Synaptic vesicle glycoprotein 2 A in serum is an ideal biomarker for early diagnosis of Alzheimer's disease.

BACKGROUND: Previous studies have demonstrated that early intervention was the best plan to inhibit the progression of Alzheimer's disease (AD), which relied on the discovery of early diagnostic biomarkers. In this study, synaptic vesicle glycoprotein 2 A (SV2A) was examined to improve the early diagnostic efficiency in AD. METHODS: In this study, biomarker testing was performed through the single-molecule array (Simoa). A total of 121 subjects including cognitively unimpaired controls, amnestic mild cognitive impairment (aMCI), AD and other types of dementia underwent cerebrospinal fluid (CSF) SV2A testing; 430 subjects including health controls, aMCI, AD and other types of dementia underwent serum SV2A, glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL) and p-tau217 testing; 92 subjects including aMCI and AD underwent both CSF SV2A and serum SV2A testing; 115 cognitively unimpaired subjects including APOE ε4 carriers and APOE ε4 non-carriers were tested for serum SV2A, GFAP, NfL and p-tau217. Then, the efficacy of SV2A for the early diagnosis of AD and its ability to identify those at high risk of AD from a cognitively unimpaired population were further analyzed. RESULTS: Both CSF and serum SV2A significantly and positively correlated with cognitive performance in patients with AD, and their levels gradually decreased with the progression of AD. Serum SV2A demonstrated excellent diagnostic efficacy for aMCI, with a sensitivity of 97.8%, which was significantly higher than those of NfL, GFAP, and p-tau217. The SV2A-positive rates ranged from 92.86 to 100% in aMCI cases that were negative for the above three biomarkers. Importantly, of all the biomarkers tested, serum SV2A had the highest positivity rate (81.82%) in individuals at risk for AD. CONCLUSIONS: Serum SV2A was demonstrated to be a novel and ideal biomarker for the early diagnosis of AD, which can effectively distinguish those at high risk of AD in cognitively unimpaired populations.

Different states of synaptic vesicle priming explain target cell type-dependent differences in neurotransmitter release.

Pronounced differences in neurotransmitter release from a given presynaptic neuron, depending on the synaptic target, are among the most intriguing features of cortical networks. Hippocampal pyramidal cells (PCs) release glutamate with low probability to somatostatin expressing oriens-lacunosum-moleculare (O-LM) interneurons (INs), and the postsynaptic responses show robust short-term facilitation, whereas the release from the same presynaptic axons onto fast-spiking INs (FSINs) is ~10-fold higher and the excitatory postsynaptic currents (EPSCs) display depression. The mechanisms underlying these vastly different synaptic behaviors have not been conclusively identified. Here, we applied a combined functional, pharmacological, and modeling approach to address whether the main difference lies in the action potential-evoked fusion or else in upstream priming processes of synaptic vesicles (SVs). A sequential two-step SV priming model was fitted to the peak amplitudes of unitary EPSCs recorded in response to complex trains of presynaptic stimuli in acute hippocampal slices of adult mice. At PC-FSIN connections, the fusion probability (Pfusion) of well-primed SVs is 0.6, and 44% of docked SVs are in a fusion-competent state. At PC-O-LM synapses, Pfusion is only 40% lower (0.36), whereas the fraction of well-primed SVs is 6.5-fold smaller. Pharmacological enhancement of fusion by 4-AP and priming by PDBU was recaptured by the model with a selective increase of Pfusion and the fraction of well-primed SVs, respectively. Our results demonstrate that the low fidelity of transmission at PC-O-LM synapses can be explained by a low occupancy of the release sites by well-primed SVs.

Phospholipids Differentially Regulate Ca2+ Binding to Synaptotagmin-1.

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca2+ and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca2+ to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca2+ with a KD ∼ 45 µM. Each subsequent binding affinity (n ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca2+ binding affinity, we explored the extent that Ca2+ binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and dioleoylphosphatidylserine (DOPS) positively modulated Ca2+ binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) was reduced with increasing [Ca2+]. Overall, we find that specific lipids differentially modulate Ca2+ binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca2+, and anionic phospholipids that may also control some aspects of vesicular exocytosis.

Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release.

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

Super-Resolution Imaging of Tau Proteins in Isolated and Immobilized Brain Synaptosomes.

Tau protein has important physiological functions at both presynaptic and postsynaptic terminals. Pathological tau species are also associated with synaptic dysfunctions in several neurodegenerative disorders, especially Alzheimer's disease. To understand tau distribution inside synaptic compartments, super-resolution imaging is required. Here, we describe a facile protocol to immobilize and image brain synaptosomes without aggregation artefacts, by substituting the standard fixative paraformaldehyde with ethylene glycol bis(succinimidyl succinate) (EGS). Super-resolution imaging of tau proteins is achieved through three-color direct stochastic optical reconstruction microscopy (dSTORM). Tau protein is found to colocalize with synaptic vesicles as well as postsynaptic densities.

Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery.

Neurons rely on the long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, as well as how the efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule (MT)-severing enzyme spastin in locally enhancing MT polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new MT growth and synaptic vesicle precursor (SVP) pausing/retention. The disruption of neuronal spastin level or activity, by CRISPRi-mediated depletion, transient overexpression, or pharmacologic inhibition of enzymatic activity, interrupts the localized enrichment of dynamic MT plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of MT polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.

PTPN11/Corkscrew Activates Local Presynaptic Mapk Signaling to Regulate Synapsin, Synaptic Vesicle Pools, and Neurotransmission Strength, with a Dual Requirement in Neurons and Glia.

Cytoplasmic protein tyrosine phosphatase nonreceptor type 11 (PTPN11) and Drosophila homolog Corkscrew (Csw) regulate the mitogen-activated protein kinase (MAPK) pathway via a conserved autoinhibitory mechanism. Disease-causing loss-of-function (LoF) and gain-of-function (GoF) mutations both disrupt this autoinhibition to potentiate MAPK signaling. At the Drosophila neuromuscular junction glutamatergic synapse, LoF/GoF mutations elevate transmission strength and reduce activity-dependent synaptic depression. In both sexes of LoF/GoF mutations, the synaptic vesicles (SV)-colocalized synapsin phosphoprotein tether is highly elevated at rest, but quickly reduced with stimulation, suggesting a larger SV reserve pool with greatly heightened activity-dependent recruitment. Transmission electron microscopy of mutants reveals an elevated number of SVs clustered at the presynaptic active zones, suggesting that the increased vesicle availability is causative for the elevated neurotransmission. Direct neuron-targeted extracellular signal-regulated kinase (ERK) GoF phenocopies both increased local presynaptic MAPK/ERK signaling and synaptic transmission strength in mutants, confirming the presynaptic regulatory mechanism. Synapsin loss blocks this elevation in both presynaptic PTPN11 and ERK mutants. However, csw null mutants cannot be rescued by wild-type Csw in neurons: neurotransmission is only rescued by expressing Csw in both neurons and glia simultaneously. Nevertheless, targeted LoF/GoF mutations in either neurons or glia alone recapitulate the elevated neurotransmission. Thus, PTPN11/Csw mutations in either cell type are sufficient to upregulate presynaptic function, but a dual requirement in neurons and glia is necessary for neurotransmission. Taken together, we conclude that PTPN11/Csw acts in both neurons and glia, with LoF and GoF similarly upregulating MAPK/ERK signaling to enhance presynaptic Synapsin-mediated SV trafficking.

Reduced SV2A and GABAA receptor levels in the brains of type 2 diabetic rats revealed by [18F]SDM-8 and [18F]flumazenil PET.

