RESUMEN
Snake envenoming is a major, but neglected, tropical disease. Among venomous snakes, those inducing neurotoxicity such as kraits (Bungarus genus) cause a potentially lethal peripheral neuroparalysis with respiratory deficit in a large number of people each year. In order to prevent the development of a deadly respiratory paralysis, hospitalization with pulmonary ventilation and use of antivenoms are the primary therapies currently employed. However, hospitals are frequently out of reach for envenomated patients and there is a general consensus that additional, non-expensive treatments, deliverable even long after the snake bite, are needed. Traumatic or toxic degenerations of peripheral motor neurons cause a neuroparalysis that activates a pro-regenerative intercellular signaling program taking place at the neuromuscular junction (NMJ). We recently reported that the intercellular signaling axis melatonin-melatonin receptor 1 (MT1) plays a major role in the recovery of function of the NMJs after degeneration of motor axon terminals caused by massive Ca2+ influx. Here we show that the small chemical MT1 agonists: Ramelteon and Agomelatine, already licensed for the treatment of insomnia and depression, respectively, are strong promoters of the neuroregeneration after paralysis induced by krait venoms in mice, which is also Ca2+ mediated. The venom from a Bungarus species representative of the large class of neurotoxic snakes (including taipans, coral snakes, some Alpine vipers in addition to other kraits) was chosen. The functional recovery of the NMJ was demonstrated using electrophysiological, imaging and lung ventilation detection methods. According to the present results, we propose that Ramelteon and Agomelatine should be tested in human patients bitten by neurotoxic snakes acting presynaptically to promote their recovery of health. Noticeably, these drugs are commercially available, safe, non-expensive, have a long bench life and can be administered long after a snakebite even in places far away from health facilities.
Asunto(s)
Antivenenos , Indenos , Mordeduras de Serpientes , Humanos , Ratones , Animales , Antivenenos/uso terapéutico , Mordeduras de Serpientes/complicaciones , Mordeduras de Serpientes/tratamiento farmacológico , Receptores de Melatonina/uso terapéutico , Venenos de Serpiente , Recuperación de la Función , Calcio , Serpientes , BungarusRESUMEN
Regeneration of the neuromuscular junction (NMJ) leverages on extensive exchange of factors released from motor axon terminals (MATs), muscle fibers and perisynaptic Schwann cells (PSCs), among which hydrogen peroxide (H2O2) is a major pro-regenerative signal. To identify critical determinants of NMJ remodeling in response to injury, we performed temporal transcriptional profiling of NMJs from 2 month-old mice during MAT degeneration/regeneration, and cross-referenced the differentially expressed genes with those elicited by H2O2 in SCs. We identified an enrichment in extracellular matrix (ECM) transcripts, including Connective Tissue Growth Factor (Ctgf), which is usually expressed during development. We discovered that Ctgf levels are increased in a Yes-associated protein (YAP)-dependent fashion in response to rapid, local H2O2 signaling generated by stressed mitochondria in the injured sciatic nerve, a finding highlighting the importance of signals triggered by mechanical force to motor nerve repair. Through sequestration of Ctgf or inactivation of H2O2, we delayed the recovery of neuromuscular function by impairing SC migration and, in turn, axon-oriented re-growth. These data indicate that H2O2 and its downstream effector Ctgf are pro-regenerative factors that enable axonal growth, and reveal a striking ECM remodeling process during nerve regeneration upon local H2O2 signaling. Our study identifies key transcriptomic changes at the regenerating NMJ, providing a rich source of pro-regenerative factors with potential for alleviating the consequences of peripheral nerve injuries.
Asunto(s)
Axones , Factor de Crecimiento del Tejido Conjuntivo , Peróxido de Hidrógeno , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos , Animales , Ratones , Axones/fisiología , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Peróxido de Hidrógeno/metabolismo , Ratones Transgénicos , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/fisiopatología , Células de Schwann/metabolismoRESUMEN
Compound muscle action potential (CMAP) recordings provide a sensitive electromyographic approach to measure nerve conduction and assess neuromuscular junction functionality in humans and rodents. In humans, it represents a diagnostic tool for neuromuscular disorders. In rodents, this approach is widely employed to dissect the molecular mechanisms driving peripheral nerve degeneration/regeneration, as well as to evaluate the effect of candidate pro-regenerative compounds. The method described here allows recording CMAP from the gastrocnemius muscle of mice after sciatic nerve stimulation. We report some representative traces of CMAP recorded from adult, healthy mice, after sciatic nerve compression and during neurotransmission recovery stimulated by melatonin administration.
