RESUMO
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.
Assuntos
Antídotos/farmacologia , Antídotos/uso terapêutico , Antitoxinas/farmacologia , Antitoxinas/uso terapêutico , Toxinas Bacterianas/antagonistas & inibidores , Animais , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Linhagem Celular Tumoral/efeitos dos fármacos , Clostridium botulinum , Modelos Animais de Doenças , Endopeptidases , Ensaios de Triagem em Larga Escala , Humanos , Índia , Concentração Inibidora 50 , Masculino , Camundongos , Neuroblastoma/tratamento farmacológico , Extratos Vegetais/farmacologia , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , TermolisinaRESUMO
Transient receptor potential (TRP) vallinoid 1 (TRPV1) and ankyrin 1 (TRPA1) are two transducing channels expressed on peripheral sensory nerves involved in pain sensation. Upregulation of their expression, stimulated by inflammatory cytokines and growth factors in animal pain models, correlate with the induction of nociceptive hyper-sensitivity. Herein, we firstly demonstrate by immuno-cytochemical labelling that TNFα augments the surface content of these channels on rat cultured dorsal root ganglion (DRG) neurons which, in turn, enhances the electrophysiological and functional responses of the latter to their specific agonists. A molecular basis underlying this TNFα-dependent enhancement was unveiled by pre-treating DRGs with a recently-published chimeric protein, consisting of the protease light chain (LC) of botulinum neurotoxin (BoNT) serotype E fused to full-length BoNT/A (LC/E-BoNT/A). This cleaves synaptosomal-associated protein of Mr 25k (SNAP-25) and reported previously to exhibit anti-nociceptive activity in a rat model of neuropathic pain. Low pM concentrations of this chimera were found to prevent the TNFα-stimulated delivery of TRPV1/A1 to the neuronal plasmalemma and, accordingly, decreased their incremental functional activities relative to those of control cells, an effect accompanied by SNAP-25 cleavage. Advantageously, LC/E-BoNT/A did not reduce the basal surface contents of the two channels or their pharmacological responses. Thus, use of multiple complementary methodologies provides evidence that LC/E-BoNT/A abolishes the TNFα-dependent augmented, but not resting, surface trafficking of TRPV1/A1. As TNFα is known to induce nociceptive hyper-sensitivity in vivo, our observed inhibition by LC/E-BoNT/A of its action in vitro could contribute to its potential alleviation of pain.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Toxinas Botulínicas/farmacologia , Gânglios Espinais/efeitos dos fármacos , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Capsaicina/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Escherichia coli , Gânglios Espinais/metabolismo , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Canais de Cátion TRPV/agonistasRESUMO
Luteal phase defects (LPD) are an important etiology of infertility which has increased in recent years. Studies have shown that bu-shen-zhu-yun decoction (BSZY-D) can lower the expression of estrogen receptor and progesterone receptor, in rats endometrium of embryonic implantation period, which upregulated by mifepristone, and improve uterine receptivity. The aim of present study was to determine the effect of BSZY-D on the synthesis and secretion of gonadotropic hormones in the anterior pituitary cells of rats. Rats were treated with saline (control) or BSZY-D two times/day for three estrous cycles by gavage. The cerebrospinal fluid (CSF) were collected for further cell treatment. The components in BSZY-D, serum and CSF were analysed by High Performance Liquid Chromatography (HPLC). Cells were either pretreated with normal CSF or BSZY-D/CSF before being stimulated with or without cetrorelix. The mRNA and proteins levels of receptors, hormones, and transcription factors were detected by RT-PCR, western blot analysis and immunostaining. We show that non-toxic concentrations of cetrorelix, a GnRH antagonist, can reduce the mRNA and protein levels of GnRHR, LH, and FSH. This effect could be reversed by the addition of BSZY-D/CSF. We also show decreased mRNA and protein expression of transcription factors, such as CREB, and Egr-1 and secretory vescicles, including SNAP-25 and Munc-18 upon treatment with cetrorelix could be reversed post co-treatment with BSZY-D/CSF. These results indicate that BSZY-D/CSF treatment led to increased levels of GnRHR, transcription factors, and secretory vesicles leading to increased secretion of FSH and LH. Thus, BSZY-D presents a promising candidate to treat luteal phase defects and infertility.