ABSTRACT
INTRODUCTION: Synaptosomal associated proteins of 25 kDa (SNAP-25), as a member of stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, is critical for membrane fusion and required for the release of neurotransmitters. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is implicated in pathologic pain. This study aimed to investigate whether and how SNAP-25 regulated AMPA receptors in neuropathic pain. METHODS: Male Sprague-Dawley rats underwent L4 spinal nerve ligation (SNL) or the sham procedure. After assessing mechanical allodynia and thermal sensitivity, the ipsilateral portion of the L4-5 spinal cord was harvested. The expression level of SNAP-25 was analyzed by Western blot analysis and real-time quantitative polymerase chain reaction. SNAP-25 phosphorylation and AMPA receptor membrane trafficking levels were evaluated with Western blot analysis. An association between SNAP-25 and AMPA membrane trafficking was confirmed by SNAP-25 expression or phosphorylation inhibition. RESULTS: The SNL procedure induced and maintained mechanical allodynia and thermal hyperalgesia. SNL increased the expression and phosphorylation of SNAP-25 and the membrane trafficking of AMPA receptors in the spinal cord. SNAP-25 expression or phosphorylation inhibition alleviated neuropathic pain and downregulated membrane trafficking of AMPA receptors after SNL. GluA1-containing AMPA receptor inhibition relieved mechanical allodynia and thermal hyperalgesia after SNL. CONCLUSIONS: The upregulation of SNAP-25-dependent membrane trafficking of AMPA receptors via SNAP-25 phosphorylation at Ser187 contributed to SNL-induced neuropathic pain. Thus, the inhibition of SNAP-25 expression or phosphorylation might serve as a treatment for neuropathic pain. However, the mechanism of GluA1-containing AMPA receptor membrane trafficking mediated by SNAP-25 phosphorylation in neuropathic pain deserves further exploration.
Subject(s)
Neuralgia/metabolism , Receptors, AMPA/metabolism , Spinal Injuries/metabolism , Spinal Injuries/physiopathology , Synaptosomal-Associated Protein 25/physiology , Animals , Male , Neuralgia/prevention & control , Pain Measurement , Phosphorylation/drug effects , RNA, Small Interfering/pharmacology , Rats , Receptors, AMPA/antagonists & inhibitors , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/biosynthesisABSTRACT
Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE-Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE-Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE-Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.
Subject(s)
Apoptosis , Endosomes/metabolism , Mitochondria/metabolism , Angiostatins/genetics , Angiostatins/metabolism , Apoptosis/drug effects , Cell Membrane/metabolism , Endocytosis , Endoplasmic Reticulum Chaperone BiP , Fibronectins/pharmacology , Fluorescence Recovery After Photobleaching , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Microscopy, Fluorescence , Neovascularization, Physiologic/drug effects , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Transport/drug effects , RNA Interference , RNA, Small Interfering/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolism , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/genetics , Synaptosomal-Associated Protein 25/metabolism , Thrombospondins/genetics , Thrombospondins/metabolismABSTRACT
Somatodendritically released peptides alter synaptic function through a variety of mechanisms, including autocrine actions that liberate retrograde transmitters. Cholecystokinin (CCK) is a neuropeptide expressed in neurons in the dorsomedial hypothalamic nucleus (DMH), a region implicated in satiety and stress. There are clear demonstrations that exogenous CCK modulates food intake and neuropeptide expression in the DMH, but there is no information on how endogenous CCK alters synaptic properties. Here, we provide the first report of somatodendritic release of CCK in the brain in male Sprague Dawley rats. CCK is released from DMH neurons in response to repeated postsynaptic depolarizations, and acts in an autocrine fashion on CCK2 receptors to enhance postsynaptic NMDA receptor function and liberate the retrograde transmitter, nitric oxide (NO). NO subsequently acts presynaptically to enhance GABA release through a soluble guanylate cyclase-mediated pathway. These data provide the first demonstration of synaptic actions of somatodendritically released CCK in the hypothalamus and reveal a new form of retrograde plasticity, depolarization-induced potentiation of inhibition. Significance statement: Somatodendritic signaling using endocannabinoids or nitric oxide to alter the efficacy of afferent transmission is well established. Despite early convincing evidence for somatodendritic release of neurohypophysial peptides in the hypothalamus, there is only limited evidence for this mode of release for other peptides. Here, we provide the first evidence for somatodendritic release of the satiety peptide cholecystokinin (CCK) in the brain. We also reveal a new form of synaptic plasticity in which postsynaptic depolarization results in enhancement of inhibition through the somatodendritic release of CCK.
