Identification of Key Genes and Imbalanced SNAREs Assembly in the Comorbidity of Polycystic Ovary Syndrome and Depression.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) have increased odds of concurrent depression, indicating that the relationship between PCOS and depression is more likely to be comorbid. However, the underlying mechanism remains unclear. Here, we aimed to use bioinformatic analysis to screen for the genetic elements shared between PCOS and depression. METHODS: Differentially expressed genes (DEGs) were screened out through GEO2R using the PCOS and depression datasets in NCBI. Protein-protein interaction (PPI) network analysis and enrichment analysis were performed to identify the potential hub genes. After verification using other PCOS and depression datasets, the associations between key gene polymorphism and comorbidity were further studied using data from the UK biobank (UKB) database. RESULTS: In this study, three key genes, namely, SNAP23, VTI1A, and PRKAR1A, and their related SNARE interactions in the vesicular transport pathway were identified in the comorbidity of PCOS and depression. The rs112568544 at SNAP23, rs11077579 and rs4458066 at PRKAR1A, and rs10885349 at VTI1A might be the genetic basis of this comorbidity. CONCLUSIONS: Our study suggests that the SNAP23, PRKAR1A, and VTI1A genes can directly or indirectly participate in the imbalanced assembly of SNAREs in the pathogenesis of the comorbidity of PCOS and depression. These findings may provide new strategies in diagnosis and therapy for this comorbidity.

Coronary artery disease patients with rs7904519 (TCF7L2) are at a persistent risk of type 2 diabetes.

AIMS: Type 2 diabetes (T2D) and coronary artery disease (CAD) often coexist and share genetic factors.This study aimed to investigate the common genetic factors underlying T2D and CAD in patients with CAD. METHODS: A three-step association approach was conducted: a) a discovery step involving 943 CAD patients with T2D and 1,149 CAD patients without T2D; b) an eliminating step to exclude CAD or T2D specific variants; and c) a replication step using the UK Biobank data. RESULTS: Ten genetic loci were associated with T2D in CAD patients. Three variants were specific to either CAD or T2D. Five variants lost significance after adjusting for covariates, while two SNPs remained associated with T2D in CAD patients (rs7904519*G: TCF7L2 and rs17608766*C: GOSR2). The T2D susceptibility rs7904519*G was associated with increased T2D risk, while the CAD susceptibility rs17608766*C was negatively associated with T2D in CAD patients. These associations were replicated in a UK Biobank data, confirming the results. CONCLUSIONS: No significant common T2D and CAD susceptibility genetic association was demonstrated indicating distinct disease pathways. However, CAD patients carrying the T2D susceptibility gene TCF7L2 remain at higher risk for developing T2D emphasizing the need for frequent monitoring in this subgroup.

Novel Genetic and Phenotypic Expansion in GOSR2-Related Progressive Myoclonus Epilepsy.

Biallelic variants in the Golgi SNAP receptor complex member 2 gene (GOSR2) have been reported in progressive myoclonus epilepsy with neurodegeneration. Typical clinical features include ataxia and areflexia during early childhood, followed by seizures, scoliosis, dysarthria, and myoclonus. Here, we report two novel patients from unrelated families with a GOSR2-related disorder and novel genetic and clinical findings. The first patient, a male compound heterozygous for the GOSR2 splice site variant c.336+1G>A and the novel c.364G>A,p.Glu122Lys missense variant showed global developmental delay and seizures at the age of 2 years, followed by myoclonus at the age of 8 years with partial response to clonazepam. The second patient, a female homozygous for the GOSR2 founder variant p.Gly144Trp, showed only mild fine motor developmental delay and generalized tonic-clonic seizures triggered by infections during adolescence, with seizure remission on levetiracetam. The associated movement disorder progressed atypically slowly during adolescence compared to its usual speed, from initial intention tremor and myoclonus to ataxia, hyporeflexia, dysmetria, and dystonia. These findings expand the genotype-phenotype spectrum of GOSR2-related disorders and suggest that GOSR2 should be included in the consideration of monogenetic causes of dystonia, global developmental delay, and seizures.

Tumor-derived extracellular vesicles delivering TNF-α promotes colorectal cancer metastasis via the NF-kB/LAMB3/AKT axis by targeting SNAP23.