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Subunidade beta do Hormônio Folículoestimulante/biossíntese , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Hormônio Luteinizante Subunidade beta/biossíntese , Hormônio Luteinizante Subunidade beta/metabolismo , Adeno-Hipófise/citologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/farmacologia , Proteínas Munc18/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptores LHRH/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Background: Premature infants are deprived of prenatal accumulation of brain docosahexaenoic acid [DHA (22:6n-3)], an omega-3 fatty acid [ω-3 FA (n-3 FA)] important for proper development of cognitive function. The resulting brain DHA deficit can be reversed by ω-3 FA supplementation.Objective: The objective was to test whether there is a critical period for providing ω-3 FA to correct cognitive deficits caused by developmental ω-3 FA deprivation in mice.Methods: Twelve timed-pregnant mice [embryonic day 14 (E14), C57/BL6NCr] were fed an ω-3 FA-deficient diet containing 0.04% α-linolenic acid [ALA (18:3n-3)], and their offspring were fed the same deficient diet (Def group) or changed to an ω-3 FA-adequate diet containing 3.1% ALA at 3 wk, 2 mo, or 4 mo of age. In parallel, 3 E14 pregnant mice were fed the adequate diet and their offspring were fed the same diet (Adeq group) throughout the experiment. Brain FA composition, learning and memory, and hippocampal synaptic protein expression were evaluated at 6 mo by gas chromatography, the Morris water maze test, and western blot analysis, respectively.Results: Maternal dietary ω-3 FA deprivation decreased DHA by >50% in the brain of their offspring at 3 wk of age. The Def group showed significantly worse learning and memory at 6 mo than those groups fed the adequate diet. These pups also had decreased hippocampal expression of postsynaptic density protein 95 (43% of Adeq group), Homer protein homolog 1 (21% of Adeq group), and synaptosome-associated protein of 25 kDa (64% of Adeq group). Changing mice to the adequate diet at 3 wk, 2 mo, or 4 mo of age restored brain DHA to the age-matched adequate concentration. However, deficits in hippocampal synaptic protein expression and spatial learning and memory were normalized only when the diet was changed at 3 wk.Conclusion: Developmental deprivation of brain DHA by dietary ω-3 FA depletion in mice may have a lasting impact on cognitive function if not corrected at an early age.
Assuntos
Encéfalo/efeitos dos fármacos , Cognição/efeitos dos fármacos , Deficiências Nutricionais/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/administração & dosagem , Recém-Nascido Prematuro , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Deficiências Nutricionais/complicações , Dieta , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Gorduras na Dieta/uso terapêutico , Proteína 4 Homóloga a Disks-Large , Ácidos Docosa-Hexaenoicos/deficiência , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Feminino , Guanilato Quinases/metabolismo , Proteínas de Arcabouço Homer/metabolismo , Humanos , Lactente , Recém-Nascido Prematuro/crescimento & desenvolvimento , Recém-Nascido Prematuro/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Proteínas de Membrana/metabolismo , Camundongos , Gravidez , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de TempoRESUMO
A pressing need exists for long-acting, non-addictive medicines to treat chronic pain, a major societal burden. Botulinum neurotoxin type A (BoNT/A) complex - a potent, specific and prolonged inhibitor of neuro-exocytosis - gives some relief in several pain disorders, but not for all patients. Our study objective was to modify BoNT/A to overcome its inability to block transmitter release elicited by high [Ca2+]i and increase its limited analgesic effects. This was achieved by fusing a BoNT/A gene to that for the light chain (LC) of type/E. The resultant purified protein, LC/E-BoNT/A, entered cultured sensory neurons and, unlike BoNT/A, inhibited release of calcitonin gene-related peptide evoked by capsaicin. Western blotting revealed that this improvement could be due to a more extensive truncation by LC/E of synaptosomal-associated protein of Mr = 25 k, essential for neuro-exocytosis. When tested in a rat spared nerve injury (SNI) model, a single intra-plantar (IPL) injection of LC/E-BoNT/A alleviated for â¼2 weeks mechanical and cold hyper-sensitivities, in a dose-dependent manner. The highest non-paralytic dose (75 U/Kg, IPL) proved significantly more efficacious than BoNT/A (15 U/Kg, IPL) or repeated systemic pregabalin (10 mg/Kg, intraperitoneal), a clinically-used pain modulator. Effects of repeated or delayed injections of this fusion protein highlighted its analgesic potential. Attenuation of mechanical hyperalgesia was extended by a second administration when the effect of the first had diminished. When injected 5 weeks after injury, LC/E-BoNT/A also reversed fully-established mechanical and cold hyper-sensitivity. Thus, combining advantageous features of BoNT/E and/A yields an efficacious, locally-applied and long-acting anti-hyperalgesic.
