Syntaxin Research Articles

Syntaxin 4 (STX4)‐ More than just a SNARE protein: Elevating STX4 content in skeletal muscle to prevent insulin resistance/pre‐diabetes

Type 2 diabetes results from a combination of peripheral insulin resistance plus insulin secretion defects. Insulin resistance is characterized by a failure of skeletal muscle to clear excess blood glucose from the circulation, and is central to what is commonly called ‘pre‐diabetes'. While type 2 diabetes afflicts ~10% of the US population, pre‐diabetes is estimated to impact more than a third of our population. Over 80% of glucose clearance occurs via the skeletal muscle, there are no directed therapeutics to restore its function in pre‐diabetics. Several studies have identified correlations between insulin resistance and reduced levels of a SNARE protein named Syntaxin 4 (STX4) in human skeletal muscle. STX4 interacts with other SNARE proteins to facilitate the fusion of glucose transporter 4 (GLUT4) vesicles with the plasma membrane, to then channel extracellular glucose to the interior of the muscle cell, ultimately ‘clearing glucose’ from the circulation. Towards understanding how STX4 content is regulated in muscle, we mimicked the pre/diabetic milieu of high glucose and high lipid content in the L6‐GLUT4myc skeletal muscle cells and demonstrated the same STX4 depletion reported in the human studies. Having established this model system, we next assessed the potential for STX4 overexpression to prevent insulin resistance, and found STX4‐overexpressing cells show less Annexin V and Propidium iodide staining compared with sham‐transfected cells, suggesting that STX4 overexpression exerted a protective effect against diabetogenic stimuli‐induced apoptosis. To next test this in vivo, we have developed an inducible skeletal muscle specific STX4 overexpressing mouse model. Remarkably, under standard non‐diabetogenic conditions, female mice overexpressing STX4 show heightened glucose tolerance and insulin sensitivity compared to control mice. Whether STX4‐overexpressing mice are protected from obesity‐induced insulin resistance, or perhaps whether STX4 upregulation in already‐obese mice might reverse insulin resistance and through which mechanism, are questions currently under pursuit. Taken together, these results suggest that STX4 functions in the skeletal muscle to facilitate glucose uptake function and to protect myocytes from diabetogenic stress induced apoptosis. This suggests that STX4 could be a viable therapeutic target for treatment and prevention of pre‐diabetes.Support or Funding InformationThis research was supported by the National Institutes of Health (DK067912, DK102233) to D.C.T. and also by the H.N. & Frances C. Berger Foundation Fellowship to K.MThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Correlation between miRNAs and target genes in response to Campylobacter jejuni inoculation in chicken

Campylobacter jejuni (C. jejuni) is one of the main causes of human campylobacteriosis worldwide. Chicken is the main reservoir of C. jejuni. The cecum is the primary site of C. jejuni colonization. Our previous study has shown that the CLOCK gene is important to regulate the response to C. jejuni inoculation and has a cross-talking with immune-related genes. Correlation networks between the CLOCK gene and microRNAs (miRNAs) play vital roles in colorectal tumorigenesis. MiRNAs play crucial roles in bacterial invasion through regulating multiple target genes. To study the regulatory correlation between miRNAs and target genes, 5 miRNAs that regulate the expression of the CLOCK gene (CLOCK-interacted miRNAs) and 5 immune-related target genes predicted through Targetscan and miRDB were selected to analyze the expression pattern in chicken ceca at 4, 8, 12, 16, 20, and 24 h post C. jejuni inoculation using quantitative PCR. The results showed that the expression of gga-miR-148a, gga-miR-1416–5p, gga-miR-30b, and gga-miR-30c were significantly up-regulated at 8 hpi, and gga-miR-30a-5p, gga-miR-30b, and gga-miR-30c were significantly down-regulated at 24 hpi, and gga-miR-1416–5p was significantly down-regulated at 20 hpi (P < 0.05). The expression levels of BCL9 (B-cell CLL/lymphoma 9), STX16 (syntaxin16), IL4R (interleukin 4 receptor), and IRF4 (interferon regulatory factor 4) genes were significantly up-regulated at 8 hpi, while SOCS3 (suppressor of cytokine signaling 3) was significantly down-regulated. Furthermore, the expression pattern of gga-miR-30b was consistent with gga-miR-30c. Both miRNAs and mRNAs showed an oscillation expression pattern in both inoculated and non-inoculated groups. The regulatory direction of SOCS3 was contrary to those of gga-miR-148a, gga-miR-1416–5p, gga-miR-30b, and gga-miR-30c at 8 hpi. In summary, MiR-30 and the miR-148/miR-152 family time-dependently regulate the response to C. jejuni inoculation in chicken ceca. BCL9, STX16, IRF4, and IL4R play important roles in the response to C. jejuni inoculation. The CLOCK gene-interacted miRNA and immune-related target genes had a correlative response to C. jejuni inoculation. The SOCS3 gene interacts with gga-miR-30b, gga-miR-30c, gga-miR-148a, and gga-miR-1416–5p in response to C. jejuni inoculation at 8 hpi. The correlations between CLOCK-interacted miRNA and immune-related target genes play vital roles in the response to C. jejuni inoculation. The results herein will lay the foundation for further study of a regulatory mechanism of chicken miRNAs and their target genes in response to C. jejuni inoculation.