Accumulating evidence have demonstrated that cytokines are enriched in tumor-derived extracellular vesicles (EVs) and widely involved in tumorigenesis of various types of carcinomas, including colorectal cancer (CRC). Nevertheless, the functions of cytokines in EVs secreted from colorectal cancer cells remain largely unknown. In the present study, we found that TNF-α was elevated in EVs from CRC patient serum samples and CRC cell lines, of which the expression was associated with aggressive features of colorectal cancer. EV TNF-α secretion is dependent on synaptosome-associated protein 23 (SNAP23). Functional experiments revealed that EV TNF-α promotes CRC cell metastasis via the NF-κB pathway by targeting SNAP23. Mechanistically, SNAP23 was transcriptionally upregulated by EV TNF-α/NF-κB axis to enhance the expression of laminin subunit beta-3 (LAMB3), thereby activating the PI3K/AKT signaling pathway and consequently facilitate CRC progression. Based on our findings, we could conclude that EV TNF-α plays an oncogenic role in CRC progression through SNAP23, which in turn promotes EV TNF-α secretion, suggesting that EV TNF-α/SNAP23 axis may serve as a diagnostic biomarker and potential therapeutic target for CRC.

SNAP23 decreases insulin secretion by competitively inhibiting the interaction between SNAP25 and STX1A.

SNAP25 is a core protein of the SNARE complex, which mediates stimulus-dependent secretion of insulin from the pancreatic ß cells. SNAP23 is a SNAP25 homolog, however, the functional role of SNAP23 in the exocytic secretion of insulin is not known. Therefore, in the present study, we investigated the functional role of SNAP23 in the insulin secretory pathway. Our results demonstrated that over-expression of SNAP23 inhibited the secretion of insulin from the INS-1 cells. Conversely, SNAP23 depletion increased insulin secretion. Mechanistically, overexpression of SNAP23 decreased SNARE complex formation by blocking the binding of SNAP25 to STX1A. The full-length SNAP23 protein with the N-terminal and C-terminal SNARE binding domains was required for competition. Moreover, SNAP23 serine 95 phosphorylation plays a crucial function in insulin secretion by enhancing the interaction between SNAP23 and STX1A. The present study presents a new pathway regulating insulin secretion. Therefore, SNAP23 may be a potential therapeutic target for diabetes mellitus.

Snap29 Is Dispensable for Self-Renewal Maintenance but Required for Proper Differentiation of Mouse Embryonic Stem Cells.

Pluripotent embryonic stem cells (ESCs) can self-renew indefinitely and are able to differentiate into all three embryonic germ layers. Synaptosomal-associated protein 29 (Snap29) is implicated in numerous intracellular membrane trafficking pathways, including autophagy, which is involved in the maintenance of ESC pluripotency. However, the function of Snap29 in the self-renewal and differentiation of ESCs remains elusive. Here, we show that Snap29 depletion via CRISPR/Cas does not impair the self-renewal and expression of pluripotency-associated factors in mouse ESCs. However, Snap29 deficiency enhances the differentiation of ESCs into cardiomyocytes, as indicated by heart-like beating cells. Furthermore, transcriptome analysis reveals that Snap29 depletion significantly decreased the expression of numerous genes required for germ layer differentiation. Interestingly, Snap29 deficiency does not cause autophagy blockage in ESCs, which might be rescued by the SNAP family member Snap47. Our data show that Snap29 is dispensable for self-renewal maintenance, but required for the proper differentiation of mouse ESCs.

Snapshots from within the cell: Novel trafficking and non trafficking functions of Snap29 during tissue morphogenesis.

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.

SNAP23-Mediated Perturbation of Cholesterol-Enriched Membrane Microdomain Promotes Extracellular Vesicle Production in Src-Activated Cancer Cells.

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.

The synaptosome-associated protein 23 (SNAP23) is necessary for proper myogenesis.

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.

CEDNIK syndrome with phenotypic variability.

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.

Computational estimates of annular diameter reveal genetic determinants of mitral valve function and disease.

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.

CEDNIK syndrome in a Brazilian patient with compound heterozygous pathogenic variants.

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.

SNAP47 Interacts with ATG14 to Promote VP1 Conjugation and CVB3 Propagation.

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.

Myopathy can be a key phenotype of membrin (GOSR2) deficiency.

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.

Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts.

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.

LINC00511 drives invasive behavior in hepatocellular carcinoma by regulating exosome secretion and invadopodia formation.

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.

SNAP23 is essential for platelet and mast cell development and required in connective tissue mast cells for anaphylaxis.

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.

Decreased neuronal synaptosome associated protein 29 contributes to poststroke cognitive impairment by disrupting presynaptic maintenance.

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.

Compound heterozygous variants in GOSR2 associated with congenital muscular dystrophy: A case report.

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.