SNAP-25 Research Articles

Repeated oxytocin prevents central sensitization by regulating synaptic plasticity via oxytocin receptor in a chronic migraine mouse model

BackgroundCentral sensitization is one of the characters of chronic migraine (CM). Aberrant synaptic plasticity can induce central sensitization. Oxytocin (OT), which is a hypothalamic hormone, plays an important antinociceptive role. However, the antinociceptive effect of OT and the underlying mechanism in CM remains unclear. Therefore, we explored the effect of OT on central sensitization in CM and its implying mechanism, focusing on synaptic plasticity.MethodsA CM mouse model was established by repeated intraperitoneal injection of nitroglycerin (NTG). Von Frey filaments and radiant heat were used to measure the nociceptive threshold. Repeated intranasal OT and intraperitoneal L368,899, an oxytocin receptor (OTR) antagonist, were administered to investigate the effect of OT and the role of OTR. The expression of calcitonin gene-related peptide (CGRP) and c-fos were measured to assess central sensitization. N-methyl D-aspartate receptor subtype 2B (NR2B)-regulated synaptic-associated proteins and synaptic plasticity were explored by western blot (WB), transmission electron microscope (TEM), and Golgi-Cox staining.ResultsOur results showed that the OTR expression in the trigeminal nucleus caudalis (TNC) of CM mouse was significantly increased, and OTR was colocalized with the postsynaptic density protein 95 (PSD-95) in neurons. Repeated intranasal OT alleviated the NTG-induced hyperalgesia and prevented central sensitization in CM mouse. Additionally, the OT treatment inhibited the overexpression of phosphorylated NR2B and synaptic-associated proteins including PSD-95, synaptophysin-1 (syt-1), and synaptosomal-associated protein 25 (snap25) in the TNC of CM mouse and restored the abnormal synaptic structure. The protective effect of OT was prevented by L368,899. Furthermore, the expression of adenylyl cyclase 1 (AC1)/ protein kinase A (PKA)/ phosphorylation of cyclic adenosine monophosphate response element-binding protein (pCREB) pathway was depressed by OT and restored by L368,899.ConclusionsOur findings demonstrate that repeated intranasal OT eliminates central sensitization by regulating synaptic plasticity via OTR in CM. The effect of OT has closely associated with the down-regulation of AC1/PKA/pCREB signaling pathway, which is activated in CM model. Repeated intranasal OT may be a potential candidate for CM prevention.

Comparison of CSF biomarkers in Down syndrome and autosomal dominant Alzheimer's disease: a cross-sectional study

Due to trisomy of chromosome 21 and the resultant extra copy of the amyloid precursor protein gene, nearly all adults with Down syndrome develop Alzheimer's disease pathology by the age of 40 years and are at high risk for dementia given their increased life expectancy compared with adults with Down syndrome in the past. We aimed to compare CSF biomarker patterns in Down syndrome with those of carriers of autosomal dominant Alzheimer's disease mutations to enhance our understanding of disease mechanisms in these two genetic groups at high risk for Alzheimer's disease. We did a cross-sectional study using data from adults enrolled in the Alzheimer's Biomarker Consortium-Down Syndrome (ABC-DS) study, a multisite longitudinal study of Alzheimer's disease in Down syndrome, as well as a cohort of carriers of autosomal dominant Alzheimer's disease mutations and non-carrier sibling controls enrolled in the Dominantly Inherited Alzheimer Network (DIAN) study. For ABC-DS, participants with baseline CSF, available clinical diagnosis, and apolipoprotein E genotype as of Jan 31, 2019, were included in the analysis. DIAN participants with baseline CSF, available clinical diagnosis, and apolipoprotein E genotype as of June 30, 2018, were evaluated as comparator groups. CSF samples obtained from adults with Down syndrome, similarly aged carriers of autosomal dominant Alzheimer's disease mutations, and non-carrier siblings (aged 30-61 years) were analysed for markers of amyloid β (Aβ1-40, Aβ1-42); tau phosphorylated at threonine 181-related processes; neuronal, axonal, or synaptic injury (total tau, visinin-like protein 1, neurofilament light chain [NfL], synaptosomal-associated protein 25); and astrogliosis and neuroinflammation (chitinase-3-like protein 1 [YKL-40]) via immunoassay. Biomarker concentrations were compared as a function of dementia status (asymptomatic or symptomatic), and linear regression was used to evaluate and compare the relationship between biomarker concentrations and age among groups. We assessed CSF samples from 341 individuals (178 [52%] women, 163 [48%] men, aged 30-61 years). Participants were adults with Down syndrome (n=41), similarly aged carriers of autosomal dominant Alzheimer's disease mutations (n=192), and non-carrier siblings (n=108). Individuals with Down syndrome had patterns of Alzheimer's disease-related CSF biomarkers remarkably similar to carriers of autosomal dominant Alzheimer's disease mutations, including reductions (all p<0·0080) in Aβ1-42 to Aβ1-40 ratio and increases in markers of phosphorylated tau-related processes; neuronal, axonal, and synaptic injury (p<0·080); and astrogliosis and neuroinflammation, with greater degrees of abnormality in individuals with dementia. Differences included overall higher concentrations of Aβ and YKL-40 (both p<0·0008) in Down syndrome and potential elevations in CSF tau (p<0·010) and NfL (p<0·0001) in the asymptomatic stage (ie, no dementia symptoms). National Institute on Aging, Eunice Kennedy Shriver National Institute of Child Health and Human Development, German Center for Neurodegenerative Diseases, and Japan Agency for Medical Research and Development.

