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To enable transmission of information in the brain, synaptic vesicles fuse to presynaptic membranes, liberating their content and exposing transiently a myriad of vesicular transmembrane proteins. However, versatile methods for quantifying the synaptic translocation of endogenous proteins during neuronal activity remain unavailable, as the fast dynamics of synaptic vesicle cycling difficult specific isolation trafficking proteins during such a transient surface exposure. Here, we developed a novel approach using synaptic cleft proximity labeling to capture and quantify activity-driven trafficking of endogenous synaptic proteins at the synapse. We show that accelerating cleft biotinylation times to match the fast dynamics of vesicle exocytosis allows capturing endogenous proteins transiently exposed at the synaptic surface during neural activity, enabling for the first time the study of the translocation of nearly every endogenous synaptic protein. As proof-of-concept, we further applied this technology to obtain direct evidence of the surface translocation of noncanonical trafficking proteins, such as ATG9A and NPTX1, which had been proposed to traffic during activity but for which direct proof had not yet been shown. The technological advancement presented here will facilitate future studies dissecting the molecular identity of proteins exocytosed at the synapse during activity, helping to define the molecular machinery that sustains neurotransmission in the mammalian brain. The fast dynamics of synaptic vesicle release present a challenge for the quantification of synaptic protein translocation during neuronal activity. This study presents a method for quantifying the translocation of endogenous neuronal proteins to the synaptic surface and visualizes the translocation of proteins of interest when neurons are firing. [ABSTRACT FROM AUTHOR]

Zinc transporters (ZnTs) act as H+/Zn2+ antiporters, crucial for zinc homeostasis. Brain‐specific ZnT3 expressed in synaptic vesicles transports Zn2+ from the cytosol into vesicles and is essential for neurotransmission, with ZnT3 dysfunction associated with neurological disorders. Ubiquitously expressed ZnT4 localized to lysosomes facilitates the Zn2+ efflux from the cytosol to lysosomes, mitigating the cell injury risk. Despite their importance, the structures and Zn2+ transport mechanisms remain unclear. We characterized the three‐dimensional structures of human ZnT3 (inward‐facing) and ZnT4 (outward‐facing) using cryo‐electron microscopy. By combining these structures, we assessed the conformational changes that could occur within the transmembrane domain during Zn2+ transport. Our results provide a structural basis for a more comprehensive understanding of the H+/Zn2+ exchange mechanisms exhibited by ZnTs. [ABSTRACT FROM AUTHOR]

UNC13 family (also known as Munc13) proteins are evolutionarily conserved proteins involved in the rapid and regulated secretion of vesicles, including synaptic vesicles and cytotoxic granules. Fast and regulated secretion at the neuronal and immunological synapses requires multiple steps, from the biogenesis of vesicles to membrane fusion, and a complex array of proteins for each step. Defects at these steps can lead to various genetic disorders. Recent studies have shown multiple roles of UNC13D in the secretion of cytotoxic granules by immune cells. Here, the molecular structure and detailed roles of UNC13D in the biogenesis, tethering, and priming of cytotoxic vesicles and in endoplasmic reticulum are summarized. Moreover, its association with immune diseases, including familial hemophagocytic lymphohistiocytosis type 3, macrophage activation syndrome, juvenile idiopathic arthritis, and autoimmune lymphoproliferative syndrome, is reviewed. Finally, the therapeutic application of CRISPR/Cas9-based gene therapy for genetic diseases is introduced. [ABSTRACT FROM AUTHOR]

Peripheral Nerve Injuries (PNI) affect more than 20 million Americans and severely impact quality of life by causing long-term disability. PNI is characterized by nerve degeneration distal to the site of nerve injury resulting in long periods of skeletal muscle denervation. During this period, muscle fibers atrophy and frequently become incapable of "accepting" innervation because of the slow speed of axon regeneration post injury. We hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. To this end, we generate a mouse model in which NANOG, a pluripotency-associated transcription factor is expressed locally upon delivery of doxycycline (Dox) in a polymeric vehicle. NANOG expression in the muscle upregulates the percentage of Pax7+ nuclei and expression of eMYHC along with other genes that are involved in muscle development. In a sciatic nerve transection model, NANOG expression leads to upregulation of key genes associated with myogenesis, neurogenesis and neuromuscular junction (NMJ) formation. Further, NANOG mice demonstrate extensive overlap between synaptic vesicles and NMJ acetylcholine receptors (AChRs) indicating restored innervation. Indeed, NANOG mice show greater improvement in motor function as compared to wild-type (WT) animals, as evidenced by improved toe-spread reflex, EMG responses and isometric force production. In conclusion, we demonstrate that reprogramming muscle can be an effective strategy to improve reinnervation and functional outcomes after PNI. Peripheral nerves slowly regrow after injury, but often fail to form functional synapses. Here, authors use NANOG, a pluripotency factor, to induce denervated muscle to a pro-regenerative state improving muscle reinnervation after nerve transection. [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]

The present study uses electron microscopy to document ultrastructural characteristics of hippocampal GABAergic inhibitory synapses under resting and stimulated conditions in three experimental systems. Synaptic profiles were sampled from stratum pyramidale and radiatum of the CA1 region from (1) perfusion fixed mouse brains, (2) immersion fixed rat organotypic slice cultures, and from (3) rat dissociated hippocampal cultures of mixed cell types. Synapses were stimulated in the brain by a 5 min delay in perfusion fixation to trigger an ischemia-like excitatory condition, and by treating the two culture systems with 90 mM high K+ for 2–3 min to depolarize the neurons. Upon such stimulation conditions, the presynaptic terminals of the inhibitory synapses exhibited similar structural changes to those seen in glutamatergic excitatory synapses, with depletion of synaptic vesicles, increase of clathrin-coated vesicles and appearance of synaptic spinules. However, in contrast to excitatory synapses, no structural differences were detected in the postsynaptic compartment of the inhibitory synapses upon stimulation. There were no changes in the appearance of material associated with the postsynaptic membrane or the length and curvature of the membrane. Also no change was detected in the labeling density of gephyrin, a GABAergic synaptic marker, lining the postsynaptic membrane. Furthermore, virtually all inhibitory synaptic clefts remained rigidly apposed, unlike in the case of excitatory synapses where ~ 20–30% of cleft edges were open upon stimulation, presumably to facilitate the clearance of neurotransmitters from the cleft. The fact that no open clefts were induced in inhibitory synapses upon stimulation suggests that inhibitory input may not need to be toned down under these conditions. On the other hand, similar to excitatory synapse, EGTA (a calcium chelator) induced open clefts in ~ 18% of inhibitory synaptic cleft edges, presumably disrupting similar calcium-dependent trans-synaptic bridges in both types of synapses. [ABSTRACT FROM AUTHOR]

