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Extracellular vesicles (EVs) originating from intraluminal vesicles (ILVs) formed within multivesicular bodies (MVBs), often referred to as small EV (sEV) or exosomes, are aberrantly produced by cancer cells and regulate the tumor microenvironment. The tyrosine kinase c-Src is upregulated in a wide variety of human cancers and is involved in promoting sEV secretion, suggesting its role in malignant progression. In this study, we found that activated Src liberated synaptosomal-associated protein 23 (SNAP23), a SNARE molecule, from lipid rafts to non-rafts on cellular membrane. We also demonstrated that SNAP23 localized in non-rafts induced cholesterol downregulation and ILV formation, resulting in the upregulation of sEV production in c-Src-transformed cells. Furthermore, the contribution of the SNAP23-cholesterol axis on sEV upregulation was confirmed in pancreatic cancer cells. High SNAP23 expression is associated with poor prognosis in patients with pancreatic cancer. These findings suggest a unique mechanism for the upregulation of sEV production via SNAP23-mediated cholesterol downregulation in Src-activated cancer cells.

Health benefits of physical activity (PA) may be mediated by DNA methylation alterations. The purpose of the current study was to comprehensively identify CpG sites whose methylation levels were associated with accelerometer-assessed total PA in a general Japanese population.The study participants were from the baseline survey of Saga Japan Multi-institutional Collaborative Cohort. PA was objectively measured by a single-axis accelerometer for 7 d. We used a two-stage strategy. In the discovery stage, we performed a meta-analysis of two epigenome-wide association studies of total PA in 898 individuals (a combination of random sample ( n = 507) and case-control study sample ( n = 391)). Peripheral blood DNA methylation levels were measured using Infinium EPIC or HM450 arrays. In the replication stage, we subsequently examined whether CpG sites significantly associated ( P1 × 10 -5 ) with total PA were replicated in another sample ( n = 1711), in which methylation levels were measured by pyrosequencing. A multiple linear regression was performed to determine the cross-sectional association between total PA and methylation levels with adjustment for potential confounders, including body mass index. A fixed-effects model was used in the meta-analysis. Correlations between total PA-associated DNA methylation and several inflammatory markers, such as high-sensitivity C-reactive protein, were also conducted.In the meta-analysis, nine CpG sites were significantly associated with total PA ( P1 × 10 -5 ). Among the nine sites, one site cg07030336 (annotated to VTI1A/ZDHHC6 gene) was successfully replicated ( P = 0.009).The current study showed that greater accelerometer-assessed total PA was associated with higher DNA methylation levels at cg07030336 ( VTI1A/ZDHHC6 ) in the general population. In addition, we found a divergent relationship between the methylation levels at cg07030336 and several inflammatory biomarkers.

Von Willebrand Factor (VWF) can promote platelet adhesion to the post-atherosclerotic regions to initiate thrombosis. The synthesis and secretion of VWF are important functions of endothelial cells (ECs). However, the mechanism through which blood flow regulates endothelial secretion of VWF remains unclear. We utilized a parallel-plate flow apparatus to apply fluid shear stress to human umbilical vein endothelial cells (HUVECs). Compared with pulsatile shear stress that mimics laminar flow in the straight parts of arteries or upstream of atherosclerotic stenosis sites, short-term exposure to oscillatory shear stress (OS) that mimics disturbed flow increased VWF secretion independent of affecting synaptosomal-associated protein 23 (SNAP23) expression and promoted the translocation of SNAP23 to the cell membrane. Vimentin associated with SNAP23, and this association was enhanced by OS or histamine. Acrylamide, a reagent that disrupts vimentin intermediate filaments, prevented histamine/OS-induced SNAP23 translocation, as well as VWF secretion. Immunofluorescence analysis revealed that the polarity of the vimentin intermediate filament network decreased after stimulation with histamine or OS. In addition, inhibition of protein kinase A (PKA) or G protein coupled receptor 68 (GPR68) eliminated the histamine/OS-induced phosphorylation of vimentin at Ser38 and secretion of VWF. Furthermore, syntaxin 7 might assist with the translocation of SNAP23 to the cell membrane, thus playing a role in promoting VWF secretion. The GPR68/PKA/vimentin signaling pathway may represent a novel mechanism for the regulation of SNAP23-mediated VWF secretion by ECs under OS and provide strategies for the prevention of atherosclerosis-related thrombosis.

Background Neurons are highly specialized cells with a complex morphology generated by various membrane trafficking steps. They contain Golgi outposts in dendrites, which are formed from somatic Golgi tubules. In trafficking membrane fusion is mediated by a specific combination of SNARE proteins. A functional SNARE complex contains four different helices, one from each SNARE subfamily (R-, Qa, Qb and Qc). Loss of the two Qb SNAREs vti1a and vti1b from the Golgi apparatus and endosomes leads to death at birth in mice with massive neurodegeneration in peripheral ganglia and defective axon tracts. Methods Hippocampal and cortical neurons were isolated from Vti1a−/−Vti1b−/− double deficient, Vti1a−/−Vti1b+/−, Vti1a+/−Vti1b−/− and Vti1a+/−Vti1b+/− double heterozygous embryos. Neurite outgrowth was determined in cortical neurons and after stimulation with several neurotrophic factors or the Rho-associated protein kinase ROCK inhibitor Y27632, which induces exocytosis of enlargeosomes, in hippocampal neurons. Moreover, postsynaptic densities were isolated from embryonic Vti1a−/−Vti1b−/− and Vti1a+/−Vti1b+/− control forebrains and analyzed by western blotting. Results Golgi outposts were present in Vti1a−/−Vti1b+/− and Vti1a+/−Vti1b−/− dendrites of hippocampal neurons but not detected in the absence of vti1a and vti1b. The length of neurites was significantly shorter in double deficient cortical neurons. These defects were not observed in Vti1a−/−Vti1b+/− and Vti1a+/−Vti1b−/− neurons. NGF, BDNF, NT-3, GDNF or Y27632 as stimulator of enlargeosome secretion did not increase the neurite length in double deficient hippocampal neurons. Vti1a−/−Vti1b−/− postsynaptic densities contained similar amounts of scaffold proteins, AMPA receptors and NMDA receptors compared to Vti1a+/−Vti1b+/−, but much more TrkB, which is the receptor for BDNF. Conclusion The absence of Golgi outposts did not affect the amount of AMPA and NMDA receptors in postsynaptic densities. Even though TrkB was enriched, BDNF was not able to stimulate neurite elongation in Vti1a−/−Vti1b−/− neurons. Vti1a or vti1b function as the missing Qb-SNARE together with VAMP-4 (R-SNARE), syntaxin 16 (Qa-SNARE) and syntaxin 6 (Qc-SNARE) in induced neurite outgrowth. Our data show the importance of vti1a or vti1b for two pathways of neurite elongation.