Corrigendum] Cryptotanshinone inhibits IgE‑mediated degranulation through inhibition of spleen tyrosine kinase and tyrosine‑protein kinase phosphorylation in mast cells

Following the publication of the above article, an interested reader drew to the authors' attention that they had mentioned that activated PKCδ phosphorylates IKKβ in order that IKKβ is relocated to the plasma membrane, resulting in the induction of mast cell degranulation; however, four references the authors had included did not seem to support this statement. The authors have re-examined their paper, and realized that the four references the reader mentioned were indeed cited incorrectly, and wish to rectify this error through revising the third paragraph in the Discussion section, the References section, and an associated figure (Fig.6C) in order to avoid any further misunderstandings on the part of the readership. First, the authors wish to revise the wording of the third and fourth paragraphs of the Discussion, as featured on pp.1101-1102, to the following (changed text is indicated in bold): 'We showed that CRT exerts anti-AD effect through inhibition of the mast cell degranulation in mast cells. Upon IgE/antigen stimulation, the immunoreceptor tyrosine-based activation motif (ITAM) region of FcεRI receptor which is on the mast cell surface is phosphorylated and the initial signalling protein kinases Lyn and Syk are recruited to the ITAM (28,29). Then, the activated Lyn and Syk leads to phosphorylation of the transmembrane adaptor linker for activation of T cells (LAT). Phosphorylated LAT which is a scaffold for multimolecular signalling complexes and activates PLCγ through phosphorylation. The activated PLCγ hydrolyses phosphatidylinositol biphosphate (PIP2) to generate second signalling molecules IP3 and DAG, which activate PKCs including PKCδ to induce the mast cell degranulation (30,31). On the other hand, cross-linking of FcεRI also activates IKKβ, which moves to the lipid raft fractions and phosphorylates synaptosomal-associated protein23 (SNAP-23) leading to degranulation (7). Since PKCδ phosphorylates IKKα, but not IKKβ (32), it is not likely that two signalling pathways are directly connected. In this study, novel function of CRT on phosphorylations of Lyn/Syk kinases in mast cells is elucidated for the first time. Furthermore, it is likely that this inhibitory effect of CRT on Lyn/Syk kinases negatively affected activities of their downstream signalling molecules including PLCγ, PKCδ, and IKKβ, which leads to decrease in mast cell degranulation by CRT treatment. Besides the inhibitory effect of CRT on mast cell degranulation, here we provide additional evidence that CRT exerts anti-AD effects through inactivation of MAPK and NF‑κB. It has been reported that CRT regulates the activities of MAPK and NF‑κB in various cell types. In rhabdomyosarcoma, hepatoma, and breast carcinoma, CRT activates MAPK p38/JNK and suppresses ERK1/2, followed by caspase-independent apoptosis (10,33,34). In chronic myeloid leukaemia cells, CRT enhances TNF‑α-induced apoptosis through the activation of MAPK p38 (35). In smooth muscle cells, CRT exerts anti-migration/invasion effect as it inhibits TNF‑α/NF‑κB signalling pathway (36).' Secondly, the authors wish to make the following changes to the Reference list: New references 30-32 have been inserted to the list, as follows: 30. Ozawa K, Szallasi Z, Kazanietz MG, BlumbergPM, MischakH, Mushinski JF and Beaven MA: Ca2+-dependent and Ca2+-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cell. J Biol Chem268: 1749-1756, 1993. 31. Cho SH, Woo CH, Yoon SB and Kim JH: Protein kinaseCδ functions downstream of Ca2+ mobilization in FcεRI signaling to degranulation in mast cells. J Allergy Clin Immunol114: 1085-1092, 2004. 32. Yamaguchi T, Miki Y and Yoshida K: Protein kinaseCδ activates IκB-kinaseα to induce the p53 tumor suppressor in response to oxidative stress. Cell Signal19: 2088-2097, 2007. The addition of these new references means that the former references 30-33 have been accordingly renumbered to references 33-36. Finally, the authors have revised Fig.6C, as it appeared on p.1102, in order to assist the understanding of the readers, and the corrected version of Fig.6 appears on the next page. All these corrections have been approved by all the authors, with the exception of the first author, Sumiyasuren Buyanravjikh, who is no longer uncontactable. The authors regret that these errors were included in the paper, even though they did not substantially alter any of the major conclusions reported in the study, are grateful to the Editor for allowing them this opportunity to publish a Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 18: 1095‑1193, 2018; DOI: 10.3892/mmr.2018.9042].

