Physiological roles of endocytosis and presynaptic scaffold in vesicle replenishment at fast and slow central synapses.

After exocytosis, release sites are cleared of vesicular residues to replenish with transmitter-filled vesicles. Endocytic and scaffold proteins are thought to underlie this site-clearance mechanism. However, the physiological significance of this mechanism at diverse mammalian central synapses remains unknown. Here, we tested this in a physiologically optimized condition using action potential evoked EPSCs at fast calyx synapse and relatively slow hippocampal CA1 synapse, in post-hearing mice brain slices at 37°C and in 1.3 mM [Ca2+]. Pharmacological block of endocytosis enhanced synaptic depression at the calyx synapse, whereas it attenuated synaptic facilitation at the hippocampal synapse. Block of scaffold protein activity likewise enhanced synaptic depression at the calyx but had no effect at the hippocampal synapse. At the fast calyx synapse, block of endocytosis or scaffold protein activity significantly enhanced synaptic depression as early as 10 ms after the stimulation onset. Unlike previous reports, neither endocytic blockers nor scaffold protein inhibitors prolonged the recovery from short-term depression. We conclude that the release-site clearance by endocytosis can be a universal phenomenon supporting vesicle replenishment at both fast and slow synapses, whereas the presynaptic scaffold mechanism likely plays a specialized role in vesicle replenishment predominantly at fast synapses.

Parallel processing of quickly and slowly mobilized reserve vesicles in hippocampal synapses.

Vesicles within presynaptic terminals are thought to be segregated into a variety of readily releasable and reserve pools. The nature of the pools and trafficking between them is not well understood, but pools that are slow to mobilize when synapses are active are often assumed to feed pools that are mobilized more quickly, in a series. However, electrophysiological studies of synaptic transmission have suggested instead a parallel organization where vesicles within slowly and quickly mobilized reserve pools would separately feed independent reluctant- and fast-releasing subdivisions of the readily releasable pool. Here, we use FM-dyes to confirm the existence of multiple reserve pools at hippocampal synapses and a parallel organization that prevents intermixing between the pools, even when stimulation is intense enough to drive exocytosis at the maximum rate. The experiments additionally demonstrate extensive heterogeneity among synapses in the relative sizes of the slowly and quickly mobilized reserve pools, which suggests equivalent heterogeneity in the numbers of reluctant and fast-releasing readily releasable vesicles that may be relevant for understanding information processing and storage.

Vesicle and reaction-diffusion hybrid modeling with STEPS.

Vesicles carry out many essential functions within cells through the processes of endocytosis, exocytosis, and passive and active transport. This includes transporting and delivering molecules between different parts of the cell, and storing and releasing neurotransmitters in neurons. To date, computational simulation of these key biological players has been rather limited and has not advanced at the same pace as other aspects of cell modeling, restricting the realism of computational models. We describe a general vesicle modeling tool that has been designed for wide application to a variety of cell models, implemented within our software STochastic Engine for Pathway Simulation (STEPS), a stochastic reaction-diffusion simulator that supports realistic reconstructions of cell tissue in tetrahedral meshes. The implementation is validated in an extensive test suite, parallel performance is demonstrated in a realistic synaptic bouton model, and example models are visualized in a Blender extension module.

Specific Extracellular Vesicles, Generated and Operating at Synapses, Contribute to Neuronal Effects and Signaling.

In all cell types, small EVs, very abundant extracellular vesicles, are generated and accumulated within MVB endocytic cisternae. Upon MVB fusion and exocytosis with the plasma membrane, the EVs are released to the extracellular space. In the central nervous system, the release of neuronal EVs was believed to occur only from the surface of the body and dendrites. About 15 years ago, MVB cisternae and EVs were shown to exist and function at synaptic boutons, the terminals' pre- and post-synaptic structures essential for canonical neurotransmitter release. Recent studies have revealed that synaptic EVs are peculiar in many respects and heterogeneous with respect to other neuronal EVs. The distribution of synaptic EVs and the effect of their specific molecules are found at critical sites of their distribution. The role of synaptic EVs could consist of the modulation of canonical neurotransmitter release or a distinct, non-canonical form of neurotransmission. Additional roles of synaptic EVs are still not completely known. In the future, additional investigations will clarify the role of synaptic EVs in pathology, concerning, for example, circuits, trans-synaptic transmission, diagnosis and the therapy of diseases.