ABSTRACT PTPRQ plays an important role in the development of inner ear hair cell stereocilia. While many autosomal recessive variants in PTPRQ have been identified as the pathogenic cause for nonsyndromic hearing loss (DFNB84A), so far only one autosomal dominant PTPRQ variant, c.6881G>A (p.Trp2294*), has been reported for late‐onset, mild‐to‐severe hearing loss (DFNA73). By using targeted next‐generation sequencing, this study identified a novel PTPRQ truncating pathogenic variant, c.3697del (p.Leu1233Phefs*11), from a Chinese Han family that co‐segregated with autosomal dominant, postlingual, progressive hearing loss. A Ptprq‐3700del knock‐in mouse model was generated by CRISPR‐Cas9 and characterized for its hearing function and inner ear morphology. While the homozygous knock‐in mice exhibit profound hearing loss at all frequencies at the age of 3 weeks, the heterozygous mutant mice resemble the human patients in mild, progressive hearing loss from age 3 to 12 weeks, primarily affecting high frequencies. At this stage, the homozygous knock‐in mice have a normal hair cell count but disorganized stereocilia. Cochlear proteosome analysis of the homozygous mutant mice revealed differentially expressed genes and pathways involved in oxidative phosphorylation, regulation of angiogenesis and synaptic vesicle cycling. Our study provides a valuable animal model for further functional studies of the pathogenic mechanisms underlying DFNA73. [ABSTRACT FROM AUTHOR]

Vagus nerve stimulation (VNS) is the subject of exploration as an adjunct treatment for neurological disorders such as epilepsy, chronic migraine, pain, and depression. A non‐invasive form of VNS is transcutaneous auricular VNS (taVNS). Combining animal models and positron emission tomography (PET) may lead to a better understanding of the elusive mechanisms of taVNS. We evaluated the acute effect of electrical stimulation of the left vagus nerve via the ear on brain synaptic vesicle glycoprotein 2A (SV2A) as a measure of presynaptic density and glucose metabolism in naïve rats. Female Sprague–Dawley rats were imaged with [11C]UCB‐J (n = 11) or [18F]fluorodeoxyglucose ([18F]FDG) PET (n = 13) on two separate days, (1) at baseline, and (2) after acute unilateral left taVNS or sham stimulation (30 min). We calculated the regional volume of distribution (VT) for [11C]UCB‐J and standard uptake values (SUV) for [18F]FDG. We observed regional reductions of [11C]UCB‐J binding in response to taVNS ranging from 36% to 59%. The changes in taVNS compared to baseline were significantly larger than those induced by sham stimulation. The differences were observed bilaterally in the frontal cortex, striatum, and midbrain. The [18F]FDG PET uptake remained unchanged following acute taVNS or sham stimulation compared to baseline values. This proof‐of‐concept study shows for the first time that acute taVNS for 30 min can modulate in vivo synaptic SV2A density in cortical and subcortical regions of healthy rats. Preclinical disease models and PET ligands of different targets can be a powerful combination to assess the therapeutic potential of taVNS. [ABSTRACT FROM AUTHOR]

The editorial delves into the intricacies of immunological and redirecting artificial synapses, emphasizing their crucial role in intercellular communication and signal transduction. It also explores the significance of microvilli in antigen sensing and effector functions, particularly in the context of cancer immunotherapy. Furthermore, the document provides a comprehensive analysis of genetic data from various animal species, including Felidae, Canidae, and Mustelidae, offering valuable insights for researchers studying genetic diversity and evolutionary relationships within these families. Additionally, it presents a systematic overview of taxonomic classifications for species within the Mustelidae, Otariidae, Phocidae, and Ursidae families, aiding in understanding the taxonomy of these animals. [Extracted from the article]

Neuropeptides and neurotrophins are stored in and released from dense core vesicles (DCVs). While DCVs and synaptic vesicles (SVs) share fundamental SNARE/SM proteins for exocytosis, a detailed understanding of DCV exocytosis remains elusive. We recently identified the RAB3-RIM1 pathway to be essential for DCV, but not SV exocytosis, highlighting a significant distinction between the SV and DCV secretory pathways. Whether RIM1 is the only RAB3 effector that is essential for DCV exocytosis is currently unknown. In this study, we show that rabphilin-3A (RPH3A), a known downstream effector of RAB3A, is a negative regulator of DCV exocytosis. Using live-cell imaging at single-vesicle resolution with RPH3A deficient hippocampal mouse neurons, we show that DCV exocytosis increased threefold in the absence of RPH3A. RAB3A-binding deficient RPH3A lost its punctate distribution, but still restored DCV exocytosis to WT levels when re-expressed. SNAP25-binding deficient RPH3A did not rescue DCV exocytosis. In addition, we show that RPH3A did not travel with DCVs, but remained stationary at presynapses. RPH3A null neurons also had longer neurites, which was partly restored when ablating all regulated secretion with tetanus neurotoxin. Taken together, these results show that RPH3A negatively regulates DCV exocytosis, potentially also affecting neuron size. Furthermore, RAB3A interaction is required for the synaptic enrichment of RPH3A, but not for limiting DCV exocytosis. Instead, the interaction of RPH3A with SNAP25 is relevant for inhibiting DCV exocytosis. [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]

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]