β3-adrenoceptor activation exhibits a dual effect on behaviors and glutamate receptor function in the prefrontal cortex

β-adrenoceptor (β-AR), especially the β1- and β2-AR subtypes, is known to participate in stress-related behavioral changes. Recently, SR58611A, a brain-penetrant β3-AR agonist, exhibits anxiolytic- and antidepressant-like effects. In this study, we sought to study the role of SR58611A in behavioral changes and its potential cellular and molecular mechanism in the prefrontal cortex (PFC). We found that rats with SR58611A (1 mg/kg) enhanced PFC-mediated recognition memory, whereas administration of higher dosage of SR58611A (20 mg/kg) caused hyperlocomotion, and exhibited an impairment effect on recognition memory. Electrophysiological data also indicated that SR58611A (1 mg/kg) selectively enhanced NMDA receptor-mediated excitatory postsynaptic currents (EPSC) through interacting with norepinephrine (NE) system and activating β3-AR, whereas higher dosage of SR58611A (20 mg/kg) reduced both AMPA receptor- and NMDA receptor-mediated EPSC. SR58611A-induced different effects on EPSC linked with the change of the surface expression quantity of NMDA receptor and/or AMPA receptor subunits. Synaptosomal-associated protein 25 (SNAP-25), which is a key soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein involved in incorporation of NMDA receptor to postsynaptic membrane, contributed to SR58611A (1 mg/kg)-induced enhancement of recognition memory and NMDA receptor function. Moreover, SR58611A (1 mg/kg) could rescue repeated stress-induced defect of both recognition memory and NMDA receptor function through a SNAP-25-dependent mechanism. These results provide a potential mechanism underlying the cognitive-enhancing effects of SR58611A (1 mg/kg).

Aedes aegypti SNAP and a calcium transporter ATPase influence dengue virus dissemination.

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world’s population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.

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

BackgroundTumor 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.MethodsThe 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.ResultsWe 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.ConclusionsOur 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.

Machine Learning Prediction of ADHD Severity: Association and Linkage to ADGRL3, DRD4, and SNAP25

Objective: To investigate whether single nucleotide polymorphisms (SNPs) in the ADGRL3, DRD4, and SNAP25 genes are associated with and predict ADHD severity in families from a Caribbean community. Method: ADHD severity was derived using latent class cluster analysis of DSM-IV symptomatology. Family-based association tests were conducted to detect associations between SNPs and ADHD severity latent phenotypes. Machine learning algorithms were used to build predictive models of ADHD severity based on demographic and genetic data. Results: Individuals with ADHD exhibited two seemingly independent latent class severity configurations. SNPs harbored in DRD4, SNAP25, and ADGRL3 showed evidence of linkage and association to symptoms severity and a potential pleiotropic effect on distinct domains of ADHD severity. Predictive models discriminate severe from non-severe ADHD in specific symptom domains. Conclusion: This study supports the role of DRD4, SNAP25, and ADGRL3 genes in outlining ADHD severity, and a new prediction framework with potential clinical use.

