RESUMEN
Voltage-gated CaV2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. CaV2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathologic pain, but a biological function of nociceptor CaV2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional CaV2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that CaV2.2 channels in TRPV1-nociceptor endings are activated by capsaicin-induced depolarization and contribute to increased intracellular calcium. Capsaicin induces hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat hypersensitivity depends on peripheral CaV2.2 channel activity, and especially a cell-type-specific CaV2.2 splice isoform. CaV2.2 channels at peripheral nerve endings might be important therapeutic targets to mitigate certain forms of chronic pain.SIGNIFICANCE STATEMENT It is generally assumed that nociceptor termini in the spinal cord dorsal horn are the functionally significant sites of CaV2.2 channel in control of transmitter release and the transmission of sensory information from the periphery to central sites. We show that peripheral CaV2.2 channels are essential for the classic heat hypersensitivity response to develop in skin following capsaicin exposure. This function of CaV2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related CaV2.1 channels. Our findings suggest that interrupting CaV2.2-dependent calcium entry in skin might reduce heat hypersensitivity that develops after noxious heat exposure and may limit the degree of heat hypersensitivity associated with certain other forms of pain.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Calcio/metabolismo , Hiperalgesia/metabolismo , Neuronas/fisiología , Nociceptores/fisiología , Terminales Presinápticos/metabolismo , Piel/inervación , Asta Dorsal de la Médula Espinal/metabolismo , Animales , Calor , Ratones , Nocicepción/fisiología , Estimulación Física , Piel/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Many cellular and physiological processes are coordinated by regulatory networks that produce a remarkable complexity of transcript isoforms. In the mammalian nervous system, alternative pre-mRNA splicing generates functionally distinct isoforms that play key roles in normal physiology, supporting development, plasticity, complex behaviors, and cognition. Neuronal splicing programs controlled by RNA-binding proteins, are influenced by chromatin modifications and can exhibit neuronal subtype specificity. As highlighted in recent publications, aberrant alternative splicing is a major contributor to disease phenotypes. Therefore, understanding the underlying mechanisms of alternative splicing regulation and identifying functional splicing isoforms with critical phenotypic roles are expected to provide a comprehensive resource for therapeutic development, as illuminated by recent successful interventions of spinal muscular atrophy. Here, we discuss the latest progress in the study of the emerging complexity of alternative splicing mechanisms in neurons, and how these findings inform new therapies to correct and control splicing defects.
Asunto(s)
Empalme Alternativo/fisiología , Trastorno del Espectro Autista/terapia , Atrofia Muscular Espinal/terapia , Neuronas/metabolismo , Animales , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Isoformas de Proteínas/metabolismo , Empalme del ARNRESUMEN
Several single nucleotide polymorphisms (SNPs) in the Mu Opioid Receptor gene (OPRM1) have been identified and associated with a wide variety of clinical phenotypes related both to pain sensitivity and analgesic requirements. The A118G and other potentially functional OPRM1 SNPs show significant differences in their allele distributions among populations. However, they have not been properly addressed in a population genetic analysis. Population stratification could lead to erroneous conclusions when they are not taken into account in association studies. The aim of our study was to analyze OPRM1 SNP variability by comparing population samples of the International Hap Map database and to analyze a new population sample from the city of Corrientes, Argentina. The results confirm that OPRM1 SNP variability differs among human populations and displays a clear ancestry genetic structure, with three population clusters: Africa, Asia, and Europe-America.
RESUMEN
Cell-specific alternative splicing modulates myriad cell functions and is disrupted in disease. The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors. In sensory neurons, cell-specific alternative splicing of the presynaptic CaV channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated is unknown. We find that cell and exon-specific DNA hypomethylation permits CTCF binding, the master regulator of mammalian chromatin structure, which, in turn, controls splicing in a DRG-derived cell line. In vivo, hypomethylation of an alternative exon specifically in nociceptors, likely permits CTCF binding and expression of CaV2.2 channel isoforms with increased opioid sensitivity in mice. Following nerve injury, exon methylation is increased, and splicing is disrupted. Our studies define the molecular mechanisms of cell-specific alternative splicing of a functionally validated exon in normal and disease states - and reveal a potential target for the treatment of chronic pain.
Asunto(s)
Empalme Alternativo , Factor de Unión a CCCTC/metabolismo , Canales de Calcio Tipo N/genética , Metilación de ADN , Exones , Neuronas/metabolismo , Animales , Canales de Calcio Tipo N/fisiología , Linaje de la Célula , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/fisiología , ADN Metiltransferasa 3A , Ganglios Espinales/metabolismo , Ratones , Traumatismos de los Nervios Periféricos/metabolismo , Canales Catiónicos TRPV/fisiologíaRESUMEN
Dynamic changes in alternative splicing during the life cycle of neurons support development and plasticity, and are implicated in disease pathology. Cell-specific alternative splicing programs coordinate exon selection across networks of functionally connected genes. In this opinion piece, we highlight recent publications that identify some of the molecular mechanisms-RNA and DNA binding proteins and epigenetic modifications-which direct cell-specific exon selection during pre-mRNA splicing. Aberrant splicing patterns are signature features of a growing number of diseases of the nervous system. Recent publications demonstrate the value of delineating basic mechanisms that dictate exon choice to inform the development of new therapeutic strategies that correct or compensate for damaging deficits in alternative splicing.