Key points Intersectin‐1 (Itsn1) is a scaffold protein that plays a key role in coupling exocytosis and endocytosis of synaptic vesicles (SVs). However, it is unclear whether and how Itsn1 regulates these processes to support efficient neurotransmission during development. To address this, we examined the calyx of Held synapse in the auditory brainstem of wild‐type and Itsn1 mutant mice before (immature) and after (mature) the onset of hearing. Itsn1 was present in the pre‐ and postsynaptic compartments at both developmental stages. Loss of function of Itsn1 did not alter presynaptic action potentials, Ca2+ entry via voltage‐gated Ca2+ channels (VGCCs), transmitter release or short‐term depression (STD) induced by depletion of SVs in the readily releasable pool (RRP) in either age group. Yet, fast Ca2+‐dependent recovery from STD was attenuated in mature mutant synapses, while it was unchanged in immature mutant synapses. This deficit at mature synapses was rescued by introducing the DH–PH domains of Itsn1 into the presynaptic terminals. Inhibition of dynamin, which interacts with Itsn1 during endocytosis, had no effect on STD recovery. Interestingly, we found a developmental enrichment of Itsn1 near VGCCs, which may underlie the Itsn1‐mediated fast replenishment of the RRP. Consequently, the absence of Itsn1 in mature synapses led to a higher failure rate of postsynaptic spiking during high‐frequency synaptic transmission. Taken together, our findings suggest that Itsn1 translocation to the vicinity of VGCCs during development is crucial for accelerating Ca2+‐dependent RRP replenishment and sustaining high‐fidelity neurotransmission. Itsn1 is expressed in the pre‐ and postsynaptic compartments of the calyx of Held synapse. Developmental upregulation of vesicular glutamate transporter‐1 is Itsn1 dependent. Itsn1 does not affect basal synaptic transmission at different developmental stages. Itsn1 is required for Ca2+‐dependent recovery from short‐term depression in mature synapses. Itsn1 mediates the recovery through its DH–PH domains, independent of its interactive partner dynamin. Itsn1 translocates to the vicinity of presynaptic Ca2+ channels during development. Itsn1 supports high‐fidelity neurotransmission by enabling rapid recovery from vesicular depletion during repetitive activity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Key points Sensory hair cells of the inner ear utilize specialized ribbon synapses to transmit sensory stimuli to the central nervous system. This transmission necessitates rapid and sustained neurotransmitter release, which depends on a large pool of synaptic vesicles at the hair‐cell presynapse. While previous work in neurons has shown that kinesin motor proteins traffic synaptic material along microtubules to the presynapse, the mechanisms of this process in hair cells remain unclear. Our study demonstrates that the kinesin motor protein Kif1a, along with an intact microtubule network, is essential for enriching synaptic vesicles at the presynapse in hair cells. Through genetic and pharmacological approaches, we disrupt Kif1a function and impair microtubule networks in hair cells of the zebrafish lateral‐line system. These manipulations led to a significant reduction in synaptic‐vesicle populations at the presynapse in hair cells. Using electron microscopy, in vivo calcium imaging, and electrophysiology, we show that a diminished supply of synaptic vesicles adversely affects ribbon‐synapse function. Kif1aa mutants exhibit dramatic reductions in spontaneous vesicle release and evoked postsynaptic calcium responses. Furthermore, kif1aa mutants exhibit impaired rheotaxis, a behaviour reliant on the ability of hair cells in the lateral line to respond to sustained flow stimuli. Overall, our results demonstrate that Kif1a‐mediated microtubule transport is critical to enrich synaptic vesicles at the active zone, a process that is vital for proper ribbon‐synapse function in hair cells. Kif1a mRNAs are present in zebrafish hair cells. Loss of Kif1a disrupts the enrichment of synaptic vesicles at ribbon synapses. Disruption of microtubules depletes synaptic vesicles at ribbon synapses. Kif1aa  mutants have impaired ribbon‐synapse and sensory‐system function. [ABSTRACT FROM AUTHOR]

Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy. [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]

Coastal marine environments are characterized by daily, seasonal and long-term changes in both O2 and CO2, driven by local biotic and abiotic factors. The neuroepithelial cells (NECs) of fish are thought to be the putative chemoreceptors for sensing oxygen and CO2, and, thus, NECs play a key role in detecting these environmental changes. However, the role of NECs as chemosensors in marine fish remains largely understudied. In this study, the NECs of marine threespine sticklebacks (Gasterosteus aculeatus) were characterized using immunohistochemistry. We then determined if there were changes in NEC size and density, and in gill morphology in response to either mild (10 kPa) or moderate (6.8 kPa) hypoxia and two levels of elevated CO2 (1,500 and 3,000 µatm). We found that the NECs of stickleback contained synaptic vesicles and were innervated, and were 50–300% larger and 2 to 4 times more abundant than in other similar sized freshwater fishes. NEC size and density were largely unaffected by exposure to hypoxia, but there was a 50% decrease in interlamellar cell mass (ILCM) in response to mild and moderate hypoxia. NECs increased in size, but not abundance in response to elevated CO2. Moreover, fish exposed to moderate or elevated CO2 had 53–78% larger ILCMs compared to control fish. Our results demonstrated that adult marine sticklebacks have NECs that can respond to environmentally relevant pCO2 and likely hypoxia, which highlights the importance of NECs in marine fishes under the heterogeneity of environmental conditions in coastal areas. [ABSTRACT FROM AUTHOR]

KIF1A/UNC-104 proteins, which are members of the kinesin superfamily of motor proteins, play a pivotal role in the axonal transport of synaptic vesicles and their precursors. Drosophila melanogaster UNC-104 (DmUNC-104) is a relatively recently discovered Drosophila kinesin. Although some point mutations that disrupt synapse formation have been identified, the biochemical properties of the DmUNC-104 protein have not been investigated. Here, we prepared recombinant full-length DmUNC-104 protein and determined its biochemical features. We analyzed the effect of a previously identified missense mutation in the forkhead-associated (FHA) domain, called bristly (bris). The bris mutation strongly promoted the dimerization of DmUNC-104 protein, whereas wild-type DmUNC-104 was a mixture of monomers and dimers. We further tested the G618R mutation near the FHA domain, which was previously shown to disrupt the autoinhibition of Caenorhabditis elegans UNC-104. The biochemical properties of the G618R mutant recapitulated those of the bris mutant. Finally, we found that disease-associated mutations also promote the dimerization of DmUNC-104. Collectively, our results suggest that the FHA domain is essential for autoinhibition of KIF1A/UNC-104 proteins, and that abnormal dimerization of KIF1A might be linked to human diseases. [ABSTRACT FROM AUTHOR]

