Moniezia benedeni drives the SNAP-25 expression of the enteric nerves in sheep's small intestine.

BACKGROUND: The neuroimmune network plays a crucial role in regulating mucosal immune homeostasis within the digestive tract. Synaptosome-associated protein 25 (SNAP-25) is a presynaptic membrane-binding protein that activates ILC2s, initiating the host's anti-parasitic immune response. METHODS: To investigate the effect of Moniezia benedeni (M. benedeni) infection on the distribution of SNAP-25 in the sheep's small intestine, the recombinant plasmid pET-28a-SNAP-25 was constructed and expressed in BL21, yielding the recombinant protein. Then, the rabbit anti-sheep SNAP-25 polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of SNAP-25 in the intestines of normal and M. benedeni-infected sheep were detected by ELISA. RESULTS: The results showed that the SNAP-25 recombinant protein was 29.3 KDa, the titer of the prepared immune serum reached 1:128,000. It was demonstrated that the rabbit anti-sheep SNAP-25 polyclonal antibody could bind to the natural protein of sheep SNAP-25 specifically. The expression levels of SNAP-25 in the sheep's small intestine revealed its primary presence in the muscular layer and lamina propria, particularly around nerve fibers surrounding the intestinal glands. Average expression levels in the duodenum, jejunum, and ileum were 130.32 pg/mg, 185.71 pg/mg, and 172.68 pg/mg, respectively. Under conditions of M. benedeni infection, the spatial distribution of SNAP-25-expressing nerve fibers remained consistent, but its expression level in each intestine segment was increased significantly (P < 0.05), up to 262.02 pg/mg, 276.84 pg/mg, and 326.65 pg/mg in the duodenum, jejunum, and ileum, and it was increased by 101.06%, 49.07%, and 89.16% respectively. CONCLUSIONS: These findings suggest that M. benedeni could induce the SNAP-25 expression levels in sheep's intestinal nerves significantly. The results lay a foundation for further exploration of the molecular mechanism by which the gastrointestinal nerve-mucosal immune network perceives parasites in sheep.

PTPRS is a novel marker for early Tau pathology and synaptic integrity in Alzheimer's disease.

We examined the role of protein tyrosine phosphatase receptor sigma (PTPRS) in the context of Alzheimer's disease and synaptic integrity. Publicly available datasets (BRAINEAC, ROSMAP, ADC1) and a cohort of asymptomatic but "at risk" individuals (PREVENT-AD) were used to explore the relationship between PTPRS and various Alzheimer's disease biomarkers. We identified that PTPRS rs10415488 variant C shows features of neuroprotection against early Tau pathology and synaptic degeneration in Alzheimer's disease. This single nucleotide polymorphism correlated with higher PTPRS transcript abundance and lower p(181)Tau and GAP-43 levels in the CSF. In the brain, PTPRS protein abundance was significantly correlated with the quantity of two markers of synaptic integrity: SNAP25 and SYT-1. We also found the presence of sexual dimorphism for PTPRS, with higher CSF concentrations in males than females. Male carriers for variant C were found to have a 10-month delay in the onset of AD. We thus conclude that PTPRS acts as a neuroprotective receptor in Alzheimer's disease. Its protective effect is most important in males, in whom it postpones the age of onset of the disease.

All SNAP25 molecules in the vesicle-plasma membrane contact zone change conformation during vesicle priming.

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.

GLP-1 signaling-regulated SNAP25 is involved in improved insulin secretion in diabetic GK rats after Roux-en-Y gastric bypass surgery.