Subject(s)
Cholecystokinin/metabolism , Dorsomedial Hypothalamic Nucleus/cytology , Inhibitory Postsynaptic Potentials/drug effects , Neuronal Plasticity/drug effects , Neurons/drug effects , gamma-Aminobutyric Acid/metabolism , Animals , Animals, Newborn , Cholecystokinin/pharmacology , GABA Agents/pharmacology , Guanosine Diphosphate/analogs & derivatives , Guanosine Diphosphate/pharmacology , In Vitro Techniques , Male , Patch-Clamp Techniques , Peptides/pharmacology , Proglumide/analogs & derivatives , Proglumide/pharmacology , Quinazolinones/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, Cholecystokinin B/antagonists & inhibitors , Receptor, Cholecystokinin B/metabolism , Signal Transduction/drug effects , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/metabolism , Thionucleotides/pharmacology , gamma-Aminobutyric Acid/pharmacologyABSTRACT
The goal of this study was to develop and characterize a novel intravaginal film platform for targeted delivery of small interfering RNA (siRNA)-loaded nanoparticles (NP) to dendritic cells as a potential gene therapy for the prevention of sexually transmitted human immunodeficiency virus (HIV) infection. Poly(ethylene glycol) (PEG)-functionalized poly(D, L-lactic-co-glycolic acid) (PLGA)/polyethylenimine (PEI)/siRNA NP (siRNA-NP) were fabricated using a modified emulsion-solvent evaporation method and characterized for particle size, zeta potential, encapsulation efficiency (EE), and siRNA release. siRNA-NP were decorated with anti-HLA-DR antibody (siRNA-NP-Ab) for targeting delivery to HLA-DR+ dendritic cells (DCs) and homogeneously dispersed in a biodegradable film consisting of poly vinyl alcohol (PVA) and λ-carrageenan. The siRNA-NP-Ab-loaded film (siRNA-NP-Ab-film) was transparent, displayed suitable physicomechanical properties, and was noncytotoxic. Targeting activity was evaluated in a mucosal coculture model consisting of a vaginal epithelial monolayer (VK2/E6E7 cells) and differentiated KG-1 cells (HLA-DR+ DCs). siRNA-NP-Ab were rapidly released from the film and were able to penetrate the epithelial layer to be taken up by differentiated KG-1 cells. siRNA-NP-Ab demonstrated higher targeting activity and significantly higher knockdown of synaptosome-associated 23-kDa protein (SNAP-23) mRNA and protein when compared to siRNA-NP without antibody conjugation. Overall, these data suggest that our novel siRNA-NP-Ab-film may be a promising platform for preventing HIV infection within the female genital tract.
Subject(s)
Dendritic Cells/immunology , Leukemia, Myeloid, Acute/immunology , Nanoparticles/administration & dosage , RNA, Small Interfering/genetics , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Vagina/immunology , Carrageenan/chemistry , Cell Differentiation , Cell Survival , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/metabolism , Female , Genetic Therapy , HIV Infections/prevention & control , Humans , Lactic Acid/chemistry , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Nanoparticles/chemistry , Particle Size , Polyethylene Glycols/chemistry , Polyethyleneimine/chemistry , Polyglycolic Acid/chemistry , Synaptosomal-Associated Protein 25/genetics , Vagina/cytology , Vagina/metabolismABSTRACT
Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A's location, distribution, and movement within a cell. Due to BoNT/A's high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25197) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25197, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25197 rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25197 in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25197-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.