Assuntos
Toxinas Botulínicas/uso terapêutico , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Neuralgia/complicações , Peptídeo Hidrolases/uso terapêutico , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Toxinas Botulínicas/química , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Masculino , Modelos Moleculares , Atividade Motora/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Pregabalina/toxicidade , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/uso terapêutico , Células Receptoras Sensoriais/efeitos dos fármacos , Fatores de Tempo , Gânglio Trigeminal/citologiaRESUMO
Alzheimer's Disease (AD) is a progressive neurodegenerative disorder characterized by extracellular plaques containing abnormal Amyloid Beta (Aß) aggregates, intracellular neurofibrillary tangles containing hyperphosphorylated tau protein, microglia-dominated neuroinflammation, and impairments in synaptic plasticity underlying cognitive deficits. Therapeutic strategies for the treatment of AD are currently limited. In this study, we investigated the effects of dietary supplementation of 4% pomegranate extract to a standard chow diet on neuroinflammation, and synaptic plasticity in APPsw/Tg2576 mice brain. Treatment with a custom mixed diet (pellets) containing 4% pomegranate for 15 months ameliorated the loss of synaptic structure proteins, namely PSD-95, Munc18-1, and SNAP25, synaptophysin, phosphorylation of Calcium/Calmodulin Dependent Protein Kinase IIα (p-CaMKIIα/ CaMKIIα), and phosphorylation of Cyclic AMP-Response Element Binding Protein (pCREB/CREB), inhibited neuroinflammatory activity, and enhanced autophagy, and activation of the phophoinositide-3-kinase-Akt-mammalian target of rapamycin signaling pathway. These neuroprotective effects were associated with reduced ß-site cleavage of Amyloid Precursor Protein in APPsw/Tg2576 mice. Therefore, long-term supplementation with pomegranates can attenuate AD pathology by reducing inflammation, and altering APP-dependent processes.
