RESUMEN
Spinal muscular atrophy (SMA) is a lethal human disease characterized by motor neuron dysfunction and muscle deterioration due to depletion of the ubiquitous survival motor neuron (SMN) protein. Drosophila SMN mutants have reduced muscle size and defective locomotion, motor rhythm, and motor neuron neurotransmission. Unexpectedly, restoration of SMN in either muscles or motor neurons did not alter these phenotypes. Instead, SMN must be expressed in proprioceptive neurons and interneurons in the motor circuit to nonautonomously correct defects in motor neurons and muscles. SMN depletion disrupts the motor system subsequent to circuit development and can be mimicked by the inhibition of motor network function. Furthermore, increasing motor circuit excitability by genetic or pharmacological inhibition of K(+) channels can correct SMN-dependent phenotypes. These results establish sensory-motor circuit dysfunction as the origin of motor system deficits in this SMA model and suggest that enhancement of motor neural network activity could ameliorate the disease.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Neuronas Colinérgicas/metabolismo , Modelos Animales de Enfermedad , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/genética , Humanos , Larva/metabolismo , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/metabolismo , Mutación , Proteínas de Unión al ARN/genética , Células Receptoras Sensoriales/metabolismoRESUMEN
Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Atrofia Muscular Espinal/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Humanos , Proteínas de la Membrana/genética , Ratones , Células 3T3 NIH , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.
Asunto(s)
Analgesia , Receptor de Adenosina A1/metabolismo , Adenosina/química , Adenosina/metabolismo , Regulación Alostérica/efectos de los fármacos , Analgesia/métodos , Animales , Sitios de Unión , Modelos Animales de Enfermedad , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2/química , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Hiperalgesia/tratamiento farmacológico , Lípidos , Masculino , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Estabilidad Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A1/química , Transducción de Señal/efectos de los fármacosRESUMEN
The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK1R), Gαq/i, and ßarrestin-2. Whereas the FDA-approved NK1R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK1R+ve neurons in knockin mice expressing human NK1R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK1R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK1R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK1R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of diverse diseases.
Asunto(s)
Endosomas , Receptores de Neuroquinina-1 , Ratones , Humanos , Animales , Receptores de Neuroquinina-1/genética , Aprepitant/farmacología , Sustancia P/farmacología , Receptores Acoplados a Proteínas G , Dolor/tratamiento farmacológicoRESUMEN
G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.
Asunto(s)
Proteína Similar al Receptor de Calcitonina/genética , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Nocicepción/fisiología , Dolor/fisiopatología , Transmisión Sináptica/efectos de los fármacos , Antagonistas Adrenérgicos/farmacología , Animales , Péptido Relacionado con Gen de Calcitonina/farmacología , Proteína Similar al Receptor de Calcitonina/antagonistas & inhibidores , Proteína Similar al Receptor de Calcitonina/metabolismo , Capsaicina/antagonistas & inhibidores , Capsaicina/farmacología , Colestanoles/farmacología , Clatrina/antagonistas & inhibidores , Clatrina/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Endosomas/efectos de los fármacos , Formaldehído/antagonistas & inhibidores , Formaldehído/farmacología , Adyuvante de Freund/antagonistas & inhibidores , Adyuvante de Freund/farmacología , Regulación de la Expresión Génica , Inyecciones Espinales , Masculino , Ratones , Microtomía , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nocicepción/efectos de los fármacos , Dolor/inducido químicamente , Dolor/genética , Dolor/prevención & control , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Ratas , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Inhibitory glycinergic neurotransmission in the spinal cord dorsal horn plays an important role in regulating nociceptive signalling by inhibiting neuronal excitation. Blocking glycinergic transmission in the dorsal horn causes normally innocuous stimuli to become painful (allodynia) and increases sensitivity to noxious stimuli (hyperalgesia). Loss of inhibitory signalling is thought to contribute to the development of pathological pain. Management of neuropathic pain with current therapeutics is challenging and there is a great need for more effective treatments. Preclinical studies using drugs that increase glycinergic signalling by potentiating glycine receptor activity or inhibiting transporter activity suggest that targeting this system is a good therapeutic strategy. The spatially restricted expression of glycine receptors and transporters is an advantage for targeting specific pathologies such as pain. However, until recently there have been few pharmacological modulators identified and most of which do not specifically target glycinergic signalling. This mini-review provides an overview of recent advances in the development of therapeutics and novel approaches that aim to increase glycinergic neurotransmission for the treatment of persistent pain.