Subject(s)
Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Botulinum Toxins, Type A/immunology , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/pharmacology , Blotting, Western , Botulinum Toxins, Type A/metabolism , Botulinum Toxins, Type A/pharmacology , Cells, Cultured , Humans , Immunohistochemistry , Male , Mice , Microscopy, Confocal , Neurons/drug effects , Neurons/immunology , Neurons/metabolism , Peptide Fragments/immunology , Protein Transport , Rats, Sprague-Dawley , Recombinant Proteins , Skin/drug effects , Skin/immunology , Skin/metabolism , Substrate Specificity , Urinary Bladder/drug effects , Urinary Bladder/immunology , Urinary Bladder/metabolismABSTRACT
SNAP-25 is a key component of the synaptic-vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP-25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP-25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP-25 levels at 13-14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage-gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13-14 DIV neurons with reduced SNAP-25 expression show paired-pulse depression as opposed to paired-pulse facilitation occurring in their wild-type counterparts. This phenotype disappears with synapse maturation. As alterations in short-term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP-25.
Subject(s)
Glutamic Acid/metabolism , Neuronal Plasticity/physiology , Neurons/physiology , Synaptic Transmission/physiology , Synaptosomal-Associated Protein 25/metabolism , Action Potentials/drug effects , Action Potentials/physiology , Animals , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Gene Expression Regulation, Developmental , Gene Silencing , Glutamic Acid/pharmacology , Hippocampus/cytology , Hippocampus/drug effects , Humans , Mice , Neuronal Plasticity/drug effects , Neurons/cytology , Neurons/drug effects , Primary Cell Culture , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Synaptic Transmission/drug effects , Synaptic Vesicles/drug effects , Synaptic Vesicles/physiology , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/genetics , gamma-Aminobutyric Acid/metabolism , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Rapid endocytosis, which takes only a few seconds, is widely observed in secretory cells. Although it is more efficient in recycling vesicles than in slow clathrin-mediated endocytosis, its underlying mechanism, thought to be clathrin independent, is largely unclear. Here, we report that cleavage of three SNARE proteins essential for exocytosis, including synaptobrevin, SNAP-25, and syntaxin, inhibited rapid endocytosis at the calyx of Held nerve terminal, suggesting the involvement of the three SNARE proteins in rapid endocytosis. These SNARE proteins were also involved in slow endocytosis. In addition, SNAP-25 and syntaxin facilitated vesicle mobilization to the readily releasable pool, most likely via their roles in endocytosis and/or exocytosis. We conclude that both rapid and slow endocytosis share the involvement of SNARE proteins. The dual role of three SNARE proteins in exo- and endocytosis suggests that SNARE proteins may be molecular substrates contributing to the exocytosis-endocytosis coupling, which maintains exocytosis in secretory cells.
Subject(s)
Endocytosis/physiology , Qa-SNARE Proteins/metabolism , R-SNARE Proteins/metabolism , Synapses/metabolism , Synaptosomal-Associated Protein 25/metabolism , Animals , Botulinum Toxins/pharmacology , Endocytosis/drug effects , Exocytosis/drug effects , Female , Male , Patch-Clamp Techniques , Peptides/pharmacology , Qa-SNARE Proteins/antagonists & inhibitors , R-SNARE Proteins/chemistry , Rats , Rats, Wistar , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Tetanus Toxin/pharmacologyABSTRACT
BACKGROUND AND PURPOSE: Injectable botulinum neurotoxin (BoNT) is the principal effective treatment for blepharospasm (BSP). This trial explores the safety and efficacy of topical acetyl hexapeptide-8 (AH8), a competitive SNAP25 inhibitor, as a potential new therapy in BSP. METHODS: Double-blind, placebo-controlled, randomized trial of daily topical application of AH8 in 24 patients with BSP. The primary outcome was time to return to baseline Jankovic Blepharospasm Rating Scale (JBRS) after a BoNT injection simultaneously with the initiation of AH8. Patients displaying a strictly regular pattern of response to 3-monthly injections of BoNT were included. RESULTS: There were no significant adverse events. There was a trend for longer time until return to baseline JBRS after injection in the active group compared to placebo (3.7 months vs. 3.0 months), and for better scores in the active group. One-third (4/12) of the patients in the active group had a considerable extension of symptom control after BoNT (range: 3.3-7.1 months). CONCLUSIONS: Topical AH8 is safe and promising for extending the duration of action of BoNT therapy for BSP.
