis upon intense activity probably due to alterations in membrane properties, synapsin 1 levels and Ca2+ kinetics could be an early sign of nascent NMJ pathology, which leads to neuromuscular contact disorganization. [ABSTRACT FROM AUTHOR]

Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43−/−), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43−/− mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy. [ABSTRACT FROM AUTHOR]

α-Synuclein carries multiple functions in neurotransmitter release at neuronal synapses. Its net impact on synaptic release is likely a balance between an inhibitory effect mediated by synaptic vesicle clustering, and a release-promoting effect mediated by SNARE-complex chaperoning and fusion pore opening. Pathologically, α-synuclein aggregates are a hallmark of synucleinopathies including Parkinson's disease. α-Synuclein dosage and disease outcome are strongly linked, which has led to the gain-of-toxic function hypothesis. However, loss-of-function of α-synuclein, through sequestering of physiological α-synuclein into aggregates, has recently gained attention as a potential pathogenic mechanism, supported by reduced functional α-synuclein in human PD tissue. Current therapeutic strategies have mostly aimed at reducing α-synuclein levels, with little benefit so far. If α-synuclein loss-of-function contributes to disease pathogenesis, these strategies may be even expected to worsen disease outcome. α-Synuclein is a neuronal protein that is enriched in presynaptic terminals. Under physiological conditions, it binds to synaptic vesicle membranes and functions in neurotransmitter release, although the molecular details remain unclear, and it is controversial whether α-synuclein inhibits or facilitates neurotransmitter release. Pathologically, in synucleinopathies including Parkinson's disease (PD), α-synuclein forms aggregates that recruit monomeric α-synuclein and spread throughout the brain, which triggers neuronal dysfunction at molecular, cellular, and organ levels. Here, we present an overview of the effects of α-synuclein on SNARE-complex assembly, neurotransmitter release, and synaptic vesicle pool homeostasis, and discuss how the observed divergent effects of α-synuclein on neurotransmitter release can be reconciled. We also discuss how gain-of-function versus loss-of-function of α-synuclein may contribute to pathogenesis in synucleinopathies. [ABSTRACT FROM AUTHOR]

Many synaptic vesicles undergo exocytosis in motor nerve terminals during neuromuscular communication. Endocytosis then recovers the synaptic vesicle pool and presynaptic membrane area. The kinetics of endocytosis may shape neuromuscular transmission, determining its long-term reliability. Here, using fluorescent dyes, the time course of endocytosis induced by intense activity of the phrenic nerve was studied at the mouse diaphragm neuromuscular junction. It was found that a significant portion of endocytic events occurs after the end of tetanic stimulation. Pitstop 2, clathrin inhibitor, and more profoundly dynole 34–2, dynamin antagonist, suppressed endocytic FM1-43 dye uptake both during and after tetanus. Furthermore, synaptic vesicles formed in the presence of the endocytic blockers released FM-dye during subsequent evoked exocytosis at a lower rate. 25-Hydroxycholesterol (25HC) is an oxysterol, ubiquitously synthetized from excessive cholesterol. In addition, its production greatly increases by activated macrophages. 25HC accelerated FM-dye endocytosis and its sequential evoked exocytosis, and dynole (but not pitstop) prevented 25HC-mediated enhancement of endocytic FM-dye uptake. The positive effects of 25HC were interfered with chelation of cytosolic Ca2+ with a slow Ca2+ buffer EGTA-AM, Ca2+ antagonist TMB8, and sphingomyelin-hydrolyzing enzyme. In contrast to amphiphilic FM1-43 dye capture, 25HC reduced uptake of hydrophilic high molecular weight markers (labeled dextrans and toxin), which utilize bulk endocytosis to enter into nerve terminals. Thus, synaptic vesicle endocytosis had a relatively slow kinetics following the tetanic activity and can be accelerated by 25HC. The positive effect of 25HC on endocytosis engages a dynamin-dependent pathway, interconnected with cytoplasmic Ca2+ and sphingomyelin integrity. [ABSTRACT FROM AUTHOR]

Joensuu and colleagues have recently shown that botulinum neurotoxin (BoNT) type A exploits a heterotrimeric complex in the presynaptic membrane to bind to and enter neurons using a Trojan horse-like mechanism. Similar processes may be relevant to the neuronal entry of different botulinum toxin serotypes and other neuropathogens. [ABSTRACT FROM AUTHOR]

ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating early endosome trafficking and maturation. It has been demonstrated that ALS2 forms a tetramer, and this oligomerization is essential for its GEF activity and endosomal localization in established cancer cells. However, despite that ALS2 deficiency is implicated in neurological diseases, neither the subcellular distribution of ALS2 nor the form of its complex in the central nervous system (CNS) has been investigated. In this study, we showed that ALS2 in the brain was enriched both in synaptosomal and cytosolic fractions, while those in the liver were almost exclusively present in cytosolic fraction by differential centrifugation. Gel filtration chromatography revealed that cytosolic ALS2 prepared both from the brain and liver formed a tetramer. Remarkably, synaptosomal ALS2 existed as a high-molecular weight complex in addition to a tetramer. Such complex was also observed not only in embryonic brain but also several neuronal and glial cultures, but not in fibroblast-derived cell lines. Thus, the high-molecular weight ALS2 complex represents a unique form of ALS2-homophilic oligomers in the CNS, which may play a role in the maintenance of neural function. [Display omitted] • ALS2 forms a unique homophilic high-molecular weight complex in synaptosomes. • ALS2 exists as a tetramer in cytosol in the brain and liver. • Formation of the ALS2 complex is independent of neural development stages. [ABSTRACT FROM AUTHOR]

Alpha-synuclein is a presynaptic protein linked to Parkinson's disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1. [ABSTRACT FROM AUTHOR]

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3‐phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2‐mediated hyperactivation of Cdk5 downstream of receptor‐ and activity‐dependent calcium influx. Our results unravel an unexpected function for PI(3)P‐containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P‐producing VPS34 kinase in neurological disease and neurodegeneration. Synopsis: Neural circuit function requires mechanisms for the control of neurotransmitter release. We Here, presynaptic vesicle cycling and neurotransmission are found to be feedback‐regulated by the endosomal lipid phosphatidylinositol 3‐phosphate (PI(3)P). Neuronal activity inhibits endosomal PI(3)P synthesis in neurons.Reduced PI(3)P levels repress neurotransmission and impair vesicle recycling.Loss of PI(3)P triggers calpain and Cdk5 activation, a key negative control switch for neurotransmission.PI(3)P loss prevents hyperactivity of neuronal networks independent of synaptic inhibition. [ABSTRACT FROM AUTHOR]

Glutamate is a principal neurotransmitter used extensively by the nervous systems of all vertebrate and invertebrate animals. It is primarily an excitatory neurotransmitter that has been implicated in nervous system development, as well as a myriad of brain functions from the simple transmission of information between neurons to more complex aspects of nervous system function including synaptic plasticity, learning, and memory. Identification of glutamatergic neurons and their sites of glutamate release are thus essential for understanding the mechanisms of neural circuit function and how information is processed to generate behavior. Here, we describe and characterize smFLAG-vGlut, a conditional marker of glutamatergic synaptic vesicles for the Drosophila model system. smFLAG-vGlut is validated for functionality, conditional expression, and specificity for glutamatergic neurons and synaptic vesicles. The utility of smFLAG-vGlut is demonstrated by glutamatergic neurotransmitter phenotyping of 26 different central complex neuron types of which nine were established to be glutamatergic. This illumination of glutamate neurotransmitter usage will enhance the modeling of central complex neural circuitry and thereby our understanding of information processing by this region of the fly brain. The use of smFLAG for glutamatergic neurotransmitter phenotyping and identification of glutamate release sites can be extended to any Drosophila neuron(s) represented by a binary transcription system driver. [ABSTRACT FROM AUTHOR]

Mutations in the ESCRT‐III subunit CHMP2B cause frontotemporal dementia (FTD) and lead to impaired endolysosomal trafficking and lysosomal storage pathology in neurons. We investigated the effect of mutant CHMP2B on synaptic pathology, as ESCRT function was recently implicated in the degradation of synaptic vesicle (SV) proteins. We report here that expression of C‐terminally truncated mutant CHMP2B results in a novel synaptopathy. This unique synaptic pathology is characterised by selective retention of presynaptic SV trafficking proteins in aged mutant CHMP2B transgenic mice, despite significant loss of postsynaptic proteins. Furthermore, ultrastructural analysis of primary cortical cultures from transgenic CHMP2B mice revealed a significant increase in the number of presynaptic endosomes, while neurons expressing mutant CHMP2B display defective SV recycling and alterations to functional SV pools. Therefore, we reveal how mutations in CHMP2B affect specific presynaptic proteins and SV recycling, identifying CHMP2B FTD as a novel synaptopathy. This novel synaptopathic mechanism of impaired SV physiology may be a key early event in multiple forms of FTD, since proteins that mediate the most common genetic forms of FTD all localise at the presynapse. [ABSTRACT FROM AUTHOR]