Depletion of the membrane-fusion regulator Munc18c attenuates caerulein hyperstimulation–induced pancreatitis

Epithelial pancreatic acinar cells perform crucial functions in food digestion, and acinar cell homeostasis required for secretion of digestive enzymes relies on SNARE-mediated exocytosis. The ubiquitously expressed Sec1/Munc18 protein mammalian uncoordinated-18c (Munc18c) regulates membrane fusion by activating syntaxin-4 (STX-4) to bind cognate SNARE proteins to form a SNARE complex that mediates exocytosis in many cell types. However, in the acinar cell, Munc18c's functions in exocytosis and homeostasis remain inconclusive. Here, we found that pancreatic acini from Munc18c-depleted mice (Munc18c+/-) and human pancreas (lenti-Munc18c-shRNA-treated) exhibit normal apical exocytosis of zymogen granules (ZGs) in response to physiologic stimulation with the intestinal hormone cholecystokinin (CCK-8). However, when stimulated with supraphysiologic CCK-8 levels to mimic pancreatitis, Munc18c-depleted (Munc18c+/-) mouse acini exhibited a reduction in pathological basolateral exocytosis of ZGs resulting from a decrease in fusogenic STX-4 SNARE complexes. This reduced basolateral exocytosis in part explained the less severe pancreatitis observed in Munc18c+/- mice after hyperstimulation with the CCK-8 analog caerulein. Likely as a result of this secretory blockade, Munc18c-depleted acini unexpectedly activated a component of the endoplasmic reticulum (ER) stress response that contributed to autophagy induction, resulting in downstream accumulation of autophagic vacuoles and autolysosomes. We conclude that Munc18c's role in mediating ectopic basolateral membrane fusion of ZGs contributes to the initiation of CCK-induced pancreatic injury, and that blockade of this secretory process could increase autophagy induction.

Pancreatitis-Induced Depletion of Syntaxin 2 Promotes Autophagy and Increases Basolateral Exocytosis

Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy. We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-μm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays. Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen. In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

PMicroRNA-124a regulates LPS-induced septic cardiac dysfunction by targeting STX2.

To examine the role of miR-124a in LPS-induced septic cardiac insufficiency where underlying mechanism is unclear. Expression of miR-124a was decreased in myocardium of LPS-induced septic cardiac dysfunction model. miR-124a antagomiR or agomiR were injected via tail vein to induce miR-124a-dysregulated model. miR-124a antagomiR aggravated LPS-induced cardiac dysfunction and apoptosis, while miR-124a agomiR had the opposite effect. Syntaxin-2 (STX2) was indicated as a candidate target gene by bioinformatic software. Further experiments confirmed that STX2 was downregulated in miR-124a agomiR-treated rats but upregulated in miR-124a antagomiR-treated rats, and STX2 inhibition could strongly block the miR-124a antagomiR-associated increase in cell apoptosis. Luciferase reporter activity assay indicated that STX2 was a direct target of miR-124a. Serological detection reveled that miR-124a was down-regulated in the plasma of septic cardiac dysfunction rats. miR-124a aggravates LPS-induced cardiac dysfunction and the miR-124a/STX2 pathway might serve as the potential diagnostic and therapeutic targets for septic cardiac dysfunction.