The axonal transport of synaptic vesicle precursors relies on KIF1A and UNC-104 ortholog motors. In mammals, KIF1Bβ is also responsible for the axonal transport of synaptic vesicle precursors. Mutations in KIF1A and KIF1Bβ lead to a wide range of neuropathies. Although previous studies have revealed the biochemical, biophysical and cell biological properties of KIF1A, and its defects in neurological disorders, the fundamental properties of KIF1Bβ remain elusive. In this study, we determined the motile parameters of KIF1Bβ through single-molecule motility assays. We found that the C-terminal region of KIF1Bβ has an inhibitory role in motor activity. AlphaFold2 prediction suggests that the C-terminal region blocks the motor domain. Additionally, we established simple methods for testing the axonal transport activity of human KIF1Bβ using Caenorhabditis elegans genetics. Taking advantage of these methods, we demonstrated that these assays enable the detection of reduced KIF1Bβ activities, both in vitro and in vivo, caused by a Charcot–Marie–Tooth disease-associated Q98L mutation. [ABSTRACT FROM AUTHOR]

The presynaptic nerve terminal is a specialized structure that contains various subcellular compartments, such as the synaptic vesicle (SV) cluster, the active zone (AZ), and the endocytic zone for synaptic transmission. The liquid–liquid phase separation (LLPS) principle could account for known fluid-like properties of numerous presynaptic molecules in each component of the nerve terminal. Numerous presynaptic proteins whose primary functions are attributed to synaptic vesicle clusters, the active zone and the endocytic zone can undergo LLPS. Several neurodegenerative disease-associated proteins expressed in nerve terminals undergo LLPS, and their liquid-to-solid transition can be crucial for the progression of these diseases with connections to synaptic dysfunction. The presynaptic nerve terminal is crucial for transmitting signals to the adjacent cell. To fulfill this role, specific proteins with distinct functions are concentrated in spatially confined areas within the nerve terminals. A recent concept termed liquid–liquid phase separation (LLPS) has provided new insights into how this process may occur. In this review, we aim to summarize the LLPS of proteins in different parts of the presynaptic nerve terminals, including synaptic vesicle (SV) clusters, the active zone (AZ), and the endocytic zone, with an additional focus on neurodegenerative diseases (NDDs), where the functional relevance of these properties is explored. Last, we propose new perspectives and future directions for the role of LLPS in presynaptic nerve terminals. [ABSTRACT FROM AUTHOR]

• Poly(I:C)-induced maternal immune activation (MIA) affects the synaptic proteome. • The synaptic proteome of adult MIA offspring is changed in a sex-specific manner. • Synaptic proteome changes after MIA in embryonic offspring are not affected by sex. • Overlap in embryonic and adult changes might provide potential intervention targets. Maternal immune activation (MIA) triggers neurobiological changes in offspring, potentially reshaping the molecular synaptic landscape, with the hippocampus being particularly vulnerable. However, critical details regarding developmental timing of these changes and whether they differ between males and females remain unclear. We induced MIA in C57BL/6J mice on gestational day nine using the viral mimetic poly(I:C) and performed mass spectrometry-based proteomic analyses on hippocampal synaptoneurosomes of embryonic (E18) and adult (20 ± 1 weeks) MIA offspring. In the embryonic synaptoneurosomes, MIA led to lipid, polysaccharide, and glycoprotein metabolism pathway disruptions. In the adult synaptic proteome, we observed a dynamic shift toward transmembrane trafficking, intracellular signalling cascades, including cell death and growth, and cytoskeletal organisation. In adults, many associated pathways overlapped between males and females. However, we found distinct sex-specific enrichment of dopaminergic and glutamatergic pathways. We identified 50 proteins altered by MIA in both embryonic and adult samples (28 with the same directionality), mainly involved in presynaptic structure and synaptic vesicle function. We probed human phenome-wide association study data in the cognitive and psychiatric domains, and 49 of the 50 genes encoding these proteins were significantly associated with the investigated phenotypes. Our data emphasise the dynamic effects of viral-like MIA on developing and mature hippocampi and provide novel targets for study following prenatal immune challenges. The 22 proteins that changed directionality from the embryonic to adult hippocampus, suggestive of compensatory over-adaptions, are particularly attractive for future investigations. [ABSTRACT FROM AUTHOR]

Botulism is a deadly neuroparalytic condition caused by the botulinum neurotoxin (BoNT) produced by Clostridium botulinum and related species. Toxin‐neutralizing antibodies are the most effective treatments for BoNT intoxication. We generated human monoclonal antibodies neutralizing type B botulinum neurotoxin (BoNT/B), designated M2 and M4. The combination of these antibodies exhibited a strong neutralizing effect against BoNT/B toxicity. In this study, we analyzed the mechanisms of action of these antibodies in vitro. M4 binds to the C‐terminus of the heavy chain (the receptor‐binding domain) and inhibits BoNT/B binding to neuronal PC12 cells. Although M2 recognized the light (L) chain (the metalloprotease domain), it did not inhibit substrate (VAMP2) cleavage in the cleavage assay. M2 increased the surface localization of BoNT/B in PC12 cells at a later time point, suggesting that M2 inhibits the translocation of the L chain from synaptic vesicles to the cytosol. These results indicate that M2 and M4 inhibit the different processes of BoNT/B individually and that multistep inhibition is important for the synergistic effect of the combination of monoclonal antibodies. Our findings may facilitate the development of effective therapeutic antibodies against BoNTs. [ABSTRACT FROM AUTHOR]

The microtubule‐associated protein tau participates in neurotransmission regulation via its interaction with synaptic vesicles (SVs). The precise nature and mechanics of tau's engagement with SVs, especially regarding alterations in vesicle dynamics, remain a matter of discussion. We report an electrochemical method using a synapse‐mimicking nanopipette to monitor vesicle dynamics induced by tau. A model vesicle of ~30 nm is confined within a lipid‐modified nanopipette orifice with a comparable diameter to mimic the synaptic lipid environment. Both tau and phosphorylated tau (p‐tau) present two‐state dynamic behavior in this biomimetic system, showing typical ionic current oscillation, induced by lipid‐tau interaction. The results indicate that p‐tau has a stronger affinity to the lipid vesicles in the confined environment, blocking the vesicle movement to a higher degree. Taken together, this method bridges a gap for sensing synaptic vesicle dynamics in a confined lipid environment, mimicking vesicle movement near the synaptic membrane. These findings contribute to understanding how different types of tau protein regulate synaptic vesicle motility and to underlying its functional and pathological behaviours in disease. [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]