Recent work has revealed that spontaneous release plays critical roles in the central nervous system, but how it is regulated remains elusive. Here we report that synaptotagmin-11 (Syt11), a Ca2+ -independent Syt isoform associated with schizophrenia and Parkinson's disease, suppressed spontaneous release. Syt11-knockout hippocampal neurons showed an increased frequency of miniature excitatory postsynaptic currents while overexpression of Syt11 inversely decreased the frequency. Neither knockout nor overexpression of Syt11 affected the average amplitude, suggesting the presynaptic regulation of spontaneous neurotransmission by Syt11. Glutathione-S-transferase pull-down, co-immunoprecipitation, and affinity-purification experiments demonstrated a direct interaction of Syt11 with vps10p-tail-interactor-1a (vti1a), a non-canonical SNARE protein that maintains spontaneous release. Importantly, knockdown of vti1a reversed the phenotype of Syt11 knockout, identifying vti1a as the main target of Syt11 inhibition. Domain analysis revealed that the C2A domain of Syt11 bound vti1a with high affinity. Consistently, expression of the C2A domain alone rescued the phenotype of elevated spontaneous release in Syt11-knockout neurons similar to the full-length protein. Altogether, our results suggest that Syt11 inhibits vti1a-containing vesicles during spontaneous release.

Insulin stimulates translocation of glucose transporter 4 (GLUT4) from the perinuclear location to the plasma membrane. In the unstimulated state, intracellular vesicles containing GLUT4 are sequestered into specialized storage vesicles that have come to be known as the insulin-responsive compartment (IRC). The IRC is a functional compartment in the perinuclear region that is a target of the insulin signaling cascade, although its precise nature is unclear. Here, we report a novel molecular mechanism facilitating formation of the IRC.We determined synaptosomal-associated protein of 29 kDa (SNAP29) by mass spectrometry to be an EH domain-containing protein 1 (EHD1)-binding protein. Then, its expression was confirmed by western blotting. Subcellular localization of SNAP29 was determined by immunofluorescent microscopy. Interactions between SNAP29 and syntaxins were determined by immunoprecipitation. We measured glucose uptake and GLUT4 translocation in 3T3-L1 adipocyte expressing SNAP29 or silencing SNAP29.We found SNAP29 to be localized in the perinuclear region and to show partial co-localization with GLUT4 under basal conditions. We also found that SNAP29 binds to syntaxin6, a Qc-SNARE, in adipocytes. In SNAP29-expressing cells, vesicles containing GLUT4 were observed to aggregate around the perinuclear region. In contrast, when SNAP29 was silenced, perinuclear GLUT4 vesicles were dispersed throughout the cytosol. Insulin-stimulated glucose uptake was inhibited in both SNAP29-expressing and SNAP29-silenced cells.These data suggest that SNAP29 sequesters and anchors GLUT4-containing vesicles in the perinuclear region, and might have a role in the biogenesis of the perinuclear IRC.

CEDNIK syndrome is a rare autosomal recessive syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma of which 25 cases from 19 families have been reported to date. It is a progressive neurodegenerative disorder caused by the loss-of-function pathogenic variant of the SNAP29 gene encoding a member of the SNARE family of proteins. We describe two female siblings from a Syrian parent-related family with CEDNIK syndrome due to homozygous pathogenic variant in SNAP29 [c.223delG(p.Val75Serf*28)]. Palmoplantar keratoderma, reported as a cardinal sign in CEDNIK syndrome, was absent in both patients as of the last follow-up, and one of our patients had a verrucous venous malformation, a finding that has not been previously reported.

T1-weighted, cross-sectional MR images showing shoulder girdle, abdominal, paraspinal, gluteal and thigh muscles almost completely replaced by fat, whereas lower leg muscles are almost unaffected i a patient who is compound heterozygous for pathogenic variants in GOSR2.

Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein 23 (SNAP23) is a member of the SNARE protein family and mediates the vesicle docking and membrane fusion steps of secretion during exocytosis. Skeletal muscle has been established as a secretory organ; however, the role of SNAP23 in the context of skeletal muscle development is still unknown. Here, we show that depletion of SNAP23 in C2C12 mouse myoblasts reduces their ability to differentiate into myotubes as a result of premature cell cycle exit and early activation of the myogenic transcriptional program. This effect is rescued when cells are seeded at a high density or when cultured in conditioned medium from wild type cells. Proteomic analysis of collected medium indicates that SNAP23 depletion leads to a misregulation of exocytosis, including decreased secretion of the insulin-like growth factor 1 (IGF1), a critical protein for muscle growth, development, and function. We further demonstrate that treatment of SNAP23-depleted cells with exogenous IGF1 rescues their myogenic capacity. We propose that SNAP23 mediates the secretion of specific proteins, such as IGF1, that are important for achieving proper differentiation of skeletal muscle cells during myogenesis. This work highlights the underappreciated role of skeletal muscle as a secretory organ and contributes to the understanding of factors necessary for myogenesis.