PURPOSE: Type 2 diabetes mellitus (T2DM) is associated with a greater risk of Alzheimer's disease. Synaptic impairment and protein aggregates have been reported in the brains of T2DM models. Here, we assessed whether neurodegenerative changes in synaptic vesicle 2 A (SV2A), γ-aminobutyric acid type A (GABAA) receptor, amyloid-ß, tau and receptor for advanced glycosylation end product (RAGE) can be detected in vivo in T2DM rats. METHODS: Positron emission tomography (PET) using [18F]SDM-8 (SV2A), [18F]flumazenil (GABAA receptor), [18F]florbetapir (amyloid-ß), [18F]PM-PBB3 (tau), and [18F]FPS-ZM1 (RAGE) was carried out in 12-month-old diabetic Zucker diabetic fatty (ZDF) and SpragueDawley (SD) rats. Immunofluorescence staining, Thioflavin S staining, proteomic profiling and pathway analysis were performed on the brain tissues of ZDF and SD rats. RESULTS: Reduced cortical [18F]SDM-8 uptake and cortical and hippocampal [18F]flumazenil uptake were observed in 12-month-old ZDF rats compared to SD rats. The regional uptake of [18F]florbetapir and [18F]PM-PBB3 was comparable in the brains of 12-month-old ZDF and SD rats. Immunofluorescence staining revealed Thioflavin S-negative, phospho-tau-positive inclusions in the cortex and hypothalamus in the brains of ZDF rats and the absence of amyloid-beta deposits. The level of GABAA receptors was lower in the cortex of ZDF rats than SD rats. Proteomic analysis further demonstrated that, compared with SD rats, synaptic-related proteins and pathways were downregulated in the hippocampus of ZDF rats. CONCLUSION: These findings provide in vivo evidence for regional reductions in SV2A and GABAA receptor levels in the brains of aged T2DM ZDF rats.

Identification and Characterization of Synaptic Vesicle Membrane Protein VAT-1 Homolog as a New Catechin-Binding Protein.

(-)-Epigallocatechin-3-gallate (EGCg), a major constituent of green tea extract, is well-known to exhibit many beneficial actions for human health by interacting with numerous proteins. In this study we identified synaptic vesicle membrane protein VAT-1 homolog (VAT1) as a novel EGCg-binding protein in human neuroglioma cell extracts using a magnetic pull-down assay and LC-tandem mass spectrometry. We prepared recombinant human VAT1 and analyzed its direct binding to EGCg and its alkylated derivatives using surface plasmon resonance. For EGCg and the derivative NUP-15, we measured an association constant of 0.02-0.85 ×103 M-1s-1 and a dissociation constant of nearly 8 × 10-4 s-1. The affinity Km(affinity) of their binding to VAT1 was in the 10-20 µM range and comparable with that of other EGCg-binding proteins reported previously. Based on the common structure of the compounds, VAT1 appeared to recognize a catechol or pyrogallol moiety around the B-, C- and G-rings of EGCg. Next, we examined whether VAT1 mediates the effects of EGCg and NUP-15 on expression of neprilysin (NEP). Treatments of mock cells with these compounds upregulated NEP, as observed previously, whereas no effect was observed in the VAT1-overexpressing cells, indicating that VAT1 prevented the effects of EGCg or NUP-15 by binding to and inactivating them in the cells overexpressing VAT1. Further investigation is required to determine the biological significance of the VAT1-EGCg interaction.

Endoplasmic reticulum stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.

Dense core vesicles (DCVs) and synaptic vesicles are specialised secretory vesicles in neurons and neuroendocrine cells, and abnormal release of their cargo is associated with various pathophysiologies. Endoplasmic reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using thapsigargin. DCV exocytosis was severely compromised in ER-stressed PC12 cells and was reversed to varying magnitudes by ER stress attenuators. Experiments with tunicamycin, an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in insulin-secreting INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB-binding site. The effects of loss of function of ATF4 in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the eIF2α (EIF2A)-ATF4 axis.

Tracing synaptic loss in Alzheimer's brain with SV2A PET-tracer UCB-J.