Synaptic Vesicle Glycoprotein 2C (SV2C), characterized by its selective expression in discrete brain regions such as the midbrain, has recently emerged as a promising player in Parkinson's Disease (PD) - a debilitating neurodegenerative disorder affecting millions worldwide. This review aims to consolidate our current understanding of SV2C's function, its involvement in PD pathogenesis, and to evaluate its potential as a therapeutic target. Integrating previous findings of SV2C, from genetics to molecular studies, and drawing on insights from the largest East Asian genome-wide association study that highlights SV2C as a novel risk factor for PD, we explore the potential pathways through which SV2C may influence the disease. Our discussion extends to the implications of SV2C's role in synaptic vesicle trafficking, neurotransmitter release, and α-synuclein homeostasis, thereby laying the groundwork for future investigations that could pave the way for novel therapeutic strategies in combating PD. [ABSTRACT FROM AUTHOR]

Serotonin is distinct among synaptic neurotransmitters because it is amphipathic and released from synaptic vesicles at concentrations superior to its water solubility limit (270 mM in synaptic vesicles for a solubility limit of 110 mM). Hence, serotonin is mostly aggregated in the synaptic cleft, due to extensive aromatic stacking. This important characteristic has received scant attention, as most representations of the serotonergic synapse take as warranted that serotonin molecules are present as monomers after synaptic vesicle exocytosis. Using a combination of in silico and physicochemical approaches and a new experimental device mimicking synaptic conditions, we show that serotonin aggregates are efficiently dissolved by gangliosides (especially GM1) present in postsynaptic membranes. This initial interaction, driven by electrostatic forces, attracts serotonin from insoluble aggregates and resolves micelles into monomers. Serotonin also interacts with cholesterol via a set of CH-π and van der Waals interactions. Thus, gangliosides and cholesterol act together as a functional serotonin-collecting funnel on brain cell membranes. Based on this unique mode of interaction with postsynaptic membranes, we propose a new model of serotonergic transmission that takes into account the post-exocytosis solubilizing effect of gangliosides and cholesterol on serotonin aggregates. [ABSTRACT FROM AUTHOR]

Background: The connection between diabetes-associated cognitive dysfunction (DACD) and Alzheimer's disease (AD) has been shown in several observational studies. However, it remains controversial as to how the two related. Objective: To explore shared genes and pathways between DACD and AD using bioinformatics analysis combined with biological experiment. Methods: We analyzed GEO microarray data to identify DEGs in AD and type 2 diabetes mellitus (T2DM) induced-DACD datasets. Weighted gene co-expression network analysis was used to find modules, while R packages identified overlapping genes. A robust protein-protein interaction network was constructed, and hub genes were identified with Gene ontology enrichment and Kyoto Encyclopedia of Genome and Genome pathway analyses. HT22 cells were cultured under high glucose and amyloid-β 25–35 (Aβ25-35) conditions to establish DACD and AD models. Quantitative polymerase chain reaction with reverse transcription verification analysis was then performed on intersection genes. Results: Three modules each in AD and T2DM induced-DACD were identified as the most relevant and 10 hub genes were screened, with analysis revealing enrichment in pathways such as synaptic vesicle cycle and GABAergic synapse. Through biological experimentation verification, 6 key genes were identified. Conclusions: This study is the first to use bioinformatics tools to uncover the genetic link between AD and DACD. GAD1, UCHL1, GAP43, CARNS1, TAGLN3, and SH3GL2 were identified as key genes connecting AD and DACD. These findings offer new insights into the diseases' pathogenesis and potential diagnostic and therapeutic targets. [ABSTRACT FROM AUTHOR]

Human vesicular monoamine transporter 2 (VMAT2), a member of the SLC18 family, plays a crucial role in regulating neurotransmitters in the brain by facilitating their uptake and storage within vesicles, preparing them for exocytotic release. Because of its central role in neurotransmitter signalling and neuroprotection, VMAT2 is a target for neurodegenerative diseases and movement disorders, with its inhibitor being used as therapeutics. Despite the importance of VMAT2 in pharmacophysiology, the molecular basis of VMAT2-mediated neurotransmitter transport and its inhibition remains unclear. Here we show the cryo-electron microscopy structure of VMAT2 in the substrate-free state, in complex with the neurotransmitter dopamine, and in complex with the inhibitor tetrabenazine. In addition to these structural determinations, monoamine uptake assays, mutational studies, and pKa value predictions were performed to characterize the dynamic changes in VMAT2 structure. These results provide a structural basis for understanding VMAT2-mediated vesicular transport of neurotransmitters and a platform for modulation of current inhibitor design. VMAT2 regulates neurotransmitter uptake into synaptic vesicles. Here, the authors determined the cryo-EM structures of VMAT2, providing a structural basis for understanding VMAT2- mediated vesicular transport of neurotransmitters. [ABSTRACT FROM AUTHOR]

Background: A novel positron emission tomography (PET) imaging tracer, [18F] SynVesT-1, targeting synaptic vesicle glycoprotein 2 (SV2A), has been developed to meet clinical demand. Utilizing the Trasis AllinOne-36 (AIO) module, we've automated synthesis to Good Manufacturing Practice (GMP) standards, ensuring sterile, pyrogen-free production. The fully GMP-compliant robust synthesis of [18F] SynVesT-1 boosting reliability and introducing a significant degree of simplicity and its comprehensive validation for routine human use. Results: [18F] SynVesT-1 was synthesized by small modifications to the original [18F] SynVesT-1 synthesis protocol to better fit AIO module using an in-house designed cassette and sequence. With a relatively small precursor load of 5 mg, [18F] SynVesT-1 was obtained with consistently high radiochemical yields (RCY) of 20.6 ± 1.2% (the decay-corrected RCY, n = 3) at end of synthesis. Each of the final formulated batches demonstrated radiochemical purity (RCP) and enantiomeric purity surpassing 99%. The entire synthesis process was completed within a timeframe of 80 min (75 ± 3.1 min, n = 3), saves 11 min compared to reported GMP automated synthesis procedures. The in-human PET imaging of total body PET/CT and time-of-flight (TOF) PET/MR showed that [18F] SynVesT-1 is an excellent tracer for SV2A. It is advantageous for decentralized promotion and application in multi-center studies. Conclusion: The use of AIO synthesizer maintains high production yields and increases reliability, reduces production time and allows rapid training of production staff. Besides, the as-prepared [18F] SynVesT-1 displays excellent in vivo binding properties in humans and holds great potential for the imaging and quantification of synaptic density in vivo. [ABSTRACT FROM AUTHOR]