The superfamily of Ras proteins comprises different molecules belonging to the GTPase family. They normally cycle between an active state bound to GTP which activates effectors while the protein is membrane-associated, and an inactive GDP-bound state. They regulate the intracellular trafficking and other cellular processes. The family of Rab proteins includes several members and they have been found, among other Ras proteins, to be fundamental for important biological processes, such as endocytosis and exocytosis. SNARE proteins control the fusion of vesicles by forming quaternary complexes which are divided into two small groups on the two different compartments. Generally, the association of three SNARE proteins on the donor compartment with the one on the target compartment determines the formation of the SNARE complex, the opening of the fusion pore and the formation of one single bigger vesicle. Interestingly, novel interactions between other molecules involved in intracellular trafficking, endosomal fusion and maturation have recently been found, such as the interaction between invariant chain and the Qb SNARE vti1b, and more functional connections between Rab proteins and SNAREs are supposed to be fundamental for the regulation of membrane fusion.

Macroautophagy/autophagy is an evolutionarily conserved intracellular pathway for the degradation of cytoplasmic materials. Under stress conditions, autophagy is upregulated and double-membrane autophagosomes are formed by the expansion of phagophores. The ATG16L1 precursor fusion contributes to development of phagophore structures and is critical for the biogenesis of autophagosomes. Here, we discovered a novel role of the protein tyrosine phosphatase PTPN9 in the regulation of homotypic ATG16L1 vesicle fusion and early autophagosome formation. Depletion of PTPN9 and its Drosophila homolog Ptpmeg2 impaired autophagosome formation and autophagic flux. PTPN9 colocalized with ATG16L1 and was essential for homotypic fusion of ATG16L1(+) vesicles during starvation-induced autophagy. We further identified the Q-SNARE VTI1B as a substrate target of PTPN9 phosphatase. Like PTPN9, the VTI1B nonphosphorylatable mutant but not the phosphomimetic mutant enhanced SNARE complex assembly and autophagic flux. Our findings highlight the important role of PTPN9 in the regulation of ATG16L1(+) autophagosome precursor fusion and autophagosome biogenesis through modulation of VTI1B phosphorylation status. Abbreviations: csw: corkscrew; EBSS: Earle’s balanced salt solution; ERGIC: ER-Golgi intermediate compartment; ESCRT: endosomal sorting complexes required for transport; mop: myopic; NSF: N-ethylmaleimide-sensitive factor; PAS: phagophore assembly site; PolyQ: polyglutamine; PtdIns3P: phosphatidylinositol-3-phosphate; PTK: protein tyrosine kinase; PTM: posttranslational modification; PTP: protein tyrosine phosphatase; PTPN23/HD-PTP: protein tyrosine phosphatase non-receptor type 23; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; STX7: syntaxin 7; STX8: syntaxin 8; STX17: syntaxin 17; VAMP3: vesicle associated membrane protein 3; VAMP7: vesicle associated membrane protein 7; VTI1B: vesicle transport through interaction with t-SNAREs 1B; YKT6: YKT6 v-SNARE homolog; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Previously we reported that adipocyte SNAP23 (synaptosome-associated protein of 23 kDa) deficiency blocks the activation of macroautophagy, leading to an increased abundance of BAX, a pro-death Bcl-2 family member, and activation and adipocyte cell death both in vitro and in vivo. Here, we found that knockdown of SNAP23 inhibited the association of the autophagosome regulators ATG16L1 and ATG9 compartments by nutrient depletion and reduced the formation of ATG16L1 membrane puncta. ATG16L1 knockdown inhibited autophagy flux and increased BAX protein levels by suppressing BAX degradation. The elevation in BAX protein had no effect on BAX activation or cell death in the nutrient-replete state. However, following nutrient depletion, BAX was activated with a concomitant induction of cell death. Co-immunoprecipitation analyses demonstrated that SNAP23 and ATG16L1 proteins form a stable complex independent of nutrient condition, whereas in the nutrient-depleted state, BAX binds to SNAP23 to form a ternary BAX–SNAP23–ATG16L1 protein complex. Taken together, these data support a model in which SNAP23 plays a crucial function as a scaffold for ATG16L1 necessary for the suppression of BAX activation and induction of the intrinsic cell death program.

Syntaxin17, a key autophagosomal N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR–GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 3(10) helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17–SNAP29–VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome–lysosome fusion process.

Membrane trafficking is a core cellular process that supports diversification of cell shapes and behaviors relevant to morphogenesis during development and in adult organisms. However, how precisely trafficking components regulate specific differentiation programs is incompletely understood. Snap29 is a multifaceted Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptor, involved in a wide range of trafficking and non-trafficking processes in most cells. A body of knowledge, accrued over more than two decades since its discovery, reveals that Snap29 is essential for establishing and maintaining the operation of a number of cellular events that support cell polarity and signaling. In this review, we first summarize established functions of Snap29 and then we focus on novel ones in the context of autophagy, Golgi trafficking and vesicle fusion at the plasma membrane, as well as on non-trafficking activities of Snap29. We further describe emerging evidence regarding the compartmentalisation and regulation of Snap29. Finally, we explore how the loss of distinct functions of human Snap29 may lead to the clinical manifestations of congenital disorders such as CEDNIK syndrome and how altered SNAP29 activity may contribute to the pathogenesis of cancer, viral infection and neurodegenerative diseases.