Asunto(s)
Dolor Crónico/tratamiento farmacológico , Glicinérgicos/farmacología , Glicinérgicos/uso terapéutico , Transmisión Sináptica/efectos de los fármacos , Animales , Dolor Crónico/metabolismo , Humanos , Proteínas de Transporte de Membrana/metabolismo , Receptores de Glicina/metabolismoRESUMEN
In the spinal cord and periphery, adenosine inhibits neuronal activity through activation of the adenosine A1 receptor (A1R), resulting in antinociception and highlighting the potential of therapeutically targeting the receptor in the treatment of neuropathic pain. This study investigated the changes in adenosine tone and A1R signaling, together with the actions of a novel A1R positive allosteric modulator (PAM), VCP171 [(2-amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone], on excitatory and inhibitory neurotransmission at spinal cord superficial dorsal horn synapses in a rat partial nerve-injury model of neuropathic pain. In the absence of A1R agonists, superfusion of the A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 µM), produced a significantly greater increase in electrically evoked α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic current (eEPSC) amplitude in both lamina I and II neurons from nerve-injured animals than in controls, suggesting that endogenous adenosine tone is increased in the dorsal horn. Inhibitory GABAergic and glycinergic synaptic currents were also significantly increased by DPCPX in controls but there was no difference after nerve injury. The A1R agonist, N6-cyclopentyladenosine, produced greater inhibition of eEPSC amplitude in lamina II but not lamina I of the spinal cord dorsal horn in nerve-injured versus control animals, suggesting a functional increase in A1R sensitivity in lamina II neurons after nerve injury. The A1R PAM, VCP171, produced a greater inhibition of eEPSC amplitude of nerve-injury versus control animals in both lamina I and lamina II neurons. Enhanced adenosine tone and A1R sensitivity at excitatory synapses in the dorsal horn after nerve injury suggest that new generation PAMs of the A1R can be effective treatments for neuropathic pain.
Asunto(s)
Agonistas del Receptor de Adenosina A1/farmacología , Potenciales Postsinápticos Excitadores , Neuralgia/tratamiento farmacológico , Neuronas Aferentes/efectos de los fármacos , Tiofenos/farmacología , Agonistas del Receptor de Adenosina A1/uso terapéutico , Regulación Alostérica , Animales , Hiperalgesia/tratamiento farmacológico , Masculino , Neuronas Aferentes/metabolismo , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A1/metabolismo , Tiofenos/uso terapéutico , Xantinas/farmacologíaRESUMEN
Inherited metabolic disorders are a group of genetic conditions that can cause severe neurological impairment and child mortality. Uniquely, these disorders respond to dietary treatment; however, this option remains largely unexplored because of low disorder prevalence and the lack of a suitable paradigm for testing diets. Here, we screened 35 Drosophila amino acid disorder models for disease-diet interactions and found 26 with diet-altered development and/or survival. Using a targeted multi-nutrient array, we examine the interaction in a model of isolated sulfite oxidase deficiency, an infant-lethal disorder. We show that dietary cysteine depletion normalizes their metabolic profile and rescues development, neurophysiology, behavior, and lifelong fly survival, thus providing a basis for further study into the pathogenic mechanisms involved in this disorder. Our work highlights the diet-sensitive nature of metabolic disorders and establishes Drosophila as a valuable tool for nutrigenomic studies for informing potential dietary therapies.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Enfermedades Metabólicas , Lactante , Niño , Animales , Humanos , Nutrigenómica , Drosophila , Dieta , Enfermedades Metabólicas/genéticaRESUMEN
Pre-mRNA alternative splicing is an important mechanism for the generation of synaptic protein diversity, but few factors governing this process have been identified. From a screen for Drosophila mutants with aberrant synaptic development, we identified beag, a mutant with fewer synaptic boutons and decreased neurotransmitter release. Beag encodes a spliceosomal protein similar to splicing factors in humans and Caenorhabditis elegans. We find that both beag mutants and mutants of an interacting gene dsmu1 have changes in the synaptic levels of specific splice isoforms of Fasciclin II (FasII), the Drosophila ortholog of neural cell adhesion molecule. We show that restoration of one splice isoform of FasII can rescue synaptic morphology in beag mutants while expression of other isoforms cannot. We further demonstrate that this FasII isoform has unique functions in synaptic development independent of transsynaptic adhesion. beag and dsmu1 mutants demonstrate an essential role for these previously uncharacterized splicing factors in the regulation of synapse development and function.
Asunto(s)
Empalme Alternativo/fisiología , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de Drosophila/fisiología , Terminales Presinápticos/fisiología , Empalme Alternativo/genética , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mutación , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Empalmosomas/metabolismoRESUMEN
ABSTRACT: Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1 to 3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized nonevoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibitors of clathrin, Dnm, and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm, and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment.