Myelination of axons is a key determinant of fast action potential propagation, axonal health and circuit function. Previously considered a static structure, it is now clear that myelin is dynamically regulated in response to neuronal activity in the central nervous system (CNS). However, how activity-dependent signals are conveyed to oligodendrocytes remains unclear. Here, we review the potential mechanisms by which neurons could communicate changing activity levels tomyelin, with a focus on the accumulating body of evidence to support activity-dependent vesicular signalling directly onto myelin sheaths. We discuss recent in vivo findings of activity-dependent fusion of neurotransmitter vesicles from non-synaptic axonal sites, and how modulation of this vesicular fusion regulates the stability and growth of myelin sheaths. We also consider the potential mechanisms by which myelin could sense and respond to axon-derived signals to initiate remodelling, and the relevance of these adaptations for circuit function. We propose that axonal vesicular signalling represents an important and underappreciated mode of communication by which neurons can transmit activity-regulated signals to myelinating oligodendrocytes and, potentially, more broadly to other cell types in the CNS. [ABSTRACT FROM AUTHOR]

The accumulation of β‐amyloid in Alzheimer's disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti‐aggregant behavior against alpha‐synuclein and β‐amyloid fibrillation in vitro and is up‐regulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti‐aggregant role in Alzheimer's progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up‐ and down‐regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control and/or altered protein degradation. ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild‐type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Importantly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS‐encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimer's disease progression. [ABSTRACT FROM AUTHOR]

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 (Syt1K326A/K328A) and SV2A knockdown. SV2A co‐clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2AT84A). 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. [ABSTRACT FROM AUTHOR]

The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na+, Cl−, and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post‐translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short‐lived S‐acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl‐Rac binding and IP‐ABE) confirmed with positive and negative controls (palmitoylated and non‐palmitoylated proteins). According to a short‐lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2‐bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation. [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]

This study aimed to elucidate the genetic causes underlying the juvenile parkinsonism (JP) diagnosed in a girl with several family members diagnosed with spinocerebellar ataxia type 2 (SCA2). To achieve this, whole-exome sequencing, analysis of CAG repeats, RNA sequencing analysis on fibroblasts, and metabolite identification were performed. As a result, a homozygous missense mutation SNP T>C (rs2254562) in synaptojamin 1 (SYNJ1), which has been implicated in the regulation of membrane trafficking in the synaptic vesicles, was identified. Additionally, we observed overexpression of L1 cell adhesion molecule (L1CAM), Cdc37, GPX1, and GPX4 and lower expression of ceruloplasmin in the patient compared to the control. We also found changes in sphingolipid, inositol, and inositol phosphate metabolism. These findings help to clarify the mechanisms of JP and suggest that the etiology of JP in the patient may be multifactorial. This is the first report of the rs2254562 mutation in the SYNJ gene identified in a JP patient with seizures and cognitive impairment. [ABSTRACT FROM AUTHOR]

Purpose: Here, we evaluate a PET displacement model with a Single-step and Numerical solution in healthy individuals using the synaptic vesicle glycoprotein (SV2A) PET-tracer [11C]UCB-J and the anti-seizure medication levetiracetam (LEV). We aimed to (1) validate the displacement model by comparing the brain LEV-SV2A occupancy from a single PET scan with the occupancy derived from two PET scans and the Lassen plot and (2) determine the plasma LEV concentration-SV2A occupancy curve in healthy individuals. Methods: Eleven healthy individuals (five females, mean age 35.5 [range: 25–47] years) underwent two 120-min [11C]UCB-J PET scans where an LEV dose (5–30 mg/kg) was administered intravenously halfway through the first PET scan to partially displace radioligand binding to SV2A. Five individuals were scanned twice on the same day; the remaining six were scanned once on two separate days, receiving two identical LEV doses. Arterial blood samples were acquired to determine the arterial input function and plasma LEV concentrations. Using the displacement model, the SV2A-LEV target engagement was calculated and compared with the Lassen plot method. The resulting data were fitted with a single-site binding model. Results: SV2A occupancies and VND estimates derived from the displacement model were not significantly different from the Lassen plot (p = 0.55 and 0.13, respectively). The coefficient of variation was 14.6% vs. 17.3% for the Numerical and the Single-step solution in Bland-Altman comparisons with the Lassen plot. The average half maximal inhibitory concentration (IC50), as estimated from the area under the curve of the plasma LEV concentration, was 12.5 µg/mL (95% CI: 5–25) for the Single-Step solution, 11.8 µg/mL (95% CI: 4–25) for the Numerical solution, and 6.3 µg/mL (95% CI: 0.08-21) for the Lassen plot. Constraining Emax to 100% did not significantly improve model fits. Conclusion: Plasma LEV concentration vs. SV2A occupancy can be determined in humans using a single PET scan displacement model. The average concentration of the three computed IC50 values ranges between 6.3 and 12.5 µg/mL. The next step is to use the displacement model to evaluate LEV occupancy and corresponding plasma concentrations in relation to treatment efficacy. Clinical trial registration: NCT05450822. Retrospectively registered 5 July 2022 https://clinicaltrials.gov/ct2/results? term=NCT05450822&Search=Search. [ABSTRACT FROM AUTHOR]

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high-frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a distinct population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

In contrast to humans, lampreys spontaneously recover their swimming capacity after a complete spinal cord injury (SCI). This recovery process involves the regeneration of descending axons. Spontaneous axon regeneration in lampreys has been mainly studied in giant descending neurons. However, the regeneration of neurochemically distinct descending neuronal populations with small-caliber axons, as those found in mammals, has been less studied. Cholecystokinin (CCK) is a regulatory neuropeptide found in the brain and spinal cord that modulates several processes such as satiety, or locomotion. CCK shows high evolutionary conservation and is present in all vertebrate species. Work in lampreys has shown that all CCKergic spinal cord axons originate in a single neuronal population located in the caudal rhombencephalon. Here, we investigate the spontaneous regeneration of CCKergic descending axons in larval lampreys following a complete SCI. Using anti-CCK-8 immunofluorescence, confocal microscopy and lightning adaptive deconvolution, we demonstrate the partial regeneration of CCKergic axons (81% of the number of axonal profiles seen in controls) 10 weeks after the injury. Our data also revealed a preference for regeneration of CCKergic axons in lateral spinal cord regions. Regenerated CCKergic axons exhibit colocalization with synaptic vesicle marker SV2, indicative of functional synaptic connections. We also extracted swimming dynamics in injured animals by using DeepLabCut. Interestingly, the degree of CCKergic reinnervation correlated with improved swimming performance in injured animals, suggesting a potential role in locomotor recovery. These findings open avenues for further exploration into the role of specific neuropeptidergic systems in post-SCI spinal locomotor networks.