Progressive myoclonic epilepsies (PMEs) comprise a group of rare disorders of different genetic aetiologies, leading to childhood-onset myoclonus, myoclonic seizures and subsequent neurological decline. One of the genetic causes for PME, a mutation in the gene coding for Golgi SNAP receptor 2 (GOSR2), gives rise to a PME-subtype prevalent in Northern Europe and hence referred to as North Sea Progressive Myoclonic Epilepsy (NS-PME). Treatment for NS-PME, as for all PME subtypes, is symptomatic; the pathophysiology of NS-PME is currently unknown, precluding targeted therapy. Here, we investigated the pathophysiology of NS-PME. By means of chart review in combination with interviews with patients (n = 14), we found heat to be an exacerbating factor for a majority of NS-PME patients (86%). To substantiate these findings, we designed a NS-PME Drosophila melanogaster model. Downregulation of the Drosophila GOSR2-orthologue Membrin leads to heat-induced seizure-like behaviour. Specific downregulation of GOSR2/Membrin in glia but not in neuronal cells resulted in a similar phenotype, which was progressive as the flies aged and was partially responsive to treatment with sodium barbital. Our data suggest a role for GOSR2 in glia in the pathophysiology of NS-PME. (C) 2019 IBRO. Published by Elsevier Ltd. All rights reserved.

Vti proteins are conserved from yeast to humans and regulate intracellular membrane trafficking by providing one specific SNARE domain, the Qb SNARE, to the four helical SNARE bundle that drives membrane fusion. Two mammalian Vti genes, Vti1a and Vti1b are reported to regulate distinct aspects of endolysosomal trafficking and retrograde transport to the Golgi, but have also been implicated in synaptic vesicle secretion. In this review, we summarize the current evidence for the role of Vti proteins in intracellular trafficking in different cells. We propose that, despite some unique aspects, the two mammalian VTI genes have largely redundant functions in neurosecretory cells and recycle molecules required for the sorting of regulated cargo to the Golgi. Defects in this recycling also lead to defects in synaptic transmission and dense core vesicle secretion.

Successive fusion events between transport vesicles and their target membranes mediate trafficking of secreted, membrane- and organelle-localised proteins. During the initial steps of this process, termed the secretory pathway, COPII vesicles bud from the endoplasmic reticulum (ER) and fuse with the cis-Golgi membrane, thus depositing their cargo. This fusion step is driven by a quartet of SNARE proteins that includes the cis-Golgi t-SNARE Membrin, encoded by the GOSR2 gene. Mis-sense mutations in GOSR2 result in Progressive Myoclonus Epilepsy (PME), a severe neurological disorder characterised by ataxia, myoclonus and seizures in the absence of significant cognitive impairment. However, given the ubiquitous and essential function of ER-to-Golgi transport, why GOSR2 mutations cause neurological dysfunction and not lethality or a broader range of developmental defects has remained an enigma. Here we highlight new work that has shed light on this issue and incorporate insights into canonical and non-canonical secretory trafficking pathways in neurons to speculate as to the cellular and molecular mechanisms underlying GOSR2 PME. This article is part of a Special Issue entitled: SNARE proteins: a long journey of science in brain physiology and pathology: from molecular.

BET1 is required, together with its SNARE complex partners GOSR2, SEC22b, and Syntaxin‐5 for fusion of endoplasmic reticulum‐derived vesicles with the ER‐Golgi intermediate compartment (ERGIC) and the cis‐Golgi. Here, we report three individuals, from two families, with severe congenital muscular dystrophy (CMD) and biallelic variants in BET1 (P1 p.(Asp68His)/p.(Ala45Valfs*2); P2 and P3 homozygous p.(Ile51Ser)). Due to aberrant splicing and frameshifting, the variants in P1 result in low BET1 protein levels and impaired ER‐to‐Golgi transport. Since in silico modeling suggested that p.(Ile51Ser) interferes with binding to interaction partners other than SNARE complex subunits, we set off and identified novel BET1 interaction partners with low affinity for p.(Ile51Ser) BET1 protein compared to wild‐type, among them ERGIC‐53. The BET1/ERGIC‐53 interaction was validated by endogenous co‐immunoprecipitation with both proteins colocalizing to the ERGIC compartment. Mislocalization of ERGIC‐53 was observed in P1 and P2’s derived fibroblasts; while in the p.(Ile51Ser) P2 fibroblasts specifically, mutant BET1 was also mislocalized along with ERGIC‐53. Thus, we establish BET1 as a novel CMD/epilepsy gene and confirm the emerging role of ER/Golgi SNAREs in CMD., This study describes three individuals with a progressive early‐onset congenital muscular dystrophy, and additional epilepsy in one, caused by biallelic variants in the BET1 gene. BET1, along with its SNARE complex partners, is essential for ER‐to‐Golgi trafficking.