Syntaxin 4 mediates endosome recycling for lytic granule exocytosis in cytotoxic T-lymphocytes.

Adaptive and innate immunity utilize the perforin-killing pathway to eliminate virus-infected or cancer cells. Cytotoxic T-lymphocytes (CTLs) and natural killer cells mediate this process by releasing toxic proteins at the contact area with target cells known as immunological synapse (IS). Formation of a stable IS and exocytosis of toxic proteins requires persistent fusion of Rab11a recycling endosomes with the plasma membrane (PM) that may assure the delivery of key effector proteins. Despite the importance of the recycling endosomal compartment, the membrane fusion proteins that control this process at the IS remain elusive. Here, by performing knockdown experiments we found that syntaxin 4 (STX4) is necessary for cytotoxic activity and CD107a degranulation against target cells in a similar fashion to syntaxin 11, which is involved in lytic granule (LG) exocytosis and immunodeficiency when it is mutated. Using total internal reflection fluorescent microscopy we identified that STX4 mediates fusion of EGFP-Rab11a vesicles at the IS. Immunoprecipitation experiments in lysates of activated CTLs indicate that endogenous STX4 may drive this fusion step by interacting with cognate proteins: Munc18-3/SNAP23/VAMP7 and/or VAMP8. These results reveal the role of STX4 in mediating fusion of Rab11a endosomes upstream of lytic granules (LGs) exocytosis and further demonstrate the importance of this pathway in controlling CTL-mediated cytotoxicity.

A novel Drosophila model of TDP-43 proteinopathies: N-terminal sequences combined with the Q/N domain induce protein functional loss and locomotion defects.

ABSTRACTTransactive response DNA-binding protein 43 kDa (TDP-43, also known as TBPH in Drosophila melanogaster and TARDBP in mammals) is the main protein component of the pathological inclusions observed in neurons of patients affected by different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The number of studies investigating the molecular mechanisms underlying neurodegeneration is constantly growing; however, the role played by TDP-43 in disease onset and progression is still unclear. A fundamental shortcoming that hampers progress is the lack of animal models showing aggregation of TDP-43 without overexpression. In this manuscript, we have extended our cellular model of aggregation to a transgenic Drosophila line. Our fly model is not based on the overexpression of a wild-type TDP-43 transgene. By contrast, we engineered a construct that includes only the specific TDP-43 amino acid sequences necessary to trigger aggregate formation and capable of trapping endogenous Drosophila TDP-43 into a non-functional insoluble form. Importantly, the resulting recombinant product lacks functional RNA recognition motifs (RRMs) and, thus, does not have specific TDP-43-physiological functions (i.e. splicing regulation ability) that might affect the animal phenotype per se. This novel Drosophila model exhibits an evident degenerative phenotype with reduced lifespan and early locomotion defects. Additionally, we show that important proteins involved in neuromuscular junction function, such as syntaxin (SYX), decrease their levels as a consequence of TDP-43 loss of function implying that the degenerative phenotype is a consequence of TDP-43 sequestration into the aggregates. Our data lend further support to the role of TDP-43 loss-of-function in the pathogenesis of neurodegenerative disorders. The novel transgenic Drosophila model presented in this study will help to gain further insight into the molecular mechanisms underlying neurodegeneration and will provide a valuable system to test potential therapeutic agents to counteract disease.