SV2A, an essential transporter‐like synaptic vesicle protein, is a major target for antiepileptic drugs and a receptor for clostridial neurotoxins including Botox. While SV2A is required for normal levels of evoked neurotransmitter release, the mechanism underlying this role remains unclear. Here, we introduce a new chemogenetic approach for all‐optical monitoring of excitation–secretion coupling, and we demonstrate its use in characterizing the SV2A knockout (KO) phenotype in cultured hippocampal neurons. This method employs the HaloTag system to target a robust small‐molecule Ca2+ indicator, JF646–BAPTA, to the presynaptic compartment. The far‐red fluorescence of this indicator enables multiplexing with the fluorescent glutamate sensor iGluSnFR for detection of presynaptic Ca2+ influx and glutamate release at the same axonal boutons. Evoked glutamate release probability was reduced in SV2A KO neurons without a change in presynaptic Ca2+ entry, suggesting that SV2A supports vesicle fusion by increasing the functional availability, or efficiency, of the Ca2+‐regulated membrane fusion machinery. Key points: One of the most prescribed antiepileptic medications, levetiracetam, acts by binding a protein of uncertain molecular function.This transporter‐like protein, SV2A, is trafficked to synaptic vesicles and acts to support neurotransmitter release, but the mechanism underlying this function has not been determinedIn this study, we sought to establish whether SV2A changes Ca2+ signalling at nerve terminals, which is a key regulatory system for synaptic vesicle exocytosis.To do so, we adapted new chemogenetic tools to perform all‐optical measurements of presynaptic Ca2+ and glutamate release in neurons lacking SV2A.Our measurements showed that loss of SV2A reduces glutamate release without reducing Ca2+ influx at hippocampal nerve terminals, demonstrating that SV2A increases the likelihood that Ca2+ will trigger synaptic vesicle fusion. [ABSTRACT FROM AUTHOR]

Background: The cellular and molecular mechanisms by which general anaesthesia occurs is poorly understood. Hippocampal interneurone subpopulations, which are critical regulators of cognitive function, have diverse neurophysiological and synaptic properties, but their responses to anaesthetics are unclear.Methods: We used live-cell imaging of fluorescent biosensors expressed in mouse hippocampal neurones to delineate interneurone subtype-specific effects of isoflurane on synaptic vesicle exocytosis. The role of voltage-gated sodium channel (Nav) subtype expression in determining isoflurane sensitivity was probed by overexpression or knockdown of specific Nav subtypes in identified interneurones.Results: Clinically relevant concentrations of isoflurane differentially inhibited synaptic vesicle exocytosis: to 83.1% (11.7%) of control in parvalbumin-expressing interneurones, and to 58.6% (13.3%) and 64.5% (8.5%) of control in somatostatin-expressing interneurones and glutamatergic neurones, respectively. The relative expression of Nav1.1 (associated with lower sensitivity) and Nav1.6 (associated with higher sensitivity) determined the sensitivity of exocytosis to isoflurane.Conclusions: Isoflurane inhibits synaptic vesicle exocytosis from hippocampal glutamatergic neurones and GABAergic interneurones in a cell-type-specific manner depending on their expression of voltage-gated sodium channel subtypes. [ABSTRACT FROM AUTHOR]

Fragile X mental retardation protein (FMRP) is a neuronal protein mediating multiple functions, with its absence resulting in one of the most common monogenic causes of autism, Fragile X syndrome (FXS). Analyses of FXS pathophysiology have identified a range of aberrations in synaptic signaling pathways and plasticity associated with group I metabotropic glutamate (mGlu) receptors. These studies, however, have mostly focused on the post‐synaptic functions of FMRP and mGlu receptor activation, and relatively little is known about their presynaptic effects. Neurotransmitter release is mediated via multiple forms of synaptic vesicle (SV) fusion, each of which contributes to specific neuronal functions. The impacts of mGlu receptor activation and loss of FMRP on these SV fusion events remain unexplored. Here we combined electrophysiological and fluorescence imaging analyses on primary hippocampal cultures prepared from an Fmr1 knockout (KO) rat model. Compared to wild‐type (WT) hippocampal neurons, KO neurons displayed an increase in the frequency of spontaneous excitatory post‐synaptic currents (sEPSCs), as well as spontaneous SV fusion events. Pharmacological activation of mGlu receptors in WT neurons caused a similar increase in spontaneous SV fusion and sEPSC frequency. Notably, this increase in SV fusion was not observed when spontaneous activity was blocked using the sodium channel antagonist tetrodotoxin. Importantly, the effect of mGlu receptor activation on spontaneous SV fusion was occluded in Fmr1 KO neurons. Together, our results reveal that FMRP represses spontaneous presynaptic SV fusion, whereas mGlu receptor activation increases this event. This reciprocal control appears to be mediated via their regulation of intrinsic neuronal excitability. [ABSTRACT FROM AUTHOR]

