A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.

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.

SYT4 binds to SNAP25 to facilitate exosomal secretion and prostate cancer enzalutamide resistance.

Prostate carcinoma represents a predominant malignancy affecting the male population, with androgen deprivation therapy (ADT) serving as a critical therapeutic modality for advanced disease states, but it often leads to the development of resistance. Enzalutamide (Enz), a second-generation antiandrogen drug, initially offers substantial therapeutic benefit, but its efficacy wanes as drug resistance ensues. In this study, we found that synaptotagmin 4 (SYT4) is an upregulated gene in enzalutamide-resistant (EnzR) cell lines. The downregulation of SYT4, in combination with enzalutamide therapy, substantially enhances the antiproliferative effect on resistant prostate cancer cells beyond the capacity of enzalutamide monotherapy. SYT4 promotes vesicle efflux by binding to the synaptosome-associated protein 25 (SNAP25), thereby contributing to cell resistance against enzalutamide. The elevated expression of SYT4 is mediated by bromodomain-containing protein 4 (BRD4), and BRD4 inhibition effectively suppressed the expression of SYT4. Treatment with a therapeutic dose of enzalutamide combined with ASO-1, an antisense oligonucleotide drug targeting SYT4, shows promising results in reversing the resistance of prostate cancer to enzalutamide.

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.

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.

In vitro evaluation of exocytosis-associated SNARE molecules in human granulosa cells in polycystic ovary syndrome.

PURPOSE: Patients with polycystic ovarian morphology (PCOM) make up 20% cases for assisted reproductive technology (ART). Folliculogenesis is impaired in PCOS. Signaling molecules are involved in follicle development. Dysregulations of intrafollicular environment and signaling molecules are observed in PCOS. Granulosa cells (GCs) and oocytes secrete molecules into follicular fluid by exocytosis of SNAREs. The aim of this study is to evaluate vesicle transport and vesicle fusion proteins (SNAREs) in GCs from PCOS patients who have undergone IVF treatment. METHODS: Follicular fluids were collected from patients who undergo IVF/ICSI with the diagnosis of male factor (n = 10) and PCOS (n = 10) patients. GCs were separated and cultured. Each group of GCs was stimulated with FSH-hCG. The cells were examined under electron microscope. Immunofluorescent labeling was performed on cells for Stx6, SNAP25, StxBP1, FSHr, and KITL. Integrated density was analyzed from images of Stx6, SNAP25, StxBP1, FSHr, and KITL. RESULTS: Intercellular communication occurs by signal molecules; Stx6, SNAP25, and StxBP1 fusion proteins involved in exocytosis were decreased in the GCs of PCOS. There was no increase in in vitro stimulation with FSH-hCG either. In the electron microscope, it was observed that exocytosis of the vesicles was disrupted. CONCLUSIONS: Exocytosis and vesicular dynamics are among the basic physiological functions of human steroidogenic granulosa cells. Follicle development is necessary for production of competent oocytes and ovulation. Understanding the pathophysiology of PCOS at follicular level is important for disease management. According to our findings, deficits in vesicular dynamics of human granulosa cells in may be central to the treatment strategy for PCOS patients.

Detection of botulinum neurotoxin A (BoNT/A) enzymatic activity by pregnancy test strips based on hCG-modified magnetic nanoparticles.

The detection of botulinum neurotoxin A (BoNT/A) endopeptidase activity by pregnancy test paper based on human chorionic gonadotropin (hCG)-functionalized peptide-modified magnetic nanoparticles (MNs) is described for the first time. HCG-functionalized SNAP-25 peptide substrate with hydrolysis recognition sites was optimally designed. HCG can be recognized by pregnancy test strips. BoNT/A light chain (BoNT-LcA) is the central part of the endopeptidase function in holotoxin, which can specifically hydrolyze SNAP-25 peptide to release the hCG-peptide probe, and the hCG-peptide probe released can be quantitatively detected by pregnancy test strips, achieving indirect determination of BoNT/A. By quantifying the T-line color intensity of test strips, the visual detection limit for BoNT-LcA is 12.5 pg/mL, and the linear range of detection for BoNT-LcA and BoNT/A holotoxin was 100 pg/mL to 1 ng/mL and 25 to 250 ng/mL. The ability of the method to quantify BoNT/A was validated in human serum samples. This method shows the potential for sensitive detecting BoNT/A and has prospects for the diagnosis and prognosis of clinical botulism.

Tirbanibulin (KX2-391) analog KX2-361 inhibits botulinum neurotoxin serotype A mediated SNAP-25 cleavage in pre- and post-intoxication models in cells.

Botulinum neurotoxins (BoNT) inhibit neuroexocytosis, leading to the potentially lethal disease botulism. BoNT serotype A is responsible for most human botulism cases, and there are no approved therapeutics to treat already intoxicated patients. A growing body of research has demonstrated that BoNT/A can escape into the central nervous system, and therefore, identification of BoNT/A inhibitors that can penetrate BBB and neutralize the toxin within intoxicated neurons would be important. We previously identified an FDA-approved, orally bioavailable compound, KX2-391 (Tirbanibulin) that inhibits BoNT/A in motor neuron assays. Recently, a structural analog of KX2-391, KX2-361, has been shown to exhibit good oral bioavailability and cross BBB with high efficiency in mouse experiments. Therefore, in this work, we evaluated the inhibitory effects of KX2-361 against BoNT/A. Toward this goal, we first evaluated the compound for its effects on cell viability in PC12 cells, via MTT assay, and in mouse embryonic stem cell (mESC)-derived motor neurons, with imaging-based assays. Following, we tested KX2-361 in mESC-derived motor neurons intoxicated with BoNT/A holotoxin, and the compound exhibited activity against the toxin in both pre- and post-intoxication conditions. Excitingly, KX2-361 also inhibited BoNT/A enzymatic component (light chain; LC) in PC12 cells transfected with BoNT/A LC. Furthermore, our molecular docking analyses suggested that KX2-361 can directly bind to BoNT/A LC. Medicinal chemistry approaches to develop structural analogs of KX2-361 to increase its efficacy against BoNT/A may provide a critical lead compound with BBB penetration capacity for drug development efforts against BoNT/A intoxication.

Correlation of Presynaptic and Postsynaptic Proteins with Pathology in Alzheimer's Disease.

Synaptic transmission is essential for nervous system function and the loss of synapses is a known major contributor to dementia. Alzheimer's disease dementia (ADD) is characterized by synaptic loss in the mesial temporal lobe and cerebral neocortex, both of which are brain areas associated with memory and cognition. The association of synaptic loss and ADD was established in the late 1980s, and it has been estimated that 30-50% of neocortical synaptic protein is lost in ADD, but there has not yet been a quantitative profiling of different synaptic proteins in different brain regions in ADD from the same individuals. Very recently, positron emission tomography (PET) imaging of synapses is being developed, accelerating the focus on the role of synaptic loss in ADD and other conditions. In this study, we quantified the densities of two synaptic proteins, the presynaptic protein Synaptosome Associated Protein 25 (SNAP25) and the postsynaptic protein postsynaptic density protein 95 (PSD95) in the human brain, using enzyme-linked immunosorbent assays (ELISA). Protein was extracted from the cingulate gyrus, hippocampus, frontal, primary visual, and entorhinal cortex from cognitively unimpaired controls, subjects with mild cognitive impairment (MCI), and subjects with dementia that have different levels of Alzheimer's pathology. SNAP25 is significantly reduced in ADD when compared to controls in the frontal cortex, visual cortex, and cingulate, while the hippocampus showed a smaller, non-significant reduction, and entorhinal cortex concentrations were not different. In contrast, all brain areas showed lower PSD95 concentrations in ADD when compared to controls without dementia, although in the hippocampus, this failed to reach significance. Interestingly, cognitively unimpaired cases with high levels of AD pathology had higher levels of both synaptic proteins in all brain regions. SNAP25 and PSD95 concentrations significantly correlated with densities of neurofibrillary tangles, amyloid plaques, and Mini Mental State Examination (MMSE) scores. Our results suggest that synaptic transmission is affected by ADD in multiple brain regions. The differences were less marked in the entorhinal cortex and the hippocampus, most likely due to a ceiling effect imposed by the very early development of neurofibrillary tangles in older people in these brain regions.

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation.

Protein S-acylation is a reversible post-translational modification that modulates the localization and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC (Asp-His-His-Cys) domain-containing (zDHHC) proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyze S-acylation in a two-step process involving "autoacylation" of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. Here, we developed and implemented a novel FRET-based high-throughput assay for the discovery of compounds that interfere with autoacylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. Our screen of >350,000 compounds identified two related tetrazole-containing compounds (TTZ-1 and TTZ-2) that inhibited both zDHHC2 autoacylation and substrate S-acylation in cell-free systems. These compounds were also active in human embryonic kidney 293T cells, where they inhibited the S-acylation of two substrates (SNAP25 and PSD95 [postsynaptic density protein 95]) mediated by different zDHHC enzymes, with some apparent isoform selectivity. Furthermore, we confirmed activity of the hit compounds through resynthesis, which provided sufficient quantities of material for further investigations. The assays developed provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating cellular activities of zDHHC enzymes in S-acylation.

Toward Antifragility: Social Defeat Stress Enhances Learning and Memory in Young Mice Via Hippocampal Synaptosome Associated Protein 25.

Social adversity not only causes severe psychological diseases but also may improve people's ability to learn and grow. However, the beneficial effects of social adversity are often ignored. In this study, we investigated whether and how social adversity affects learning and memory in a mouse social defeat stress (SDS) model. A total of 652 mice were placed in experimental groups of six to 23 mice each. SDS enhanced spatial, novelty, and fear memory with increased synaptosome associated protein 25 (SNAP-25) level and dendritic spine density in hippocampal neurons among young but not middle-aged mice. Chemogenetic inhibition of hippocampal CaMK2A+ neurons blocked SDS-induced enhancement of learning or memory. Knockdown of SNAP-25 or blockade of N-methyl-D-aspartate (NMDA) receptor subunit GluN2B in the hippocampus prevented SDS-induced learning memory enhancement in an emotion-independent manner. These findings suggest that social adversity promotes learning and memory ability in youths and provide a neurobiological foundation for biopsychological antifragility.

Neuronal-specific TNFAIP1 ablation attenuates postoperative cognitive dysfunction via targeting SNAP25 for K48-linked ubiquitination.

BACKGROUND: Synaptosomal-associated protein 25 (SNAP25) exerts protective effects against postoperative cognitive dysfunction (POCD) by promoting PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy and repressing caspase-3/gasdermin E (GSDME)-mediated pyroptosis. However, the regulatory mechanisms of SNAP25 protein remain unclear. METHODS: We employed recombinant adeno-associated virus 9 (AAV9)-hSyn to knockdown tumor necrosis factor α-induced protein 1 (TNFAIP1) or SNAP25 and investigate the role of TNFAIP1 in POCD. Cognitive performance, hippocampal injury, mitophagy, and pyroptosis were assessed. Co-immunoprecipitation (co-IP) and ubiquitination assays were conducted to elucidate the mechanisms by which TNFAIP1 stabilizes SNAP25. RESULTS: Our results demonstrated that the ubiquitin ligase TNFAIP1 was upregulated in the hippocampus of mice following isoflurane (Iso) anesthesia and laparotomy. The N-terminal region (residues 1-96) of TNFAIP1 formed a conjugate with SNAP25, leading to lysine (K) 48-linked polyubiquitination of SNAP25 at K69. Silencing TNFAIP1 enhanced SH-SY5Y cell viability and conferred antioxidant, pro-mitophagy, and anti-pyroptosis properties in response to Iso and lipopolysaccharide (LPS) challenges. Conversely, TNFAIP1 overexpression reduced HT22 cell viability, increased reactive oxygen species (ROS) accumulation, impaired PINK1/Parkin-dependent mitophagy, and induced caspase-3/GSDME-dependent pyroptosis by suppressing SNAP25 expression. Neuron-specific knockdown of TNFAIP1 ameliorated POCD, restored mitophagy, and reduced pyroptosis, which was reversed by SNAP25 depletion. CONCLUSIONS: In summary, our findings demonstrated that inhibiting TNFAIP1-mediated degradation of SNAP25 might be a promising therapeutic approach for mitigating postoperative cognitive decline. Video Abstract.

Differential dysregulation of CREB and synaptic genes in transgenic Drosophila melanogaster expressing shaggy (GSK3), TauWT, or Amyloid-beta.

BACKGROUND: Tau, Amyloid-beta (Aß42), and Glycogen synthase kinase 3 (GSK3) contribute to synaptic dysfunction observed in Alzheimer's disease (AD), the most common form of dementia. In the current study, the effect of pan-neuronal expression of TauWT, Aß42, or shaggy (orthologue of GSK3) in Drosophila melanogaster was assessed on the locomotor function, ethanol sensitivity, synaptic genes and CREB expression. The effect of TauWT and Aß42 on the expression of shaggy was also determined. METHODS AND RESULTS: Gene expression analysis was performed using quantitative real-time RT-PCR method. While syt1, SNAP25 and CREB (upstream transcription factor of syt1 and SNAP25) were upregulated in flies expressing TauWT or Aß42, a prominent decline was observed in those genes in shaggy expressing flies. Although all transgenic flies showed climbing disability and higher sensitivity to ethanol, abnormality in these features was significantly more prominent in transgenic flies expressing shaggy compared to TauWT or Aß42. Despite a significant upregulation of shaggy transcription in TauWT expressing flies, Aß42 transgenic flies witnessed no significant changes. CONCLUSIONS: TauWT, Aß42, and shaggy may affect synaptic plasticity through dysregulation of synaptic genes and CREB, independently. However shaggy has more detrimental effect on synaptic genes expression, locomotor ability and sensitivity to ethanol. It is important when it comes to drug discovery. It appears that CREB is a direct effector of changes in synaptic genes expression as they showed similar pattern of alteration and it is likely to be a part of compensatory mechanisms independent of the GSK3/CREB pathway in TauWT or Aß42 expressing flies.

Synaptic retrograde regulation of the PKA-induced SNAP-25 and Synapsin-1 phosphorylation.

BACKGROUND: Bidirectional communication between presynaptic and postsynaptic components contribute to the homeostasis of the synapse. In the neuromuscular synapse, the arrival of the nerve impulse at the presynaptic terminal triggers the molecular mechanisms associated with ACh release, which can be retrogradely regulated by the resulting muscle contraction. This retrograde regulation, however, has been poorly studied. At the neuromuscular junction (NMJ), protein kinase A (PKA) enhances neurotransmitter release, and the phosphorylation of the molecules of the release machinery including synaptosomal associated protein of 25 kDa (SNAP-25) and Synapsin-1 could be involved. METHODS: Accordingly, to study the effect of synaptic retrograde regulation of the PKA subunits and its activity, we stimulated the rat phrenic nerve (1 Hz, 30 min) resulting or not in contraction (abolished by µ-conotoxin GIIIB). Changes in protein levels and phosphorylation were detected by western blotting and cytosol/membrane translocation by subcellular fractionation. Synapsin-1 was localized in the levator auris longus (LAL) muscle by immunohistochemistry. RESULTS: Here we show that synaptic PKA Cß subunit regulated by RIIß or RIIα subunits controls activity-dependent phosphorylation of SNAP-25 and Synapsin-1, respectively. Muscle contraction retrogradely downregulates presynaptic activity-induced pSynapsin-1 S9 while that enhances pSNAP-25 T138. Both actions could coordinately contribute to decreasing the neurotransmitter release at the NMJ. CONCLUSION: This provides a molecular mechanism of the bidirectional communication between nerve terminals and muscle cells to balance the accurate process of ACh release, which could be important to characterize molecules as a therapy for neuromuscular diseases in which neuromuscular crosstalk is impaired.

Structural and mechanistic insights into secretagogin-mediated exocytosis.

Secretagogin (SCGN) is a hexa-EF-hand protein that is highly expressed in the pancreas, brain, and gastrointestinal tract. SCGN is known to modulate regulated exocytosis in multiple cell lines and tissues; however, its exact functions and underlying mechanisms remain unclear. Here, we report that SCGN interacts with the plasma membrane SNARE SNAP-25, but not the assembled SNARE complex, in a Ca2+-dependent manner. The crystal structure of SCGN in complex with a SNAP-25 fragment reveals that SNAP-25 adopts a helical structure and binds to EF-hands 5 and 6 of SCGN. SCGN strongly inhibits SNARE-mediated vesicle fusion in vitro by binding to SNAP-25. SCGN promotes the plasma membrane localization of SNAP-25, but not Syntaxin-1a, in SCGN-expressing cells. Finally, SCGN controls neuronal growth and brain development in zebrafish, likely via interacting with SNAP-25 or its close homolog, SNAP-23. Our results thus provide insights into the regulation of SNAREs and suggest that aberrant synapse functions underlie multiple neurological disorders caused by SCGN deficiency.

Biology activity and characterization of the functional L-HN fragment derivative of botulinum neurotoxin serotype E.

OBJECTIVES: The mature botulinum neurotoxin (BoNT) is a long peptide chain consisting of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). In this study, we further explored the biology activity and characteristics of recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E in vivo and in vitro. METHODS: Neurotoxicity of L-HN fragments from botulinum neurotoxins was assessed in mice. Cleavage of dichain EL-HN in vitro and in neuro-2a cells was assessed and compared with that of single chain EL-HN. Interaction of HN domain and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored in vitro and in neuro-2a cells only expressing SV2C. RESULTS: We found that the 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL-HN-DC) was 0.5 µg and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT (A/B/E/F). The cleavage efficiency of EL-HN-DC toward synaptosome associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL-HN-DC fragment might enter neuro-2a cells via binding to SV2C to efficiently cleave SNAP25. CONCLUSIONS: The EL-HN fragment showed good biological activities in vivo and in vitro, and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport.

Effect of exercise engagement and cardiovascular risk on neuronal injury.

INTRODUCTION: Neuronal health as a potential underlying mechanism of the beneficial effects of exercise has been understudied in humans. Furthermore, there has been limited consideration of potential moderators (e.g., cardiovascular health) on the effects of exercise. METHODS: Clinically normal middle-aged and older adults completed a validated questionnaire about exercise engagement over a 10-year period (n = 75; age 63 ± 8 years). A composite estimate of neuronal injury was formulated that included cerebrospinal fluid-based measures of visinin-like protein-1, neurogranin, synaptosomal-associated protein 25, and neurofilament light chain. Cardiovascular risk was estimated using the Framingham Risk Score. RESULTS: Cross-sectional analyses showed that greater exercise engagement was associated with less neuronal injury in the group with lower cardiovascular risk (p = 0.008), but not the group with higher cardiovascular risk (p = 0.209). DISCUSSION: Cardiovascular risk is an important moderator to consider when examining the effects of exercise on cognitive and neural health, and may be relevant to personalized exercise recommendations. HIGHLIGHTS: We examined the association between exercise engagement and neuronal injury. Vascular risk moderated the association between exercise and neuronal injury. Cardiovascular risk may be relevant to personalized exercise recommendations.

Sex-specific effects of SNAP-25 genotype on verbal memory and Alzheimer's disease biomarkers in clinically normal older adults.

INTRODUCTION: We tested sex-dependent associations of variation in the SNAP-25 gene, which encodes a presynaptic protein involved in hippocampal plasticity and memory, on cognitive and Alzheimer's disease (AD) neuroimaging outcomes in clinically normal adults. METHODS: Participants were genotyped for SNAP-25 rs1051312 (T > C; SNAP-25 expression: C-allele > T/T). In a discovery cohort (N = 311), we tested the sex by SNAP-25 variant interaction on cognition, Aß-PET positivity, and temporal lobe volumes. Cognitive models were replicated in an independent cohort (N = 82). RESULTS: In the discovery cohort, C-allele carriers exhibited better verbal memory and language, lower Aß-PET positivity rates, and larger temporal volumes than T/T homozygotes among females, but not males. Larger temporal volumes related to better verbal memory only in C-carrier females. The female-specific C-allele verbal memory advantage was evidenced in the replication cohort. CONCLUSIONS: In females, genetic variation in SNAP-25 is associated with resistance to amyloid plaque formation and may support verbal memory through fortification of temporal lobe architecture. HIGHLIGHTS: The SNAP-25 rs1051312 (T > C) C-allele results in higher basal SNAP-25 expression. C-allele carriers had better verbal memory in clinically normal women, but not men. Female C-carriers had higher temporal lobe volumes, which predicted verbal memory. Female C-carriers also exhibited the lowest rates of amyloid-beta PET positivity. The SNAP-25 gene may influence female-specific resistance to Alzheimer's disease (AD).

Cerebrospinal fluid biomarkers in the Longitudinal Early-onset Alzheimer's Disease Study.

INTRODUCTION: One goal of the Longitudinal Early Onset Alzheimer's Disease Study (LEADS) is to define the fluid biomarker characteristics of early-onset Alzheimer's disease (EOAD). METHODS: Cerebrospinal fluid (CSF) concentrations of Aß1-40, Aß1-42, total tau (tTau), pTau181, VILIP-1, SNAP-25, neurogranin (Ng), neurofilament light chain (NfL), and YKL-40 were measured by immunoassay in 165 LEADS participants. The associations of biomarker concentrations with diagnostic group and standard cognitive tests were evaluated. RESULTS: Biomarkers were correlated with one another. Levels of CSF Aß42/40, pTau181, tTau, SNAP-25, and Ng in EOAD differed significantly from cognitively normal and early-onset non-AD dementia; NfL, YKL-40, and VILIP-1 did not. Across groups, all biomarkers except SNAP-25 were correlated with cognition. Within the EOAD group, Aß42/40, NfL, Ng, and SNAP-25 were correlated with at least one cognitive measure. DISCUSSION: This study provides a comprehensive analysis of CSF biomarkers in sporadic EOAD that can inform EOAD clinical trial design.

STXBP6 Gene Mutation: A New Form of SNAREopathy Leads to Developmental Epileptic Encephalopathy.

Syntaxin-binding protein 6 (STXBP6), also known as amysin, is an essential component of the SNAP receptor (SNARE) complex and plays a crucial role in neuronal vesicle trafficking. Mutations in genes encoding SNARE proteins are often associated with a broad spectrum of neurological conditions defined as "SNAREopathies", including epilepsy, intellectual disability, and neurodevelopmental disorders such as autism spectrum disorders. The present whole exome sequencing (WES) study describes, for the first time, the occurrence of developmental epileptic encephalopathy and autism spectrum disorders as a result of a de novo deletion within the STXBP6 gene. The truncated protein in the STXBP6 gene leading to a premature stop codon could negatively modulate the synaptic vesicles' exocytosis. Our research aimed to elucidate a plausible, robust correlation between STXBP6 gene deletion and the manifestation of developmental epileptic encephalopathy.