2,2',4,4'-tetrabromodiphenyl ether (PBDE-47), the widely used brominated flame retardant, has remarkable neurotoxicity which is associated with autophagy disorder. However, the mechanism remains unclear. The results showed that PBDE-47 damaged lysosomal biogenesis and interfered with autophagy-lysosome fusion both in vivo and in vitro. Our investigation further demonstrated that PBDE-47 could downregulate TFEB expression and inhibit the nuclear translocation of TFEB. Knockdown of TFEB in PC12 cells increased the reduction of lysosomal-associated proteins and the expression of STX17-SNAP29-VAMP8 proteins involved in autophagy-lysosomal fusion. Conversely, Overexpression TFEB in vitro significantly improved lysosomal abundance and ameliorated the autophagosome-lysosome fusion inhibition, thus restoring autophagic flux and improving PC12 cells survival. In addition, TFEB biologically interacted with STX17 by not inducing or inducing TFEB overexpression. Collectively, our results indicate that the autophagy flux compromised by PBDE-47 is related to the defective fusion of autophagosome and lysosome. TFEB may serve as a promising molecular target for future study of PBDE-47 developmental neurotoxicity.

The mammalian target of rapamycin (mTORC1) has been shown to regulate autophagy at different steps. However, how mTORC1 regulates the N-ethylmaleimide-sensitive protein receptor (SNARE) complex remains elusive. Here we show that mTORC1 inhibits formation of the SNARE complex (STX17-SNAP29-VAMP8) by phosphorylating VAMP8, thereby blocking autophagosome-lysosome fusion. A VAMP8 phosphorylation mimic mutant is unable to promote autophagosome-lysosome fusion in vitro. Furthermore, we identify SCFD1, a Sec1/Munc18-like protein, that localizes to the autolysosome and is required for SNARE complex formation and autophagosome-lysosome fusion. VAMP8 promotes SCFD1 recruitment to autolysosomes when dephosphorylated. Consistently, phosphorylated VAMP8 or SCFD1 depletion inhibits autophagosome-lysosome fusion, and expression of phosphomimic VAMP8 leads to increased lipid droplet accumulation when expressed in mouse liver. Thus, our study supports that mTORC1-mediated phosphorylation of VAMP8 blocks SCFD1 recruitment, thereby inhibiting STX17-SNAP29-VAMP8 complex formation and autophagosome-lysosome fusion., Autophagy relies on coordinated fusion of organelle membranes, although the interplay between the regulatory machinery is not well studied. Here, the authors show that SNARE complex formation is inhibited by mTORC1 phosphorylation of VAMP8, which prevents autophagosome-lysosome fusion.

Dopaminergic (DA) cell death in Parkinson’s disease (PD) is associated with the gradual appearance of neuronal protein aggregates termed Lewy bodies (LBs) that are comprised of vesicular membrane structures and dysmorphic organelles in conjunction with the protein alpha-Synuclein (α-Syn). Although the exact mechanism of neuronal aggregate formation and death remains elusive, recent research suggests α-Syn-mediated alterations in the lysosomal degradation of aggregated proteins and organelles – a process termed autophagy. Here, we used a combination of molecular biology and immunochemistry to investigate the effect of α-Syn on autophagy turnover in cultured human DA neurons and in human post-mortem brain tissue. We found α-Syn overexpression to reduce autophagy turnover by compromising the fusion of autophagosomes with lysosomes, thus leading to a decrease in the formation of autolysosomes. In accord with a compensatory increase in the plasma membrane fusion of autophagosomes, α-Syn enhanced the number of extracellular vesicles (EV) and the abundance of autophagy-associated proteins in these EVs. Mechanistically, α-Syn decreased the abundance of the v-SNARE protein SNAP29, a member of the SNARE complex mediating autophagolysosome fusion. In line, SNAP29 knockdown mimicked the effect of α-Syn on autophagy whereas SNAP29 co-expression reversed the α-Syn-induced changes on autophagy turnover and EV release and ameliorated DA neuronal cell death. In accord with our results from cultured neurons, we found a stage-dependent reduction of SNAP29 in SNc DA neurons from human post-mortem brain tissue of Lewy body pathology (LBP) cases. In summary, our results thus demonstrate a previously unknown effect of α-Syn on intracellular autophagy-associated SNARE proteins and, as a consequence, a reduced autolysosome fusion. As such, our findings will therefore support the investigation of autophagy-associated pathological changes in PD

Ubiquitin conjugating enzyme 9 (UBC9), the sole small ubiquitin-like modifier(SUMO) conjugating enzyme, is considered to be a vital regulator of the mechanism of SUMOylation and is likely to participate in the progression of Alzheimer's disease (AD). Our previous studies found that UBC9 is highly mobile in neurons, but the underlying mechanism is still unknown. We designed to investigate the underlying mechanism of the synaptic redistribution of UBC9 in AD. We found that β-amyloid peptide (Aβ) significantly decreased presynaptic UBC9 expression and increased postsynaptic UBC9 expression but did not affect UBC9 expression in synaptosomes. Moreover, there is evidence that extracellular vesicles (EVs) may facilitate synaptic gap transmission. Immunoprecipitation assays showed that flotillin, which is specifically expressed in EVs, co-immunoprecipitated with UBC9 in cell lysates and media and that Aβ treatment enhanced this interaction. Additionally, epidermal growth factor and oleanolic acid have been found to either promote or inhibit the release of EVs, thus blocking the Aβ-induced presynaptic release of UBC9. However, knockdown of synaptosome associated protein 23 (SNAP-23) or inhibition of SNAP23 phosphorylation was found to secure Aβ-induced presynaptic release of UBC9. These results suggest that Aβ induces redistribution of UBC9 from presynaptic to postsynaptic terminals, which may mediate through EVs associated with SNAP23. Our study reveals the cellular mechanisms of EVs as an essential component of the presynaptic release of UBC9.