Munc13‐1 is a large banana‐shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest that multiple copies of Munc13‐1 form nano‐assemblies in active zones of neurons. However, it is not known whether such clustering of Munc13‐1 is correlated with multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13‐1 clustering is also unknown. Here, we report that nano‐clustering is an inherent property of Munc13‐1 and is indeed required for vesicle binding to bilayers containing Munc13‐1. Purified Munc13‐1 protein reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ˜ 20 copies as revealed by a combination of quantitative TIRF microscopy and step‐wise photobleaching. Surprisingly, only clusters containing a minimum of 6 copies of Munc13‐1 were capable of efficiently capturing and retaining small unilamellar vesicles. The C‐terminal C2C domain of Munc13‐1 is not required for Munc13‐1 clustering, but is required for efficient vesicle capture. This capture is largely due to a combination of electrostatic and hydrophobic interactions between the C2C domain and the vesicle membrane. [ABSTRACT FROM AUTHOR]

Recent studies show that alcohol exposure can induce glial production of neuroimmune factors in the CNS. Of these, IL-6 has gained attention because it is involved in a number of important physiological and pathophysiological processes that could be affected by alcohol-induced CNS production of IL-6, particularly under conditions of excessive alcohol use. For example, IL-6 has been shown to play a role in hippocampal behaviors and synaptic plasticity (long-term potentiation; LTP) associated with memory and learning. Surprisingly, in our in vitro studies of LTP at the Schaffer collateral to CA1 pyramidal neuron synapse in hippocampus from transgenic mice that express elevated levels of astrocyte produced IL-6 (TG), LTP was not altered by the increased levels of IL-6. However, exposure to acute alcohol revealed neuroadaptive changes that served to protect LTP against the alcohol-induced reduction of LTP observed in hippocampus from non-transgenic control mice (WT). Here we examined the induction phase of LTP to assess if presynaptic neuroadaptive changes occurred in the hippocampus of TG mice that contributed to the resistance of LTP to alcohol. Results are consistent with a role for IL-6-induced neuroadaptive effects on presynaptic mechanisms involved in transmitter release in the resistance of LTP to alcohol in hippocampus from the TG mice. These actions are important with respect to a role for IL-6 in physiological and pathophysiological processes in the CNS and in CNS actions of alcohol, especially when excessive alcohol used is comorbid with conditions associated with elevated levels of IL-6 in the CNS. [ABSTRACT FROM AUTHOR]

In Caenorhabditis elegans, the cha-1 gene encodes choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine. We have analyzed a large number of cha-1 hypomorphic mutants, most of which are missense alleles. Some homozygous cha-1 mutants have approximately normal ChAT immunoreactivity; many other alleles lead to consistent reductions in synaptic immunostaining, although the residual protein appears to be stable. Regardless of protein levels, neuromuscular function of almost all mutants is temperature-sensitive, i.e., neuromuscular function is worse at 25° than at 14°. We show that the temperature effects are not related to acetylcholine release, but specifically to alterations in acetylcholine synthesis. This is not a temperature-dependent developmental phenotype, because animals raised at 20° to young adulthood and then shifted for 2 h to either 14° or 25° had swimming and pharyngeal pumping rates similar to animals grown and assayed at either 14° or 25°, respectively. We also show that the temperature-sensitive phenotypes are not limited to missense alleles; rather, they are a property of most or all severe cha-1 hypomorphs. We suggest that our data are consistent with a model of ChAT protein physically, but not covalently, associated with synaptic vesicles; and there is a temperature-dependent equilibrium between vesicle-associated and cytoplasmic (i.e., soluble) ChAT. Presumably, in severe cha-1 hypomorphs, increasing the temperature would promote dissociation of some of the mutant ChAT protein from synaptic vesicles, thus removing the site of acetylcholine synthesis (ChAT) from the site of vesicular acetylcholine transport. This, in turn, would decrease the rate and extent of vesicle-filling, thus increasing the severity of the behavioral deficits. [ABSTRACT FROM AUTHOR]

