Loss of VAMP5 in mice results in duplication of the ureter and insufficient expansion of the lung.

BACKGROUND: Vesicle-associated membrane protein 5 (VAMP5) is a member of the SNARE protein family, which regulates the docking and fusion of membrane vesicles within cells. Previously, we reported ubiquitous expression of VAMP5 proteins in various organs except the brain and small intestine. However, the precise roles of VAMP5 in each organ remain unclear. To explore the roles of VAMP5 in vivo, we generated VAMP5 knockout (KO) mice. RESULTS: VAMP5 KO mice showed low birth rate and low body weight. KO embryos grew normally in the uterus, and tended to die around birth. Anatomical analysis revealed that viable KO mice often exhibited duplication of the ureter, and dead KO mice showed insufficient expansion of the lung. VAMP5 was localized in the epithelial cells of the ureter and terminal bronchiole. CONCLUSIONS: VAMP5 KO mice showed a low birth rate and abnormalities of the urinary and respiratory systems. VAMP5 KO mice died around birth, possibly due to defects in vesicoureteral flow and breathing. The results presented could provide a basis for future studies to understand the roles of VAMP5 protein. Developmental Dynamics 247:754-762, 2018. © 2018 Wiley Periodicals, Inc.

Entamoeba histolytica-Induced Mucin Exocytosis Is Mediated by VAMP8 and Is Critical in Mucosal Innate Host Defense.

Intestinal mucus secretion is critical in maintaining mucosal host defense against a myriad of pathogens by preventing direct association with the epithelium. Entamoeba histolytica specifically binds colonic MUC2 mucin and also induces potent hypersecretion from goblet cells; however, characterization of the nature of the mechanisms controlling mucus release remains elusive. In this report, we identify vesicle SNARE vesicle-associated membrane protein 8 (VAMP8) present on mucin granules as orchestrating regulated exocytosis in human goblet cells in response to the presence of E. histolytica VAMP8 was specifically activated during E. histolytica infection, and ablation of VAMP8 led to impaired mucin secretion. As a consequence, loss of VAMP8 increased E. histolytica adherence to epithelial cells associated with enhanced cell death through apoptosis characterized by caspase 3 and 9 cleavages and DNA fragmentation. With the mucosal barrier compromised in Vamp8-/- animals, E. histolytica induced an aggressive proinflammatory response with elevated levels of interleukin-1 alpha (IL-1α), IL-1ß, and tumor necrosis factor alpha (TNF-α) secretion. This report is the first to characterize regulated mucin exocytosis in intestinal goblet cells in response to a pathogen and the downstream consequences of improper mucin secretion in mucosal barrier defense.IMPORTANCE The intestinal tract is exposed to countless substances and pathogens, and yet homeostasis is maintained, in part by the mucus layer that houses the microbiota and spatially separates potential threats from the underlying single layer of epithelium. Despite the critical role of mucus in innate host defense, characterization of the mechanisms by which mucus is secreted from specialized goblet cells in the gut remains elusive. Here, we describe the machinery that regulates mucus secretion as well as the consequence during infection with the colonic pathogen Entamoeba histolytica Abolishment of the key machinery protein VAMP8 abrogated mucus release in cultured human colonic goblet cells and during E. histolytica infection in Vamp8-/- mice, which showed enhanced amoeba contact and killing of epithelial cells, triggering a potent proinflammatory response. This report highlights the importance of the VAMP8 secretory machinery in facilitating mucus release from intestinal goblet cells and the dire consequences that occur during disease pathogenesis if these pathways are not functional.

VAMP7 Regulates Autophagy to Maintain Mitochondrial Homeostasis and to Control Insulin Secretion in Pancreatic ß-Cells.

VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic ß-cells is largely unknown. To elucidate the physiological role of VAMP7 in ß-cells, we generated pancreatic ß-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient ß-cells showed defective autophagosome formation and reduced mitochondrial function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient ß-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre ß-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in ß-cells.

VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.

Tomosyn-2 is required for normal motor performance in mice and sustains neurotransmission at motor endplates.