Glia Maturation Factor-γ Regulates Monocyte Migration through Modulation of β1-Integrin

Monocyte migration requires the dynamic redistribution of integrins through a regulated endo-exocytosis cycle, but the complex molecular mechanisms underlying this process have not been fully elucidated. Glia maturation factor-γ (GMFG), a novel regulator of the Arp2/3 complex, has been shown to regulate directional migration of neutrophils and T-lymphocytes. In this study, we explored the important role of GMFG in monocyte chemotaxis, adhesion, and β1-integrin turnover. We found that knockdown of GMFG in monocytes resulted in impaired chemotactic migration toward formyl-Met-Leu-Phe (fMLP) and stromal cell-derived factor 1α (SDF-1α) as well as decreased α5β1-integrin-mediated chemoattractant-stimulated adhesion. These GMFG knockdown impaired effects could be reversed by cotransfection of GFP-tagged full-length GMFG. GMFG knockdown cells reduced the cell surface and total protein levels of α5β1-integrin and increased its degradation. Importantly, we demonstrate that GMFG mediates the ubiquitination of β1-integrin through knockdown or overexpression of GMFG. Moreover, GMFG knockdown retarded the efficient recycling of β1-integrin back to the plasma membrane following normal endocytosis of α5β1-integrin, suggesting that the involvement of GMFG in maintaining α5β1-integrin stability may occur in part by preventing ubiquitin-mediated degradation and promoting β1-integrin recycling. Furthermore, we observed that GMFG interacted with syntaxin 4 (STX4) and syntaxin-binding protein 4 (STXBP4); however, only knockdown of STXBP4, but not STX4, reduced monocyte migration and decreased β1-integrin cell surface expression. Knockdown of STXBP4 also substantially inhibited β1-integrin recycling in human monocytes. These results indicate that the effects of GMFG on monocyte migration and adhesion probably occur through preventing ubiquitin-mediated proteasome degradation of α5β1-integrin and facilitating effective β1-integrin recycling back to the plasma membrane.

The localisation of the apical Par/Cdc42 polarity module is specifically affected in microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a genetic disorder affecting intestinal absorption. It is caused by mutations in MYO5B or syntaxin 3 (STX3) affecting apical membrane trafficking. Morphologically, MVID is characterised by a depletion of apical microvilli and the formation of microvillus inclusions inside the cells, suggesting a loss of polarity. To investigate this hypothesis, we examined the location of essential apical polarity determinants in five MVID patients. We found that the polarity determinants Cdc42, Par6B, PKCζ/ι and the structural proteins ezrin and phospho-ezrin were lost from the apical membrane and accumulated either in the cytoplasm or on the basal side of enterocytes in patients, which suggests an inversion of cell polarity. Moreover, microvilli-like structures were observed at the basal side as per electron microscopy analysis. We next performed Myo5B depletion in three dimensional grown human Caco2 cells forming cysts and found a direct link between the loss of Myo5B and the mislocalisation of the same apical proteins; furthermore, we observed that a majority of cysts displayed an inverted polarity phenotype as seen in some patients. Finally, we found that this loss of polarity was specific for MVID: tissue samples of patients with Myo5B-independent absorption disorders showed normal polarity but we identified Cdc42 as a potentially essential biomarker for trichohepatoenteric syndrome. Our findings indicate that the loss of Myo5B induces a strong loss of enterocyte polarity, potentially leading to polarity inversion. Our results show that polarity determinants could be useful markers to help establishing a diagnosis in patients. Furthermore, they could be used to characterise other rare intestinal absorption diseases.

SNAP23 Regulates Endothelial Exocytosis of von Willebrand Factor.

Endothelial exocytosis regulates vascular thrombosis and inflammation. The trafficking and release of endothelial vesicles is mediated by SNARE (Soluble NSF Attachment protein REceptors) molecules, but the exact identity of endothelial SNAREs has been unclear. Three SNARE molecules form a ternary complex, including isoforms of the syntaxin (STX), vesicle-associated membrane protein (VAMP), and synaptosomal-associated protein (SNAP) families. We now identify SNAP23 as the predominant endothelial SNAP isoform that mediates endothelial exocytosis of von Willebrand Factor (VWF). SNAP23 was localized to the plasma membrane. Knockdown of SNAP23 decreased endothelial exocytosis, suggesting it is important for endothelial exocytosis. SNAP23 interacted with the endothelial exocytic machinery, and formed complexes with other known endothelial SNARE molecules. Taken together, these data suggest that SNAP23 is a key component of the endothelial SNARE machinery that mediates endothelial exocytosis.