Subject(s)
Blepharospasm/drug therapy , Botulinum Toxins, Type A/administration & dosage , Neuromuscular Agents/administration & dosage , Oligopeptides/administration & dosage , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Administration, Topical , Adult , Aged , Double-Blind Method , Drug Therapy, Combination , Humans , Middle Aged , Pilot ProjectsABSTRACT
INTRODUCTION. Chronic migraine is the most frequent complication of migraine. Its management is complex and difficult, and is based essentially on preventive measures. AIM. To analyse the development of the use of Onabotulinumtoxin A (OnabotA) in migraine, especially in its chronic form, the method of administration, its mechanism of action, its safety profile and its possible indications in clinical practice. DEVELOPMENT. The study conducts a thorough review of all the clinical trials in the literature that have used OnabotA in the prevention of migraine, both in its episodic and its chronic forms, and the outcomes in the chronic form are analysed in detail. CONCLUSIONS. In studies in phase III, OnabotA has proved to be effective in the treatment of patients with chronic migraine, with significant reductions in the mean frequency of days with headaches, the number of headache episodes, the days with migraine or the proportion of patients with severe disability, in addition to other parameters. It is also effective in the subgroup of patients with symptomatic headache due to medication abuse. OnabotA has proved to be safe and well tolerated in this indication, with foreseeable, usually mild or moderate, transitory side effects. In sum, OnabotA is a safe, well-tolerated alternative in the preventive treatment of chronic migraine.
Subject(s)
Botulinum Toxins, Type A/therapeutic use , Migraine Disorders/drug therapy , Acetylcholine/metabolism , Botulinum Toxins, Type A/administration & dosage , Botulinum Toxins, Type A/adverse effects , Botulinum Toxins, Type A/pharmacology , Cervical Plexus/physiopathology , Chronic Disease , Clinical Trials as Topic , Double-Blind Method , Headache Disorders, Secondary/etiology , Headache Disorders, Secondary/prevention & control , Humans , Injections, Intramuscular , Multicenter Studies as Topic , Muscle Weakness/chemically induced , Neuromuscular Blockade , Patient Selection , Randomized Controlled Trials as Topic , Sympathectomy, Chemical , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Treatment Outcome , Trigeminal Nerve/physiopathologyABSTRACT
The widely used botulinum neurotoxin A (BoNT/A) blocks neurotransmission via cleavage of the synaptic protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Recent evidence demonstrating long-distance propagation of SNAP-25 proteolysis has challenged the idea that BoNT/A remains localized to the injection site. However, the extent to which distant neuronal networks are impacted by BoNT/A retrograde trafficking remains unknown. Importantly, no studies have addressed whether SNAP-25 cleavage translates into structural and functional changes in distant intoxicated synapses. Here we show that the BoNT/A injections into the adult rat optic tectum result in SNAP-25 cleavage in retinal neurons two synapses away from the injection site, such as rod bipolar cells and photoreceptors. Retinal endings displaying cleaved SNAP-25 were enlarged and contained an abnormally high number of synaptic vesicles, indicating impaired exocytosis. Tectal injection of BoNT/A in rat pups resulted in appearance of truncated-SNAP-25 in cholinergic amacrine cells. Functional imaging with calcium indicators showed a clear reduction in cholinergic-driven wave activity, demonstrating impairments in neurotransmission. These data provide the first evidence for functional effects of the retrograde trafficking of BoNT/A, and open the possibility of using BoNT/A fragments as drug delivery vehicles targeting the central nervous system.