Asunto(s)
Melatonina , Potenciales de Acción/fisiología , Adulto , Animales , Electromiografía/métodos , Humanos , Melatonina/farmacología , Ratones , Músculo Esquelético/fisiología , Nervio Ciático/fisiología , Transmisión SinápticaRESUMEN
Snake envenoming is a major but neglected human disease in tropical and subtropical regions. Among venomous snakes in the Americas, coral snakes of the genus Micrurus are particularly dangerous because they cause a peripheral neuroparalysis that can persist for many days or, in severe cases, progress to death. Ventilatory support and the use of snake species-specific antivenoms may prevent death from respiratory paralysis in most cases. However, there is a general consensus that additional and non-expensive treatments that can be delivered even long after the snake bite are needed. Neurotoxic degeneration of peripheral motor neurons activates pro-regenerative intercellular signaling programs, the greatest of which consist of the chemokine CXCL12α, produced by perisynaptic Schwann cells, which act on the CXCR4 receptor expressed on damaged neuronal axons. We recently found that the CXCR4 agonist NUCC-390 promotes axonal growth. Here, we show that the venom of the highly neurotoxic snake Micrurus nigrocinctus causes a complete degeneration of motor axon terminals of the soleus muscle, followed by functional regeneration whose time course is greatly accelerated by NUCC-390. These results suggest that NUCC-390 is a potential candidate for treating human patients envenomed by Micrurus nigrocinctus as well as other neurotoxic Micrurus spp. in order to improve the recovery of normal neuromuscular physiology, thus reducing the mortality and hospital costs of envenoming.
Asunto(s)
Serpientes de Coral , Mordeduras de Serpientes , Animales , Antivenenos , Venenos Elapídicos/toxicidad , Elapidae , Humanos , Receptores CXCR4 , Venenos de SerpienteRESUMEN
We used α-Latrotoxin (α-LTx), the main neurotoxic component of the black widow spider venom, which causes degeneration of the neuromuscular junction (NMJ) followed by a rapid and complete regeneration, as a molecular tool to identify by RNA transcriptomics factors contributing to the structural and functional recovery of the NMJ. We found that Urocortin 2 (UCN2), a neuropeptide involved in the stress response, is rapidly expressed at the NMJ after acute damage and that inhibition of CRHR2, the specific receptor of UCN2, delays neuromuscular transmission rescue. Experiments in neuronal cultures show that CRHR2 localises at the axonal tips of growing spinal motor neurons and that its expression inversely correlates with synaptic maturation. Moreover, exogenous UCN2 enhances the growth of axonal sprouts in cultured neurons in a CRHR2-dependent manner, pointing to a role of the UCN2-CRHR2 axis in the regulation of axonal growth and synaptogenesis. Consistently, exogenous administration of UCN2 strongly accelerates the regrowth of motor axon terminals degenerated by α-LTx, thereby contributing to the functional recovery of neuromuscular transmission after damage. Taken together, our results posit a novel role for UCN2 and CRHR2 as a signalling axis involved in NMJ regeneration.
Asunto(s)
Axones/fisiología , Neuronas Motoras/citología , Regeneración Nerviosa , Enfermedades de la Unión Neuromuscular/prevención & control , Unión Neuromuscular/patología , Venenos de Araña/toxicidad , Urocortinas/metabolismo , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Unión Neuromuscular/efectos de los fármacos , Enfermedades de la Unión Neuromuscular/inducido químicamente , Enfermedades de la Unión Neuromuscular/metabolismo , Enfermedades de la Unión Neuromuscular/patología , Terminales Presinápticos , Ratas , Ratas Sprague-Dawley , Urocortinas/genéticaRESUMEN
Melatonin is an ancient multi-tasking molecule produced by the pineal gland and by several extrapineal tissues. A variety of activities has been ascribed to this hormone in different physiological and pathological contexts, but little is known about its role in peripheral neuroregeneration. Here, we have exploited two different types of injury to test the capability of melatonin to stimulate regeneration of motor axons: (a) the acute and reversible presynaptic degeneration induced by the spider neurotoxin α-Latrotoxin and (b) the compression/transection of the sciatic nerve. We found that in both cases melatonin administration accelerates the process of nerve repair. This pro-regenerative action is MT1 -mediated, and at least in part due to a sustained activation of the ERK1/2 pathway. These findings reveal a receptor-mediated, pro-regenerative action of melatonin in vivo that holds important clinical implications, as it posits melatonin as a safe candidate molecule for the treatment of a number of peripheral neurodegenerative conditions.