INTRODUCTION: Synaptic loss is an early prominent feature of Alzheimer's disease (AD). The recently developed novel synaptic vesicle 2A protein (SV2A) PET-tracer UCB-J has shown great promise in tracking synaptic loss in AD. However, there have been discrepancies between the findings and a lack of mechanistic insight. METHODS: Here we report the first extensive pre-clinical validation studies for UCB-J in control (CN; n = 11) and AD (n = 11) brains using a multidimensional approach of post-mortem brain imaging techniques, radioligand binding, and biochemical studies. RESULTS AND DISCUSSION: We demonstrate that UCB-J could target SV2A protein with high specificity and depict synaptic loss at synaptosome levels in AD brain regions compared to CNs. UCB-J showed highest synaptic loss in AD hippocampus followed in descending order by frontal cortex, temporal cortex, parietal cortex, and cerebellum. 3H-UCB-J large brain-section autoradiography and cellular/subcellular fractions binding studies indicated potential off-target interaction with phosphorylated tau (p-tau) species in AD brains, which could have subsequent clinical implications for imaging studies. HIGHLIGHTS: Synaptic positron emission tomography (PET)-tracer UCB-J could target synaptic vesicle 2A protein (SV2A) with high specificity in Alzheimer's disease (AD) and control brains. Synaptic PET-tracer UCB-J could depict synaptic loss at synaptosome levels in AD brain regions compared to control. Potential off-target interaction of UCB-J with phosphorylated tau (p-tau) species at cellular/subcellular levels could have subsequent clinical implications for imaging studies, warranting further investigations.

Transport and inhibition mechanism for VMAT2-mediated synaptic vesicle loading of monoamines.

Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington's Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

A plant flavonol and genetic suppressors rescue a pathogenic mutation associated with kinesin in neurons.

KIF1A, a microtubule-based motor protein responsible for axonal transport, is linked to a group of neurological disorders known as KIF1A-associated neurological disorder (KAND). Current therapeutic options for KAND are limited. Here, we introduced the clinically relevant KIF1A(R11Q) variant into the Caenorhabditis elegans homolog UNC-104, resulting in uncoordinated animal behaviors. Through genetic suppressor screens, we identified intragenic mutations in UNC-104's motor domain that rescued synaptic vesicle localization and coordinated movement. We showed that two suppressor mutations partially recovered motor activity in vitro by counteracting the structural defect caused by R11Q at KIF1A's nucleotide-binding pocket. We found that supplementation with fisetin, a plant flavonol, improved KIF1A(R11Q) worms' movement and morphology. Notably, our biochemical and single-molecule assays revealed that fisetin directly restored the ATPase activity and processive movement of human KIF1A(R11Q) without affecting wild-type KIF1A. These findings suggest fisetin as a potential intervention for enhancing KIF1A(R11Q) activity and alleviating associated defects in KAND.

Biallelic variants in the synaptic vesicle glycoprotein 2 A are associated with epileptic encephalopathy.

Synaptic Vesicle Glycoprotein 2 A (SV2A) is a membrane protein of synaptic vesicles and the binding site of antiepileptic drug levetiracetam. Biallelic Arg383Gln is reported in a family with intractable epilepsy earlier. Here, we report on the second family with early onset drug resistant epilepsy. We identified homozygous Arg289Ter variant by exome sequencing that segregated with the phenotype in the family. The affected children in these two families are normal at birth and developed recurrent seizures beginning in the second month of life and developed secondary failure of growth and development. Knock out mice models earlier had replicated the human phenotype observed in these two families. These findings support that biallelic loss of function variants in SV2A result in early onset intractable epilepsy in humans.

Interpretation of presynaptic phenotypes of synaptic plasticity in terms of a two-step priming process.

Studies on synaptic proteins involved in neurotransmitter release often aim at distinguishing between their roles in vesicle priming (the docking of synaptic vesicles to the plasma membrane and the assembly of a release machinery) as opposed to the process of vesicle fusion. This has traditionally been done by estimating two parameters, the size of the pool of fusion-competent vesicles (the readily releasable pool, RRP) and the probability that such vesicles are released by an action potential, with the aim of determining how these parameters are affected by molecular perturbations. Here, it is argued that the assumption of a homogeneous RRP may be too simplistic and may blur the distinction between vesicle priming and fusion. Rather, considering priming as a dynamic and reversible multistep process allows alternative interpretations of mutagenesis-induced changes in synaptic transmission and suggests mechanisms for variability in synaptic strength and short-term plasticity among synapses, as well as for interactions between short- and long-term plasticity. In many cases, assigned roles of proteins or causes for observed phenotypes are shifted from fusion- to priming-related when considering multistep priming. Activity-dependent enhancement of priming is an essential element in this alternative view and its variation among synapse types can explain why some synapses show depression and others show facilitation at low to intermediate stimulation frequencies. Multistep priming also suggests a mechanism for frequency invariance of steady-state release, which can be observed in some synapses involved in sensory processing.