Subject(s)
Botulinum Toxins, Type A/pharmacology , Synaptic Transmission/drug effects , Synaptic Vesicles/metabolism , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Animals , Calcium Signaling , Rats , Rats, Long-Evans , Retinal Neurons/drug effects , Retinal Neurons/metabolism , Retinal Neurons/ultrastructure , Synaptic Transmission/physiology , Synaptic Vesicles/drug effects , Synaptic Vesicles/ultrastructure , Synaptosomal-Associated Protein 25/metabolismABSTRACT
The light chain of botulinum neurotoxin A (BoNT/A-LC) is a Zn-dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C-terminus of SNAP25 (residues 141-206) retains full activity for BoNT/A-LC-catalyzed cleavage at P1-P1' (Gln197-Arg198). Using the structure of a complex between the C-terminus of SNAP25 and BoNT/A-LC as a model to design SNAP25-derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala-Asn-Gln-Arg (residues 195-198) at the substrate cleavage site, with the intent to identify possible side-chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1-P1' residues to explore the spatial tolerance within the active-site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A-LC. Among the SNAPIs tested, several known cleavage-resistant, single-point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly-insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A-LC. Two of the Gly-insertion mutants exhibited 10-fold lower IC50 values than the wildtype 66-mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.
Subject(s)
Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/metabolism , Glycine/chemistry , Synaptosomal-Associated Protein 25/chemistry , Binding Sites/physiology , Catalytic Domain , Models, Molecular , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/genetics , Synaptosomal-Associated Protein 25/metabolismABSTRACT
Neurotransmitter release from presynaptic terminals is under the tight control of various metabotropic receptors. We report here that in addition to the regulation of Ca(2+) channel activity, metabotropic GABA(B) receptors (GABA(B)Rs) at murine hippocampal glutamatergic synapses utilize an inhibitory pathway that directly targets the synaptic vesicle release machinery. Acute application of the GABA(B)R agonist baclofen rapidly and reversibly inhibits vesicle fusion, which occurs independently of the SNAP-25 C-terminus. Using applications of hypertonic sucrose solutions, we find that the size of the readily releasable pool remains unchanged by GABA(B)R activation, but the sensitivity of primed vesicles to hypertonic stimuli appears lowered as the response amplitudes at intermediate sucrose concentrations are smaller and release kinetics are slowed. These data show that presynaptic GABA(B)Rs can inhibit neurotransmitter release directly by increasing the energy barrier for vesicle fusion.
Subject(s)
Hippocampus/pathology , Presynaptic Terminals/metabolism , Receptors, GABA-B/metabolism , Receptors, Metabotropic Glutamate/metabolism , Synaptic Transmission , Animals , Baclofen/pharmacology , Botulinum Toxins, Type A/pharmacology , Cells, Cultured , Electromagnetic Radiation , GABA-B Receptor Agonists/pharmacology , Membrane Fusion/drug effects , Mice , Mice, Inbred C57BL , Presynaptic Terminals/drug effects , Presynaptic Terminals/pathology , Rats , Rats, Sprague-Dawley , Synaptic Transmission/drug effects , Synaptic Vesicles/drug effects , Synaptic Vesicles/pathology , Synaptosomal-Associated Protein 25/antagonists & inhibitorsABSTRACT
Clostridium botulinum produces the most lethal toxins known to man, as such they are high risk terrorist threats, and alarmingly there is no approved therapeutic. We report the first cross-over small molecule inhibitor of these neurotoxins and propose a mechanism by which it may impart its inhibitory activity.