Asunto(s)
Empalme Alternativo , Neuronas , Exones , Empalme del ARNRESUMEN
Cell-specific regulation of gene expression is important for maintaining cortical excitatory/inhibitory balance. In this issue of Neuron, Vuong et al. (2018) reveal an unlikely role for a broadly expressed RNA binding protein, Rbfox1, in protecting inhibitory transmission in the hippocampus.
Asunto(s)
Proteína 1 de Membrana Asociada a Vesículas , Animales , Neuronas , Factores de Empalme de ARN , Proteínas de Unión al ARN/genética , Transmisión SinápticaRESUMEN
The synaptic adhesion molecules Neurexin and Neuroligin alter the development and function of synapses and are linked to autism in humans. In C. elegans, post-synaptic Neurexin (NRX-1) and pre-synaptic Neuroligin (NLG-1) mediate a retrograde synaptic signal that inhibits acetylcholine (ACh) release at neuromuscular junctions. Here, we show that the retrograde signal decreases ACh release by inhibiting the function of pre-synaptic UNC-2/CaV2 calcium channels. Post-synaptic NRX-1 binds to an auxiliary subunit of pre-synaptic UNC-2/CaV2 channels (UNC-36/α2δ), decreasing UNC-36 abundance at pre-synaptic elements. Retrograde inhibition is mediated by a soluble form of NRX-1's ectodomain, which is released from the post-synaptic membrane by the SUP-17/ADAM10 protease. Mammalian Neurexin-1α binds α2δ-3 and decreases CaV2.2 current in transfected cells, whereas Neurexin-1α has no effect on CaV2.2 reconstituted with α2δ-1 and α2δ-2. Collectively, these results suggest that α-Neurexin binding to α2δ is a conserved mechanism for regulating synaptic transmission.
Asunto(s)
Fenómenos Biofísicos/fisiología , Canales de Calcio Tipo N/metabolismo , Glicoproteínas/metabolismo , Neuropéptidos/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Acetilcolina/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Subunidades de Proteína/metabolismoRESUMEN
Spinal Muscular Atrophy (SMA) is caused by diminished Survival of Motor Neuron (SMN) protein, leading to neuromuscular junction (NMJ) dysfunction and spinal motor neuron (MN) loss. Here, we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs. Loss of the C. elegans SMN ortholog, SMN-1, causes NMJ defects. We found that increased levels of the C. elegans Gemin3 ortholog, MEL-46, ameliorates these defects. Increased MEL-46 levels also restored perturbed microRNA (miR-2) function in smn-1(lf) animals. We determined that miR-2 regulates expression of the C. elegans M2 muscarinic receptor (m2R) ortholog, GAR-2. GAR-2 loss ameliorated smn-1(lf) and mel-46(lf) synaptic defects. In an SMA mouse model, m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects. Collectively, these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation, followed by increased m2R expression, and neuronal dysfunction in SMA.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , MicroARNs/metabolismo , Atrofia Muscular Espinal/fisiopatología , Receptor Muscarínico M2/análisis , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Animales , ARN Helicasas DEAD-box/metabolismo , Modelos Animales de EnfermedadRESUMEN
The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G protein-coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the voltage-gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and agonist-dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Ghrelina/metabolismo , Hipotálamo/fisiología , Neuronas/fisiología , Receptores de Ghrelina/metabolismo , Animales , Secuencia de Bases , Calcio/metabolismo , Señalización del Calcio/fisiología , Células Cultivadas , Células HEK293 , Humanos , Activación del Canal Iónico/fisiología , Ratones , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Receptores de Ghrelina/genéticaRESUMEN
Nervous system (NS) activity participates in metabolic homeostasis by detecting peripheral signal molecules derived from food intake and energy balance. High quality diets are thought to include fiber-rich foods like whole grain rice, breads, cereals, and grains. Several studies have associated high consumption of fiber-enriched diets with a reduced risk of diabetes, obesity, and gastrointestinal disorders. In the lower intestine, anaerobic fermentation of soluble fibers by microbiota produces short chain fatty acids (SCFAs), key energy molecules that have a recent identified leading role in the intestinal gluconeogenesis, promoting beneficial effects on glucose tolerance and insulin resistance. SCFAs are also signaling molecules that bind to specific G-protein coupled receptors (GPCRs) named Free Fatty Acid Receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43). However, how SCFAs impact NS activity through their GPCRs is poorly understood. Recently, studies have demonstrated the presence of FFA2 and FFA3 in the sympathetic NS of rat, mouse and human. Two studies have showed that FFA3 activation by SCFAs increases firing and norepinephrine (NE) release from sympathetic neurons. However, the recent study from the Ikeda Laboratory revealed that activation of FFA3 by SCFAs impairs N-type calcium channel (NTCC) activity, which contradicts the idea of FFA3 activation leading to increased action potential evoked NE release. Here we will discuss the scope of the latter study and the putative physiological role of SCFAs and FFAs in the sympathetic NS.