Coxsackievirus B3 (CVB3), an enterovirus (EV) in the family of Picornaviridae, is a global human pathogen for which effective antiviral treatments and vaccines are lacking. Previous research demonstrated that EV-D68 downregulated the membrane fusion protein SNAP47 (synaptosome associated protein 47) and SNAP47 promoted EV-D68 replication via regulating autophagy. In the current study, we investigated the interplay between CVB3 and cellular SNAP47 using HEK293T/HeLa cell models. We showed that, upon CVB3 infection, protein levels of SNAP47 decreased independent of the activity of virus-encoded proteinase 3C. We further demonstrated that the depletion of SNAP47 inhibited CVB3 infection, indicating a pro-viral function of SNAP47. Moreover, we found that SNAP47 co-localizes with the autophagy-related protein ATG14 on the cellular membrane fractions together with viral capsid protein VP1, and expression of SNAP47 or ATG14 enhanced VP1 conjugation. Finally, we revealed that disulfide interactions had an important role in strengthening VP1 conjugation. Collectively, our study elucidated a mechanism by which SNAP47 and ATG14 promoted CVB3 propagation through facilitating viral capsid assembly.

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.

Background Extracellular vesicles (EVs) harbor thousands of proteins that hold promise for biomarker development. Usually difficult to purify, EVs in urine are relatively easily obtained and have demonstrated efficacy for kidney disease prediction. Herein, we further characterize the proteome of urinary EVs to explore the potential for biomarkers unrelated to kidney dysfunction, focusing on Parkinson's disease (PD). Methods Using a quantitative mass spectrometry approach, we measured urinary EV proteins from a discovery cohort of 50 subjects. EVs in urine were classified into subgroups and EV proteins were ranked by abundance and variability over time. Enriched pathways and ontologies in stable EV proteins were identified and proteins that predict PD were further measured in a cohort of 108 subjects. Findings Hundreds of commonly expressed urinary EV proteins with stable expression over time were distinguished from proteins with high variability. Bioinformatic analyses reveal a striking enrichment of endolysosomal proteins linked to Parkinson's, Alzheimer's, and Huntington's disease. Tissue and biofluid enrichment analyses show broad representation of EVs from across the body without bias towards kidney or urine proteins. Among the proteins linked to neurological diseases, SNAP23 and calbindin were the most elevated in PD cases with 86% prediction success for disease diagnosis in the discovery cohort and 76% prediction success in the replication cohort. Interpretation Urinary EVs are an underutilized but highly accessible resource for biomarker discovery with particular promise for neurological diseases like PD.

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ–activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane–localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane–localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.

CEDNIK (CErebral Dysgenesis, Neuropathy, Ichthyosis, and Keratoderma) syndrome is a neuroichthyotic syndrome characterized by a constellation of clinical features including severe developmental retardation, microcephaly, and facial dysmorphism. Here, we report the first case of CEDNIK syndrome from India presenting with characteristic clinical features and harboring a novel mutation of SNAP29 gene.

Hepatitis B virus (HBV) replication and envelopment is dependent on cellular autophagy. Previously, we have provided evidence for the extensive lysosomal degradation of HBV virions and the hepatitis B surface antigen (HBsAg), which is likely controlled by autophagosome-lysosome fusion. Synaptosomal-associated protein 29 (SNAP29) has been identified as a protein specifically mediating autophagosome-lysosome fusion. Thus, in the present study, we addressed the hypothesis that SNAP29 is required for the autophagic degradation of HBV virions and HBsAg. We found that silencing SNAP29 significantly increased the number of autophagosomes and concomitantly promoted HBV replication and HBsAg production. Conversely, SNAP29 overexpression decreased HBV production. Consistent with this, SNAP29 modulated HBV production by interacting with vesicle-associated membrane protein 8 (VAMP8) and synergistically regulated HBV replication with Rab7 complexes. Moreover, the production and release of the small HBsAg is strongly regulated by SNAP29 expression, suggesting that its export occurs partly through the autophagic pathway. Our findings provide new evidence, strongly suggesting that autophagic degradation critically determines the production of HBV virions and HBsAg and that this is controlled by the SNAP29-VAMP8 interaction.-Lin, Y., Wu, C., Wang, X., Liu, S., Kemper, T., Li, F., Squire, A., Zhu, Y., Zhang, J., Chen, X., Lu, M. Synaptosomal-associated protein 29 is required for the autophagic degradation of hepatitis B virus.

Background Tumor cells are known to release large numbers of exosomes containing active substances that participate in cancer progression. Abnormally expressed long noncoding RNAs (lncRNAs) have been confirmed to regulate multiple processes associated with tumor progression. However, the mechanism by which lncRNAs affect exosome secretion remains unclear. Methods The underlying mechanisms of long noncoding RNA LINC00511 (LINC00511) regulation of multivesicular body (MVB) trafficking, exosome secretion, invadopodia formation, and tumor invasion were determined through gene set enrichment analysis (GSEA), immunoblotting, nanoparticle tracking analysis, confocal colocalization analysis, electron microscopy, and invasion experiments. Results We revealed that the tumorigenesis process is associated with a significant increase in vesicle secretion in hepatocellular carcinoma (HCC). Additionally, LINC00511 was significantly more highly expressed in HCC tissues and is related to vesicle trafficking and MVB distribution. We also found that in addition to the formation of invadopodia in HCC progression, abnormal LINC00511 induces invadopodia formation in HCC cells by regulating the colocalization of vesicle associated membrane protein 7 (VAMP7) and synaptosome associated protein 23 (SNAP23) to induce the invadopodia formation, which are key secretion sites for MVBs and control exosome secretion. Finally, we revealed that LINC0051-induced invadopodia and exosome secretion were involved in tumor progression. Conclusions Our experiments revealed novel findings on the relationship between LINC00511 dysregulation in HCC and invadopodia production and exosome secretion. This is a novel mechanism by which LINC00511 regulates invadopodia biogenesis and exosome secretion to further promote cancer progression.