Mechanisms of Intestinal Serotonin Transporter (SERT) Upregulation by TGF-β1 Induced Non-Smad Pathways.

TGF-β1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-β1 on serotonin transporter (SERT) activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT), is not known. Current studies were designed to examine acute effects of TGF-β1 on SERT. Model human intestinal Caco-2 cells grown as monolayer’s or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-β1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-β1 receptor (TGFRI) as SB-431542, a specific TGF-βRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-β1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 μM) attenuated the TGF-β1-mediated increase in SERT function. TGF-β1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3) and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-β1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-β1 (10 ng/ml, 1h). These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-β1 offers a novel therapeutic strategy to treat GI disorders.

Cacna1f gene decreased contractility of skeletal muscle in rat model with congenital stationary night blindness

The CACNA1F gene encodes a member of the alpha-1F subunit family in the voltage-dependent calcium channel (Cav1.4) complex. Mutations in this gene result in incomplete congenital stationary night blindness (iCSNB2) in humans. And Cav1.4 mutation could affect the functions of the skeletal muscle. This study investigated the role of Cacna1f mutations in alteration of the skeletal muscle functions in a Cacna1f mutation rat model (Cacna1fCSNB2 rat). We found that the muscle endurance behaviors of Cacna1fCSNB2 rats were significantly lower than those of the wild-type rats. The high-frequency fatigue resistance of the soleus muscle was decreased in Cacna1fCSNB2 rats under continuous tetanic stimulation. The expression levels of the syntaxin (SYN) proteins in the soleus of the Cacna1fCSNB2 rats were lower than those of wild-type rats. SYN was expressed in the soleus muscle, but not in the extensor digitorum longus. The Cav1.4 protein was not detected in the skeletal muscle of Cacna1fCSNB2 rats. The Cacna1f mRNA level in the soleus of Cacna1fCSNB2 rats was decreased compared with that in wild-type rats. This study demonstrated for the first time that the Cacna1f mutation reduces the function of slow-twitch skeletal muscle. And it also demonstrated that the Cacna1f gene affects synapse-associated protein expression, which may block the signal transmission in synaptic connectivity of the retina and skeletal muscle in Cacna1f-mutant rats.

Human cytomegalovirus miR-US33-5p inhibits viral DNA synthesis and viral replication by down-regulating expression of the host Syntaxin3

During infection with human cytomegalovirus (HCMV), overexpression of hcmv-miR-US33 can inhibit the lytic viral replication and down-regulate US29 mRNA. However, it remains unknown whether inhibition of viral replication by miR-US33 is mediated by down-regulation of expression of US29 or another host gene. Here, we identified the host gene Syntaxin3 (STX3) to be a direct target of hcmv-miR-US33-5p using Hybrid-PCR and luciferase-reporter assays. It was further demonstrated that the levels of STX3 protein were down-regulated in hcmv-miR-US33-5p-overexpressing cells. Experiments with STX3-specific siRNA, or with an inhibitor of hcmv-miR-US33-5p confirmed that hcmv-miR-US33-5p-mediated inhibition of HCMV DNA synthesis and of viral replication are specifically mediated by down-regulation of STX3 expression.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Chinese children: a primary study on clinical characteristics and gene mutations

To study the clinical features and gene mutations of EBV-HLH in Chinese children. Sixteen pediatric patients with Epstein-Barr virus-associated HLH were enrolled the study from May 2011 to September 2012, who were admitted to Capital Institute of Pediatrics. All patients' clinical features were recorded and their nucleotide sequences of all exons and their flanking intronic sequences of perforin (PRF1) gene, protein unc-13 homolog D (UNC13D) gene, two fusion protein genes of syntaxin 11 (STX11)gene, synaptic binding protein 2 (STXBP2) gene, SH2 domain 1A (SH2D1A) gene, and the X-linked inhibitor of apoptosis protein (XIAP) gene were amplified by PCR followed by direct sequencing. Based on HLH gene detection results, all patients divided into the positive and negative gene EBV-HLH subgroups. Statistical analysis was conducted using SPSS 11.5 software. Seven of sixteen pediatric patients with EBV-HLH were identified with heterozygous, hemizygous, and homozygous mutations in PRF1, UNC13D, STXBP2, and SH2D1A. No significant differences were found on the gender, age, illness duration, EBV load, the positive duration of EBV-DNA, lab results, clinical symptoms, treatment, and the outcome between the HLH gene positive and negative subgroups (P > 0.05). A considerable EBV-HLH pediatric patients have genetic defects, and HLH gene defects may not affect the clinical features and treatment of EBV-HLH pediatric patients.