Subject(s)
Biological Products/pharmacology , Botulinum Toxins/antagonists & inhibitors , Caffeic Acids/therapeutic use , Clostridium botulinum/metabolism , HIV Integrase Inhibitors/therapeutic use , Neurotoxins/antagonists & inhibitors , Succinates/therapeutic use , Amino Acid Sequence , Biological Products/therapeutic use , Botulinum Toxins/chemistry , Botulinum Toxins/metabolism , Caffeic Acids/pharmacology , Catalytic Domain , Cross-Linking Reagents/chemistry , Fluorescence Resonance Energy Transfer , HIV Integrase Inhibitors/pharmacology , Humans , Male , Molecular Sequence Data , Neurotoxins/chemistry , Neurotoxins/metabolism , Substrate Specificity , Succinates/pharmacology , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolism , Vesicle-Associated Membrane Protein 2/antagonists & inhibitors , Vesicle-Associated Membrane Protein 2/chemistry , Vesicle-Associated Membrane Protein 2/metabolismABSTRACT
Some dentists propose administering botulinum neurotoxin injections to treat orofacial pain. The scientific literature has documented there are dangerous uncontrollable effects of long-distance traveling of botulinum neurotoxin from the injection site. These distant effects are not technique-specific, not predictable, and cannot be controlled by the amount of neurotoxin, nor by the site administered. These uncontrollable distant effects of "off-label" botulinum neurotoxin injections, at the very least, must be thoroughly disclosed to patients.
Subject(s)
Botulinum Toxins, Type A/adverse effects , Neuromuscular Agents/adverse effects , Botulinum Toxins, Type A/pharmacology , Cholinergic Antagonists/pharmacology , Deglutition Disorders/chemically induced , Facial Pain/drug therapy , Humans , Neuromuscular Agents/pharmacology , Peripheral Nervous System Diseases/chemically induced , Synaptosomal-Associated Protein 25/antagonists & inhibitorsABSTRACT
SNAP-25, a synaptosome-associated exocytosis protein of 25 kd mw, plays an important role in the secretory activity of several endocrine cells. In the present study, we investigated surgically removed pituitary adenomas including 40 prolactin (PRL), 31 growth hormone, 5 adrenocorticotropic hormone, 5 thyroid-stimulating hormone, 14 follicle-stimulating hormone/luteinizing hormone/alpha-subunit-producing tumors, and 5 null cell adenomas. Among the 40 patients with PRL-producing pituitary adenoma, 16 had been preoperatively treated with the dopamine agonist bromocriptine. Similarly, of the 31 patients with GH-producing pituitary adenomas, 15 had been treated with the long-acting somatostatin analog, octreotide. All tumors were subjected to transsphenoidal surgery, formalin-fixed, routinely processed, and paraffin-embedded. Sections of 4 to 6-microm thickness were stained with hematoxylin and eosin and the periodic acid-Schiff as well as the Gordon-Sweet silver methods. Immunostaining for SNAP-25 (streptavidin-biotin peroxidase complex method) showed that 10 PRL-producing adenomas were strongly immunoreactive. Immunopositivity was mainly cell membrane in distribution but several cells showed mild cytoplasmic staining. Nuclei were immunonegative. Preoperative bromocriptine treatment markedly decreased SNAP-25 immunopositivity. Among GH-producing adenomas, SNAP-25 was seen in 5 cases; reactivity being mild-to-moderate, membrane-bound, and cytoplasmic. Octreotide caused no significant reduction in immunopositivity. Other adenoma types were virtually immunonegative. Six autopsy-derived human pituitaries and 4 surgically obtained nontumorous pituitaries were also immunonegative for SNAP-25. It is conceivable that SNAP-25 plays an important role in PRL release and is involved in the bromocriptine-induced suppression of PRL secretion from PRL-producing adenoma cells.
Subject(s)
Adenoma/metabolism , Pituitary Neoplasms/metabolism , Synaptosomal-Associated Protein 25/biosynthesis , Synaptosomal-Associated Protein 25/genetics , Adenoma/diagnosis , Adenoma/immunology , Adenoma/pathology , Bromocriptine/therapeutic use , Human Growth Hormone/biosynthesis , Humans , Immunohistochemistry , Octreotide/therapeutic use , Pituitary Neoplasms/drug therapy , Pituitary Neoplasms/immunology , Pituitary Neoplasms/pathology , Prolactin/antagonists & inhibitors , Prolactin/biosynthesis , Prolactin/metabolism , Synaptosomal-Associated Protein 25/antagonists & inhibitorsABSTRACT
During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.
Subject(s)
Chemokines/metabolism , Exocytosis/genetics , Gene Expression Regulation , Ovulation/genetics , Synaptosomal-Associated Protein 25/genetics , Animals , Chorionic Gonadotropin/pharmacology , Female , Granulosa Cells/drug effects , Granulosa Cells/metabolism , Mice , Mice, Mutant Strains , Ovulation/drug effects , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Synaptosomal-Associated Protein 25/antagonists & inhibitorsABSTRACT
SNARE proteins mediate the fusion of lipid bilayers by the directed assembly of coiled-coil domains arising from apposing membranes. We have utilized inverted cone-shaped lipids, antagonists of the necessary membrane deformation during fusion to characterize the extent and range of SNARE assembly up to the moment of stalk formation between bilayers. The inverted cone-shaped lipid family of acyl-CoAs specifically inhibits the completion of fusion in an acyl-chain length-dependent manner. Removal of acyl-CoA from the membrane relieves the inhibition and initiates a burst of membrane fusion with rates exceeding any point in the control curves lacking acyl-CoA. This burst indicates the accumulation of semi-assembled fusion complexes. These preformed complexes are resistant to cleavage by botulinum toxin B and thus appear to have progressed beyond the "loosely zippered" state of docked synaptic vesicles. Surprisingly, application of the soluble domain of VAMP2, which blocks SNARE assembly by competing for binding on the available t-SNAREs, blocks recovery from the acyl-CoA inhibition. Thus, complexes formed in the presence of a lipidic antagonist to fusion are incompletely assembled, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion. In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin.
Subject(s)
Membrane Lipids/physiology , SNARE Proteins/physiology , Acyl Coenzyme A/physiology , Cell Fusion , Humans , Liposomes , Membrane Fusion/physiology , Oleic Acid/physiology , SNARE Proteins/antagonists & inhibitors , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/metabolism , Syntaxin 1/antagonists & inhibitors , Syntaxin 1/metabolismABSTRACT
SNARE-proteins (soluble NSF-attachment protein receptor) are important for Ca(2+)-dependent exocytosis. We have used capacitance measurements and confocal imaging to dissect the role of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 in rapid exocytosis in insulin-secreting pancreatic beta-cells. Following immunoneutralization of syntaxin 1 and SNAP-25, exocytosis was strongly reduced and associated with a marked reduction in the size of the readily releasable pool (RRP) by 65% and 86% in the presence of the anti-SNAP-25 and anti-syntaxin 1 antibodies respectively. The size of the immediately releasable pool (IRP), a subset of RRP in close association with the voltage-dependent Ca(2+)-channels, was reduced to an equal extent. The reduction in IRP correlated with slowed release kinetics and the time constant (tau) increased from a control value of 16 to 36 ms and 51 ms after inclusion of anti-SNAP-25 and anti-syntaxin 1 antibodies respectively in the pipette solution. We further show that SNAP-25 and syntaxin 1 aggregate in clusters along the plasma membrane. The size of these clusters was estimated to be approximately 300 nm and every beta-cell contained approximately 400 SNAP-25/syntaxin 1 clusters. Whereas the inhibitory action of the anti-syntaxin 1 antibody on exocytosis could be attributed almost entirely to suppression of the voltage-dependent Ca(2+)-current (-40%), the effect of the anti-SNAP-25 antibody was not mediated by decreased Ca(2+)-entry and is more likely due to a direct interference with the exocytotic machinery. Our data are consistent with the concept that both syntaxin 1 and SNAP-25 are required for rapid exocytosis in beta-cells.