Assuntos
Doença de Alzheimer/dietoterapia , Encéfalo/imunologia , Sinapses Elétricas/metabolismo , Inflamação/dietoterapia , Lythraceae , Extratos Vegetais/uso terapêutico , Placa Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/patologia , Dieta , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Munc18/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
"Mirror pain" or mirror-image pain (MP) is pain opposite to the side of injury. Mechanism and frequency in humans are not known. There is no consent on therapy. Here we report that unilaterally injected botulinum toxin type A (BT-A) has bilateral effect in experimental MP, thus deserves to be investigated as therapy for this condition. We examined the localization of BT-A's bilateral antinociceptive action in MP induced by 3 % carrageenan intramuscular injection in Wistar rats. BT-A was applied peripherally (5 U/kg), into ipsilateral or contralateral hind paw pad (i.pl.) and centrally (1 U/kg), at spinal (intrathecally, i.t.) or supraspinal (intracisternally, i.c.) level. Additionally, we examined the involvement of central opioid and GABAergic systems, as well as the contribution of peripheral capsaicin-sensitive neurons to BT-A's bilateral antinociceptive effect. Ipsilateral i.pl. and i.t. BT-A reduced the bilateral mechanical sensitivity to von Frey filaments, while contralateral i.pl. and i.c. treatments had no effect on either tested side. Bilateral antinociceptive effect of ipsilateral i.pl. BT-A was prevented by µ-opioid antagonist naloxonazine (1.5 µg/10 µl) and GABAA antagonist bicuculline (1 µg/10 µl) if applied at the spinal level, in contrast to supraspinal application of the same doses. Local treatment of sciatic nerve with 2 % capsaicin 5 days following BT-A i.pl. injection caused desensitization of sciatic capsaicin-sensitive fibers, but did not affect bilateral antinociceptive effect of BT-A and the presence of cleaved SNAP-25 at the spinal cord slices. Present experiments suggest segmental actions of peripheral BT-A at spinal level, which are probably not solely dependent on capsaicin-sensitive neurons.
Assuntos
Analgésicos/uso terapêutico , Toxinas Botulínicas Tipo A/farmacologia , Toxinas Botulínicas Tipo A/uso terapêutico , Lateralidade Funcional/efeitos dos fármacos , Dor/tratamento farmacológico , Analgésicos/farmacologia , Animais , Bicuculina/uso terapêutico , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/toxicidade , Carragenina/toxicidade , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Masculino , Naloxona/análogos & derivados , Naloxona/uso terapêutico , Dor/induzido quimicamente , Dor/patologia , Medição da Dor , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Botulinum toxin type A (BTX-A) selectively cleaves synaptosomal-associated protein of 25 kDa (SNAP-25) and results in inhibition of the fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane to undergo exocytosis and release. The aim of this study was to investigate whether BTX-A inhibited the pyloric smooth muscle contractility induced by acetylcholine (ACh) after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin (TSN). Three groups of rat pyloric muscle strips were studied in vitro. All strips were allowed to equilibrate for 52 min under a basal loading tension of 1 g in Krebs solution and spontaneous contractile waves were recorded as their own controls before adding each drug. According to experimental protocols, 100 µM ACh, 1 µM atropine, 29.6 µM TSN and 10 U/ml BTX-A was added, respectively. BTX-A directly inhibited pyloric spontaneous contraction and ACh-induced contractile response. Addition of 10 U/ml BTX-A still inhibited pyloric smooth muscle contractility following incubation of TSN, while subsequent administration of 100 µM ACh had no effect. BTX-A inhibits pyloric smooth muscle contractility in our study suggesting BTX-A inhibits not only ACh release from cholinergic nerves but also muscarinic cholinergic muscular transmission.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Contração Muscular/efeitos dos fármacos , Piloro/efeitos dos fármacos , Acetilcolina , Animais , Toxinas Botulínicas Tipo A/uso terapêutico , Neurônios Colinérgicos/efeitos dos fármacos , Medicamentos de Ervas Chinesas , Gastroparesia/tratamento farmacológico , Técnicas In Vitro , Ratos Sprague-Dawley , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Synaptosomal-associated protein of 25 kDa (SNAP-25) is a key molecule in the soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE) complex mediating fast Ca(2+)-triggered release of hormones and neurotransmitters, and both splice variants, SNAP-25a and SNAP-25b, can participate in this process. Here we explore the hypothesis that minor alterations in the machinery mediating regulated membrane fusion can increase the susceptibility for metabolic disease and precede obesity and type 2 diabetes. Thus, we used a mouse mutant engineered to express normal levels of SNAP-25 but only SNAP-25a. These SNAP-25b-deficient mice were exposed to either a control or a high-fat/high-sucrose diet. Monitoring of food intake, body weight, hypothalamic function, and lipid and glucose homeostases showed that SNAP-25b-deficient mice fed with control diet developed hyperglycemia, liver steatosis, and adipocyte hypertrophy, conditions dramatically exacerbated when combined with the high-fat/high-sucrose diet. Thus, modified SNARE function regulating stimulus-dependent exocytosis can increase the vulnerability to and even provoke metabolic disease. When combined with a high-fat/high-sucrose diet, this vulnerability resulted in diabesity. Our SNAP-25b-deficient mouse may represent a diabesity model.