Excess phosphoserine-129 α-synuclein induces synaptic vesicle trafficking and declustering defects at a vertebrate synapse.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein aberrantly accumulates throughout neurons, including at synapses. During neuronal activity, α-synuclein is reversibly phosphorylated at serine 129 (pS129). While pS129 comprises ∼4% of total α-synuclein under physiological conditions, it dramatically increases in PD and DLB brains. The impacts of excess pS129 on synaptic function are currently unknown. We show here that compared with wild-type (WT) α-synuclein, pS129 exhibits increased binding and oligomerization on synaptic membranes and enhanced vesicle "microclustering" in vitro. Moreover, when acutely injected into lamprey reticulospinal axons, excess pS129 α-synuclein robustly localized to synapses and disrupted SV trafficking in an activity-dependent manner, as assessed by ultrastructural analysis. Specifically, pS129 caused a declustering and dispersion of SVs away from the synaptic vicinity, leading to a significant loss of total synaptic membrane. Live imaging further revealed altered SV cycling, as well as microclusters of recently endocytosed SVs moving away from synapses. Thus, excess pS129 caused an activity-dependent inhibition of SV trafficking via altered vesicle clustering/reclustering. This work suggests that accumulation of pS129 at synapses in diseases like PD and DLB could have profound effects on SV dynamics.

Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells.

In the ear, inner hair cells (IHCs) employ sophisticated glutamatergic ribbon synapses with afferent neurons to transmit auditory information to the brain. The presynaptic machinery responsible for neurotransmitter release in IHC synapses includes proteins such as the multi-C2-domain protein otoferlin and the vesicular glutamate transporter 3 (VGluT3). Yet, much of this likely unique molecular machinery remains to be deciphered. The scarcity of material has so far hampered biochemical studies which require large amounts of purified samples. We developed a subcellular fractionation workflow combined with immunoisolation of VGluT3-containing membrane vesicles, allowing for the enrichment of glutamatergic organelles that are likely dominated by synaptic vesicles (SVs) of IHCs. We have characterized their protein composition in mice before and after hearing onset using mass spectrometry and confocal imaging and provide a fully annotated proteome with hitherto unidentified proteins. Despite the prevalence of IHC marker proteins across IHC maturation, the profiles of trafficking proteins differed markedly before and after hearing onset. Among the proteins enriched after hearing onset were VAMP-7, syntaxin-7, syntaxin-8, syntaxin-12/13, SCAMP1, V-ATPase, SV2, and PKCα. Our study provides an inventory of the machinery associated with synaptic vesicle-mediated trafficking and presynaptic activity at IHC ribbon synapses and serves as a foundation for future functional studies.

Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles.

Neuronal communication relies on the release of neurotransmitters from various populations of synaptic vesicles. Despite displaying vastly different release probabilities and mobilities, the reserve and recycling pool of vesicles co-exist within a single cluster suggesting that small synaptic biomolecular condensates could regulate their nanoscale distribution. Here, we performed a large-scale activity-dependent phosphoproteome analysis of hippocampal neurons in vitro and identified Tau as a highly phosphorylated and disordered candidate protein. Single-molecule super-resolution microscopy revealed that Tau undergoes liquid-liquid phase separation to generate presynaptic nanoclusters whose density and number are regulated by activity. This activity-dependent diffusion process allows Tau to translocate into the presynapse where it forms biomolecular condensates, to selectively control the mobility of recycling vesicles. Tau, therefore, forms presynaptic nano-biomolecular condensates that regulate the nanoscale organization of synaptic vesicles in an activity-dependent manner.