Background: A recently published East Asian genome‐wide association study of Parkinson;s disease (PD) reported 2 novel risk loci, SV2C and WBSCR17. Objectives: The objective of this study were to determine whether recently reported novel SV2C and WBSCR17 loci contribute to the risk of developing PD in European and East Asian ancestry populations. Methods: We report an association analysis of recently reported variants with PD in the COURAGE‐PD cohort (9673 PD patients; 8465 controls) comprising individuals of European and East Asian ancestries. In addition, publicly available summary data (41,386 PD patients; 476,428 controls) were pooled. Results: Our findings confirmed the role of the SV2C variant in PD pathogenesis (rs246814, COURAGE‐PD PEuropean = 6.64 × 10−4, pooled PD P = 1.15 × 10−11). The WBSCR17 rs9638616 was observed as a significant risk marker in the East Asian pooled population only (P = 1.16 × 10−8). Conclusions: Our comprehensive study provides an up‐to‐date summary of recently detected novel loci in different PD populations and confirmed the role of SV2C locus as a novel risk factor for PD irrespective of the population or ethnic group analyzed. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society [ABSTRACT FROM AUTHOR]

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles (SV) and released at the synaptic active zone (AZ). While in recent years major progress has been made in unraveling the molecular machinery responsible for SV docking, fusion and exocytosis, the mechanisms governing AZ protein and SV trafficking through axons still remain unclear. Here, we performed stop-flow nerve ligation to examine axonal trafficking of endogenous AZ and SV proteins. Rat sciatic nerves were collected 1 h, 3 h and 8 h post ligation and processed for immunohistochemistry and electron microscopy. First, we followed the transport of an integral synaptic vesicle protein, SV2A and a SV-associated protein involved in SV trafficking, Rab3a, and observed that while SV2A accumulated on both sides of ligation, Rab3a was only noticeable in the proximal segment of the ligated nerve indicating that only SV trans -membrane protein SV2A displayed a bi-directional axonal transport. We then demonstrate that multiple AZ proteins accumulate rapidly on either side of the ligation with a timescale similar to that of SV2A. Overall, our data uncovers an unexpected robust bi-directional, coordinated -trafficking of SV and AZ proteins in peripheral nerves. This implies that pathological disruption of axonal trafficking will not only impair trafficking of newly synthesized proteins to the synapse but will also affect retrograde transport, leading to neuronal dysfunction and likely neurodegeneration. • Axonal transport of active zone proteins in time dependent. • Active zone proteins are transported in robust bi-directional way along nerves. • Active zone proteins and synaptic vesicle proteins are trafficked together. • Active zone proteins colocalize with synaptic vesicle proteins on a single vesicle. [ABSTRACT FROM AUTHOR]

The Rho kinase (ROCK) signaling pathway is an attractive therapeutic target in neurodegeneration since it has been linked to the prevention of neuronal death and neurite regeneration. The isoquinoline derivative fasudil is a potent ROCK inhibitor, which is already approved for chronic clinical treatment in humans. However, the effects of chronic fasudil treatments on neuronal function are still unknown. We analyzed here chronic fasudil treatment in primary rat hippocampal cultures. Neurons were stimulated with 20 Hz field stimulation and we investigated pre‐synaptic mechanisms and parameters regulating synaptic transmission after fasudil treatment by super resolution stimulated emission depletion (STED) microscopy, live‐cell fluorescence imaging, and western blotting. Fasudil did not affect basic synaptic function or the amount of several synaptic proteins, but it altered the chronic dynamics of the synaptic vesicles. Fasudil reduced the proportion of the actively recycling vesicles, and shortened the vesicle lifetime, resulting overall in a reduction of the synaptic response upon stimulation. We conclude that fasudil does not alter synaptic structure, accelerates vesicle turnover, and decreases the number of released vesicles. This broadens the known spectrum of effects of this drug, and suggests new potential clinical uses. [ABSTRACT FROM AUTHOR]

The synapse is formed between a presynapse (which releases neurotransmitter) and the postsynapse (which transduces this chemical signal). Over the past decade, presynaptic dysfunction has emerged as a key mediator of a series of neurodevelopmental and neurodegenerative disorders. This special issue will highlight some of the important presynaptic molecules and mechanisms that are disrupted in these conditions and reveal potential routes for therapy. [ABSTRACT FROM AUTHOR]

Over the course of the last few decades it has become clear that many neurodevelopmental and neurodegenerative disorders have a synaptic defect, which contributes to pathogenicity. A rise in new techniques, and in particular '‐omics'‐based methods providing large datasets, has led to an increase in potential proteins and pathways implicated in synaptic function and related disorders. Additionally, advancements in imaging techniques have led to the recent discovery of alternative modes of synaptic vesicle recycling. This has resulted in a lack of clarity over the precise role of different pathways in maintaining synaptic function and whether these new pathways are dysfunctional in neurodevelopmental and neurodegenerative disorders. A greater understanding of the molecular detail of pre‐synaptic function in health and disease is key to targeting new proteins and pathways for novel treatments and the variety of new techniques currently available provides an ideal opportunity to investigate these functions. This review focuses on techniques to interrogate pre‐synaptic function, concentrating mainly on synaptic vesicle recycling. It further examines techniques to determine the underlying molecular mechanism of pre‐synaptic dysfunction and discusses methods to identify molecular targets, along with protein–protein interactions and cellular localization. In combination, these techniques will provide an expanding and more complete picture of pre‐synaptic function. With the application of recent technological advances, we are able to resolve events with higher spatial and temporal resolution, leading research towards a greater understanding of dysfunction at the presynapse and the role it plays in pathogenicity. [ABSTRACT FROM AUTHOR]