Assuntos
Doenças Metabólicas/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Adiposidade , Animais , Glicemia/metabolismo , Peso Corporal , Dislipidemias/patologia , Ingestão de Energia , Metabolismo Energético , Comportamento Alimentar , Feminino , Homeostase , Hipertrofia , Hipotálamo/metabolismo , Insulina/metabolismo , Secreção de Insulina , Leptina/sangue , Fígado/metabolismo , Fígado/patologia , Masculino , Doenças Metabólicas/sangue , Camundongos Obesos , Fenótipo , Receptores para Leptina/metabolismo , Proteína 25 Associada a Sinaptossoma/deficiênciaRESUMO
Alzheimer's disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid ß peptide (Aß) and microglia-dominated neuroinflammation. The therapeutic options for AD are currently limited. In this study, we investigated the antiinflammatory effects and the underlying molecular mechanisms of Ginkgo biloba extract EGb 761 when administered to TgCRND8 AD mice, which overexpress human Alzheimer's amyloid precursor protein (APP) specifically in neurons. We gave APP-transgenic mice EGb 761 as a dietary supplement for 2 or 5months. Plasma concentrations of EGb 761 components in mice were in the same range as such concentrations in humans taking EGb 761 at the recommended dose (240mg daily). Treatment with EGb 761 for 5months significantly improved the cognitive function of the mice as measured by the Barnes Maze test. It also attenuated the loss of synaptic structure proteins, such as PSD-95, Munc18-1, and SNAP25. Treatment with EGb 761 for 5months inhibited microglial inflammatory activation in the brain. The effects of treatment with EGb 761 for 2months were weak and not statistically significant. Moreover, EGb 761 activated autophagy in microglia. Treatment with EGb 761 decreased Aß-induced microglial secretion of TNF-α and IL-1ß and activation of caspase-1, both of which were abolished by the inhibition of autophagy. Treatment with EGb 761 also reduced the concentrations of NLRP3 protein that colocalized with LC3-positive autophagosomes or autolysosomes in microglia. Additionally, long-term treatment with EGb 761 may reduce cerebral Aß pathology by inhibiting ß-secretase activity and Aß aggregation. Therefore, long-term treatment with G. biloba extract EGb 761, a clinically available and well-tolerated herbal medication, ameliorates AD pathology by antiinflammatory and Aß-directed mechanisms.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Ginkgo biloba , Aprendizagem em Labirinto/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Munc18/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/farmacologia , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
The aim of the present study was to analyze a role of the ERK1/2 signaling pathway in the regulation of excitation and inhibitory neurons in the hippocampus and the temporal cortex of Krushinsky-Molodkina rats during seizure development finalizing with ataxia. Analysis was done by Western bloting as well as by immunohistochemistry. The results demonstrated significant up-regulation of ERK1/2 activity in the hippocampus in several seconds after sound stimulation. At the same time increased ERK1/2 activity was correlated with enhanced level of SNARE protein SNAP-25 and activation of synapsin I, the proteins which regulate exocytosis machinery. Decreased level of VGLUT2 associated with activation of ERK1/2 and exocytosis proteins supposed activation of glutamate release in the hippocampus, while in the temporal cortex diminished activity of ERK1/2 and synapsin I associated with VGLUT2 up-regulation assumed inhibition of glutamatergic transmission. Our data let us supposed that decreasing of glutamate release in th& temporal cortex could be a trigger for the inhibition of hippocampal glutamatergic system and the beginning of further ataxia stage. Our data demonstrated correlation between expression and activity of exocytosis proteins and ERK1/2 mainly in the glutamategic neurons of the hippocampus and the temporal cortex that let us proposed significant role of ERK1/2 kinases as a positive regulator of glutamate release and as a result initiation of seizure expression.