Abstract 181: VAMP8 Mediates Endothelial Granule Exocytosis

Endothelial cells maintain the integrity of the vasculature. In response to injury, endothelial cells undergo exocytosis, releasing various compounds into the blood stream by vesicle fusion with the plasma membrane. Endothelial exocytosis provokes inflammation and thrombosis as one of the earliest responses to vascular injury. Enhanced understanding of these events may lead to novel treatments for vascular diseases such as atherosclerosis, stroke, myocardial infarction, and thrombosis. However, the exocytic machinery that regulates granule secretion from endothelial cells is not completely defined. We tested the hypothesis that specific proteins in the soluble N-ethylmaleimide sensitive factor receptor attachment protein (SNARE) superfamily are associated with the endothelial-specific secretory organelles, Weibel-Palade bodies (WPBs), and mediates endothelial exocytosis. We found that human endothelial cells express members of the SNARE superfamily, including vesicle associated membrane protein 3 (VAMP3), VAMP8, syntaxin 4 (STX4), and synaptosomal associated protein 23 (SNAP23). Out of all these proteins, we found that only VAMP8 co-localizes with WPBs containing von Willebrand Factor (VWF), the major component of endothelial granules. Furthermore, VAMP8 also interacts with STX4, another SNARE component of the exocytic machinery. To explore the role of VAMP8 in regulating endothelial granule exocytosis, we knocked down VAMP8 expression with siRNA in endothelial cells. Decreasing VAMP8 expression impairs exocytosis; conversely, rescue of VAMP8 expression after VAMP8 knockdown restores exocytosis to normal level. Mice lacking VAMP8 have decreased endothelial exocytosis, as measured by decreased plasma VWF levels and decreased leukocyte rolling along vessels. In conclusion, our data suggest that VAMP8 plays a critical role in mediating endothelial exocytosis and may be a potential therapeutic target for vascular inflammatory and thrombotic diseases.

Loss of Syntaxin 3 Causes Variant Microvillus Inclusion Disease

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.

Syntaxin 1B, but Not Syntaxin 1A, Is Necessary for the Regulation of Synaptic Vesicle Exocytosis and of the Readily Releasable Pool at Central Synapses

Two syntaxin 1 (STX1) isoforms, HPC-1/STX1A and STX1B, are coexpressed in neurons and function as neuronal target membrane (t)-SNAREs. However, little is known about their functional differences in synaptic transmission. STX1A null mutant mice develop normally and do not show abnormalities in fast synaptic transmission, but monoaminergic transmissions are impaired. In the present study, we found that STX1B null mutant mice died within 2 weeks of birth. To examine functional differences between STX1A and 1B, we analyzed the presynaptic properties of glutamatergic and GABAergic synapses in STX1B null mutant and STX1A/1B double null mutant mice. We found that the frequency of spontaneous quantal release was lower and the paired-pulse ratio of evoked postsynaptic currents was significantly greater in glutamatergic and GABAergic synapses of STX1B null neurons. Deletion of STX1B also accelerated synaptic vesicle turnover in glutamatergic synapses and decreased the size of the readily releasable pool in glutamatergic and GABAergic synapses. Moreover, STX1A/1B double null neurons showed reduced and asynchronous evoked synaptic vesicle release in glutamatergic and GABAergic synapses. Our results suggest that although STX1A and 1B share a basic function as neuronal t-SNAREs, STX1B but not STX1A is necessary for the regulation of spontaneous and evoked synaptic vesicle exocytosis in fast transmission.