Tomosyn-1 (STXBP5) is a soluble NSF attachment protein receptor complex-binding protein that inhibits vesicle fusion, but the role of tomosyn-2 (STXBP5L) in the mammalian nervous system is still unclear. Here we generated tomosyn-2 null (Tom2(KO/KO)) mice, which showed impaired motor performance. This was accompanied by synaptic changes at the neuromuscular junction, including enhanced spontaneous acetylcholine release frequency and faster depression of muscle motor endplate potentials during repetitive stimulation. The postsynaptic geometric arrangement and function of acetylcholine receptors were normal. We conclude that tomosyn-2 supports motor performance by regulation of transmitter release willingness to sustain synaptic strength during high-frequency transmission, which makes this gene a candidate for involvement in neuromuscular disorders.

Absence of TI-VAMP/Vamp7 leads to increased anxiety in mice.

Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 of Vamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.

VAMP8 is a vesicle SNARE that regulates mucin secretion in airway goblet cells.

Mucin secretion is an innate defence mechanism, which is noxiously upregulated in obstructive lung diseases (e.g. chronic obstructive pulmonary disease (COPD), cystic fibrosis and asthma). Mucin granule exocytosis is regulated by specific protein complexes, but the SNARE exocytotic core has not been defined in airway goblet cells. In this study, we identify VAMP8 as one of the SNAREs regulating mucin granule exocytosis. VAMP8 mRNA was present in human airway and lung epithelial cells, and deep-sequencing and expression analyses of airway epithelial cells revealed that VAMP8 transcripts were expressed at 10 times higher levels than other VAMP mRNAs. In human airway epithelial cell cultures and freshly excised tissues, VAMP8 immunolocalised mainly to goblet cell mucin granules. The function of VAMP8 in airway mucin secretion was tested by RNA interference techniques. Both VAMP8 short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) reduced mucin secretion induced by PAR agonists, neutrophil elastase and ATP in two airway epithelial cell culture models. Notably, basal (non-agonist elicited) mucin secretion was also reduced in these experiments. VAMP8 knockdown was also effective in decreasing mucin secretion in airway epithelial cell cultures with induced mucous metaplasia/mucin hypersecretion. Unlike VAMP8 silencing, knockdown of VAMP2 or VAMP3 did not affect mucin secretion. Importantly, in VAMP8 knock-out (KO) mice with IL-13-induced mucous metaplasia, mucin content in the bronchoalveolar lavage (BAL) and ATP-stimulated mucin secretion in the trachea were reduced compared to WT-matched littermates. Our data indicate that VAMP8 is an essential SNARE in airway mucin granule exocytosis. Reduction of VAMP8 activity/expression may provide a novel therapeutic target to ameliorate airway mucus obstruction in lung diseases.

Enhanced energy expenditure, glucose utilization, and insulin sensitivity in VAMP8 null mice.

OBJECTIVE: Previous studies have demonstrated that the VAMP8 protein plays a complex role in the control of granule secretion, transport vesicle trafficking, phagocytosis, and endocytosis. The present study was aimed to investigate the role of VAMP8 in mediating GLUT4 trafficking and therefore insulin action in mice. RESEARCH DESIGN AND METHODS: Physiological parameters were measured using Oxymax indirect calorimetry system in 12-week-old VAMP8 null mice. Dynamic analysis of glucose homeostasis was assessed using euglycemic-hyperinsulinemic clamp coupled with tracer radioactively labeled 2-deoxyglucose. Insulin stimulated GLUT4 protein expressions on muscle cell surface were examined by immunofluorescence microscopy. RESULTS: VAMP8 null mice display reduced adiposity with increased energy expenditure despite normal food intake and reduced spontaneous locomotor activity. In parallel, the VAMP8 null mice also had fasting hypoglycemia (84 ± 11 vs. 115 ± 4) and enhanced glucose tolerance with increased insulin sensitivity due to increases in both basal and insulin-stimulated glucose uptake in skeletal muscle (0.19 ± 0.04 vs. 0.09 ± 0.01 mmol/kg/min during basal, 0.6 ± 0.04 vs. 0.31 ± 0.06 mmol/kg/min during clamp in red-gastrocnemius muscle, P < 0.05). Consistent with a role for VAMP8 in the endocytosis of the insulin-responsive GLUT4, sarcolemma GLUT4 protein levels were increased in both the basal and insulin-stimulated states without any significant change in the total amount of GLUT4 protein or related facilitative glucose transporters present in skeletal muscle, GLUT1, GLUT3, and GLUT11. CONCLUSIONS: These data demonstrate that, in the absence of VAMP8, the relative subcellular distribution of GLUT4 is altered, resulting in increased sarcolemma levels that can account for increased glucose clearance and insulin sensitivity.