Genetic factors undoubtedly affect the development of congenital heart disease (CHD) but still remain ill defined. We sought to identify genetic risk factors associated with CHD and to accomplish a functional analysis of SNP-carrying genes. We performed a genome-wide association study (GWAS) of 4034 White patients with CHD and 8486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified 4 highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence the expression of WNT3, and the variant rs870142 related to septal defects is proposed to influence the expression of MSX1. We analyzed the expression of all 4 genes during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNA-Seq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3, and MSX1 play an essential functional role in heart development at the embryonic and newborn stages.

Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

Degranulation, a fundamental effector response from mast cells (MCs) and platelets, is an example of regulated exocytosis. This process is mediated by SNARE proteins and their regulators. We have previously shown that several of these proteins are essential for exocytosis in MCs and platelets. Here, we assessed the role of the SNARE protein SNAP23 using conditional knockout mice, in which SNAP23 was selectively deleted from either the megakaryocyte/platelet or connective tissue MC lineages. We found that removal of SNAP23 in platelets results in severe defects in degranulation of all three platelet secretory granule types, i.e., alpha, dense, and lysosomal granules. The mutation also induces thrombocytopenia, abnormal platelet morphology and activation, and reduction in the number of alpha granules. Therefore, the degranulation defect might not be secondary to an intrinsic failure of the machinery mediating regulated exocytosis in platelets. When we removed SNAP23 expression in MCs, there was a complete developmental failure in vitro and in vivo. The developmental defects in platelets and MCs and the abnormal translocation of membrane proteins to the surface of platelets indicate that SNAP23 is also involved in constitutive exocytosis in these cells. The MC conditional deletant animals lacked connective tissue MCs, but their mucosal MCs were normal and expanded in response to an antigenic stimulus. We used this mouse to show that connective tissue MCs are required and mucosal MCs are not sufficient for an anaphylactic response.

Cortical morphogenesis entails several neurobiological events, including proliferation and differentiation of progenitors, migration of neuroblasts, and neuronal maturation leading to functional neural circuitry. These neurodevelopmental processes are delicately regulated by many factors. Endosomal SNAREs have emerged as formidable modulators of neuronal growth, aside their well-known function in membrane/vesicular trafficking. However, our understanding of their influence on cortex formation is limited. Here, we report that the SNAREs Vti1a and Vti1b (Vti1a/1b) are critical for proper cortical development. Following null mutation of Vti1a/1b in mouse, the late-embryonic mutant cortex appeared dysgenic, and the cortical progenitors therein were depleted beyond normal. Notably, cortical layer 5 (L5) is distinctively disorganized in the absence of Vti1a/1b. The latter defect, coupled with an overt apoptosis of Ctip2-expressing L5 neurons, likely contributed to the substantial loss of corticospinal and callosal projections in the Vti1a/1b-deficient mouse brain. These findings suggest that Vti1a/1b serve key neurodevelopmental functions during cortical histogenesis, which when mechanistically elucidated, can lend clarity to how endosomal SNAREs regulate brain development, or how their dysfunction may have implications for neurological disorders. Copyright © 2021 IBRO. Published by Elsevier Ltd. All rights reserved.

Botulinum neurotoxin (BoNT) serotype A inhibits neurotransmitter release by cleaving SNAP-25 and represents an established pharmaceutical for treating medical conditions caused by hyperactivity of cholinergic nerves. Oversecretion from non-neuronal cells is often also the cause of diseases. Notably, excessive release of inflammatory messengers is thought to contribute to diseases such as chronic obstructive pulmonary disease, asthma, diabetes etc. The expansion of its application to these medical conditions is prevented because the major non-neuronal SNAP-25 isoform responsible for exocytosis, SNAP-23, is, in humans, virtually resistant to BoNT/A. Based on previous structural data and mutagenesis studies of SNAP-23 we optimized substrate binding pockets of the enzymatic domain for interaction with SNAP-23. Systematic mutagenesis and rational design yielded the mutations E148Y, K166F, S254A, and G305D, each of which individually increased the activity of LC/A against SNAP-23 between 3- to 23-fold. The assembled quadruple mutant showed approximately 2000-fold increased catalytic activity against human SNAP-23 in in vitro cleavage assays. A comparable increase in activity was recorded for the full-length BoNT/A quadruple mutant tested in cultivated primary neurons transduced with a fluorescently tagged-SNAP-23 encoding gene. Equipped with a suitable targeting domain this quadruple mutant promises to complete successfully tests in cells of the immune system.

The fibrous annulus of the mitral valve plays an important role in valvular function and cardiac physiology, while normal variation in the size of cardiovascular anatomy may share a genetic link with common and rare disease. We derived automated estimates of mitral valve annular diameter in the 4-chamber view from 32,220 MRI images from the UK Biobank at ventricular systole and diastole as the basis for GWAS. Mitral annular dimensions corresponded to previously described anatomical norms, and GWAS inclusive of 4 population strata identified 10 loci, including possibly novel loci (GOSR2, ERBB4, MCTP2, MCPH1) and genes related to cardiac contractility (BAG3, TTN, RBFOX1). ATAC-Seq of primary mitral valve tissue localized multiple variants to regions of open chromatin in biologically relevant cell types and rs17608766 to an algorithmically predicted enhancer element in GOSR2. We observed strong genetic correlation with measures of contractility and mitral valve disease and clinical correlations with heart failure, cerebrovascular disease, and ventricular arrhythmias. Polygenic scoring of mitral valve annular diameter in systole was predictive of risk mitral valve prolapse across 4 cohorts. In summary, genetic and clinical studies of mitral valve annular diameter revealed genetic determinants of mitral valve biology, while highlighting clinical associations. Polygenic determinants of mitral valve annular diameter may represent an independent risk factor for mitral prolapse. Overall, computationally estimated phenotypes derived at scale from medical imaging represent an important substrate for genetic discovery and clinical risk prediction.