Parkinson's disease is a common neurodegenerative disorder and is clinically characterized by bradykinesia, rigidity, and resting tremor. Missense mutations in the leucine‐rich repeat protein kinase‐2 gene (LRRK2) are a recognized cause of inherited Parkinson's disease. The physiological and pathological impact of LRRK2 is still obscure, but accumulating evidence indicates that LRRK2 orchestrates diverse aspects of membrane trafficking, such as membrane fusion and vesicle formation and transport along actin and tubulin tracks. In the present review, we focus on the special relation between LRRK2 and synaptic vesicles. LRRK2 binds and phosphorylates key actors within the synaptic vesicle cycle. Accordingly, alterations in dopamine and glutamate transmission have been described upon LRRK2 manipulations. However, the different modeling strategies and phenotypes observed require a critical approach to decipher the outcome of LRRK2 at the pre‐synaptic site. [ABSTRACT FROM AUTHOR]

Neurotransmitter release during synaptic transmission comprises a tightly orchestrated sequence of molecular events, and Munc13-1 is a cornerstone of the fusion machinery. A forward genetic screen for defects in neurotransmitter release in Caenorhabditis elegans identified a mutation in the Munc13-1 ortholog UNC-13 that eliminated its unique and deeply conserved C-terminal module (referred to as HC2M) containing a Ca2+-insensitive C2 domain flanked by membrane-binding helices. The HC2M module could be functionally replaced in vivo by protein domains that localize to synaptic vesicles but not to the plasma membrane. HC2M is broadly conserved in other Unc13 family members and is required for efficient synaptic vesicle priming. We propose that the HC2M domain evolved as a vesicle/endosome adaptor and acquired synaptic vesicle specificity in the Unc13ABC protein family. [ABSTRACT FROM AUTHOR]

Na+(K+)/H+ exchanger 6 (NHE6) on synaptic vesicle (SV) is critical for the presynaptic regulation of quantal size at the glutamatergic synapses by converting the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane. We recently found that NHE6 directly interacts with secretory carrier membrane protein 5 (SCAMP5), and SCAMP5-dependent recruitment of NHE6 to SVs controls the strength of synaptic transmission by modulation of quantal size of glutamate release at rest. It is, however, unknown whether NHE6 recruitment by SCAMP5 plays a role during synaptic plasticity. Here, we found that the number of NHE6-positive presynaptic boutons was significantly increased by the chemical long-term potentiation (cLTP). Since cLTP involves new synapse formation, our results indicated that NHE6 was recruited not only to the existing presynaptic boutons but also to the newly formed presynaptic boutons. Knock down of SCAMP5 completely abrogated the enhancement of NHE6 recruitment by cLTP. Interestingly, despite an increase in the number of NHE6-positive boutons by cLTP, the quantal size of glutamate release at the presynaptic terminals remained unaltered. Together with our recent results, our findings indicate that SCAMP5-dependent recruitment of NHE6 plays a critical role in manifesting presynaptic efficacy not only at rest but also during synaptic plasticity. Since both are autism candidate genes, reduced presynaptic efficacy by interfering with their interaction may underlie the molecular mechanism of synaptic dysfunction observed in autism. [ABSTRACT FROM AUTHOR]

The BAD-BAX-caspase-3 cascade is a canonical apoptosis pathway. Macroautophagy ("autophagy" hereinafter) is a process by which organelles and aggregated proteins are delivered to lysosomes for degradation. Here, we report a new function of the BAD-BAX-caspase-3 cascade and autophagy in the control of synaptic vesicle pools. We found that, in hippocampal neurons of male mice, the BAD-BAX-caspase-3 pathway regulates autophagy, which in turn limits the size of synaptic vesicle pools and influences the kinetics of activity-induced depletion and recovery of synaptic vesicle pools. Moreover, the caspase-autophagy pathway is engaged by fear conditioning to facilitate associative fear learning and memory. This work identifies a new mechanism for controlling synaptic vesicle pools, and a novel, nonapoptotic, presynaptic function of the BAD-BAX-caspase-3 cascade. [ABSTRACT FROM AUTHOR]