Assuntos
Ataxia/genética , Epilepsia Reflexa/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Convulsões/genética , Estimulação Acústica , Animais , Animais Endogâmicos , Ataxia/metabolismo , Ataxia/patologia , Epilepsia Reflexa/metabolismo , Epilepsia Reflexa/patologia , Exocitose/genética , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Regulação da Expressão Gênica , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ratos , Convulsões/metabolismo , Convulsões/patologia , Transdução de Sinais , Sinapsinas/genética , Sinapsinas/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Lobo Temporal/metabolismo , Lobo Temporal/patologia , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
It has recently been proposed that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. We hypothesized that inhibition of ERK1/2 activity could prevent audiogenic seizures by altering GABA and glutamate release mechanisms. Krushinsky-Molodkina rats, genetically prone to audiogenic seizure, were recruited in the experiments. Animals were i.p. injected with an inhibitor of ERK1/2 SL 327 at different doses 60 min before audio stimulation. We demonstrated for the first time that inhibition of ERK1/2 activity by SL 327 injections prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. The obtained data also demonstrated unchanged levels of GABA production, and an increase in the level of vesicular glutamate transporter 2. The study of exocytosis protein expression showed that SL 327 treatment leads to downregulation of vesicle-associated membrane protein 2 and synapsin I, and accumulation of synaptosomal-associated protein 25 (SNAP-25). The obtained data indicate that the inhibition of ERK1/2 blocks seizure behavior presumably by altering the exocytosis machinery, and identifies ERK1/2 as a potential target for the development of new strategies for seizure treatment. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. Here we report that inhibition of ERK1/2 by SL 327 prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. Accumulation of VGLUT2 was associated with differential changing of synaptic proteins VAMP2, SNAP-25 and synapsin I. The obtained data indicate that the inhibition of ERK1/2 alters neurotransmitter release by changing the exocytosis machinery, thus preventing seizures.
Assuntos
Aminoacetonitrila/análogos & derivados , Epilepsia Reflexa/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Estimulação Acústica/efeitos adversos , Aminoacetonitrila/farmacologia , Aminoacetonitrila/uso terapêutico , Animais , Encéfalo/metabolismo , Proteína de Ligação a CREB/metabolismo , Epilepsia Reflexa/enzimologia , Epilepsia Reflexa/genética , Exocitose/efeitos dos fármacos , Feminino , Ácido Glutâmico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Mutantes , Tempo de Reação/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapsinas/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/biossíntese , Proteína Vesicular 2 de Transporte de Glutamato/genética , Ácido gama-Aminobutírico/biossíntese , Ácido gama-Aminobutírico/metabolismoRESUMO
Pancreatic ß-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.
Assuntos
Cálcio/metabolismo , Gorduras na Dieta/farmacologia , Suplementos Nutricionais , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Desnutrição/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Taurina/farmacologia , Animais , Humanos , Secreção de Insulina , Masculino , Camundongos , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25(+/-)) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25(+/-) mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drugs.
Assuntos
Anticonvulsivantes/uso terapêutico , Transtornos Cognitivos/tratamento farmacológico , Epilepsia/tratamento farmacológico , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Aprendizagem por Associação/efeitos dos fármacos , Aprendizagem por Associação/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Carbamazepina/uso terapêutico , Transtornos Cognitivos/patologia , Transtornos Cognitivos/fisiopatologia , Epilepsia/patologia , Epilepsia/fisiopatologia , Etossuximida/uso terapêutico , Hipercinese/tratamento farmacológico , Hipercinese/patologia , Hipercinese/fisiopatologia , Ácido Caínico , Masculino , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nimodipina/uso terapêutico , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Proteína 25 Associada a Sinaptossoma/genética , Ácido Valproico/uso terapêuticoRESUMO
Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.