BACKGROUND: Roux-en-Y gastric bypass surgery (RYGB) improves glucose-stimulated insulin secretion (GSIS) in type 2 diabetes (T2D) patients. SNAP25 plays an essential role in GSIS. Clinical studies indicate that enhanced GLP-1 signaling is an important contributor to the improved ß-cell function in T2D. We aimed to explore whether GLP-1-regulated SNAP25 is involved in the enhanced secretory function of ß-cells in diabetic Goto-Kakizaki (GK) rats after RYGB. METHODS AND RESULTS: RYGB or sham surgery was conducted in GK rats. mRNA and protein expression of SNAP25 was assessed by qPCR and Western blot, respectively. Occupancy of CREB and acetyltransferase CBP and acetylation of histone H3 (ACH3) at the Snap25 promoter were determined using ChIP assay. RYGB led to increased SNAP25 expression and CREB phosphorylation in islets from GK rats. Increased SNAP25 improved GSIS in ß-cells cultured in high glucose conditions. Consistent with increased plasma GLP-1 after RYGB, GLP-1R agonist exendin4 increased SNAP25 expression and CREB phosphorylation in ß-cells. Mechanistically, exendin4 promoted the recruitment of CREB and CBP, thereby increasing ACH3 at the Snap25 promoter. Consistently, inhibition of CBP attenuated the effect of exendin4 on SNAP25 expression. Furthermore, the knockdown of SNAP25 diminished the increase of GSIS potentiated by chronic GLP-1 culture in INS-1 832/13 cells. CONCLUSIONS: Our findings unravel the novel mechanisms of RYGB-enhanced SNAP25 expression in ß-cells, and SNAP25 may contribute to the improved ß-cell secretory function induced by RYGB.

SNAP25 is a potential target for early stage Alzheimer's disease and Parkinson's disease.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD), two common irreversible neurodegenerative diseases, share similar early stage syndromes, such as olfaction dysfunction. Yet, the potential comorbidity mechanism of AD and PD was not fully elucidated. METHODS: The gene expression profiles of GSE5281 and GSE8397 were downloaded from the Gene Expression Omnibus (GEO) database. We utilized a series of bioinformatics analyses to screen the overlapped differentially expressed genes (DEGs). The hub genes were further identified by the plugin CytoHubba of Cytoscape and validated in the hippocampus (HIP) samples of APP/PS-1 transgenic mice and the substantial nigra (SN) samples of A53T transgenic mice by real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the expression of the target genes in the olfactory epithelium/bulb was detected by RT-qPCR. Finally, molecular docking was used to screen potential compounds for the target gene. RESULTS: One hundred seventy-four overlapped DEGs were identified in AD and PD. Five of the top ten enrichment pathways mainly focused on the synapse. Five hub genes were identified and further validated. As a common factor in AD and PD, the changes of synaptosomal-associated protein 25 (SNAP25) mRNA in olfactory epithelium/bulb were significantly decreased and had a strong association with those in the HIP and SN samples. Pazopanib was the optimal compound targeting SNAP25, with a binding energy of - 9.2 kcal/mol. CONCLUSIONS: Our results provided a theoretical basis for understanding the comorbidity mechanism of AD and PD and highlighted that SNAP25 in the olfactory epithelium may serve as a potential target for early detection and intervention in both AD and PD.

Role of SNAP25 on the occurrence and development of eosinophilic gastritis.

Eosinophilic gastritis is characterized by gastrointestinal symptoms accompanied by peripheral eosinophilia. This study aims to explore the association between eosinophilic gastritis and Synaptosome Associated Protein 25 (SNAP25), and provide a new direction for the diagnosis and treatment of eosinophilic gastritis. GSE54043 was downloaded from the gene expression omnibus database. Differentially expressed genes (DEGs) were screened. The functions of common DEGs were annotated by Database for Annotation, Visualization and Integrated Discovery and Metascape. The protein-protein interaction network of common DEGs was obtained by Search Tool for the Retrieval of Interacting Genes and visualized by Cytoscape. Significant modules were identified from the protein-protein interaction network. A total of 186 patients with eosinophilic gastritis were recruited. The clinical data were recorded and the expression levels of CPE, SST, PCSK2, SNAP25, and SYT4 were detected. Pearson chi-square test and Spearman correlation coefficient were used to analyze the relationship between eosinophilic gastritis and related parameters. Univariate and multivariate Logistic regression were used for further analysis. 353 DEGs were presented. The top 10 genes screened by cytoHubb were shown, and Veen diagram figured out 5 mutual genes. Pearson's chi-square test showed that SNAP25 (P < .001) was significantly associated with eosinophilic gastritis. Spearman correlation coefficient showed a significant correlation between eosinophilic gastritis and SNAP25 (ρ = -0.569, P < .001). Univariate logistic regression analysis showed that SNAP25 (OR = 0.046, 95% CI: 0.018-0.116, P < .001) was significantly associated with eosinophilic gastritis. Multivariate logistic regression analysis showed that SNAP25 (OR = 0.024, 95% CI: 0.007-0.075, P < .001) was significantly associated with eosinophilic gastritis. The low expression of SNAP25 gene in eosinophilic gastritis is associated with a higher risk of eosinophilic gastritis.