Tarabykin highlights work from Kunii and colleagues demonstrating a role for a SNARE complex in the regulation of neuronal progenitor polarity., SNARE vesicle targeting complex controls the polarity of neuronal progenitors. Kunii et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201910080) show that the SNAP23–VAMP8–Syntaxin1B complex is required for membrane targeting of N-cadherin and formation of adherence junction complexes in radial glia neuronal progenitors, the major prerequisite of cell polarity establishment.

Tumor Necrosis Factor α (TNFα) is best known as a mediator of inflammation and immunity, and also plays important roles in tumor biology. However, the role of TNFα in tumor biology is complex and not completely understood. In a human melanoma cell line, M2, and a lung carcinoma cell line, A549, TNFα up-regulates prion protein (PrP) level, and promotes tumor cell migration in a PrP dependent manner. Silencing PRNP abrogates TNFα induced tumor cell migration; this phenotype is reversed when PRNP is re-introduced. Treatment with TNFα activates nuclear factor kappa B (NF-κB) signaling, which then mitigates autophagy by reducing the expression of Forkhead Box P3 (FOXP3). Down regulation of FOXP3 reduces the transcription of synaptosome associated protein 29 (SNAP29), which is essential in the fusion of autophagosome and lysosome creating autolysosome. FOXP3 being a bona fide transcription factor for SNAP29 is confirmed in a promoter binding assay. Accordingly, silencing SNAP29 in these cell lines also up-regulates PrP, and promotes tumor cell migration without TNFα treatment. But, when SNAP29 or FOXP3 is silenced in these cells, they are no longer respond to TNFα. Thus, a reduction in autophagy is the underlying mechanism by which expression of PrP is up-regulated, and tumor cell migration is enhanced upon TNFα treatment. Disrupting the TNFα-NF-κB-FOXP3-SNAP29 signaling axis may provide a therapeutic approach to mitigate tumor cell migration. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-020-00320-4) contains supplementary material, which is available to authorized users.

CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis, and Keratoderma) syndrome is a neuro ichthyotic syndrome characterized by a clinical constellation of features including severe developmental delay, microcephaly, and facial dysmorphism. Here, we report the clinical and molecular characterization of a patient with CEDNIK syndrome harboring two compound heterozygous variants in the SNAP29 gene. The patient presents a combination of a loss-of-function SNAP29 mutation and a ∼370 kb 22q11.2 deletion, each of these genetic variants inherited from one of the parents. This report provides detailed data of a patient with unprecedented genetic events leading to the CEDNIK phenotype and may contribute to the elucidation of this rare condition.

The homozygous missense variant in the GOSR2 gene (c.430G > T) is known to be associated with progressive myoclonic epilepsy (PME). The clinical presentation of GOSR2-related PME involves the development of ataxia, seizures, scoliosis, areflexia, and mildly elevated creatine kinase. Recently, it has been suggested that some compound heterozygous variants in GOSR2 are associated with a predominant muscular dystrophy phenotype. Here we report a case of a now 22 month old female who presented with congenital hypotonia and persistently elevated creatine kinase levels. Whole exome sequencing showed pathogenic compound heterozygous variants in GOSR2 (c.430G > T and c.82C > T). This case contributes to the expanding clinical spectrum of GOSR2 variants with PME representing the milder end and congenital muscular dystrophy representing the more severe end of the spectrum.

Background: Poststroke cognitive impairments are common in stroke survivors, and pose a high risk of incident dementia. However, the cause of these cognitive impairments is obscure and required an investigation. Methods: Oxygen-glucose deprivation (OGD) model and middle cerebral artery occlusion (MCAO) model were used to imitate in vitro or in vivo acute cerebral ischemia, respectively. The differentially expressed synaptosome associated protein 29 (SNAP29)-interacting proteins upon ischemia and reperfusion were analyzed with bioinformatics analysis and the results indicated that the changes of SNAP29 after acute ischemia were mainly involved in the synaptic functions. The outcomes of SNAP29 reduction were assessed with SNAP29 knockdown, which mimicked the distribution of SNAP29 along neuronal processes after acute ischemia. Using the whole-cell patch clamp recording method and transmission electron microscope, the pre-synaptic function and readily releasable pool (RRP) were observed after SNAP29 knock down. Using photogenetic manipulations and behavioral tests, the neuronal projection and cognitive functions of mice with SNAP29 knock down in hippocampus CA1 region were evaluated. Results: It was found that SNAP29 protein levels decreased in both in vitro and in vivo ischemic models. Further, the SNAP29 reduction wasn't associated with impaired autophagy flux and neuronal survival. When SNAP29 was knocked down in primary cortical neurons, the frequency of AMPARs-mediated mEPSCs, but not the amplitude, significantly decreased. Meanwhile, the mice with SNAP29 knockdown at CA1 region of hippocampus developed an impairment in hippocampus-mPFC (middle prefrontal cortex) circuit and behavioral dysfunctions. Moreover, the size of RRP at presynaptic sites was diminished. Conclusion: Since SNAP29 protein levels didn't significantly influence the neuronal survival and its decrease was sufficient to disturb the neural circuit via a presynaptic manner, the SNAP29-associated strategies may be an efficient target against poststroke synaptic dysfunction and cognitive deficits.

Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.