Novel Applications of Non-Invasive Intravesical Botulinum Toxin a Delivery in the Treatment of Functional Bladder Disorders.

Although intravesical botulinum toxin type A (BoNT-A) injection for functional bladder disorders is effective, the injection-related problems—such as bladder pain and urinary tract infection—make the procedure invasive and inconvenient. Several vehicles have recently been developed to deliver BoNT-A without injection, thereby making the treatment less or non-invasive. Laboratory evidence revealed that liposome can carry BoNT-A across the uroepithelium and act on sub-urothelial nerve endings. A randomized placebo controlled study revealed that intravesical administration of liposome-encapsulated BoNT-A and TC-3 hydrogel embedded BoNT-A can improve urinary frequency, urgency, and reduce incontinence in patients with overactive bladders. A single-arm prospective study also revealed that intravesical administration of TC-3 hydrogel embedded BoNT-A can relieve bladder pain in patients with interstitial cystitis/bladder pain syndrome (IC/BPS). We recently administered suprapubic energy shock wave (ESW) after BoNT-A intravesical administration in six patients with IC/BPS. Although pain reduction and symptom improvement were not significant, immunochemical staining showed cleaved synaptosome-associated protein 25 in the bladder after the procedure. This suggests that ESW can promote passage of BoNT-A across the uroepithelium. In conclusion, using vehicles to intra-vesically deliver BoNT-A for functional bladder disorders is promising. Further studies are necessary to confirm the efficacy and explore novel applications.

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.

Integrative Structural Dynamics Probing of the Conformational Heterogeneity in Synaptosomal-Associated Protein 25

SNAP-25 (synaptosomal-associated protein of 25 kDa) is a prototypical intrinsically disordered protein (IDP) that is unstructured in its unbound form but forms coiled-coil helices in the SNARE complex. With high conformational heterogeneity, detailed structural dynamics of unbound SNAP-25 remains elusive. Here, we present an integrative method to probe the structural dynamics of SNAP-25 by combining replica-exchange discrete molecular dynamics (rxDMD) simulations and label-based experiments at ensemble and single-molecule levels. The rxDMD simulation allows us to systematically characterize the coil-to-molten-globular transition and reconstruct structural ensemble consistent with prior ensemble experiments. Label-based experiments using Forster Resonance Energy Transfer and Double Electron Electron Resonance further probe the conformational dynamics of SNAP-25. Agreements between simulations and experiments under both ensemble and single-molecule conditions allow us to assign specific helix-coil transitions in SNAP-25 that occur in sub-millisecond timescales and potentially play a vital role in forming the SNARE complex. We expect this integrative approach would help further our understanding of IDPs.

Differential requirement of NPHP1 for compartmentalized protein localization during photoreceptor outer segment development and maintenance.

Nephrocystin (NPHP1) is a ciliary transition zone protein and its ablation causes nephronophthisis (NPHP) with partially penetrant retinal dystrophy. However, the precise requirements of NPHP1 in photoreceptors are not well understood. Here, we characterize retinal degeneration in a mouse model of NPHP1 and show that NPHP1 is required to prevent infiltration of inner segment plasma membrane proteins into the outer segment during the photoreceptor maturation. We demonstrate that Nphp1 gene-trap mutant mice, which were previously described as null, are likely hypomorphs due to the production of a small quantity of functional mRNAs derived from nonsense-associated altered splicing and skipping of two exons including the one harboring the gene-trap. In homozygous mutant animals, inner segment plasma membrane proteins such as syntaxin-3 (STX3), synaptosomal-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (IMPG2) accumulate in the outer segment when outer segments are actively elongating. This phenotype, however, is spontaneously ameliorated after the outer segment elongation is completed. Consistent with this, some photoreceptor cell loss (~30%) occurs during the photoreceptor maturation period but it stops afterward. We further show that Nphp1 genetically interacts with Cep290, another NPHP gene, and that a reduction of Cep290 gene dose results in retinal degeneration that continues until adulthood in Nphp1 mutant mice. These findings demonstrate that NPHP1 is required for the confinement of inner segment plasma membrane proteins during the outer segment development, but its requirement diminishes as photoreceptors mature. Our study also suggests that additional mutations in other NPHP genes may influence the penetrance of retinopathy in human NPHP1 patients.