Assuntos
Toxinas Botulínicas Tipo A/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/imunologia , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Isolated islets from low-protein (LP) diet rats showed decreased insulin secretion in response to glucose and carbachol (Cch). Taurine (TAU) increases insulin secretion in rodent islets with a positive effect upon the cholinergic pathway. Here, we investigated the effect of TAU administration upon glucose tolerance and insulin release in rats fed on a normal protein diet (17%) without (NP) or with 2.5% of TAU in their drinking water (NPT), and LP diet fed rats (6%) without (LP) or with TAU (LPT). Glucose tolerance was found to be higher in LP, compared to NP rats. However, plasma glucose levels, during ipGTT, in LPT rats were similar to those of controls. Isolated islets from LP rats secreted less insulin in response to increasing glucose concentrations (2.8-22.2 mmol/L) and to 100 µmol/L Cch. This lower secretion was accompanied by a reduction in Cch-induced internal Ca(2+) mobilization. TAU supplementation prevents these alterations, as judged by the higher secretion induced by glucose or Cch in LPT islets. In addition, Ach-M3R, syntaxin 1 and synaptosomal associated protein of 25 kDa protein expressions in LP were lower than in NP islets. The expressions of these proteins in LPT were normalized. Finally, the sarcoendoplasmatic reticulum Ca(2+)-ATPase 3 protein expression was higher in LPT and NPT, compared with controls. In conclusion, TAU supplementation to LP rats prevented alterations in glucose tolerance as well as in insulin secretion from isolated islets. The latter effect involves the normalization of the cholinergic pathway, associated with the preservation of exocytotic proteins.
Assuntos
Dieta com Restrição de Proteínas , Suplementos Nutricionais , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Taurina/farmacologia , Animais , Western Blotting , Carbacol/administração & dosagem , Regulação da Expressão Gênica , Glucose/administração & dosagem , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , Taurina/sangueRESUMO
Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins, there are elevated concerns regarding their possible use as bioterrorism agents. Moreover, their widespread use for cosmetic purposes, and as medical treatments, has increased the potential risk of accidental overdosing and environmental exposure. Hence, there is an urgent need to develop novel modalities to counter BoNT intoxication. Mammalian motoneurons are the main target of BoNTs; however, due to the difficulty and poor efficiency of the procedures required to isolate the cells, they are not suitable for high-throughput drug screening assays. Here, we explored the suitability of embryonic stem (ES) cell-derived motoneurons as a renewable, reproducible, and physiologically relevant system for BoNT studies. We found that the sensitivity of ES-derived motoneurons to BoNT/A intoxication is comparable to that of primary mouse spinal motoneurons. Additionally, we demonstrated that several BoNT/A inhibitors protected SNAP-25, the BoNT/A substrate, in the ES-derived motoneuron system. Furthermore, this system is compatible with immunofluorescence-based high-throughput studies. These data suggest that ES-derived motoneurons provide a highly sensitive system that is amenable to large-scale screenings to rapidly identify and evaluate the biological efficacies of novel therapeutics.