Asunto(s)
Axones/efectos de los fármacos , Melatonina/farmacología , Neuronas Motoras/efectos de los fármacos , Regeneración Nerviosa/efectos de los fármacos , Traumatismos de los Nervios Periféricos/tratamiento farmacológico , Receptor de Melatonina MT1/agonistas , Nervio Ciático/efectos de los fármacos , Animales , Axones/metabolismo , Axones/patología , Células Cultivadas , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/patología , Fosforilación , Ratas Wistar , Receptor de Melatonina MT1/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Nervio Ciático/patología , Transducción de Señal , Venenos de Araña/toxicidad , Factores de TiempoRESUMEN
Envenomation by snakes is a major neglected human disease. Hospitalization and use of animal-derived antivenom are the primary therapeutic supports currently available. There is consensus that additional, not expensive, treatments that can be delivered even long after the snake bite are needed. We recently showed that the drug dubbed NUCC-390 shortens the time of recovery from the neuroparalysis caused by traumatic or toxic degeneration of peripheral motor neurons. These syndromes are characterized by the activation of a pro-regenerative molecular axis, consisting of the CXCR4 receptor expressed at the damaged site in neuronal axons and by the release of its ligand CXCL12α, produced by surrounding Schwann cells. This intercellular signaling axis promotes axonal growth and functional recovery from paralysis. NUCC-390 is an agonist of CXCR4 acting similarly to CXCL12α. Here, we have tested its efficacy in a murine model of neuroparalytic envenoming by a Papuan Taipan (Oxyuranus scutellatus) where a degeneration of the motor axon terminals caused by the presynaptic PLA2 toxin Taipoxin, contained in the venom, occurs. Using imaging of the neuromuscular junction and electrophysiological analysis, we found that NUCC-390 administration after injection of either the purified neuroparalytic Taipoxin or the whole Taipan venom, significantly accelerates the recovery from paralysis. These results indicate that NUCC-390, which is non-toxic in mice, should be considered for trials in humans to test its efficacy in accelerating the recovery from the peripheral neuroparalysis induced by Taipans. NUCC-390 should be tested as well in the envenomation by other snakes that cause neuroparalytic syndromes in humans. NUCC-390 could become an additional treatment, common to many snake envenomings, that can be delivered after the bite to reduce death by respiratory deficits and to shorten and improve functional recovery.
Asunto(s)
Venenos Elapídicos/toxicidad , Indazoles/farmacología , Unión Neuromuscular/efectos de los fármacos , Parálisis/terapia , Piperidinas/farmacología , Piridinas/farmacología , Receptores CXCR4/agonistas , Potenciales de Acción/efectos de los fármacos , Animales , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/efectos de los fármacosRESUMEN
Schwann cells are key players in neuro-regeneration: They sense "alarm" signals released by degenerating nerve terminals and differentiate toward a proregenerative phenotype, with phagocytosis of nerve debris and nerve guidance. At the murine neuromuscular junction, hydrogen peroxide (H2O2) is a key signal of Schwann cells' activation in response to a variety of nerve injuries. Here we report that Schwann cells exposed to low doses of H2O2 rewire the expression of several RNAs at both transcriptional and translational levels. Among the genes positively regulated at both levels, we identified an enriched cluster involved in cytoskeleton remodeling and cell migration, with the Annexin (Anxa) proteins being the most represented family. We show that both Annexin A2 (Anxa2) transcript and protein accumulate at the tips of long pseudopods that Schwann cells extend upon H2O2 exposure. Interestingly, Schwann cells reply to this signal and to nerve injury by locally translating Anxa2 in pseudopods, and undergo an extensive cytoskeleton remodeling. Our results show that, similarly to neurons, Schwann cells take advantage of local protein synthesis to change shape and move toward damaged axonal terminals to facilitate axonal regeneration.