Metformin facilitates mesenchymal stem cell-derived extracellular nanovesicles release and optimizes therapeutic efficacy in intervertebral disc degeneration

Extracellular vesicles (EVs) are extracellular nanovesicles that deliver diverse cargoes to the cell and participate in cell communication. Mesenchymal stem cell (MSCs)-derived EVs are considered a therapeutic approach in musculoskeletal degenerative diseases, including intervertebral disc degeneration. However, limited production yield and unstable quality have impeded the clinical application of EVs. In the present study, it is indicated that metformin promotes EVs release and alters the protein profile of EVs. Metformin enhances EVs production via an autophagy-related pathway, concomitantly with the phosphorylation of synaptosome-associated protein 29. More than quantity, quality of MSCs-derived EVs is influenced by metformin treatment. Proteomics analysis reveals that metformin increases the protein content of EVs involved in cell growth. It is shown that EVs derived from metformin-treated MSCs ameliorate intervertebral disc cells senescence in vitro and in vivo. Collectively, these findings demonstrate the great promise of metformin in EVs-based intervertebral disc regeneration.

The Vesicle‐Mediated Transport Genes, Snap23 , Tmed2 , and Trip10 , are Alternatively Spliced During Striated Muscle Development

The reprogramming of alternative splicing patterns during development is a hallmark of tissue maturation and identity. Heart and skeletal (striated) muscle tissues exhibit the most highly conserved splicing programs, which substantially change during postnatal development. Notably, alterations of these splicing events are associated with severe cardiac and muscular diseases. In striated muscle, a collection of genes encoding membrane trafficking proteins are spliced in a developmental stage-specific manner. However, few splice isoforms have been characterized, and little is known about their functional roles during heart and skeletal muscle development. We hypothesize that coordinated splicing of membrane trafficking genes represents a splicing network, which functions to regulate the transport of cargo that is critical for muscle development and homeostasis. Here, we characterize three proteins involved in vesicle-mediated transport that are alternatively spliced in striated muscle: SNAP23, TMED2, and TRIP10. The synaptosome-associated protein 23 (SNAP23) is a SNARE protein that mediates vesicle fusion with the plasma membrane during exocytosis, the transmembrane emp23 domain-containing protein 2 (TMED2) regulates vesicle budding during secretion, and the thyroid hormone receptor interactor 10 (TRIP10) is involved in endocytosis and membrane tubulation. First, we found that during striated muscle development, the Snap23, Tmed2, and Trip10 pre-mRNAs undergo splicing changes of a single exon. Second, these three splicing transitions are characterized by a shift from short to long isoform expression that are regulated by the RBPs quaking (QKI) and polypyrimidine tract binding protein 1 (PTBP1). Third, depletion of SNAP23 in muscle cells results in reduced cell viability and differentiation, which can be rescued with conditioned media from differentiated cells or by seeding cells on collagen-coated plates. Finally, exclusive expression of the short SNAP23 isoform leads to increased fusion of myoblasts into myotubes that exhibit greater myotube area and a higher nuclei-per-myotube ratio. Taken together, our data suggest that splicing of Snap23 is an important regulator of myogenesis, where SNAP23 isoform expression controls secretion of cargo that is necessary for muscle cell fusion and myotube size. Preliminary studies show that disruption of TMED2 and TRIP10 isoform levels in muscle cells likewise affects differentiation. Elucidating the mechanisms of regulation and the functional consequences of splicing networks will be fundamental to our understanding of muscle development and disease.

Association Analyses of SNAP25, HNMT, FCHSD1, and DBH Single-Nucleotide Polymorphisms with Parkinson's Disease in a Northern Chinese Population.