Communication in the central nervous system is based on the transmission of electrical signals at specialized junctions between nerve cells termed synapses. During chemical neurotransmission, tiny membrane spheres called synaptic vesicles that are packed with neurotransmitters elicit a postsynaptic response by fusing with the presynaptic membrane and releasing their content into the synaptic cleft. Synaptic vesicle fusion is followed by the reuptake of the membrane by endocytosis and the local reformation of functional synaptic vesicles within the presynaptic compartment to sustain further rounds of neurotransmitter release. Here, we provide an overview of the clathrin-associated endocytic adaptor proteins that help to sort and recycle synaptic vesicles during presynaptic activity. These adaptors also serve additional functions in the turnover of defective or aged synaptic components and in the retrograde axonal transport of important signaling molecules by regulating the formation or transport of autophagosomes. Endocytic adaptors thus play multiple roles in the maintenance of synaptic function. Defects in their expression or function can lead to neurodegenerative and neurological diseases. [ABSTRACT FROM AUTHOR]

Synaptic vesicle proteins, including N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), Synaptotagmin‐1 and Complexin, are responsible for controlling the synchronised fusion of synaptic vesicles with the presynaptic plasma membrane in response to elevated cytosolic calcium levels. A range of structures of SNAREs and their regulatory proteins have been elucidated, but the exact organisation of these proteins at synaptic junction membranes remains elusive. Here, we have used cryoelectron tomography to investigate the arrangement of synaptic proteins in an in vitro reconstituted fusion system. We found that the separation between vesicle and target membranes strongly correlates with the organisation of protein complexes at junctions. At larger membrane separations, protein complexes assume a 'clustered' distribution at the docking site, inducing a protrusion in the target membrane. As the membrane separation decreases, protein complexes become displaced radially outwards and assume a 'ring‐like' arrangement. Our findings indicate that docked vesicles can possess a wide range of protein complex numbers and be heterogeneous in their protein arrangements. [ABSTRACT FROM AUTHOR]

Introduction: Edible bird nest (EBN) is a natural food product produced from edible nest swiftlet's saliva which consists of glycoproteins as one of its main components; these glycoproteins contain an abundant of sialic acid. The dietary EBN supplementation has been reported to enhance brain functions in mammals and that the bioactivities and nutritional value of EBN are important during periods of rapid brain growth particularly for preterm infant. However, the effects of EBN in maternal on multigeneration learning and memory function still remain unclear. Thus, the present study aimed to determine the effects of maternal EBN supplementation on learning and memory function of their first (F1)‐ and second (F2)‐generation mice. Methods: CJ57BL/6 breeder F0 mice were fed with EBN (10 mg/kg) from different sources. After 6 weeks of diet supplementations, the F0 animals were bred to produce F1 and F2 animals. At 6 weeks of age, the F1 and F2 animals were tested for spatial recognition memory using a Y‐maze test. The sialic acid content from EBN and brain gene expression were analyzed using HPLC and PCR, respectively. Results: All EBN samples contained glycoprotein with high level of sialic acid. Dietary EBN supplementation also showed an upregulation of GNE, ST8SiaIV, SLC17A5, and BDNF mRNA associated with an improvement in Y‐maze cognitive performance in both generations of animal. Qualitatively, the densities of synaptic vesicles in the presynaptic terminal were higher in the F1 and F2 animals which might derive from maternal EBN supplementation. Conclusion: This study provided a solid foundation toward the growing research on nutritional intervention from dietary EBN supplementation on cognitive and neurological development in the generation of mammals. [ABSTRACT FROM AUTHOR]