Non-coding variants in VAMP2 and SNAP25 affect gene expression: potential implications in migraine susceptibility.

Migraine is a common and complex neurological disease potentially caused by a polygenic interaction of multiple gene variants. Many genes associated with migraine are involved in pathways controlling the synaptic function and neurotransmitters release. However, the molecular mechanisms underpinning migraine need to be further explored.Recent studies raised the possibility that migraine may arise from the effect of regulatory non-coding variants. In this study, we explored the effect of candidate non-coding variants potentially associated with migraine and predicted to lie within regulatory elements: VAMP2_rs1150, SNAP25_rs2327264, and STX1A_rs6951030. The involvement of these genes, which are constituents of the SNARE complex involved in membrane fusion and neurotransmitter release, underscores their significance in migraine pathogenesis. Our reporter gene assays confirmed the impact of at least two of these non-coding variants. VAMP2 and SNAP25 risk alleles were associated with a decrease and increase in gene expression, respectively, while STX1A risk allele showed a tendency to reduce luciferase activity in neuronal-like cells. Therefore, the VAMP2_rs1150 and SNAP25_rs2327264 non-coding variants affect gene expression, which may have implications in migraine susceptibility. Based on previous in silico analysis, it is plausible that these variants influence the binding of regulators, such as transcription factors and micro-RNAs. Still, further studies exploring these mechanisms would be important to shed light on the association between SNAREs dysregulation and migraine susceptibility.

miR-23a-3p and miR-181a-5p modulate SNAP-25 expression.

SNAP-25 protein is a key protein of the SNARE complex that is involved in synaptic vesicles fusion with plasma membranes and neurotransmitter release, playing a fundamental role in neural plasticity. Recently the concentration of three specific miRNAs-miR-27b-3p, miR-181a-5p and miR-23a-3p -was found to be associated with a specific SNAP-25 polymorphism (rs363050). in silico analysis showed that all the three miRNAs target SNAP-25, but the effect of the interaction between these miRNAs and the 3'UTR of SNAP-25 mRNA is currently unknown. For this reason, we verified in vitro whether miR-27b-3p, miR-181a-5p and miR-23a-3p modulate SNAP-25 gene and protein expression. Initial experiments using miRNAs-co-transfected Vero cells and SNAP-25 3'UTR luciferase reporter plasmids showed that miR-181a-5p (p≤0.01) and miR-23a-3p (p<0.05), but not miR-27b-3p, modulate the luciferase signal, indicating that these two miRNAs bind the SNAP-25 3'UTR. Results obtained using human oligodendroglial cell line (MO3.13) transfected with miR-181a-5p or miR-27b-3p confirmed that miR-181a-5p and miR-23a-3p regulate SNAP-25 gene and protein expression. Interestingly, the two miRNAs modulate in an opposite way SNAP-25, as miR-181a-5p significantly increases (p<0.0005), whereas miR-23a-3p decreases (p<0.0005) its expression. These results for the first time describe the ability of miR-181a-5p and miR-23a-3p to modulate SNAP-25 expression, suggesting their possible use as biomarkers or as therapeutical targets for diseases in which SNAP-25 expression is altered.

Rapid genome sequencing identifies a novel de novo SNAP25 variant for neonatal congenital myasthenic syndrome.

Congenital myasthenic syndrome (CMS) is a group of 32 disorders involving genetic dysfunction at the neuromuscular junction resulting in skeletal muscle weakness that worsens with physical activity. Precise diagnosis and molecular subtype identification are critical for treatment as medication for one subtype may exacerbate disease in another (Engel et al., Lancet Neurol 14: 420 [2015]; Finsterer, Orphanet J Rare Dis 14: 57 [2019]; Prior and Ghosh, J Child Neurol 36: 610 [2021]). The SNAP25-related CMS subtype (congenital myasthenic syndrome 18, CMS18; MIM #616330) is a rare disorder characterized by muscle fatigability, delayed psychomotor development, and ataxia. Herein, we performed rapid whole-genome sequencing (rWGS) on a critically ill newborn leading to the discovery of an unreported pathogenic de novo SNAP25 c.529C > T; p.Gln177Ter variant. In this report, we present a novel case of CMS18 with complex neonatal consequence. This discovery offers unique insight into the extent of phenotypic severity in CMS18, expands the reported SNAP25 variant phenotype, and paves a foundation for personalized management for CMS18.

Role of SNAP-25 MnlI variant in impaired working memory and brain functions in attention deficit/hyperactivity disorder.

INTRODUCTION: Attention deficit/hyperactivity disorder (ADHD) is a hereditary neurodevelopmental disorder characterized by working memory (WM) deficits. The MnlI variant (rs3746544) of the synaptosomal-associated protein 25 (SNAP-25) gene is associated with ADHD. In this study, we investigated the role and underlying mechanism of SNAP-25 MnlI variant in cognitive impairment and brain functions in boys with ADHD. METHOD: We performed WM capacity tests using the fourth version of the Wechsler Intelligence Scale for Children (WISC-IV) and regional homogeneity (ReHo) analysis for the resting-state functional magnetic resonance imaging data of 56 boys with ADHD divided into two genotypic groups (TT homozygotes and G-allele carriers). Next, Spearman's rank correlation analysis between the obtained ReHo values and the WM index (WMI) calculated for each participant. RESULTS: Compared with G-allele carrier group, there were higher ReHo values for the left medial prefrontal cortex (mPFC) and higher WM capacity in TT homozygote group. Contrary to TT homozygote group, the WM capacity was negatively correlated with the peak ReHo value for the left mPFC in G-allele carrier group. CONCLUSION: These findings suggest that SNAP-25 MnlI variant may underlie cognitive and brain function impairments in boys with ADHD, thus suggesting its potential as a new target for ADHD treatment.

Investigation of possible associations of the BDNF, SNAP-25 and SYN III genes with the neurocognitive measures: BDNF and SNAP-25 genes might be involved in attention domain, SYN III gene in executive function.

OBJECTIVES: Attention-deficit/hyperactivity disorder (ADHD) is a heterogeneous disorder and Sluggish Cognitive Tempo (SCT) might be a second inattention disorder that might be even affected by different attention pathways. SCT is characterized by daydreaming, mental confusion, staring blankly and hypoactivity. In the present study, we evaluated 5 common variants (rs6265, rs3746544, rs1051312, rs133946 and rs133945) located in 3 candidate genes (BDNF, SNAP25 and SYN III) that are known to take part in synaptic plasticity and neurotransmitter transmission. METHODS: We tested the effects of these variants on neuropsychological findings assessed by a computer-based neuropsychological test battery in children with inattention symptoms (SCT and/or ADHD). RESULTS: BDNF (rs6265), SNAP25 (rs3746544 and rs1051312) and SYN III (rs133946 and rs133945) polymorphisms were associated with variable cognitive measures. BDNF gene (rs6265) polymorphism Met allele carriers and SNAP25 gene (rs3746544) T allele carriers had an association with the attention domain. SNAP25 gene (rs1051312) C allele carriers were only associated with reaction time scores. Cognitive flexibility, which is one of the key components of executive function evaluation and shifting attention test scores were associated with BDNF (rs6265) Met allele and SYN III (rs133946) gene G allele. SYN III (rs133945) gene C allele carriers had an association with verbal memory correct hit scores. CONCLUSIONS: As a conclusion, BDNF, SNAP25 and SYN III genes were associated with specific neurocognitive outcomes in children with inattention symptoms. It is important to note that exploring genotyping effects on neurocognitive functions instead of a heterogeneous psychiatric diagnosis can improve our understanding of psychopathologies.

SNAP25 mutation disrupts metabolic homeostasis, steroid hormone production and central neurobehavior.

OBJECTIVE: SNAP-25 is one of the key proteins involved in formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes that are at the core of hormonal secretion and synaptic transmission. Altered expression or function of SNAP-25 can contribute to the development of neuropsychiatric and metabolic disease. A dominant negative (DN) I67T missense mutation in the b-isoform of SNAP-25 (DN-SNAP25mut) mice leads to abnormal interactions within the SNARE complex and impaired exocytotic vesicle recycling, yet the significance of this mutation to any association between the central nervous system and metabolic homeostasis is unknown. METHODS: Here we explored aspects of metabolism, steroid hormone production and neurobehavior of DN-SNAP25mut mice. RESULTS: DN-SNAP25mut mice displayed enhanced insulin function through increased Akt phosphorylation, alongside increased adrenal and gonadal hormone production. In addition, increased anxiety behavior and beigeing of white adipose tissue with increased energy expenditure were observed in mutants. CONCLUSIONS: Our results show that SNAP25 plays an important role in bridging central neurological systems with peripheral metabolic homeostasis, and provide potential insights between metabolic disease and neuropsychiatric disorders in humans.

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.

Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells.

Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules ("on-granule clusters"). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin's Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE-SNARE interactions.

SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin clusters in a SNARE-domain-dependent manner. Our data suggest that SNARE domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose these mechanisms preserve active syntaxin 1A-SNAP25 complexes at the plasma membrane.

Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release.

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.

miR-128 as a Regulator of Synaptic Properties in 5xFAD Mice Hippocampal Neurons.

Alzheimer's disease (AD) is characterized by progressive synaptic dysfunction, deterioration of neuronal transmission, and consequently neuronal death. Although there is no treatment for AD, exposure to enriched environment (EE) in mice, as well as physical and mental activity in human subjects have been shown to have a protective effect by slowing the disease's progression and reducing AD-like cognitive impairment. However, the molecular mechanism of this mitigating effect is still not understood. One of the mechanisms that has recently been shown to be involved in neuronal degeneration is microRNAs (miRNAs) regulation, which act as a post-transcriptional regulators of gene expression. miR-128 has been shown to be significantly altered in individuals with AD and in mice following exposure to EE. Here, we focused on elucidating the possible role of miR-128 in AD pathology and found that miR-128 regulates the expression of two proteins essential for synaptic transmission, SNAP-25, and synaptotagmin1 (Syt1). Clinically relevant, in 5xFAD mouse model for AD, this miRNA's expression was found as downregulated, resembling the alteration found in the hippocampi of individuals with AD. Interestingly, exposing WT mice to EE also resulted in downregulation of miR-128 expression levels, although EE and AD conditions demonstrate opposing effects on neuronal functioning and synaptic plasticity. We also found that miR-128 expression downregulation in primary hippocampal cultures from 5xFAD mice results in increased neuronal network activity and neuronal excitability. Altogether, our findings place miR-128 as a synaptic player that may contribute to synaptic functioning and plasticity through regulation of synaptic protein expression and function.

miR-128 regulates epilepsy sensitivity in mice by suppressing SNAP-25 and SYT1 expression in the hippocampus.

Epilepsy is accompanied by abnormal neurotransmission, and microRNAs, as versatile players in the modulation of gene expression, are important in epilepsy pathology. Here, we found that miR-128 expression was elevated in the acute seizure phase and decreased during the recurrent seizure phase after status epilepticus in mice. Both SNAP-25 and SYT1 are regulated by miR-128 in vitro and in vivo. Overexpressing miR-128 in cultured neurons decreased neurotransmitter released by suppressing SNAP-25 and SYT1 expression. Anti-miR-128 injection before kainic acid (KA) injection increased the sensitivity of mice to KA-induced seizures, while overexpressing miR-128 at the latent and recurrent phases had a neuroprotective effect in KA-induced seizures. Our study shows for the first time that miR-128, a key regulator of neurotransmission, plays an important role in epilepsy pathology and that miR-128 might be a potential candidate molecular target for epilepsy therapy.

Synaptotagmin-1-, Munc18-1-, and Munc13-1-dependent liposome fusion with a few neuronal SNAREs.

Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca2+ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca2+-dependent liposome fusion between synaptobrevin liposomes and syntaxin-1-SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca2+ binding to the two C2 domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca2+-dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca2+ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons.