RESUMEN
NEW FINDINGS: What is the central question of this study? We investigated the effects of intrathecal administration of a novel toxin, CTK 01512-2, in a mouse model of Huntington's disease. We asked whether spinal cord neurons can represent a therapeutic target, given that the spinal cord seems to be involved in motor symptoms of Huntington's disease. Pharmacological approaches focusing on the spinal cord and skeletal muscles might represent a more feasible strategy than a high-risk brain intervention. What is the main finding and its importance? We provided evidence of a novel, local, neuroprotective effect of CTK 01512-2, paving a path for the development of approaches to treat motor symptoms of Huntington's disease beyond the brain. ABSTRACT: Phα1ß is a neurotoxin from the venom of the Phoneutria nigriventer spider, available as CTK 01512-2, a recombinant peptide. Owing to its antinociceptive and analgesic properties, CTK 01512-2 has been described to alleviate neuroinflammatory responses. Despite the diverse actions of CTK 01512-2 on the nervous system, little is known regarding its neuroprotective effect, especially in neurodegenerative conditions such as Huntington's disease (HD), a genetic movement disorder without cure. Here, we investigated whether CTK 01512-2 has a neuroprotective effect in a mouse model of HD. We hypothesized that spinal cord neurons might represent a therapeutic target, because the spinal cord seems to be involved in the motor symptoms of HD (BACHD) mice. We treated BACHD mice with CTK 01512-2 by intrathecal injection and performed in vivo motor behavioural and morphological analyses in the CNS (brain and spinal cord) and muscles. Our data showed that intrathecal injection of CTK 01512-2 significantly improved motor performance in the open field task. CTK 01512-2 protected neurons in the spinal cord (but not in the brain) from death, suggesting a local effect. CTK 01512-2 exerted its neuroprotective effect by inhibiting BACHD neuronal apoptosis, as revealed by a reduction in caspase-3 in the spinal cord. CTK 01512-2 was also able to revert BACHD muscle atrophy. In conclusion, our data suggest a novel role for CTK 01512-2 acting directly in the spinal cord to ameliorate morphofunctional aspects of spinal cord neurons and muscles and improve the performance of BACHD mice in motor behavioural tests. Given that HD shares similar symptoms with many neurodegenerative conditions, the findings presented herein might also be applicable to other disorders.
Asunto(s)
Enfermedad de Huntington , Fármacos Neuroprotectores , Animales , Modelos Animales de Enfermedad , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Ratones , Ratones Transgénicos , Neuronas , Fármacos Neuroprotectores/farmacología , Médula EspinalRESUMEN
PURPOSE: Abdominal hysterectomy is one of the most commonly performed gynecologic surgical procedures and is frequently associated with moderate to severe pain. The present study compared the effects of morphine and ketamine on postoperative analgesia, hemodynamic stability, and postoperative adverse effects in patients who underwent abdominal hysterectomy. DESIGN: This randomized controlled trial compares the effects of morphine plus adjuvants to those of ketamine plus adjuvants, administered as spinal anesthetic agents in patients who underwent abdominal hysterectomy. METHODS: Eighty patients were randomly assigned to two different groups: group M (morphine, 40 mcg) and group K (ketamine, 20 mg); the anesthetic agents were combined with equal quantities of other adjuvants. Postoperative analgesia was evaluated by means of a numeric pain rating scale; adverse effects (pruritus, nausea and vomiting, urinary retention, respiratory depression, and changes in bowel habits) at specific postoperative time intervals of T1 (4 hours), T2 (12 hours), and T3 (24 hours) were documented and compared. Hemodynamic stability was assessed intraoperatively. FINDINGS: Both groups displayed similar patient characteristics, comorbidities, paravertebral block level, and intraoperative hemodynamics. The present study observed a significant difference in postoperative analgesia between the two groups, 12 hours after the surgery, with group M exhibiting better results, compared with group K (P = .004). The pain scores obtained from group K were consistent with the amount of rescue medication (tramadol) administered to the subjects in the group, which showed a concomitant higher consumption of tramadol, compared with group M (42.5 and 71.8 mg in group M and group K, respectively, P = .011). Group M showed a higher incidence of pruritus, changes in bowel habits, and constipation compared with group K. CONCLUSIONS: Compared with ketamine, intrathecal morphine obtained better postoperative analgesia up to 12 hours after surgery, with a higher incidence of pruritus without any significant change in other variables.
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Ketamina , Morfina/uso terapéutico , Dolor Postoperatorio/tratamiento farmacológico , Analgesia Controlada por el Paciente , Analgésicos Opioides , Método Doble Ciego , Femenino , Humanos , Histerectomía/efectos adversos , Ketamina/uso terapéuticoRESUMEN
It is well-established that bacterial lipopolysaccharides (LPS) can promote neuroinflammation through receptor Toll-like 4 activation and induces sickness behavior in mice. This phenomenon triggers changes in membranes lipid dynamics to promote the intracellular cell signaling. Desorption electrospray ionization mass spectrometry (DESI-MS) is a powerful technique that can be used to image the distribution of lipids in the brain tissue directly. In this work, we characterize the LPS-induced neuroinflammation and the lipid dynamics in C57BL/6 mice at 3 and 24â¯h after LPS injection. We have observed that intraperitoneal administration of LPS (5â¯mg/kg body weight) induces sickness behavior and triggers a peripheral and cerebral increase of pro- and anti-inflammatory cytokine levels after 3â¯h, but only IL-10 was upregulated after 24â¯h. Morphological analysis of hypothalamus, cortex and hippocampus demonstrated that microglial activation was present after 24â¯h of LPS injection, but not at 3â¯h. DESI-MS revealed a total of 14 lipids significantly altered after 3 and 24â¯h and as well as their neuroanatomical distribution. Multivariate statistical analyzes have shown that ions associated with phosphatidylethanolamine [PE(38:4)] and docosatetraenoic acid [FA (22:4)] could be used as biomarkers to distinguish samples from the control or LPS treated groups. Finally, our data demonstrated that monitoring cerebral lipids dynamics and its neuroanatomical distribution can be helpful to understand sickness behavior and microglial activation after LPS administration.
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Lípidos/inmunología , Inflamación Neurogénica/inmunología , Neuroinmunomodulación/inmunología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/metabolismo , Citocinas/metabolismo , Hipocampo/diagnóstico por imagen , Hipocampo/metabolismo , Hipotálamo/diagnóstico por imagen , Hipotálamo/metabolismo , Conducta de Enfermedad/fisiología , Lipopolisacáridos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/inmunología , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
The venom of the Brazilian armed spider Phoneutria nigriventer is a rich source of biologically active peptides that have potential as analgesic drugs. In this study, we investigated the analgesic and adverse effects of peptide 3-5 (Tx3-5), purified from P. nigriventer venom, in several mouse models of pain. Tx3-5 was administered by intrathecal injection to mice selected as models of postoperative (plantar incision), neuropathic (partial sciatic nerve ligation) and cancer-related pain (inoculation with melanoma cells) in animals that were either sensitive or tolerant to morphine. Intrathecal administration of Tx3-5 (3-300 fmol/site) in mice could either prevent or reverse postoperative nociception, with a 50 % inhibitory dose (ID50) of 16.6 (3.2-87.2) fmol/site and a maximum inhibition of 87 ± 10 % at a dose of 30 fmol/site. Its effect was prevented by the selective activator of L-type calcium channel Bay-K8644 (10 µg/site). Tx3-5 (30 fmol/site) also produced a partial antinociceptive effect in a neuropathic pain model (inhibition of 67 ± 10 %). Additionally, treatment with Tx3-5 (30 fmol/site) nearly abolished cancer-related nociception with similar efficacy in both morphine-sensitive and morphine-tolerant mice (96 ± 7 and 100 % inhibition, respectively). Notably, Tx3-5 did not produce visible adverse effects at doses that produced antinociception and presented a TD50 of 1125 (893-1418) fmol/site. Finally, Tx3-5 did not alter the normal mechanical or thermal sensitivity of the animals or cause immunogenicity. Our results suggest that Tx3-5 is a strong drug candidate for the treatment of painful conditions.
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Analgésicos/uso terapéutico , Dolor en Cáncer/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Neuropéptidos/uso terapéutico , Neurotoxinas/uso terapéutico , Venenos de Araña/uso terapéutico , Analgésicos/efectos adversos , Analgésicos/farmacología , Animales , Agonistas de los Canales de Calcio/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/efectos adversos , Neuropéptidos/farmacología , Neurotoxinas/efectos adversos , Neurotoxinas/farmacología , Nocicepción/efectos de los fármacos , Venenos de Araña/efectos adversos , Venenos de Araña/farmacologíaRESUMEN
The mechanical properties of the local microenvironment may have important influence on the fate and function of adult tissue progenitor cells, altering the regenerative process. This is particularly critical following a myocardial infarction, in which the normal, compliant myocardial tissue is replaced with fibrotic, stiff scar tissue. In this study, we examined the effects of matrix stiffness on adult cardiac side population (CSP) progenitor cell behavior. Ovine and murine CSP cells were isolated and cultured on polydimethylsiloxane substrates, replicating the elastic moduli of normal and fibrotic myocardium. Proliferation capacity and cell cycling were increased in CSP cells cultured on the stiff substrate with an associated reduction in cardiomyogeneic differentiation and accelerated cell ageing. In addition, culture on stiff substrate stimulated upregulation of extracellular matrix and adhesion proteins gene expression in CSP cells. Collectively, we demonstrate that microenvironment properties, including matrix stiffness, play a critical role in regulating progenitor cell functions of endogenous resident CSP cells. Understanding the effects of the tissue microenvironment on resident cardiac progenitor cells is a critical step toward achieving functional cardiac regeneration.
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Células Madre Adultas/fisiología , Dimetilpolisiloxanos/química , Mecanotransducción Celular , Miocitos Cardíacos/fisiología , Células de Población Lateral/fisiología , Nicho de Células Madre , Células Madre Adultas/metabolismo , Animales , Adhesión Celular , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Senescencia Celular , Técnicas de Cocultivo , Módulo de Elasticidad , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Fenotipo , Ovinos , Células de Población Lateral/metabolismo , Factores de TiempoRESUMEN
GABA is an inhibitory neurotransmitter that appears to be associated with the action of volatile anesthetics. These anesthetics potentiate GABA-induced postsynaptic currents by synaptic GABAA receptors, although recent evidence suggests that these agents also significantly affect extrasynaptic GABA receptors. However, the effect of volatile anesthetics on the extracellular concentration of GABA in the central nervous system has not been fully established. In the present study, rat brain cortical slices loaded with [(3)H]GABA were used to investigate the effect of halothane and sevoflurane on the extracellular accumulation of this neurotransmitter. The accumulation of [(3)H]GABA was significantly increased by sevoflurane (0.058, 0.11, 0.23, 0.46, and 0.93 mM) and halothane (0.006, 0.012, 0.024, 0.048, 0072, and 0.096 mM) with an EC50 of 0.26 mM and 35 µM, respectively. TTX (blocker of voltage-dependent Na(+) channels), EGTA (an extracellular Ca(2+) chelator) and BAPTA-AM (an intracellular Ca(2+) chelator) did not interfere with the accumulation of [(3)H]GABA induced by 0.23 mM sevoflurane and 0.048 mM halothane. SKF 89976A, a GABA transporter type 1 (GAT-1) inhibitor, reduced the sevoflurane- and halothane-induced increase in the accumulation of GABA by 57 and 63 %, respectively. Incubation of brain cortical slices at low temperature (17 °C), a condition that inhibits GAT function and reduces GABA release through reverse transport, reduced the sevoflurane- and halothane-induced increase in the accumulation of [(3)H]GABA by 82 and 75 %, respectively, relative to that at normal temperature (37 °C). Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce GABA release through reverse transport, abolished the sevoflurane and halothane effects on the accumulation of [(3)H]GABA. The effect of sevoflurane and halothane did not involve glial transporters because ß-alanine, a blocker of GAT-2 and GAT-3, did not inhibit the effect of the anesthetics. In conclusion, the present study suggests that sevoflurane and halothane increase the accumulation of GABA by inducing the reverse transport of this neurotransmitter. Therefore, volatile anesthetics could interfere with neuronal excitability by increasing the action of GABA on synaptic and extrasynaptic GABA receptors.
Asunto(s)
Anestésicos/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Anestésicos/administración & dosificación , Animales , Calcio/farmacología , Frío , Halotano/administración & dosificación , Halotano/farmacología , Éteres Metílicos/administración & dosificación , Éteres Metílicos/farmacología , Ácidos Nipecóticos/farmacología , Ouabaína/farmacología , Ratas , Sevoflurano , Tetrodotoxina/toxicidad , Tritio/metabolismo , Volatilización , beta-Alanina/farmacologíaRESUMEN
The marine snail peptide ziconotide (ω-conotoxin MVIIA) is used as an analgesic in cancer patients refractory to opioids, but may induce severe adverse effects. Animal venoms represent a rich source of novel drugs, so we investigated the analgesic effects and the side-effects of spider peptide Phα1ß in a model of cancer pain in mice with or without tolerance to morphine analgesia. Cancer pain was induced by the inoculation of melanoma B16-F10 cells into the hind paw of C57BL/6 mice. After 14 days, painful hypersensitivity was detected and Phα1ß or ω-conotoxin MVIIA (10-100 pmol/site) was intrathecally injected to evaluate the development of antinociception and side-effects in control and morphine-tolerant mice. The treatment with Phα1ß or ω-conotoxin MVIIA fully reversed cancer-related painful hypersensitivity, with long-lasting results, at effective doses 50% of 48 (32-72) or 33 (21-53) pmol/site, respectively. Phα1ß produced only mild adverse effects, whereas ω-conotoxin MVIIA induced dose-related side-effects in mice at analgesic doses (estimated toxic dose 50% of 30 pmol/site). In addition, we observed that Phα1ß was capable of controlling cancer-related pain even in mice tolerant to morphine antinociception (100% of inhibition) and was able to partially restore morphine analgesia in such animals (56 ± 5% of inhibition). In this study, Phα1ß was as efficacious as ω-conotoxin MVIIA in inducing analgesia in a model of cancer pain without producing severe adverse effects or losing efficacy in opioid-tolerant mice, indicating that Phα1ß has a good profile for the treatment of cancer pain in patients.
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Analgésicos/farmacología , Melanoma Experimental/tratamiento farmacológico , Dolor/tratamiento farmacológico , Péptidos/farmacología , Venenos de Araña/farmacología , Arañas/metabolismo , Analgésicos/efectos adversos , Animales , Línea Celular Tumoral , Tolerancia a Medicamentos , Masculino , Melanoma Experimental/complicaciones , Ratones , Ratones Endogámicos C57BL , Morfina/efectos adversos , Morfina/farmacología , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Péptidos/efectos adversos , omega-Conotoxinas/efectos adversos , omega-Conotoxinas/farmacologíaRESUMEN
We investigated the effects of cholesterol removal on spontaneous and KCl-evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl-ß-cyclodextrin (MßCD) induced an increase in the frequency of miniature end-plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1-43. Treatment with MßCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MßCD were larger than those from control. In addition, treatment with MßCD reduced the fusion of synaptic vesicles that are mobilized during KCl-evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MßCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl-evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.
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Membrana Celular/fisiología , Colesterol/metabolismo , Unión Neuromuscular/fisiología , Vesículas Sinápticas/fisiología , Animales , Membrana Celular/efectos de los fármacos , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Microelectrodos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Fármacos Neuromusculares/farmacología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/ultraestructura , Cloruro de Potasio/farmacología , Compuestos de Piridinio , Compuestos de Amonio Cuaternario , Rana catesbeiana , Receptores Nicotínicos/metabolismo , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/ultraestructura , Técnicas de Cultivo de Tejidos , beta-Ciclodextrinas/farmacologíaRESUMEN
The effects of trans-resveratrol (1) were evaluated in acute nociception models induced by capsaicin or glutamate in mice, in an attempt to further characterize its mechanism of action. The oral administration of 1 (50 and 100 mg/kg) reduced significantly the licking behavior elicited by capsaicin (1.6 µg/paw) or glutamate (10 µmol/paw). The co-administration of 1 into the mouse paw (200 µg/site) markedly prevented glutamate-induced licking, without affecting capsaicin responses. In addition, the intrathecal (it) injection of 1 (150 to 600 µg/site) greatly reduced the licking behavior caused by capsaicin, but not glutamate. Similarly, the intracerebroventricular injection of 1 (300 µg/site) caused a potent inhibition of capsaicin-induced nociception, while the glutamate responses remained unaffected. However, the co-administration of 1 (300 µg/site) reduced the biting behavior induced by spinal injection of glutamate (30 µg/site, it), leaving capsaicin (6.4 µg/site)-induced biting unaltered. Notably, the oral administration of 1 (100 mg/kg) inhibited significantly the capsaicin-induced increase of c-Fos and COX-2 labeling in the spinal cord and COX-2 expression in the cortex, but failed to affect c-Fos and COX-2 expression in the glutamate model. This study has explored the effects of 1 in both the capsaicin and glutamate models, extending current knowledge on the analgesic effects of trans-resveratrol.
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Analgésicos/farmacología , Estilbenos/farmacología , Analgésicos/administración & dosificación , Animales , Conducta Animal/efectos de los fármacos , Capsaicina/administración & dosificación , Capsaicina/farmacología , Ciclooxigenasa 2/análisis , Ciclooxigenasa 2/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ácido Glutámico/administración & dosificación , Ácido Glutámico/farmacología , Masculino , Ratones , Estructura Molecular , Nocicepción/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/genética , Resveratrol , Estereoisomerismo , Estilbenos/químicaRESUMEN
It is well known that dopamine imbalances are associated with many psychiatric disorders and that the dopaminergic receptor D2 is the main target of antipsychotics. Recently it was shown that levels of two proteins implicated in dopaminergic signaling, Neuronal calcium sensor-1 (NCS-1) and DARPP-32, are altered in the prefrontal cortex (PFC) of both schizophrenic and bipolar disorder patients. NCS-1, which inhibits D2 internalization, is upregulated in the PFC of both patients. DARPP-32, which is a downstream effector of dopamine signaling, integrates the pathways of several neurotransmitters and is downregulated in the PFC of both patients. Here, we used PC12 cells stably overexpressing NCS-1 (PC12-NCS-1 cells) to address the function of this protein in DARPP-32 signaling pathway in vitro. PC12-NCS-1 cells displayed downregulation of the cAMP/PKA pathway, with decreased levels of cAMP and phosphorylation of CREB at Ser133. We also observed decreased levels of total and phosphorylated DARPP-32 at Thr34. However, these cells did not show alterations in the levels of D2 and phosphorylation of DARPP-32 at Thr75. These results indicate that NCS-1 modulates PKA/cAMP signaling pathway. Identification of the cellular mechanisms linking NCS-1 and DARPP-32 may help in the understanding the signaling machinery with potential to be turned into targets for the treatment of schizophrenia and other debilitating psychiatric disorders.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Sensoras del Calcio Neuronal/genética , Neuropéptidos/genética , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Proteínas Sensoras del Calcio Neuronal/metabolismo , Proteínas Sensoras del Calcio Neuronal/fisiología , Neuropéptidos/metabolismo , Neuropéptidos/fisiología , Células PC12 , Fosforilación , Ratas , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Transfección , Regulación hacia Arriba/genéticaRESUMEN
Opioids are the first-line treatment for cancer pain. Incomplete pain relief and the high rate of adverse effects of these compounds bring a need to combine them with other drugs acting on different targets. AIMS: We here evaluate the antinociceptive interaction and adverse events of methadone combined with recombinant Phα1ß, an analgesic toxin from Phoneutria nigriventer. MAIN METHODS: Melanoma was produced by intraplantar inoculation of B16-F10 cells into the right paw. von Frey filaments measured the paw-withdrawal threshold after administration of methadone, Phα1ß, and their combination. The degree of interaction was evaluated using isobolographic analysis. Spontaneous performance and forced motor performance were assessed with the open-field and rotarod tests, respectively. Intestinal function was evaluated by the distance traveled by charcoal and opioid tolerance was induced by daily morphine injections. KEY FINDINGS: Co-administration of Phα1ß with methadone synergistically reverses the melanoma-induced mechanical hypersensitivity. No motor alterations were observed but mild alterations on intestinal function after treatment with the combination that was also capable of restoring morphine analgesia in the tail-flick test after an opioid-induced tolerance. SIGNIFICANCE: Combinatorial treatment with Phα1ß and methadone produces synergistic analgesic potentiation with potential implications to pain treatment even under opioid tolerance conditions.
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Analgésicos/farmacología , Dolor en Cáncer/tratamiento farmacológico , Metadona/administración & dosificación , Manejo del Dolor/métodos , Venenos de Araña/administración & dosificación , Analgésicos Opioides/farmacología , Animales , Conducta Animal , Bloqueadores de los Canales de Calcio/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Tolerancia a Medicamentos , Tracto Gastrointestinal/efectos de los fármacos , Masculino , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias/complicaciones , Factores de TiempoRESUMEN
Abnormal calcium influx and glutamatergic excitotoxicity have been extensively associated with neuronal death in Huntington's disease (HD), a genetic movement disorder. Currently, there is no effective treatment for this fatal condition. The neurotoxin Phα1ß has demonstrated therapeutic effects as a calcium channel blocker, for example during pain control. However, little is known about its neuroprotective effect in HD. Herein, we investigated if Phα1ß is effective in inhibiting neuronal cell death in the BACHD mouse model for HD. We performed intrastriatal injection of Phα1ß in WT and BACHD mice. No side effects or unusual behaviors were observed upon Phα1ß administration. Using three different motor behavior tests, we observed that injection of the toxin in BACHD mice greatly improved the animals' motor-force as seen in the Wire-hang test, and also the locomotor performance, according to the Open field test. NeuN labeling for mature neuron detection revealed that Phα1ß toxin promoted neuronal preservation in the striatum and cortex, when injected locally. Intrastriatal injection of Phα1ß was not able to preserve neurons from the spinal cord and also not revert muscle atrophy in BACHD mice. Finally, we observed that Phα1ß might, at least in part, exert its protective effect by decreasing L-glutamate, measured in cerebrospinal fluid. Our data provide evidence of a novel neuroprotector effect of Phα1ß, paving a path for the development of new approaches to treat HD motor symptoms.
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Enfermedad de Huntington/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Venenos de Araña/administración & dosificación , Animales , Modelos Animales de Enfermedad , Ácido Glutámico/metabolismo , Enfermedad de Huntington/patología , Ratones , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Neuronas/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/patologíaRESUMEN
An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.
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Encéfalo/metabolismo , Enfermedades de la Unión Neuromuscular/etiología , Unión Neuromuscular/metabolismo , Reconocimiento en Psicología/fisiología , Proteínas de Transporte Vesicular de Acetilcolina/deficiencia , Acetilcolina/análisis , Acetilcolina/metabolismo , Animales , Northern Blotting , Southern Blotting , Encéfalo/patología , Encéfalo/fisiopatología , Química Encefálica , Cromatografía Líquida de Alta Presión , Femenino , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Transgénicos , Microdiálisis , Actividad Motora/fisiología , Unión Neuromuscular/patología , Unión Neuromuscular/fisiopatología , Enfermedades de la Unión Neuromuscular/patología , Enfermedades de la Unión Neuromuscular/fisiopatología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular de Acetilcolina/genéticaRESUMEN
Vascular dementia (VD) is a major cognitive disorder originated from a blood flow disruption in the brain. This process leads to chronic cerebral ischemia that deeply affects neuronal tissues and lipid homeostasis. The understanding of cerebral lipid dynamics during chronic ischemia can reveal biomarkers and novel pharmacological targets for the treatment of VD. In this study, we used the Desorption Electrospray Ionization - imaging mass spectrometry (DESI-IMS) technique to map lipids in the rat brain tissues after bilateral common carotid artery occlusion (BCCAO) rat model of chronic cerebral hypoperfusion. The brain imaging enabled the detection of differences in lipids from ischemic and non-ischemic brains. The analysis demonstrated that arachidonic acid (ARA), docosahexaenoic acid (DHA), dihomo-γ-linolenic acid, hydroxyeicosatetraenoic (HETE)-Ala and glycerophosphoethanolamine levels were significantly reduced in the hippocampus and cortex of animals submitted to BCCAO model when compared to control animals. Decanoic acid was increased after 30â¯days of BCCAO model. Partial least squares discriminant analysis (PLS-DA) could discriminate between BCCAO group and the control group, in which γ-linolenic acid (m/z 277) ion and stearic acid (m/z 283) had the highest discrimination potential. Taken together, these findings indicate that lipid dynamics are altered in chronic ischemia-induced by BCCAO in rats and indicate potential biomarkers and pharmacological targets for VD.
Asunto(s)
Isquemia Encefálica/diagnóstico por imagen , Corteza Cerebral/patología , Hipocampo/patología , Lípidos/análisis , Animales , Enfermedades de las Arterias Carótidas/patología , Enfermedad Crónica , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Masculino , Neuronas/patología , Ratas WistarRESUMEN
The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrP(C)) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrP(C) trafficking and tested whether this process controls PrP(C)-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrP(C), induced PrP(C) endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrP(C); however, heterologous expression of PrP(C) reconstituted both PKA and ERK1/2 activation. In contrast, a PrP(C) mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrP(C) endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.
Asunto(s)
Encéfalo/metabolismo , Endocitosis/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Neuronas/metabolismo , Proteínas PrPC/metabolismo , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinaminas/metabolismo , Activación Enzimática/fisiología , Proteínas de Choque Térmico/genética , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Técnicas de Cultivo de Órganos , Proteínas PrPC/genética , Transporte de Proteínas/fisiologíaRESUMEN
Dopamine may alter the phosphorylation state of DARPP-32 that plays a central role in the dopaminergic neurons biology. Studies have shown that DARPP-32/protein phosphatase 1 cascade is a major target for psychostimulants drugs. Methylphenidate is a psychostimulant that acts blocking the dopamine transporter has been used as an effective treatment for Attention Deficit Hyperactivity Disorder. We investigated if methylphenidate could alter DARPP-32 expression in five brain regions (striatum, hippocampus, prefrontal cortex, cortex and cerebellum) in young and adult rats. Our results showed that methylphenidate treatment is able to alter DARPP-32 expression in rat brain. Acute methylphenidate treatment has reduced hippocampal DARPP-32 protein levels in old rats, while chronic methylphenidate treatment has decreased them in old rat hippocampus and young rat cerebellum. It was found an increased cortical expression after chronic methylphenidate administration in old rats. Our results provide the first experimental demonstration that methylphenidate induces changes in total DARPP-32 expression that are posology- and age-related in some rat brain areas, although further studies are needed to shed more light on the mechanisms behind these findings.
Asunto(s)
Envejecimiento/fisiología , Encéfalo/efectos de los fármacos , Fosfoproteína 32 Regulada por Dopamina y AMPc/efectos de los fármacos , Metilfenidato/farmacología , Animales , Trastorno por Déficit de Atención con Hiperactividad/tratamiento farmacológico , Trastorno por Déficit de Atención con Hiperactividad/metabolismo , Trastorno por Déficit de Atención con Hiperactividad/fisiopatología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Dopamina/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Esquema de Medicación , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Ratas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Methylphenidate has been used as an effective treatment for attention deficit hyperactivity disorder (ADHD). Methylphenidate (MPH) blocks dopamine and norepinephrine transporters causing an increase in extracellular levels. The use of psychomotor stimulants continues to rise due to both the treatment of ADHD and illicit abuse. Methylphenidate sensitization mechanism has still poor knowledge. Neuronal calcium sensor 1 was identified as a dopaminergic receptor interacting protein. When expressed in mammalian cells, neuronal calcium sensor 1 attenuates dopamine-induced D2 receptor internalization by a mechanism that involves a reduction in D2 receptor phosphorylation. Neuronal calcium sensor 1 appears to play a pivotal role in regulating D2 receptor function, it will be important to determine if there are alterations in neuronal calcium sensor 1 in neuropathologies associated with deregulation in dopaminergic signaling. Then, we investigated if methylphenidate could alter neuronal calcium sensor 1 expression in five brain regions (striatum, hippocampus, prefrontal cortex, cortex and cerebellum) in young and adult rats. These regions were chosen because some are located in brain circuits related with attention deficit hyperactivity disorder. Our results showed changes in neuronal calcium sensor 1 expression in hippocampus, prefrontal cortex and cerebellum mainly in adult rats. The demonstration that methylphenidate induces changes in neuronal calcium sensor 1 levels in rat brain may help to understand sensitization mechanisms as well as methylphenidate therapeutic effects to improve attention deficit hyperactivity disorder symptoms.
Asunto(s)
Química Encefálica/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Metilfenidato/farmacología , Proteínas Sensoras del Calcio Neuronal/biosíntesis , Neuropéptidos/biosíntesis , Envejecimiento/metabolismo , Animales , Western Blotting , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Densitometría , Relación Dosis-Respuesta a Droga , Proteínas Sensoras del Calcio Neuronal/genética , Neuropéptidos/genética , Ratas , Ratas WistarRESUMEN
Step-down inhibitory avoidance (IA) is usually acquired in one single trial, which makes it ideal for studying processes initiated by training, uncontaminated by prior or further trials, rehearsals, or retrievals. Biochemical events in the hippocampus related to long-term memory (LTM) formation have been extensively studied in rats using a one trial step-down IA task. DARPP-32 (dopamine and cAMP regulated phosphoprotein of Mr 32 kDa) is a cytosolic protein that is selectively enriched in medium spiny neurons in the neostriatum. It has been shown that activation of DARPP-32 and the resultant inhibition of PP-1 activity is critical for the expression of two opposing forms of brain synaptic plasticity, striatal LTD and LTP. Both forms of plasticity are also critically linked to the activation of DA receptors. It has been shown with studies in DARPP-32 KO mice an important role of this protein in mediating the effects of DA on long term changes in neuronal excitability and to our knowledge, no studies have examined the effect of IA task on DARPP-32 expression. In order to demonstrate changes in the protein expression profile we analyzed DARPP-32 levels in the striatum, prefrontal cortex (PFC), hippocampus and entorhinal cortex of Wistar rats after step-down IA learning. Our results showed that IA induced changes on DARPP-32 expression in striatum and hippocampus. DARPP-32 expression changes corroborate with changes in expression and phosphorylation of CREB, NMDA, AMPA after IA that has been reported. These changes suggest that DARPP-32 might play a central role in the IA, as previously described as an integrator of the dopaminergic signal.
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Reacción de Prevención , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Animales , Western Blotting , Fosfoproteína 32 Regulada por Dopamina y AMPc/genética , Electroforesis en Gel de Poliacrilamida , Potenciación a Largo Plazo , Masculino , Plasticidad Neuronal , Fosforilación , Ratas , Ratas WistarRESUMEN
The purpose of the present work was to investigate the pharmacological action of a calcium channel-blocking toxin from the venom of the spider Phonetic nigriventer, Tx3-4 on calcium channels coupled to exocytosis of synaptic vesicles. Tx3-4 blocked KCl-induced exocytosis of synaptic vesicles with an IC50 of 1.1 nM. To investigate whether the target of Tx3-4 overlaps with known calcium channels that mediate calcium entry and exocytosis, we used omega-toxins that interact selectively with neuronal calcium channels. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-4 is P/Q calcium channels. In conclusion, Tx3-4 is a potent inhibitor of calcium channels involved in the KCl-induced exocytosis of synaptic vesicles in brain cortical synaptosomes.
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Canales de Calcio/efectos de los fármacos , Exocitosis/efectos de los fármacos , Neurotoxinas/farmacología , Venenos de Araña/farmacología , Animales , Encéfalo/ultraestructura , Bloqueadores de los Canales de Calcio/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Neuropéptidos/farmacología , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar , Sinaptosomas/efectos de los fármacosRESUMEN
Voltage-gated calcium channels (VGCCs) play a critical role in neuroinflammatory diseases, such as multiple sclerosis (MS). CTK 01512-2 is a recombinant version of the peptide Phα1ß derived from the spider Phoneutria nigriventer, which inhibits N-type VGCC/TRPA1-mediated calcium influx. We investigated the effects of this molecule in the mouse model of experimental autoimmune encephalomyelitis (EAE). The effects of CTK 01512-2 were compared to those displayed by ziconotide-a selective N-type VGCC blocker clinically used for chronic pain-and fingolimod-a drug employed for MS treatment. The intrathecal (i.t.) treatment with CTK 01512-2 displayed beneficial effects, by preventing nociception, body weight loss, splenomegaly, MS-like clinical and neurological scores, impaired motor coordination, and memory deficits, with an efficacy comparable to that observed for ziconotide and fingolimod. This molecule displayed a favorable profile on EAE-induced neuroinflammatory changes, including inflammatory infiltrate, demyelination, pro-inflammatory cytokine production, glial activation, and glucose metabolism in the brain and spinal cord. The recovery of spatial memory, besides a reduction of serum leptin levels, allied to central and peripheral elevation of the anti-inflammatory cytokine IL-10, was solely modulated by CTK 01512-2, dosed intrathecally. The intravenous (i.v.) administration of CTK 01512-2 also reduced the EAE-elicited MS-like symptoms, similarly to that seen in animals that received fingolimod orally. Ziconotide lacked any significant effect when dosed by i.v. route. Our results indicate that CTK 01512-2 greatly improved the neuroinflammatory responses in a mouse model of MS, with a higher efficacy when compared to ziconotide, pointing out this molecule as a promising adjuvant for MS management.