The mechanism of chemical synaptic transmission was elucidated at the frog neuromuscular junction (NMJ) and at the squid giant synapse by Katz, Miledi and other researchers. Later progress in molecular biology revealed numerous types of proteins in mammalian central synapses. To establish molecular–functional correlation in synaptic transmission, it now seems essential to re-address the fundamental mechanisms at mammalian central synapses. Using patch-clamp whole-cell recording at the calyx of Held in slices of rodent brainstem, we have identified quantal EPSCs and reproduced the quantal analysis established at the NMJ. Intra-terminal whole-cell loading of the neurotransmitter, glutamate, revealed that vesicular transmitter content is an important determinant of quantal size and its variation. Regarding the Ca2+-dependence of transmitter release, the average coupling distance between Ca2+ entry sites and exocytic vesicles was estimated as tens of nanometers and was found to undergo developmental tightening at the calyx of Held. Numerical simulations suggested that this distance can determine the synaptic delay, synchronicity of vesicular transmitter release, and release probability. The super-linear input (presynaptic)–output (postsynaptic) relationship of neurotransmission is an important physiological feature discovered at squid giant synapses. However, at the calyx of Held, unlike at the squid synapse, the input–output relationship had a wide safety margin, protecting transmitter release from a diminishment of presynaptic action potentials. As in the NMJ, Ca2+ remaining in the cytosol after action potential facilitates subsequent release. As a downstream mechanism of this residual Ca2+, a Ca2+-induced Ca2+ channel activation via high-affinity Ca2+ binding proteins was discovered at mammalian central synapses. • The concept of synaptic transmission established at frog NMJ and squid synapses are re-addressed at the rodent calyx of Held. • Vesicular transmitter content is an important determinant of quantal size and its variation. • Ca2+-release coupling distance is tens of nanometers and undergoes developmental tightening at the calyx of Held. • The input–output relationship has a wide safety margin at the calyx of Held, unlike at the squid synapse. • Residual Ca2+ activates presynaptic Ca2+ channels, thereby facilitating transmitter release at mammalian central synapses. [ABSTRACT FROM AUTHOR]

Synaptic vesicles need to be recycled and refilled rapidly to maintain high-frequency synaptic transmission. However, little is known about the control of neurotransmitter transport into synaptic vesicles, which determines the contents of synaptic vesicles and the strength of synaptic transmission. Here, we report that Na+ substantially accumulated in the calyx of Held terminals of juvenile mice of either sex during high-frequency spiking. The activity-induced elevation of cytosolic Na+ activated vesicular Na+/H+ exchanger, facilitated glutamate loading into synaptic vesicles, and increased quantal size of asynchronous released vesicles but did not affect the vesicle pool size or release probability. Consequently, presynaptic Na+ increased the EPSCs and was required to maintain the reliable high-frequency signal transmission from the presynaptic calyces to the postsynaptic medial nucleus of the trapezoid body (MNTB) neurons. Blocking Na+/H+ exchange activity decreased the postsynaptic current and caused failures in postsynaptic firing. Therefore, during high-frequency synaptic transmission, when large amounts of glutamate are released, Na accumulated in the terminals, activated vesicular Na+/H+ exchanger, and regulated glutamate loading as a function of the level of vesicle release. [ABSTRACT FROM AUTHOR]

A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity. [ABSTRACT FROM AUTHOR]

The pathogenesis of degeneration in Parkinson's disease (PD) remains poorly understood but multiple lines of evidence have converged on the presynaptic protein α-synuclein (αsyn). αSyn has been shown to regulate several cellular processes, however, its normal function remains poorly understood. In this review, we will specifically focus on its role in exocytosis. [ABSTRACT FROM AUTHOR]

The readily releasable pool (RRP) of synaptic vesicles is a key determinant of phasic neurotransmission. Although the size of the RRP is tightly regulated by intracellular factors, there is little evidence for its modification by extracellular signals. By studying the homogeneous population of synapses present in autaptic microcultures, we show that pregabalin, a prototypical gabapentinoid, decreases the effective RRP size. Simultaneous imaging of presynaptic calcium influx and recording of postsynaptic responses shows that the effect is not related to a reduction of calcium entry. The main cause is the impairment of the functional coupling among N‐type calcium channels and the RRP, resembling an increase of intracellular mobile calcium buffers. The ectodomain of neurexin‐1α shows a similar action to pregabalin, acting as an endogenous ligand of α2δ‐1 that reduces the RRP size without affecting presynaptic calcium influx. The regulatory actions described for pregabalin and the ectodomain of neurexin‐1α are mutually exclusive. The overexpression of α2δ‐1 enhances the effect of pregabalin and the ectodomain of neurexin‐1α on neurotransmission by decreasing their effective concentration. In contrast, knockdown of α2δ‐1 causes a profound inhibition of synaptic transmission. These observations prompt to consider α2δ‐1 as an outside‐in signaling platform that binds exogenous and endogenous cues for regulating the coupling of voltage‐gated calcium channels to synaptic vesicles. [ABSTRACT FROM AUTHOR]

In recent years, sensory neuroscientists have made major efforts to dissect the structure and function of ribbon synapses which process sensory information in the eye and ear. This review aims to summarize our current understanding of two key aspects of ribbon synapses: 1) their mechanisms of exocytosis and endocytosis and 2) their molecular anatomy and physiology. Our comparison of ribbon synapses in the cochlea and the retina reveals convergent signaling mechanisms, as well as divergent strategies in different sensory systems. [ABSTRACT FROM AUTHOR]