PurposeSequencing potentially causal and susceptible genes and genome-wide association studies in samples from Parkinson’s disease (PD) patients has revealed several related loci. The genes for synaptosome-associated protein of 25 kDa (SNAP25), histamine-N-methyltransferase (HNMT), FCH and double SH3 domains 1 (FCHSD1) and dopamine β-hydroxylase (DBH) are candidate loci and have not been studied in a northern Chinese population. We explored the genetic distribution of four single-nucleotide polymorphisms (rs3746544, rs11558538, rs456998, rs129882) located on SNAP25, HNMT, FCHSD1 and DBH, respectively.Patients and MethodsA total of 330 patients with sporadic PD and 332 healthy controls (HCs) were recruited from a northern Chinese population. Polymerase chain reaction restriction fragment length polymorphism was used to genotype these four SNPs.ResultsAfter statistical analyses and correction of the genotyping results, the mutant-allele T in rs456998 of FCHSD1 was found to be significantly related to reducing the PD risk (P = 0.029, OR = 0.754, 95% CI = 0.586–0.971, power = 0.591). However, rs3746544, rs11558538, and rs129882 did not show an association with PD.ConclusionFCHSD1 rs456998 may have a protective role in PD in a northern Chinese population, but more studies are needed to support this suggestion.

Importance of Spatial and Temporal Regulation of SNAP‐23 Localization in Activated Mast Cells by Transient Phosphorylation

Mast cells are innate immune tissue resident cells. These cells release various inflammatory mediators like cytokines, chemokines, histamine and lysosomal enzymes upon allergen activation. Previous work revealed that Synaptosomal-associated protein 23 kDa (SNAP-23) is a t-SNARE present in mast cells, synthesized in the cytoplasm, and then associates with the plasma membrane to play a major role during mast cell exocytosis. Early, on allergen activation it gets phosphorylated at Serine95 (S95) and Serine 120(S120) and translocates to the granule membrane containing LAMP-3. Blocking either phosphorylation or dephosphorylation by introducing phosphonegative or phosphomimetic mutations in SNAP-23 caused massive inhibition in mast cell exocytosis. Therefore, we decided to unravel the kinetics of phosphorylation and dephosphorylation at specific amino acid residues and to see their effects on SNAP-23 localization in resting and activated mast cells at different time point. It was observed that (S120) gets phosphorylated first, (S95) later. These residues were mutated by site-directed mutagenesis to Aspartate (phosphomimetic) and Alanine (phosphonegative) to get SNAP-23(S95DS120D), SNAP-23(S95AS120A), SNAP-23(S95AS120D), SNAP-23(S95DS120A) and cloned into EGFP-C2 vector. All EGFP-phosphomutants and SNAP-23 wildtype (WT) were transfected in RBL MCs to study subcellular localization by confocal microscopy at an early (10 min) and late (45 min) time points after activation. It was observed that plasma membrane association for WT SNAP-23 almost decreased to half in allergen activated (49%-33% at 10min; 24%-11% at 45min) mast cells compared to resting stage (80%-75%). In various phosphomutants of SNAP-23, the plasma membrane association was greatly affected and proper localization was not achieved on activation at both time points. These results suggest that SNAP-23 dynamicity in terms of intracellular localization and phosphorylation has a tremendous role in mast cells exocytosis and can be targeted as a potential target against allergy.

SNAP29 Restricts Cardiac Arrhythmias by Insulating A Subset of Desmosomal Proteins and Connexin43 from Autophagic Degradation