Metabolic regulation of single synaptic vesicle exo- and endocytosis in hippocampal synapses.

Glucose has long been considered a primary energy source for synaptic function. However, it remains unclear to what extent alternative fuels, such as lactate/pyruvate, contribute to powering synaptic transmission. By detecting individual release events in hippocampal synapses, we find that mitochondrial ATP production regulates basal vesicle release probability and release location within the active zone (AZ), evoked by single action potentials. Mitochondrial inhibition shifts vesicle release closer to the AZ center and alters the efficiency of vesicle retrieval by increasing the occurrence of ultrafast endocytosis. Furthermore, we uncover that terminals can use oxidative fuels to maintain the vesicle cycle during trains of activity. Mitochondria are sparsely distributed along hippocampal axons, and we find that terminals containing mitochondria display enhanced vesicle release and reuptake during high-frequency trains. Our findings suggest that mitochondria not only regulate several fundamental features of synaptic transmission but may also contribute to modulation of short-term synaptic plasticity.

Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma.

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

pHusion - a robust and versatile toolset for automated detection and analysis of exocytosis.

Exocytosis is a fundamental process used by eukaryotes to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate exocytosis in neuronal morphogenesis, previously we developed computational tools with a graphical user interface to enable the automatic detection and analysis of exocytic events from fluorescence timelapse images. Although these tools were useful, we found the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis, written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested pHusion using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types and various exocytic markers, to generate a flexible and intuitive package. Using this system, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons.

A hydrophobic groove in secretagogin allows for alternate interactions with SNAP-25 and syntaxin-4 in endocrine tissues.

Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of synaptobrevins, syntaxins, and SNAP-25. Despite SNARE-mediated release being fundamental for intercellular communication in all excitable tissues, the role of auxiliary proteins modulating the import of reserve vesicles to the active zone, and thus, scaling repetitive exocytosis remains less explored. Secretagogin is a Ca2+-sensor protein with SNAP-25 being its only known interacting partner. SNAP-25 anchors readily releasable vesicles within the active zone, thus being instrumental for 1st phase release. However, genetic deletion of secretagogin impedes 2nd phase release instead, calling for the existence of alternative protein-protein interactions. Here, we screened the secretagogin interactome in the brain and pancreas, and found syntaxin-4 grossly overrepresented. Ca2+-loaded secretagogin interacted with syntaxin-4 at nanomolar affinity and 1:1 stoichiometry. Crystal structures of the protein complexes revealed a hydrophobic groove in secretagogin for the binding of syntaxin-4. This groove was also used to bind SNAP-25. In mixtures of equimolar recombinant proteins, SNAP-25 was sequestered by secretagogin in competition with syntaxin-4. Kd differences suggested that secretagogin could shape unidirectional vesicle movement by sequential interactions, a hypothesis supported by in vitro biological data. This mechanism could facilitate the movement of transport vesicles toward release sites, particularly in the endocrine pancreas where secretagogin, SNAP-25, and syntaxin-4 coexist in both α- and ß-cells. Thus, secretagogin could modulate the pace and fidelity of vesicular hormone release by differential protein interactions.

Alpha herpesvirus exocytosis from neuron cell bodies uses constitutive secretory mechanisms, and egress and spread from axons is independent of neuronal firing activity.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system, causing severe debilitating or deadly disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous activity, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. Using live-cell fluorescence microscopy, we show that Pseudorabies Virus (PRV) particles use the constitutive Rab6 post-Golgi secretory pathway to exit from the cell body of primary neurons, independent of local calcium signaling. Some PRV particles colocalize with Rab6 in the proximal axon, but we did not detect colocalization/co-transport in the distal axon. Thus, the specific secretory mechanisms used for viral egress from axons remains unclear. To address the role of neuronal activity more generally, we used a compartmentalized neuron culture system to measure the egress and spread of PRV from axons, and pharmacological and optogenetics approaches to modulate neuronal activity. Using tetrodotoxin to silence neuronal activity, we observed no inhibition, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread from axons. We conclude that PRV egress from neurons uses constitutive secretory mechanisms: generally, activity-independent mechanisms in axons, and specifically, the constitutive Rab6 post-Golgi secretory pathway in cell bodies.

OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence.

Purpose: To undertake the first ultrastructural characterization of human retinal pigment epithelial (RPE) differentiation from fetal development to adolescence. Methods: Ten fetal eyes and three eyes aged six, nine, and 17 years were examined in the temporal retina adjacent to the optic nerve head by transmission electron microscopy. The area, number, and distribution of RPE organelles were quantified and interpreted within the context of adjacent photoreceptors, Bruch's membrane, and choriocapillaris maturation. Results: Between eight to 12 weeks' gestation (WG), pseudostratified columnar epithelia with apical tight junctions differentiate to a simple cuboidal epithelium with random distribution of melanosomes and mitochondria. Between 12 to 26 WG, cells enlarge and show long apical microvilli and apicolateral junctional complexes. Coinciding with eye opening at 26 WG, melanosomes migrate apically whereas mitochondria distribute to perinuclear regions, with the first appearance of phagosomes, complex granules, and basolateral extracellular space (BES) formation. Significantly, autophagy and heterophagy, as evidenced by organelle recycling, and the gold standard of ultrastructural evidence for autophagy of double-membrane autophagosomes and mitophagosomes were evident from 32 WG, followed by basal infoldings of RPE cell membrane at 36 WG. Lipofuscin formation and deposition into the BES evident at six years increased at 17 years. Conclusions: We provide compelling ultrastructural evidence that heterophagy and autophagy begins in the third trimester of human fetal development and that deposition of cellular byproducts into the extracellular space of RPE takes place via exocytosis. Transplanted RPE cells must also demonstrate the capacity to subserve autophagic and heterophagic functions for effective disease mitigation.

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.

Escape sites from the endo-lysosomal trafficking route manipulate exocytosis of nanoparticles in polar epithelium.

The endo-lysosomal pathway is a major barrier for the trans-epithelial transport of nanoparticles (NPs), but escape strategies could facilitate trans-epithelial delivery. Based on the polarization properties of the epithelium, different escape compartments may result in different exocytosis fates of NPs and further affect the delivery efficiency. Therefore, optimizing the escape sites is critical for trans-epithelial delivery. Here, commonly used PEG-coated-poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles were fabricated as model nanoparticles (MNPs) and the intestinal epithelium was chosen as the polarized epithelium. The MNPs were incubated with different endosomolytic agents for early endosomal escape, late endosomal escape and lysosomal escape, respectively. According to in vitro and in vivo studies, MNPs escaping from early endosomes and late endosomes exhibited stronger capacity for trans-epithelial transport than those escaping from lysosomes. By further probing into the mechanism, we surprisingly found that although MNPs escaping from early endosomes quickly egressed from the apical side of epithelia, they were subsequently followed by "reuptake" via caveolae and trafficked through the endoplasmic reticulum-Golgi apparatus (ER/GA) secretory pathway, achieving efficient trans-epithelial transport; MNPs escaping from late endosomes, which were located near the nucleus, were prone to enter the ER/GA for efficient basolateral exocytosis. However, MNPs escaping from lysosomes were detained within cells by autophagosomes. Collectively, our research suggested that early endosomes and late endosomes were ideal escape sites for trans-epithelial delivery.

Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

BACKGROUND: Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. METHODS: Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. RESULTS: Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. CONCLUSIONS: Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Simultaneous TIRF imaging of subplasmalemmal Ca2+ dynamics and granule fusions in insulin-secreting INS-1 cells reveals coexistent synchronized and asynchronous release.

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca2+-dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca2+]i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca2+]i microdomains around single or clustered Ca2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca2+ spikes or incidentally overlap with subplasmalemmal Ca2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca2+ microdomains around voltage-gated Ca2+ channels.

Ykt6 functionally overlaps with vacuolar and exocytic R-SNAREs in the yeast Saccharomyces cerevisiae.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.

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.

Quantitative Proteomics Reveal That CB2R Agonist JWH-133 Downregulates NF-κB Activation, Oxidative Stress, and Lysosomal Exocytosis from HIV-Infected Macrophages.

HIV-associated neurocognitive disorders (HAND) affect 15-55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND.