RESUMO
Schwann cells (SCs) are fundamental components of the peripheral nervous system (PNS) of all vertebrates and play essential roles in development, maintenance, function, and regeneration of peripheral nerves. There are distinct populations of SCs including: (1) myelinating SCs that ensheath axons by a specialized plasma membrane, called myelin, which enhances the conduction of electric impulses; (2) non-myelinating SCs, including Remak SCs, which wrap bundles of multiple axons of small caliber, and perysinaptic SCs (PSCs), associated with motor axon terminals at the neuromuscular junction (NMJ). All types of SCs contribute to PNS regeneration through striking morphological and functional changes in response to nerve injury, are affected in peripheral neuropathies and show abnormalities and a diminished plasticity during aging. Therefore, methodological approaches to study and manipulate SCs in physiological and pathophysiological conditions are crucial to expand the present knowledge on SC biology and to devise new therapeutic strategies to counteract neurodegenerative conditions and age-derived denervation. We present here an updated overview of traditional and emerging methodologies for the study of SCs for scientists approaching this research field.
Assuntos
Traumatismos dos Nervos Periféricos , Células de Schwann , Animais , Axônios/metabolismo , Bainha de Mielina/metabolismo , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/metabolismo , Células de Schwann/metabolismoRESUMO
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.
Assuntos
Axônios/fisiologia , Neurônios Motores/citologia , Regeneração Nervosa , Doenças da Junção Neuromuscular/prevenção & controle , Junção Neuromuscular/patologia , Venenos de Aranha/toxicidade , Urocortinas/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Junção Neuromuscular/efeitos dos fármacos , Doenças da Junção Neuromuscular/induzido quimicamente , Doenças da Junção Neuromuscular/metabolismo , Doenças da Junção Neuromuscular/patologia , Terminações Pré-Sinápticas , Ratos , Ratos Sprague-Dawley , Urocortinas/genéticaRESUMO
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.
Assuntos
Axônios/efeitos dos fármacos , Melatonina/farmacologia , Neurônios Motores/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Receptor MT1 de Melatonina/agonistas , Nervo Isquiático/efeitos dos fármacos , Animais , Axônios/metabolismo , Axônios/patologia , Células Cultivadas , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Fosforilação , Ratos Wistar , Receptor MT1 de Melatonina/metabolismo , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Transdução de Sinais , Venenos de Aranha/toxicidade , Fatores de TempoRESUMO
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.
Assuntos
Anexina A2/biossíntese , Peróxido de Hidrogênio/farmacologia , Células de Schwann/metabolismo , Animais , Anexina A2/genética , Anexina A2/metabolismo , Células Cultivadas , Citoesqueleto/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Biossíntese de Proteínas , RNA/biossíntese , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Transcriptoma/efeitos dos fármacosRESUMO
An acute and highly reproducible motor axon terminal degeneration followed by complete regeneration is induced by some animal presynaptic neurotoxins, representing an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that nerve terminals exposed to spider or snake presynaptic neurotoxins degenerate as a result of calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons release signaling molecules derived from mitochondria: hydrogen peroxide, mitochondrial DNA, and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells cocultured with neurons and at neuromuscular junction in vivo through the MAPK pathway. We propose that this inter- and intracellular signaling is involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases.
Assuntos
Axônios/metabolismo , Mitocôndrias/metabolismo , Neurotoxinas/metabolismo , Células de Schwann/metabolismo , Sinapses/metabolismo , Animais , Técnicas de Cocultura , Citocromos c/metabolismo , DNA Mitocondrial/metabolismo , Fagocitose , Serpentes , AranhasRESUMO
An extraordinary property of the peripheral nervous system is that nerve terminals can regenerate after damage caused by different physical, chemical, or biological pathogens. Regeneration is the result of a complex and ill-known interplay among the nerve, the glia, the muscle, the basal lamina and, in some cases, the immune system. This phenomenon has been studied using different injury models mainly in rodents, particularly in mice, where a lesion can be produced in a chosen anatomical area. These approaches differ significantly among them for the nature of the lesion and the final outcomes. We have reviewed here the most common experimental models employed to induce motor axon injury, the relative advantages and drawbacks, and the principal read-outs used to monitor the regenerative process. Recently introduced tools for inducing reversible damage to the motor axon terminal that overcome some of the drawbacks of the more classical approaches are also discussed. Animal models have provided precious information about the cellular components involved in the regenerative process and on its electrophysiological features. Methods and tools made available recently allow one to identify and study molecules that are involved in the crosstalk among the components of the endplate. The time-course of the intercellular signaling and of the intracellular pathways activated will draw a picture of the entire process of regeneration as seen from a privileged anatomical site of observation. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
Assuntos
Axônios/metabolismo , Músculos/fisiologia , Regeneração Nervosa/fisiologia , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/fisiologia , Animais , Humanos , Neuroglia/fisiologiaRESUMO
The neuromuscular junction is a tripartite synapse composed of the presynaptic nerve terminal, the muscle and perisynaptic Schwann cells. Its functionality is essential for the execution of body movements and is compromised in a number of disorders, including Miller Fisher syndrome, a variant of Guillain-Barré syndrome: this autoimmune peripheral neuropathy is triggered by autoantibodies specific for the polysialogangliosides GQ1b and GT1a present in motor axon terminals, including those innervating ocular muscles, and in sensory neurons. Their binding to the presynaptic membrane activates the complement cascade, leading to a nerve degeneration that resembles that caused by some animal presynaptic neurotoxins. Here we have studied the intra- and inter-cellular signaling triggered by the binding and complement activation of a mouse monoclonal anti-GQ1b/GT1a antibody to primary cultures of spinal cord motor neurons and cerebellar granular neurons. We found that a membrane attack complex is rapidly assembled following antibody binding, leading to calcium accumulation, which affects mitochondrial functionality. Consequently, using fluorescent probes specific for mitochondrial hydrogen peroxide, we found that this reactive oxygen species is rapidly produced by mitochondria of damaged neurons, and that it triggers the activation of the MAP kinase pathway in Schwann cells. These results throw light on the molecular and cellular pathogenesis of Miller Fisher syndrome, and may well be relevant to other pathologies of the motor axon terminals, including some subtypes of the Guillain Barré syndrome.
Assuntos
Peróxido de Hidrogênio/metabolismo , Síndrome de Miller Fisher/complicações , Síndrome de Miller Fisher/patologia , Mitocôndrias/metabolismo , Terminações Pré-Sinápticas/metabolismo , Células de Schwann/metabolismo , Animais , Células Cultivadas , Cerebelo/citologia , Técnicas de Cocultura , Modelos Animais de Doenças , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Gangliosídeos/imunologia , Gangliosídeos/metabolismo , Imunoglobulina G/farmacologia , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
The SNARE proteins VAMP/synaptobrevin, SNAP-25 and syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex, and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine. Experiments performed with botulinum neurotoxins led to the identification of one arginine residue in SNAP-25 and one aspartate residue in syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein-protein interactions between SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Canais Iônicos/metabolismo , Junção Neuromuscular/fisiologia , Proteínas Qa-SNARE/metabolismo , Transmissão Sináptica , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Animais Geneticamente Modificados , Toxinas Botulínicas/metabolismo , Células Cultivadas , Proteínas de Drosophila/genética , Engenharia Genética , Larva , Modelos Químicos , Mutação/genética , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/genética , Proteínas Qa-SNARE/genética , Estereoisomerismo , Proteína 25 Associada a Sinaptossoma/genéticaRESUMO
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.
Assuntos
Antivenenos , Indenos , Mordeduras de Serpentes , Humanos , Camundongos , Animais , Antivenenos/uso terapêutico , Mordeduras de Serpentes/complicações , Mordeduras de Serpentes/tratamento farmacológico , Receptores de Melatonina/uso terapêutico , Venenos de Serpentes , Recuperação de Função Fisiológica , Cálcio , Serpentes , BungarusRESUMO
An analysis of SNAP-25 isoform sequences indicates that there is a highly conserved arginine residue (198 in vertebrates, 206 in the genus Drosophila) within the C-terminal region, which is cleaved by botulinum neurotoxin A, with consequent blockade of neuroexocytosis. The possibility that it may play an important role in the function of the neuroexocytosis machinery was tested at neuromuscular junctions of Drosophila melanogaster larvae expressing SNAP-25 in which Arg206 had been replaced by alanine. Electrophysiological recordings of spontaneous and evoked neurotransmitter release under different conditions as well as testing for the assembly of the SNARE complex indicate that this residue, which is at the P(1)' position of the botulinum neurotoxin A cleavage site, plays an essential role in neuroexocytosis. Computer graphic modelling suggests that this arginine residue mediates protein-protein contacts within a rosette of SNARE complexes that assembles to mediate the fusion of synaptic vesicles with the presynaptic plasma membrane.
Assuntos
Encéfalo/metabolismo , Drosophila melanogaster/fisiologia , Proteínas Mutantes/metabolismo , Junção Neuromuscular/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Animais Geneticamente Modificados , Arginina/genética , Encéfalo/patologia , Sinalização do Cálcio , Células Cultivadas , Clonagem Molecular , Eletrofisiologia , Potenciais Evocados/genética , Exocitose/genética , Larva , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Junção Neuromuscular/genética , Transmissão Sináptica/genética , Proteína 25 Associada a Sinaptossoma/genéticaRESUMO
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.
Assuntos
Melatonina , Potenciais de Ação/fisiologia , Adulto , Animais , Eletromiografia/métodos , Humanos , Melatonina/farmacologia , Camundongos , Músculo Esquelético/fisiologia , Nervo Isquiático/fisiologia , Transmissão SinápticaRESUMO
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.
Assuntos
Cobras Corais , Mordeduras de Serpentes , Animais , Antivenenos , Venenos Elapídicos/toxicidade , Elapidae , Humanos , Receptores CXCR4 , Venenos de SerpentesRESUMO
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.
Assuntos
Axônios , Fator de Crescimento do Tecido Conjuntivo , Peróxido de Hidrogênio , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Animais , Camundongos , Axônios/fisiologia , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Peróxido de Hidrogênio/metabolismo , Camundongos Transgênicos , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Células de Schwann/metabolismoRESUMO
Tetanus and botulinum neurotoxins, produced by anaerobic bacteria of the genus Clostridium, are the most toxic proteins known and are the sole responsible for the pathogenesis of tetanus and botulism. They enter peripheral cholinergic nerve terminals and cleave proteins of the neuroexocytosis apparatus causing a persistent, but reversible, inhibition of neurotransmitter release. Botulinum neurotoxins are used in the therapy of many human syndromes caused by hyperactive cholinergic nerve terminals. Here we focus on the many advances that were recently made on the understanding of their molecular mechanism of action and on their use in human therapy.
RESUMO
The peripheral nervous system has retained through evolution the capacity to repair and regenerate after assault from a variety of physical, chemical, or biological pathogens. Regeneration relies on the intrinsic abilities of peripheral neurons and on a permissive environment, and it is driven by an intense interplay among neurons, the glia, muscles, the basal lamina, and the immune system. Indeed, extrinsic signals from the milieu of the injury site superimpose on genetic and epigenetic mechanisms to modulate cell intrinsic programs. Here, we will review the main intrinsic and extrinsic mechanisms allowing severed peripheral axons to re-grow, and discuss some alarm mediators and pro-regenerative molecules and pathways involved in the process, highlighting the role of Schwann cells as central hubs coordinating multiple signals. A particular focus will be provided on regeneration at the neuromuscular junction, an ideal model system whose manipulation can contribute to the identification of crucial mediators of nerve re-growth. A brief overview on regeneration at sensory terminals is also included.
Assuntos
Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Sistema Nervoso Periférico/fisiologia , Células de Schwann/metabolismo , HumanosRESUMO
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.
Assuntos
Venenos Elapídicos/toxicidade , Indazóis/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Paralisia/terapia , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores CXCR4/agonistas , Potenciais de Ação/efeitos dos fármacos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacosRESUMO
Snake pre-synaptic phospholipase A(2) neurotoxins paralyse the neuromuscular junction by releasing phospholipid hydrolysis products that alter curvature and permeability of the pre-synaptic membrane. Here, we report results deriving from the first chemical analysis of the action of these neurotoxic phospholipases in neurons, made possible by the use of high sensitivity mass spectrometry. The time-course of the phospholipase A(2) activity (PLA(2)) hydrolysis of notexin, beta-bungarotoxin, taipoxin and textilotoxin acting in cultured neurons was determined. At variance from their enzymatic activities in vitro, these neurotoxins display comparable kinetics of lysophospholipid release in neurons, reconciling the large discrepancy between their in vivo toxicities and their in vitro enzymatic activities. The ratios of the lyso derivatives of phosphatidyl choline, ethanolamine and serine obtained here together with the known distribution of these phospholipids among cell membranes, suggest that most PLA(2) hydrolysis takes place on the cell surface. Although these toxins were recently shown to enter neurons, their intracellular hydrolytic action and the activation of intracellular PLA(2)s appear to contribute little, if any, to the phospholipid hydrolysis measured here.
Assuntos
Venenos Elapídicos/farmacologia , Neurônios/efeitos dos fármacos , Fosfolipases A2/análise , Venenos de Serpentes/farmacologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Células Cultivadas , Cerebelo/citologia , Venenos Elapídicos/classificação , Lipídeos/análise , Neuroblastoma , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipases A2/metabolismo , Ratos , Fatores de TempoRESUMO
OBJECTIVE: To test whether the signaling axis CXCL12α-CXCR4 is activated upon crush/cut of the sciatic nerve and to test the activity of NUCC-390, a new CXCR4 agonist, in promoting nerve recovery from damage. METHODS: The sciatic nerve was either crushed or cut. Expression and localization of CXCL12α and CXCR4 were evaluated by imaging with specific antibodies. Their functional involvement in nerve regeneration was determined by antibody-neutralization of CXCL12α, and by the CXCR4 specific antagonist AMD3100, using as quantitative read-out the compound muscle action potential (CMAP). NUCC-390 activity on nerve regeneration was determined by imaging and CMAP recordings. RESULTS: CXCR4 is expressed at the injury site within the axonal compartment, whilst its ligand CXCL12α is expressed in Schwann cells. The CXCL12α-CXCR4 axis is involved in the recovery of neurotransmission of the injured nerve. More importantly, the small molecule NUCC-390 is a strong promoter of the functional and anatomical recovery of the nerve, by acting very similarly to CXCL12α. This pharmacological action is due to the capability of NUCC-390 to foster elongation of motor neuron axons both in vitro and in vivo. INTERPRETATION: Imaging and electrophysiological data provide novel and compelling evidence that the CXCL12α-CXCR4 axis is involved in sciatic nerve repair after crush/cut. This makes NUCC-390 a strong candidate molecule to stimulate nerve repair by promoting axonal elongation. We propose this molecule to be tested in other models of neuronal damage, to lay the basis for clinical trials on the efficacy of NUCC-390 in peripheral nerve repair in humans.
Assuntos
Axônios/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Indazóis/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/lesões , Neuropatia Ciática/tratamento farmacológico , Animais , Benzilaminas , Ciclamos , Modelos Animais de Doenças , Compostos Heterocíclicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/antagonistas & inibidoresRESUMO
The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12α is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL12α potently stimulates the functional recovery of damaged neuromuscular junctions via interaction with CXCR4. This result prompted us to test the neuroregeneration activity of small molecules acting as CXCR4 agonists, endowed with better pharmacokinetics with respect to the natural ligand. We focused on NUCC-390, recently shown to activate CXCR4 in a cellular system. We designed a novel and convenient chemical synthesis of NUCC-390, which is reported here. NUCC-390 was tested for its capability to induce the regeneration of motor axon terminals completely degenerated by the presynaptic neurotoxin α-Latrotoxin. NUCC-390 was found to strongly promote the functional recovery of the neuromuscular junction, as assayed by electrophysiology and imaging. This action is CXCR4 dependent, as it is completely prevented by AMD3100, a well-characterized CXCR4 antagonist. These data make NUCC-390 a strong candidate to be tested in human therapy to promote nerve recovery of function after different forms of neurodegeneration.
Assuntos
Indazóis/farmacologia , Neurônios Motores/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Piperidinas/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Piridinas/farmacologia , Receptores CXCR4/agonistas , Animais , Benzilaminas , Ciclamos , Compostos Heterocíclicos/farmacologia , Camundongos , Camundongos Endogâmicos , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Degeneração Neural/induzido quimicamente , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Terminações Pré-Sinápticas/patologia , Terminações Pré-Sinápticas/fisiologia , Cultura Primária de Células , Ratos , Venenos de Aranha/toxicidadeRESUMO
The study of the effect of snake presynaptic neurotoxins with phospholipase A2 activity on nerve terminals has recently unveiled the inhibitory action of a lysophosphatidylcholine (LysoPC)/fatty acid mixture. We report here that these neurotoxins have no activity on Drosophila melanogaster nerve terminals. However, a 1:1 mixture of LysoPC and oleic acid induces an early increase, followed by an inhibition of both evoked and spontaneous neurotransmitter release. This effect is also induced by LysoPC alone. The present findings provide an indirect evidence that the lipid hemifusion-to-pore transition is a key event in neuroexocytosis in Drosophila. Moreover, these findings substantiate the use of LysoPC as a general agonist of membrane fusion at nerve terminals. This novel tool could contribute to the unraveling of the molecular steps involved in neuroexocytosis, particularly in Drosophila, where it is straightforward to combine it with electrophysiology and genetics.