VAMP8 is essential in anaphylatoxin-induced degranulation, TNF-alpha secretion, peritonitis, and systemic inflammation.

VAMP8, a member of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) family of fusion proteins, initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. VAMP8 physiological function in inflammation has not been elucidated. In this paper, we show that deficiency of VAMP8 protects mice from anaphylatoxin (C5a)-induced neutropenia, peritonitis, and systemic inflammation. We show that, in vivo, VAMP8 deletion inhibits neutropenia and phagocyte recruitment. We also show that in macrophages, VAMP8 localizes on secretory granules and degranulation is inhibited in VAMP8-deficient macrophages. Moreover, VAMP8(-/-) mice show reduced systemic inflammation with inhibition of serum TNF-alpha levels, whereas IL-1beta, IL-6, and MIP1alpha release are not affected. In wild-type macrophages, TNF-alpha colocalizes with VAMP8-positive vesicles, and in VAMP8-deficient macrophages, the TNF-alpha release is inhibited. Furthermore, VAMP8 regulates the release of TNF-alpha and beta-hexosaminidase triggered by fMLP, and VAMP8(-/-) mice are protected from fMLP-induced peritonitis. These data demonstrate that the VAMP8 vesicle-associated-SNARE is required for the proper trafficking of secretory lysosomal granules for exocytosis in macrophages and for the release of the potent proinflammatory cytokine, TNF-alpha.

Endobrevin/VAMP-8-dependent dense granule release mediates thrombus formation in vivo.

Individuals whose platelets lack dense or alpha-granules suffer various degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. In platelets, SNARE proteins mediate the membrane fusion events required for granule cargo release. Endobrevin/VAMP-8 is the primary vesicle-SNARE (v-SNARE) responsible for efficient release of dense and alpha-granule contents; thus, VAMP-8(-/-) mice are a useful model to evaluate the importance of platelet granule secretion in thrombus formation. Thrombus formation, after laser-induced vascular injury, in these mice is delayed and decreased, but not absent. In contrast, thrombus formation is almost completely abolished in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lacks dense granules. Evaluation of aggregation of VAMP-8(-/-) and ruby-eye platelets indicates that defective ADP release is the primary abnormality leading to impaired aggregation. These results demonstrate the importance of dense granule release even in the earliest phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for antiplatelet therapies.

Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release.

Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex-mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)-like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.

Vesicle-associated membrane protein-8/endobrevin negatively regulates phagocytosis of bacteria in dendritic cells.

Phagocytosis is a specialized mechanism used by mammalian cells, particularly the cells of the immune system, such as dendritic cells (DC) and macrophages, to protect the host against infection. The process involves a complex cascade of pathways, from the ligation of surface receptors of phagocytes with components of the microorganism's surface, formation of phagosomes and subsequently phagolysosomes, to the eventual presentation of foreign Ags. Vesicle-associated membrane protein (VAMP)-8/endobrevin has been shown previously to function in the endocytic pathways. Our results showed that VAMP-8 colocalized with lysosome-associated membrane protein-2, and a significant amount of VAMP-8 was recruited to the phagosomes during bacterial ingestion. However, overexpression of VAMP-8 significantly inhibited phagocytosis in DC. We also found that the phagocytic activity of VAMP-8-/- DC was significantly higher than wild-type VAMP-8+/+ DC, thus further confirming that VAMP-8 negatively regulates phagocytosis in immature DC.

VAMP8/endobrevin as a general vesicular SNARE for regulated exocytosis of the exocrine system.

The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.

Endobrevin/VAMP-8 is the primary v-SNARE for the platelet release reaction.

Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8-/- mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. In contrast, VAMP-2+/-, VAMP-3-/-, and VAMP-2+/-/VAMP-3-/- platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3-/- platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8-/- platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8-/- mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation.