Omega-3 polyunsaturated fatty acids promote SNAREs mediated GLUT4 vesicle docking and fusion.

Glucose homeostasis imbalance and insulin resistance (IR) are major contributors to the incidence of type 2 diabetes. Omega-3 polyunsaturated fatty acids (PUFAs) are key ingredients for maintaining cellular functions and improving insulin sensitivity. However, how omega-3 PUFAs modulate the dynamic process of glucose transport at the cellular level remains unclear. Here we unraveled eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may regulate the glucose transporter 4 (GLUT4) vesicle trafficking in both normal and IR adipocytes. Both omega-3 PUFAs significantly increase glucose consumption within a range of 10-32% in the basal state. Furthermore, both EPA (200 µM) and DHA (100 µM) may significantly promote the serine/threonine protein kinase (Akt) phosphorylation by 70% and 40% in the physiological state of adipocytes, respectively. Both omega-3 PUFAs significantly advanced the Akt phosphorylation in a dose-dependent way and showed a ∼2-fold increase at the dose of 200 µM in the IR pathological state. However, they could not up-regulate the expression of GLUT4 and insulin-regulated aminopeptidase protein. We further revealed that both omega-3 PUFAs dynamically promote insulin-stimulated GLUT4 vesicle translocation and soluble N-ethylmaleimide-sensitive factor attachment protein receptor mediated vesicle docking and fusion to the plasma membrane via specifically modulating the expression of vesicle-associated membrane protein 2. Understanding the mechanisms by which omega-3 PUFAs modulate cellular metabolism and IR in peripheral tissues may provide novel insights into the potential impact of omega-3 PUFAs on the metabolic function and the management of IR.

Syntaxin of plants31 (SYP31) and SYP32 is essential for Golgi morphology maintenance and pollen development.

Pollen development is a key process for the sexual reproduction of angiosperms. The Golgi plays a critical role in pollen development via the synthesis and transport of cell wall materials. However, little is known about the molecular mechanisms underlying the maintenance of Golgi integrity in plants. In Arabidopsis thaliana, syntaxin of plants (SYP) 3 family proteins SYP31 and SYP32 are the only two Golgi-localized Qa-soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) with unknown endogenous functions. Here, we demonstrate the roles of SYP31 and SYP32 in modulating Golgi morphology and pollen development. Two independent lines of syp31/+ syp32/+ double mutants were male gametophytic lethal; the zero transmission rate of syp31 syp32 mutations was restored to largely normal levels by pSYP32:SYP32 but not pSYP32:SYP31 transgenes, indicating their functional differences in pollen development. The initial arrest of syp31 syp32 pollen occurred during the transition from the microspore to the bicellular stage, where cell plate formation in pollen mitosis I (PMI) and deposition of intine were abnormal. In syp31 syp32 pollen, the number and length of Golgi cisterna were significantly reduced, accompanied by many surrounding vesicles, which could be largely attributed to defects in anterograde and retrograde trafficking routes. SYP31 and SYP32 directly interacted with COG3, a subunit of the conserved oligomeric Golgi (COG) complex and were responsible for its Golgi localization, providing an underlying mechanism for SYP31/32 function in intra-Golgi trafficking. We propose that SYP31 and SYP32 play partially redundant roles in pollen development by modulating protein trafficking and Golgi structure.

Calcium-triggered fusion of lipid membranes is enabled by amphiphilic nanoparticles.

Lipid membrane fusion is an essential process for a number of critical biological functions. The overall process is thermodynamically favorable but faces multiple kinetic barriers along the way. Inspired by nature's engineered proteins such as SNAP receptor [soluble N-ethylmale-imide-sensitive factor-attachment protein receptor (SNARE)] complexes or viral fusogenic proteins that actively promote the development of membrane proximity, nucleation of a stalk, and triggered expansion of the fusion pore, here we introduce a synthetic fusogen that can modulate membrane fusion and equivalently prime lipid membranes for calcium-triggered fusion. Our fusogen consists of a gold nanoparticle functionalized with an amphiphilic monolayer of alkanethiol ligands that had previously been shown to fuse with lipid bilayers. While previous efforts to develop synthetic fusogens have only replicated the initial steps of the fusion cascade, we use molecular simulations and complementary experimental techniques to demonstrate that these nanoparticles can induce the formation of a lipid stalk and also drive its expansion into a fusion pore upon the addition of excess calcium. These results have important implications in general understanding of stimuli-triggered fusion and the development of synthetic fusogens for biomedical applications.

Overexpressed Tomosyn Binds Syntaxins and Blocks Secretion during Pollen Development.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex formation is necessary for intracellular membrane fusion and thus has a key role in processes such as secretion. However, little is known about the regulatory factors that bind to Qa-SNAREs, which are also known as syntaxins (SYPs) in plants. Here, we characterized Arabidopsis (Arabidopsis thaliana) Tomosyn protein (AtTMS) and demonstrated that it is a conserved regulator of SYPs in plants. AtTMS binds strongly via its R-SNARE motif-containing C terminus to the Qa domain of PM-resident, pollen-expressed SYP1s (SYP111, SYP124, SYP125, SYP131, and SYP132), which were narrowed down from 12 SYPs. AtTMS is highly expressed in pollen from the bicellular stage onwards, and overexpression of AtTMS under the control of the UBIQUITIN10, MSP1, or LAT52 promoter all resulted in defective pollen after the microspore stage in which secretion was inhibited, leading to the failure of intine deposition and cell plate formation during pollen mitosis I. In tobacco (Nicotiana benthamiana) leaf epidermal cells, overexpression of AtTMS inhibited the secretion of secreted GFP. The defects were rescued by mCherry-tagged SYP124, SYP125, SYP131, or SYP132. In vivo, SYP132 partially rescued the pMSP1:AtTMS phenotype. In addition, AtTMS, lacking a transmembrane domain, was recruited to the plasma membrane by SYP124, SYP125, SYP131, and SYP132 and competed with Vesicle-Associated Membrane Protein721/722 for binding to, for example, SYP132. Together, our results demonstrated that AtTMS might serve as a negative regulator of secretion, whereby active secretion might be fine-tuned during pollen development.

Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin.

Botulinum neurotoxins (BoNTs), the most poisonous proteins known to humankind, are a family of seven (serotype A to G) immunologically distinct proteins synthesized primarily by different strains of the anaerobic bacterium Clostridium botulinum Being the causative agents of botulism, the toxins block neurotransmitter release by specifically cleaving one of the three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, thereby inducing flaccid paralysis. The development of countermeasures and therapeutics against BoNTs is a high-priority research area for public health because of their extreme toxicity and potential for use as biowarfare agents. Extensive research has focused on designing antagonists that block the catalytic activity of BoNTs. In this study, we screened 300 small natural compounds and their analogues extracted from Indian plants for their activity against BoNT serotype A (BoNT/A) as well as its light chain (LCA) using biochemical and cellular assays. One natural compound, a nitrophenyl psoralen (NPP), was identified to be a specific inhibitor of LCA with an in vitro 50% inhibitory concentration (IC50) value of 4.74 ± 0.03 µM. NPP was able to rescue endogenous synaptosome-associated protein 25 (SNAP-25) from cleavage by BoNT/A in human neuroblastoma cells with an IC50 of 12.2 ± 1.7 µM, as well as to prolong the time to the blocking of neutrally elicited twitch tensions in isolated mouse phrenic nerve-hemidiaphragm preparations.IMPORTANCE The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P) value and other favorable characteristics (P. Leeson, Nature 481:455-456, 2012, https://doi.org/10.1038/481455a). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602-2607, 2007, https://doi.org/10.1073/pnas.0611213104). The difference between the in vitro and cellular efficacy presumably results from difficulties experienced by the compounds in crossing the cell membrane, in conjunction with poor bioavailability and high cytotoxicity. The screened nitrophenyl psoralen (NPP) effectively antagonized BoNT/A in both in vitro and ex vivo assays. Importantly, NPP inhibited the BoNT/A light chain but not other general zinc endopeptidases, such as thermolysin, suggesting high selectivity for its target. Small-molecule (nonpeptidic) inhibitors have better oral bioavailability, better stability, and better tissue and cell permeation than antitoxins or peptide inhibitors.

Chronic inflammatory pain decreases the glutamate vesicles in presynaptic terminals of the nucleus accumbens.

Reward system has been proved to be important to nociceptive behavior, and the nucleus accumbens (NAc) is a key node in reward circuitry. It has been further revealed that dopamine system modulates the NAc to influence the pain sensation, whereas the role of glutamatergic projection in the NAc in the modulation of chronic pain is still elusive. In this study, we used a complete Freund's adjuvant-induced chronic inflammatory pain model to explore the changes of the glutamatergic terminals in the NAc, and we found that following the chronic inflammation, the protein level of vesicular glutamate transporter1 (VGLUT1) was significantly decreased in the NAc. Immunofluorescence staining further showed a reduced expression of VGLUT1-positive terminals in the dopamine receptor 2 (D2R) spiny projection neurons of NAc after chronic inflammatory pain. Furthermore, using a whole-cell recording in double transgenic mice, in which dopamine receptor 1- and D2R-expressing neurons can be visualized, we found that the frequency of spontaneous excitatory postsynaptic currents was significantly decreased and paired-pulse ratio of evoked excitatory postsynaptic currents was increased in D2R neurons, but not in dopamine receptor 1 neurons in NAc of complete Freund's adjuvant group. Moreover, the abnormal expression of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex contributed to the reduced formation of glutamate vesicles. Hence, our results demonstrated that decreased glutamate release in the indirect pathway of the NAc may be a critical mechanism for chronic pain and provided a novel evidence for the presynaptic mechanisms in chronic pain regulation.

Astrocytic signaling supports hippocampal-prefrontal theta synchronization and cognitive function.

Astrocytes interact with neurons at the cellular level through modulation of synaptic formation, maturation, and function, but the impact of such interaction into behavior remains unclear. Here, we studied the dominant negative SNARE (dnSNARE) mouse model to dissect the role of astrocyte-derived signaling in corticolimbic circuits, with implications for cognitive processing. We found that the blockade of gliotransmitter release in astrocytes triggers a critical desynchronization of neural theta oscillations between dorsal hippocampus and prefrontal cortex. Moreover, we found a strong cognitive impairment in tasks depending on this network. Importantly, the supplementation with d-serine completely restores hippocampal-prefrontal theta synchronization and rescues the spatial memory and long-term memory of dnSNARE mice. We provide here novel evidence of long distance network modulation by astrocytes, with direct implications to cognitive function.

Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins.

Soluble N-ethylmaleimide-sensitive Factor Attachment Receptor (SNARE) Protein Involved in the Remission of Depression by Acupuncture in Rats.

This study aims to investigate the molecular mechanisms of acupuncture in the remission of depression. A depressive disorder model was induced by exposing Sprague-Dawley rats to chronic unpredictable stress. The rats were divided into five groups: healthy (blank group) and stressed rats (model group), and stressed rats treated with acupuncture (acupuncture group), riluzole (riluzole group), acupuncture combined with botulinum toxin A (BTX-A) injection (acupuncture+BTX-A group) or riluzole combined with BTX-A injection (riluzole+BTX-A group). Behavioral analysis showed significant differences in sucrose consumption, weight, and horizontal or vertical movements between the model and both the riluzole and acupuncture groups. No obvious differences between the riluzole+BTX-A and acupuncture+BTX-A groups were found. Moreover, no significance differences in glutamate content in the hippocampus were found among the riluzole+BTX-A, acupuncture+BTX-A and model groups (p>0.05). Western blots and reverse transcription polymerase chain reactions were employed to detect protein and mRNA expressions of VGLUT2, SNAP25, VAMP1, VAMP2, VAMP7, and syntaxin1; no obvious differences among the riluzole+BTX-A, acupuncture+BTX-A and model groups were found. These data suggest that soluble N-ethylmaleimide-sensitive factor attachment receptor proteins are involved in the remission of depression in rats treated with acupuncture.

Role of the transmembrane domain in SNARE protein mediated membrane fusion: peptide nucleic acid/peptide model systems.

Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented.

Phosphatase Inhibitors Function as Novel, Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays.

There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

Microdomains of SNARE proteins in the plasma membrane.

Exocytosis is catalyzed by the engagement of SNARE proteins embedded in the plasma membrane with complementary SNAREs in the membrane of trafficking vesicles undergoing exocytosis. In most cells studied so far, SNAREs are not randomly distributed across the plasma membrane but are clustered and segregated in discrete membrane domains of defined size, composition, and stability. SNARE clusters have been intensively studied for more than a decade. Different mechanisms have been proposed to be responsible for SNARE clustering such as partitioning into cholesterol-enriched lipid rafts, hydrophobic mismatch, posttranslational modifications of the SNAREs including phosphorylation and palmitoylation, electrostatic protein-protein and protein-lipid interactions, homotypic and heterotypic protein interactions, and anchoring to the cortical cytoskeleton. Although several of these proposed mechanisms are still controversially discussed, it is becoming apparent that independent physicochemical principles must cooperate in a synergistic manner to yield SNARE microdomains. Here, we discuss the architecture and function of SNARE domains. We also discuss the various factors influencing SNARE clustering, resulting in a model that we believe may be of general use to explain domain formation of proteins in the plasma membrane.

Vitamin E prevents hyperoxia-induced loss of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins in the rat neuronal cytoplasm.

This study examines the ability of vitamin E to inhibit hyperoxia-induced loss of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins in the neuronal cytoplasm. Here, the effects of vitamin E on hyperoxia-induced changes in the expressions of N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment protein α (α-SNAP) in the rat brain were analyzed. When rats were subjected to hyperoxia, the expression of both SNARE proteins was markedly decreased compared to normal rats. Vitamin E significantly inhibited the decrease in the expression of NSF in rats subjected to hyperoxia. Rats showed the tendency to improve the loss of α-SNAP by vitamin E-supplementation, although it was not statistically significant. On the other hand, vitamin E deficient rats showed marked loss of these proteins in the brain in the absence of oxidative stress. These results suggest that hyperoxia induces a loss of SNARE proteins, which are involved in membrane docking between synaptic vesicles and pre-synaptic membranes, and that vitamin E prevents the oxidative damage of SNARE proteins. Consequently, it is implied that vitamin E inhibits impaired neurotransmission caused by oxidative stress through the prevention of oxidative damage to SNARE proteins by probably its antioxidant effect.

Polyphenols differentially inhibit degranulation of distinct subsets of vesicles in mast cells by specific interaction with granule-type-dependent SNARE complexes.

Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. In the present study, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE genes and a lipid mixing assay of 24 possible combinations of reconstituted SNAREs indicated that the only two active SNARE complexes involved in mast cell degranulation are Syn (syntaxin) 4/SNAP (23 kDa synaptosome-associated protein)-23/VAMP (vesicle-associated membrane protein) 2 and Syn4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these two SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of three distinct subsets of granules. These results suggest the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking.

A botulinum neurotoxin-like function of Potentilla chinensis extract that inhibits neuronal SNARE complex formation, membrane fusion, neuroexocytosis, and muscle contraction.

CONTEXT: Botulinum neurotoxins (BoNTs) are popularly used to treat various diseases and for cosmetic purposes. They act by blocking neurotransmission through specific cleavage of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Recently, several polyphenols were shown to interfere with SNARE complex formation by wedging into the hydrophobic core interface, thereby leading to reduced neuroexocytosis. OBJECTIVE: In order to find industrially-viable plant extract that functions like BoNT, 71 methanol extracts of flowers were screened and BoNT-like activity of selected extract was evaluated. MATERIALS AND METHODS: After evaluating the inhibitory effect of 71 flower methanol extracts on SNARE complex formation, seven candidates were selected and they were subjected to SNARE-driven membrane fusion assay. Neurotransmitter release from neuronal PC12 cells and SNARE complex formation inside the cell was also evaluated. Finally, the effect of one selected extract on muscle contraction and digit abduction score was determined. RESULTS: The extract of Potentilla chinensis Ser. (Rosaceae)(Chinese cinquefoil) flower inhibited neurotransmitter release from neuronal PC12 cells by approximately 90% at a concentration of 10 µg/mL. The extract inhibited neuroexocytosis by interfering with SNARE complex formation inside cells. It reduced muscle contraction of phrenic nerve-hemidiaphragm by approximately 70% in 60 min, which is comparable to the action of the Ca²âº-channel blocker verapamil and BoNT type A. DISCUSSION AND CONCLUSION: While BoNT blocks neuroexocytosis by cleaving SNARE proteins, the Potentilla chinensis extract exhibited the same activity by inhibiting SNARE complex formation. The extract paralyzed muscle as efficiently as BoNT, suggesting the potential versatility in cosmetics and therapeutics.

Vitamin E inhibits oxidative stress-induced denaturation of nerve terminal proteins involved in neurotransmission.

One characteristic of age-related neurodegeneration is thought to be cognitive deficits caused by oxidative stress. Neurons in the brain are considered to be particularly vulnerable to oxidative stress, leading to neuronal oxidative damage and neurodegenerative disorders such as Alzheimer's disease (AD) and senile dementia. The process of fusing synaptic plasma membranes and synaptic vesicles involves particular proteins, such as the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins for docking both membranes, and is integral to neurotransmission. To elucidate whether oxidative stress induces denaturation of SNARE proteins, and whether vitamin E can counteract this process, changes in the expression of synaptobrevin, synaptotagmin, SNAP-25, and syntaxin-1 in rat brain nerve terminals were analyzed using an immunoblotting method. The results showed that oxidative stress induced significant reductions in the levels synaptobrevin and synaptotagmin in synaptic vesicles. Similarly, marked decreases in the levels of SNAP-25 and syntaxin-1 in pre-synaptic plasma membranes were also observed. In the absence of oxidative stress, vitamin E-deficient rats exhibited similar decreases in these proteins. In contrast, it was found that decreases in SNARE proteins, except for SNAP-25, were not observed in vitamin E-supplemented rats, even when the rats were subjected to oxidative stress. These results suggest that reactive oxygen species generated by oxidative stress are detrimental to neurons, resulting in the oxidation of SNARE proteins, thereby disrupting neurotransmission. Additionally, vitamin E is capable of protecting against such neurodegeneration.

SNARE-wedging polyphenols as small molecular botox.

Most cosmetic and therapeutic applications of Clostridium botulinum neurotoxin (BoNT) are related to muscle paralysis caused by the blocking of neurotransmitter release at the neuromuscular junction. BoNT specifically cleaves SNARE proteins at the nerve terminal and impairs neuroexocytosis. Recently, we have shown that several polyphenols inhibit neurotransmitter release from neuronal PC12 cells by interfering with SNARE complex formation. Based on our previous result, we report here that myricetin, delphinidin, and cyanidin indeed paralyze muscle by inhibiting acetylcholine release at the neuromuscular junction. While the effect of myricetin on muscle paralysis was modest compared to BoNT/A, myricetin exhibited a shorter response time than BoNT/A. Intraperitoneally-injected myricetin at an extreme dose of 1000 mg/kg did not induce death of mice, alleviating the safety issue. Thus, these polyphenols might be useful in treating various human hypersecretion diseases for which BoNT/A has been the only option of choice.

Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants.

BACKGROUND: Behavioral stress is recognized as a main risk factor for neuropsychiatric diseases. Converging evidence suggested that acute stress is associated with increase of excitatory transmission in certain forebrain areas. Aim of this work was to investigate the mechanism whereby acute stress increases glutamate release, and if therapeutic drugs prevent the effect of stress on glutamate release. METHODOLOGY/FINDINGS: Rats were chronically treated with vehicle or drugs employed for therapy of mood/anxiety disorders (fluoxetine, desipramine, venlafaxine, agomelatine) and then subjected to unpredictable footshock stress. Acute stress induced marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex in superfusion, and the chronic drug treatments prevented the increase of glutamate release. Stress induced rapid increase in the circulating levels of corticosterone in all rats (both vehicle- and drug-treated), and glutamate release increase was blocked by previous administration of selective antagonist of glucocorticoid receptor (RU 486). On the molecular level, stress induced accumulation of presynaptic SNARE complexes in synaptic membranes (both in vehicle- and drug-treated rats). Patch-clamp recordings of pyramidal neurons in the prefrontal cortex revealed that stress increased glutamatergic transmission through both pre- and postsynaptic mechanisms, and that antidepressants may normalize it by reducing release probability. CONCLUSIONS/SIGNIFICANCE: Acute footshock stress up-regulated depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex. Stress-induced increase of glutamate release was dependent on stimulation of glucocorticoid receptor by corticosterone. Because all drugs employed did not block either elevation of corticosterone or accumulation of SNARE complexes, the dampening action of the drugs on glutamate release must be downstream of these processes. This novel effect of antidepressants on the response to stress, shown here for the first time, could be related to the therapeutic action of these drugs.

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.

Membrane fusion: grappling with SNARE and SM proteins.

The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion. Vesicular and target membrane-localized SNARE proteins zipper up into an alpha-helical bundle that pulls the two membranes tightly together to exert the force required for fusion. SM proteins, shaped like clasps, bind to trans-SNARE complexes to direct their fusogenic action. Individual fusion reactions are executed by distinct combinations of SNARE and SM proteins to ensure specificity, and are controlled by regulators that embed the SM-SNARE fusion machinery into a physiological context. This regulation is spectacularly apparent in the exquisite speed and precision of synaptic exocytosis, where synaptotagmin (the calcium-ion sensor for fusion) cooperates with complexin (the clamp activator) to control the precisely timed release of neurotransmitters that initiates synaptic transmission and underlies brain function.