Desmosomes are strong cell-cell adhesion junctions that connect the cytoskeleton of one cell to another (via intermediate filaments) to maintain the mechanical integrity of tissues under constant stress, such as the heart. Classically, human genetic studies and mouse models have linked components of the desmosome mutations/deficiencies to arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited cardiac disease of the desmosome that impacts cardiac structure/morphology. However, growing evidence points to a role for loss of desmosomal integrity in cardiac electrical diseases (eg., Brugada syndrome, long QT, sinus node arrhythmias) that have no impact on cardiac structure/morphology; however, how desmosomal protein loss results in these distinct forms of cardiac disease remains unclear. We exploited an unbiased yeast-two-hybrid screen using the desmosomal protein, desmoplakin (DSP) as a bait, to uncover novel desmosomal protein-protein interactions that may help further explain the pathobiology (structural versus non-structural) encompassed by desmosomal mutations/loss. Through this screen, we identified the SNARE protein, synaptosomal-associated protein 29 (SNAP29), as a novel DSP-interacting protein. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy; however, its role at the desmosome in the heart is undefined. We hypothesized that SNAP29 is a new subcomponent of the cardiac desmosome that regulates desmosomal protein degradation and thus, may have important roles in desmosomal based diseases. We show that SNAP29 co-localizes with DSP in adult mouse heart and that cardiac-specific DSP deficient mice hearts harbor loss of SNAP29, validating SNAP29 as a subcomponent of the cardiac desmosome. By generating novel cardiomyocyte-specific SNAP29 knockout (SNAP29-cKO) mice (using Xenopus myosin light chain 2-Cre), we show that SNAP29-cKO mouse hearts displayed baseline and pacing-induced ventricular arrhythmias in an age-dependent manner in the absence of cardiac structural and functional deficits. Molecular analyses further revealed that adult SNAP29-cKO hearts displayed a loss of a subset of desmosomal (DSP, plakophilin2) as well as gap junction (connexin43) proteins when compared to controls. These cell-cell junction defects were accompanied by an accumulation of autophagic markers (p62 and LC3BII), as well as machinery (autophagic vesicles), which functionally resulted in cardiac arrhythmias. We show that acute blockade of autophagy was sufficient to rescue levels of desmosomal and gap junction proteins as well as arrhythmias in SNAP29 deficient cardiomyocytes. In summary, our data suggest that SNAP29 insulates a subset of desmosomal and gap junction proteins from selective autophagy-mediated degradation to restrict cardiac arrhythmias and that loss of SNAP29-desmosome-gap junction interactome may predispose the heart to desmosomal based diseases of an electrical (non-structural) nature. Collectively, our results identify defects in cardiac autophagy in desmosomal disease pathogenesis, while providing a novel role for SNARE proteins in cardiac arrhythmias.

Slow recovery from the inactivation of voltage-gated sodium channel Nav1.3 in mouse taste receptor cells.

Action potentials play an important role in neurotransmitter release in response to taste. Here, I have investigated voltage-gated Na+ channels, a primary component of action potentials, in respective cell types of mouse fungiform taste bud cells (TBCs) with in situ whole-cell clamping and single-cell RT-PCR techniques. The cell types of TBCs electrophysiologically examined were determined immunohistochemically using the type III inositol 1,4,5-triphoshate receptor as a type II cell marker and synaptosomal-associated protein 25 as a type III cell marker. I show that type II cells, type III cells, and TBCs not immunoreactive to these markers (likely type I cells) generate voltage-gated Na+ currents. The recovery following inactivation of these currents was well fitted with double exponential curves. The time constants in type III cells (~20ms and ~ 1s) were significantly slower than respective time constants in other cell types. RT-PCR analysis indicated the expression of Nav1.3, Nav1.5, Nav1.6, and β1 subunit mRNAs in TBCs. Pharmacological inhibition and single-cell RT-PCR studies demonstrated that type II and type III cells principally express tetrodotoxin (TTX)-sensitive Nav1.3 channels and that ~ 30% of type I cells express TTX-resistant Nav1.5 channels. The auxiliary β1 subunit that modulates gating kinetics was rarely detected in TBCs. As the β1 subunit co-expressed with an α subunit is known to accelerate the recovery from inactivation, it is likely that voltage-gated Na+ channels in TBCs may function without β subunits. Slow recovery from inactivation, especially in type III cells, may limit high-frequency firing in response to taste substances.

Effect of oral nitrite administration on gene expression of SNARE proteins involved in insulin secretion from pancreatic islets of male type 2 diabetic rats

BackgroundNitrite stimulates insulin secretion from pancreatic β-cells; however, the underlying mechanisms have not been completely addressed. The aim of this study is to determine effect of nitrite on gene expression of SNARE proteins involved in insulin secretion from isolated pancreatic islets in Type 2 diabetic Wistar rats. MethodsThree groups of rats were studied (n = 10/group): Control, diabetes, and diabetes + nitrite, which treated with sodium nitrite (50 mg/L) for 8 weeks. Type 2 diabetes was induced using a low-dose of streptozotocin (25 mg/kg) combined with high-fat diet. At the end of the study, pancreatic islets were isolated and mRNA expressions of interested genes were measured; in addition, protein expression of proinsulin and C-peptide in pancreatic tissue was assessed using immunofluorescence staining. ResultsCompared with controls, in the isolated pancreatic islets of Type 2 diabetic rats, mRNA expression of glucokinase (59%), syntaxin1A (49%), SNAP25 (70%), Munc18b (48%), insulin1 (56%), and insulin2 (52%) as well as protein expression of proinsulin and C-peptide were lower. In diabetic rats, nitrite administration significantly increased gene expression of glucokinase, synaptotagmin III, syntaxin1A, SNAP25, Munc18b, and insulin genes as well as increased protein expression of proinsulin and C-peptide. ConclusionStimulatory effect of nitrite on insulin secretion in Type 2 diabetic rats is at least in part due to increased gene expression of molecules involved in glucose sensing (glucokinase), calcium sensing (synaptotagmin III), and exocytosis of insulin vesicles (syntaxin1A, SNAP25, and Munc18b) as well as increased expression of insulin genes.

A Comparative Analysis of Erythropoietin and Carbamoylated Erythropoietin Proteome Profiles.

In recent years, erythropoietin (EPO) has emerged as a useful neuroprotective and neurotrophic molecule that produces antidepressant and cognitive-enhancing effects in psychiatric disorders. However, EPO robustly induces erythropoiesis and elevates red blood cell counts. Chronic administration is therefore likely to increase blood viscosity and produce adverse effects in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered modification of EPO, is non-erythropoietic but retains the neurotrophic and neurotrophic activity of EPO. Blood profile analysis after EPO and CEPO administration showed that CEPO has no effect on red blood cell or platelet counts. We conducted an unbiased, quantitative, mass spectrometry-based proteomics study to comparatively investigate EPO and CEPO-induced protein profiles in neuronal phenotype PC12 cells. Bioinformatics enrichment analysis of the protein expression profiles revealed the upregulation of protein functions related to memory formation such as synaptic plasticity, long term potentiation (LTP), neurotransmitter transport, synaptic vesicle priming, and dendritic spine development. The regulated proteins, with roles in LTP and synaptic plasticity, include calcium/calmodulin-dependent protein kinase type 1 (Camk1), Synaptosomal-Associated Protein, 25 kDa (SNAP-25), Sectretogranin-1 (Chgb), Cortactin (Cttn), Elongation initiation factor 3a (Eif3a) and 60S acidic ribosomal protein P2 (Rplp2). We examined the expression of a subset of regulated proteins, Cortactin, Grb2 and Pleiotrophin, by immunofluorescence analysis in the rat brain. Grb2 was increased in the dentate gyrus by EPO and CEPO. Cortactin was induced by CEPO in the molecular layer, and pleiotrophin was increased in the vasculature by EPO. The results of our study shed light on potential mechanisms whereby EPO and CEPO produce cognitive-enhancing effects in clinical and preclinical studies.

Reduced Interaction of Aggregated α-Synuclein and VAMP2 by Environmental Enrichment Alleviates Hyperactivity and Anxiety in a Model of Parkinson's Disease.

Parkinson’s disease (PD) is a prevalent motor disease caused by the accumulation of mutated α-synuclein (α-Syn); however, its early stages are also characterized by non-motor symptoms, such as olfactory loss, cognitive decline, depression, and anxiety. The therapeutic effects of environmental enrichment (EE) on motor recovery have been reported, but its effects on non-motor symptoms remain unclear. Herein, we reveal the beneficial effects of EE on PD-related non-motor symptoms and changes in synaptic plasticity in the nucleus accumbens. To investigate its therapeutic effects in the early phase of PD, we randomly assigned eight-month-old mice overexpressing human A53T (hA53T) α-Syn to either the EE or standard condition groups for two months. Next, we performed behavioral tests and biochemical and histological analyses at 10 months of age. EE significantly alleviated locomotor hyperactivity and anxiety during the early stages of PD. It normalized the levels of tyrosine hydroxylase, phosphorylated and oligomeric α-Syn, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex-forming proteins, including synaptosomal-associated protein, 25 kDa, syntaxin1, and vesicle-associated membrane protein 2 (VAMP2). Moreover, the interactions between VAMP2 and pSer129 α-Syn were markedly reduced following EE. The restoration of synaptic vesicle transportation status may underlie the neuroprotective effects of EE in hA53T α-Syn mice.