Assuntos
Antitoxina Botulínica/farmacologia , Toxinas Botulínicas/antagonistas & inibidores , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Embrionárias/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Neurônios Motores/efeitos dos fármacos , Animais , Toxinas Botulínicas/toxicidade , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/instrumentação , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Ensaios de Triagem em Larga Escala/instrumentação , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Alzheimer's disease (AD) is one of the most common forms of dementia in the elderly. In AD patients, ß-amyloid peptide (Aß) plaques and neurofibrillary tangles are common features observed in the CNS. Aß deposition results in the production of reactive oxygen species (ROS) leading to the hyperphosphorylation of tau that are associated with neuronal damage. Cholinesterase inhibitors and a partial NMDA receptor antagonist (memantine) have been identified as potential treatment options for AD. However, clinical studies have found that these drugs fail to prevent the disease progression. From ancient times, garlic (Allium sativum) has been used to treat several diseases. By 'aging' of garlic, some adverse reactions of garlic can be eliminated. Recent findings suggest that 'aged garlic extract' (AGE) may be a therapeutic agent for AD because of its antioxidant and Aß lowering properties. To date, the molecular properties of AGE have been sparsely studied in vitro or in vivo. The present study tested specific biochemical and molecular effects of AGE in neuronal and AD rodent models. Furthermore, we identified S-allyl-L-cysteine (SAC) as one of the most active chemicals responsible for the AGE-mediated effect(s). We observed significant neuroprotective and neurorescue properties of AGE and one of its ingredients, SAC, from ROS (H(2)O(2))-mediated insults to neuronal cells. Treatment of AGE and SAC were found to protect neuronal cells when they were independently co-treated with ROS. Furthermore, a novel neuropreservation effect of AGE was detected in that pre-treatment with AGE alone protected â¼ 80% neuronal cells from ROS-mediated damage. AGE was also found to preserve pre-synaptic protein synaptosomal associated protein of 25 kDa (SNAP25) from ROS-mediated insult. For example, treatment with 2% AGE containing diet and SAC (20 mg/kg of diet) independently increased (â¼70%) levels of SNAP25 and synaptophysin in Alzheimer's amyloid precursor protein-transgenic mice, of which the latter was significantly decreased in AD. Taken together, the neuroprotective, including preservation of pre-synaptic proteins by AGE and SAC can be utilized in future drug development in AD.
Assuntos
Doença de Alzheimer/patologia , Cisteína/análogos & derivados , Alho/química , Neurônios/efeitos dos fármacos , Neuroprostanos/farmacologia , Extratos Vegetais/farmacologia , Sinapses/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Linhagem Celular Transformada , Cisteína/farmacologia , Cisteína/uso terapêutico , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/efeitos adversos , L-Lactato Desidrogenase/metabolismo , Memória de Curto Prazo/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Neuroprostanos/uso terapêutico , Extratos Vegetais/uso terapêutico , Ratos , Espécies Reativas de Oxigênio/efeitos adversos , Sinaptofisina/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de TempoRESUMO
Evidence indicates that accumulation of excitotoxic mediators, such as glutamate, contributes to neuronal damage after an ischaemic insult. It is not clear, however, whether this accumulation is due to excess synaptic release or to impaired uptake. To test a role for synaptic release, here we investigated the neuroprotective potential of the synaptic blocker botulinum neurotoxin E (BoNT/E), that prevents vesicle fusion via the cleavage of the SNARE (soluble NSF-attachment receptor) protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Focal ischaemia was induced in vivo by infusing the potent vasoconstricting peptide endothelin-1 (ET-1) into the CA1 area of the hippocampus in adult rats; BoNT/E or vehicle were administered into the same site 20 min later. Injection of ET-1 was found to produce a transient and massive increase in glutamate release that was potently antagonized by BoNT/E. To assess whether blocking transmitter release translates into neuroprotection, the extent of the ischaemic damage was determined 24 h and 6 weeks after the insult. We found that BoNT/E administration consistently reduced the loss of CA1 pyramidal neurons at 24 h. The neuroprotective effect of BoNT/E, however, was no longer significant at 6 weeks. These data provide evidence that blockade of synaptic transmitter release delays neuronal cell death following focal brain ischaemia, and underline the importance of assessing long-term neuroprotection in experimental stroke studies.
Assuntos
Toxinas Botulínicas/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Região CA1 Hipocampal/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Animais , Isquemia Encefálica/induzido quimicamente , Região CA1 Hipocampal/irrigação sanguínea , Região CA1 Hipocampal/patologia , Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos , Endotelina-1/toxicidade , Feminino , Ácido Glutâmico/metabolismo , Microdiálise , Células Piramidais/efeitos dos fármacos , Células Piramidais/patologia , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de TempoRESUMO
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.