Abstract Background The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. Results To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. Conclusions We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.

Alzheimer's disease (AD) is a neurodegenerative condition in which clinical symptoms are highly correlated with the loss of glutamatergic synapses. While later stages of AD are associated with markedly decreased glutamate levels due to neuronal loss, in the early stages, pathological accumulation of glutamate and hyperactivity contribute to AD pathology and cognitive dysfunction. There is increasing awareness that presynaptic dysfunction, particularly synaptic vesicle (SV) alterations, play a key role in mediating this early‐stage hyperactivity. In the current study, we sought to determine whether the 3xTg mouse model of AD that exhibits both beta‐amyloid (Aβ) and tau‐related pathology would exhibit similar presynaptic changes as previously observed in amyloid or tau models separately. Hippocampal cultures from 3xTg mice were used to determine whether presynaptic vesicular glutamate transporters (VGlut) and glutamate are increased at the synaptic level while controlling for postsynaptic activity. We observed that 3xTg hippocampal cultures exhibited increased VGlut1 associated with an increase in glutamate release, similar to prior observations in cultures from tau mouse models. However, the SV pool size was also increased in 3xTg cultures, an effect not previously observed in tau mouse models but observed in Aβ models, suggesting the changes in pool size may be due to Aβ and not tau. Second, we sought to determine whether treatment with troriluzole, a novel 3rd generation tripeptide prodrug of the glutamate modulator riluzole, could reduce VGlut1 and glutamate release to restore cognitive deficits in 8‐month‐old 3xTg mice. Treatment with troriluzole reduced VGlut1 expression, decreased basal and evoked glutamate release, and restored cognitive deficits in 3xTg mice. Together, these findings suggest presynaptic alterations are early events in AD that represent potential targets for therapeutic intervention, and these results support the promise of glutamate‐modulating drugs such as troriluzole in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Background and aim: Pathological changes in the central nervous system (CNS) begin before the clinical symptoms of Alzheimer's Disease (AD) manifest, with the hippocampus being one of the first affected structures. Current treatments fail to alter AD progression. Traditional Chinese medicine (TCM) has shown potential in improving AD pathology through multi-target mechanisms. This study investigates pathological changes in AD hippocampal tissue and explores TCM active components that may alleviate these changes. Methods: GSE5281 and GSE173955 datasets were downloaded from GEO and normalized to identify differentially expressed genes (DEGs). Key functional modules and hub genes were analyzed using Cytoscape and R. Active TCM components were identified from literature and the Pharmacopoeia of the People's Republic of China. Enrichment analyses were performed on target genes overlapping with DEGs. Result: From the datasets, 76 upregulated and 363 downregulated genes were identified. Hub genes included SLAMF, CD34, ELN (upregulated) and ATP5F1B, VDAC1, VDAC2, HSPA8, ATP5F1C, PDHA1, UBB, SNCA, YWHAZ, PGK1 (downregulated). Literature review identified 33 active components from 23 herbal medicines. Target gene enrichment and analysis were performed for six components: dihydroartemisinin, berberine, naringenin, calycosin, echinacoside, and icariside II. Conclusion: Mitochondrial to synaptic vesicle dysfunction pathways were enriched in downregulated genes. Despite downregulation, UBB and SNCA proteins accumulate in AD brains. TCM studies suggest curcumin and echinacoside may improve hippocampal pathology and cognitive impairment in AD. Further investigation into their mechanisms is needed. [ABSTRACT FROM AUTHOR]

The mechanism research of skin wrinkles, conducted on volunteers underwent high‐intensity desk work and mice subjected to partial sleep deprivation, revealed a significant reduction in dermal thickness associated with the presence of wrinkles. This can be attributed to the activation of facial nerves in a state of hysteria due to an abnormally elevated interaction between SNAP25 and RAB3A proteins involved in the synaptic vesicle cycle (SVC). Facilitated by AI‐assisted structural design, a refined peptide called RSIpep is developed to modulate this interaction and normalize SVC. Drawing inspiration from prions, which possess the ability to protect themselves against proteolysis and invade neighboring nerve cells through macropinocytosis, RSIpep is engineered to demonstrate a GSH‐responsive reversible self‐assembly into a prion‐like supermolecule (RSIprion). RSIprion showcases protease resistance, micropinocytosis‐dependent cellular internalization, and low adhesion with constituent molecules in the cuticle, thereby endowing it with the transdermic absorption and subsequent biofunction in redressing the frenzied SVC. As a facial mud mask, it effectively reduces periorbital and perinasal wrinkles in the human face. Collectively, RSIprion not only presents a clinical potential as an anti‐wrinkle prion‐like supermolecule, but also exemplifies a reproducible instance of bionic strategy‐guided drug development that bestows transdermal ability upon the pharmaceutical molecule. [ABSTRACT FROM AUTHOR]

Synapses are fundamental to the function of the central nervous system and are implicated in a number of brain disorders. Despite their pivotal role, a comprehensive imaging resource detailing the distribution of synapses in the human brain has been lacking until now. Here, we employ high-resolution PET neuroimaging in healthy humans (17F/16M) to create a 3D atlas of the synaptic marker Synaptic Vesicle glycoprotein 2A (SV2A). Calibration to absolute density values (pmol/ml) was achieved by leveraging postmortem human brain autoradiography data. The atlas unveils distinctive cortical and subcortical gradients of synapse density that reflect functional topography and hierarchical order from core sensory to higher-order integrative areas-a distribution that diverges from SV2A mRNA patterns. Furthermore, we found a positive association between IQ and SV2A density in several higher-order cortical areas. This new resource will help advance our understanding of brain physiology and the pathogenesis of brain disorders, serving as a pivotal tool for future neuroscience research. [ABSTRACT FROM AUTHOR]