Autosomal Dominant Pseudohypoparathyroidism Type Ib: A Novel Inherited Deletion AblatingSTX16Causes Loss of Imprinting at the A/B DMR

Pseudohypoparathyroidism type Ib (PHP-Ib) is a rare imprinting disorder characterized by end-organ resistance to PTH and, frequently, to thyroid-stimulating hormone. PHP-Ib familial form, with an autosomal dominant pattern of transmission (autosomal dominant pseudohypoparathyroidism type Ib [AD-PHP-Ib]), is typically characterized by an isolated loss of methylation at the guanine nucleotide-binding protein α-stimulating activity polypeptide 1 A/B differentially methylated region (DMR), secondary to genetic deletions disrupting the upstream imprinting control region in the syntaxin-16 (STX16) locus. However, deletions described up to now failed to account some cases of patients with a methylation defect limited to the A/B DMR; thus, it is expected the existence of other still unknown rearrangements, undetectable with conventional molecular diagnostic methods. We investigated a PHP-Ib patient with a methylation defect limited to the A/B DMR and no known STX16 deletions to find the underlying primary genetic defect. A PHP-Ib patient (hypocalcaemia, hyperphosphataemia, raised serum PTH levels, no vitamin D deficiency) and his unaffected relatives were investigated by methylation specific-multiplex ligand-dependent probe amplification to search for novel pathogenetic defects affecting the guanine nucleotide-binding protein α-stimulating activity polypeptide 1 and STX16 loci. We report the clinical, biochemical, and molecular analysis of an AD-PHP-Ib patient with a novel STX16 deletion overlapping with previously identified STX16 deletions but that, unlike these genetic defects associated with AD-PHP-Ib, goes unnoticed with commonly used first-level diagnostic techniques. Our work highlights the importance of performing accurate investigations in PHP-Ib patients with methylation defects to allow precise genetic counseling because, in case of deletions, the segregation ratio is about 50% and the disease phenotype is transmitted in an autosomal dominant fashion via the mother.

Munc18-2 and Syntaxin 3 Control Distinct Essential Steps in Mast Cell Degranulation

Mast cell degranulation requires N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and mammalian uncoordinated18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. In this study, we found that small interfering RNA-mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE-binding partner syntaxin 3 (STX3) also markedly inhibited degranulation, whereas combined knockdown produced an additive inhibitory effect. Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion, demonstrating unique roles of each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation, clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast cells.

TBI and sex: Crucial role of progesterone protecting the brain in an omega − 3 deficient condition

We assessed whether the protective action of progesterone on traumatic brain injury (TBI) could be influenced by the consumption of omega−3 fatty acids during early life. Pregnant Sprague–Dawley rats were fed on omega−3 adequate or deficient diet from 3rd day of pregnancy and their female offspring were kept on the same diets up to the age of 15weeks. Ovariectomy was performed at the age of 12weeks to deprive animals from endogenous steroids until the time of a fluid percussion injury (FPI). Dietary n−3 fatty acid deficiency increased anxiety in sham animals and TBI aggravated the effects of the deficiency. Progesterone replacement counteracted the effects of TBI on the animals reared under n−3 deficiency. A similar pattern was observed for markers of membrane homeostasis such as 4-Hydroxynonenal (HNE) and secreted phospholipases A2 (sPLA2), synaptic plasticity such as brain derived neurotrophic factor (BDNF), syntaxin (STX)-3 and growth associated protein (GAP)-43, and for growth inhibitory molecules such as myelin-associated glycoprotein (MAG) and Nogo-A. Results that progesterone had no effects on sham n−3 deficient animals suggest that the availability of progesterone is essential under injury conditions. Progesterone treatment counteracted several parameters related to synaptic plasticity and membrane stability reduced by FPI and n−3 deficiency suggest potential targets for therapeutic applications. These results reveal the importance of n−3 preconditioning during early life and the efficacy of progesterone therapy during adulthood to counteract weaknesses in neuronal and behavioral plasticity.