Key points Dynamins are GTPases required for pinching vesicles off the plasma membrane once a critical curvature is reached during endocytosis. Here, we probed dynamin function in central synapses by depleting all three dynamin isoforms in postnatal hippocampal neurons down to negligible levels. We found a decrease in the propensity of evoked neurotransmission as well as a reduction in synaptic vesicle numbers. Recycling of synaptic vesicles during spontaneous or low levels of evoked activity were largely impervious to dynamin depletion, while retrieval of synaptic vesicle components at higher levels of activity was partially arrested. These results suggest the existence of balancing dynamin‐independent mechanisms for synaptic vesicle recycling at central synapses. Classical dynamin‐dependent mechanisms are not essential for retrieval of synaptic vesicle proteins after quantal single synaptic vesicle fusion, but they become more relevant for membrane retrieval during intense, sustained neuronal activity. Loss of dynamin 2 does not impair synaptic transmission. Loss of all three dynamin isoforms mostly affects evoked neurotransmission. Excitatory synapse function is more susceptible to dynamin loss. Spontaneous neurotransmission is only mildly affected by loss of dynamins. Single synaptic vesicle endocytosis is largely dynamin independent. [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]

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation. [ABSTRACT FROM AUTHOR]

The synapse is a piece of information transfer machinery replacing the electrical conduction of nerve impulses at the end of the neuron. Like many biological mechanisms, its functioning is heavily affected by time constraints. The solution selected by evolution is based on chemical communication that, in theory, cannot compete with the speed of nerve conduction. Nevertheless, biochemical and biophysical compensation mechanisms mitigate this intrinsic weakness: (i) through the high concentrations of neurotransmitters inside the synaptic vesicles; (ii) through the concentration of neurotransmitter receptors in lipid rafts, which are signaling platforms; indeed, the presence of raft lipids, such as gangliosides and cholesterol, allows a fine tuning of synaptic receptors by these lipids; (iii) through the negative electrical charges of the gangliosides, which generate an attractive (for cationic neurotransmitters, such as serotonin) or repulsive (for anionic neurotransmitters, such as glutamate) electric field. This electric field controls the flow of glutamate in the tripartite synapse involving pre- and post-synaptic neurons and the astrocyte. Changes in the expression of brain gangliosides can disrupt the functioning of the glutamatergic synapse, causing fatal diseases, such as Rett syndrome. In this review, we propose an in-depth analysis of the role of gangliosides in the glutamatergic synapse, highlighting the primordial and generally overlooked role played by the electric field of synaptic gangliosides. [ABSTRACT FROM AUTHOR]

Dynamin 1 mediates fission of endocytic synaptic vesicles in the brain and has two major splice variants, Dyn1xA and Dyn1xB, which are nearly identical apart from the extended C-terminal region of Dyn1xA. Despite a similar set of binding partners, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that Dyn1xA achieves this localization by preferentially binding to Endophilin A1 through a newly defined binding site within its long C-terminal tail extension. Endophilin A1 binds this site at higher affinity than the previously reported site, and the affinity is determined by amino acids within the Dyn1xA tail but outside the binding site. This interaction is regulated by the phosphorylation state of two serine residues specific to the Dyn1xA variant. Dyn1xA and Endophilin A1 colocalize in patches near the active zone, and mutations disrupting Endophilin A binding to the long tail cause Dyn1xA mislocalization and stalled endocytic pits on the plasma membrane during ultrafast endocytosis. Together, these data suggest that the specificity for ultrafast endocytosis is defined by the phosphorylation-regulated interaction of Endophilin A1 with the C-terminal extension of Dyn1xA. Synopsis: A splice variant of Dynamin 1, Dyn1xA, is required for ultrafast endocytosis of synaptic vesicles, but how this specificity arises is unclear, given that another variant, Dyn1xB, is nearly identical. Using a combination of proteomics, nuclear magnetic resonance, superresolution microscopy, and time-resolved electron microscopy imaging, this study reveals how Dyn1xA mediates its unique function. A high-affinity Endophilin A1 binding site exists within the extended tail of Dyn1xA that spans a splice boundary and is absent in Dyn1xB. Dyn1xA-specific amino acids near the SH3-binding PxxP motif enhance Endophilin A1 affinity to the nanomolar range. Mutations disrupting the specific interaction between Dyn1xA and Endophilin A cause defective Dyn1xA molecular condensate formation around the endocytic zone and defective ultrafast endocytosis. Ultrafast endocytosis of synaptic vesicles is mediated by the unique tail extension of the splice variant Dynamin 1xA, which contains a high-affinity binding site for endophilin A1. [ABSTRACT FROM AUTHOR]

Atg9, the only transmembrane protein among many autophagy-related proteins, was first identified in the year 2000 in yeast. Two homologs of Atg9, ATG9A and ATG9B, have been found in mammals. While ATG9B shows a tissue-specific expression pattern, such as in the placenta and pituitary gland, ATG9A is ubiquitously expressed. Additionally, ATG9A deficiency leads to severe defects not only at the molecular and cellular levels but also at the organismal level, suggesting key and fundamental roles for ATG9A. The subcellular localization of ATG9A on small vesicles and its functional relevance to autophagy have suggested a potential role for ATG9A in the lipid supply during autophagosome biogenesis. Nevertheless, the precise role of ATG9A in the autophagic process has remained a long-standing mystery, especially in neurons. Recent findings, however, including structural, proteomic, and biochemical analyses, have provided new insights into its function in the expansion of the phagophore membrane. In this review, we aim to understand various aspects of ATG9 (in invertebrates and plants)/ATG9A (in mammals), including its localization, trafficking, and other functions, in nonneuronal cells and neurons by comparing recent discoveries related to ATG9/ATG9A and proposing directions for future research.Abbreviation: AP-4: adaptor protein complex 4; ATG: autophagy related; cKO: conditional knockout; CLA-1: CLArinet (functional homolog of cytomatrix at the active zone proteins piccolo and fife); cryo-EM: cryogenic electron microscopy; ER: endoplasmic reticulum; KO: knockout; PAS: phagophore assembly site; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SV: synaptic vesicle; TGN: trans-Golgi network; ULK: unc-51 like autophagy activating kinase; WIPI2: WD repeat domain, phosphoinositide interacting 2. [ABSTRACT FROM AUTHOR]