Asunto(s)
Neuralgia , Nociceptores , Ratas , Animales , Humanos , Nociceptores/fisiología , Hiperalgesia/metabolismo , Nocicepción/fisiología , Ratas Sprague-Dawley , Transmisión Sináptica , Neuralgia/metabolismo , Células del Asta Posterior/metabolismo , Asta Dorsal de la Médula Espinal , Ganglios Espinales/fisiologíaRESUMEN
The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA's unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of ß-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.
Asunto(s)
Analgesia , Depresión , Adenosina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P1RESUMEN
Excitatory and inhibitory neurotransmission within the spinal dorsal horn is tightly controlled to regulate transmission of nociceptive signals to the brain. One aspect of this control is modulation of neuronal activity through cholinergic signaling. Nociceptive neurons in the dorsal horn express both nicotinic and muscarinic cholinergic receptors and activation of these receptors reduces pain in humans, while inhibition leads to nociceptive hypersensitivity. At a cellular level, acetylcholine (ACh) has diverse effects on excitability which is dependent on the receptor and neuronal subtypes involved. In the present study we sought to characterize the electrophysiological responses of specific subsets of lamina II interneurons from rat and marmoset spinal cord. Neurons were grouped by morphology and by action potential firing properties. Whole-cell voltage-clamp recordings from lamina II dorsal horn neurons of adult rats showed that bath applied acetylcholine increased, decreased or had no effect on spontaneous synaptic current activity in a cell-type specific manner. ACh modulated inhibitory synaptic activity in 80% of neurons, whereas excitatory synaptic activity was affected in less than 50% of neurons. In whole-cell current clamp recordings, brief somatic application of ACh induced cell-type specific responses in 79% of rat lamina II neurons, which included: depolarization and action potential firing, subthreshold membrane depolarization, biphasic responses characterized by transient depolarization followed by hyperpolarization and membrane hyperpolarization alone. Similar responses were seen in marmoset lamina II neurons and the properties of each neuron group were consistent across species. ACh-induced hyperpolarization was blocked by the muscarinic antagonist atropine and all forms of acetylcholine-induced depolarization were blocked by the nicotinic antagonist mecamylamine. The cholinergic system plays an important role in regulating nociception and this study contributes to our understanding of how circuit activity is controlled by ACh at a cellular level in primate and rodent spinal cord.
Asunto(s)
Acetilcolina/farmacología , Red Nerviosa/efectos de los fármacos , Células del Asta Posterior/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Atropina/farmacología , Callithrix , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Interneuronas/efectos de los fármacos , Masculino , Mecamilamina/farmacología , Ratones , Antagonistas Muscarínicos/farmacología , Antagonistas Nicotínicos/farmacología , Nocicepción/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-DawleyRESUMEN
Trigeminal neuropathic pain is a chronic pain condition caused by damage or inflammation of the trigeminal nerve or its branches, with both peripheral and central nervous system dysfunction contributing to the disorder. Trigeminal pain conditions present with diagnostic and therapeutic challenges to healthcare providers and often require multiple therapeutic approaches for pain reduction. This review will provide the overview of pathophysiology in peripheral and central nociceptive circuits that are involved in neuropathic pain conditions involving the trigeminal nerve and the current therapeutics that are used to treat these disorders. Recent advances in treatment of trigeminal pain, including novel therapeutics that target ion channels and receptors, gene therapy and monoclonal antibodies that have shown great promise in preclinical studies and clinical trials will also be described.
RESUMEN
Nanoparticle-mediated drug delivery is especially useful for targets within endosomes because of the endosomal transport mechanisms of many nanomedicines within cells. Here, we report the design of a pH-responsive, soft polymeric nanoparticle for the targeting of acidified endosomes to precisely inhibit endosomal signalling events leading to chronic pain. In chronic pain, the substance P (SP) neurokinin 1 receptor (NK1R) redistributes from the plasma membrane to acidified endosomes, where it signals to maintain pain. Therefore, the NK1R in endosomes provides an important target for pain relief. The pH-responsive nanoparticles enter cells by clathrin- and dynamin-dependent endocytosis and accumulate in NK1R-containing endosomes. Following intrathecal injection into rodents, the nanoparticles, containing the FDA-approved NK1R antagonist aprepitant, inhibit SP-induced activation of spinal neurons and thus prevent pain transmission. Treatment with the nanoparticles leads to complete and persistent relief from nociceptive, inflammatory and neuropathic nociception and offers a much-needed non-opioid treatment option for chronic pain.
Asunto(s)
Aprepitant/administración & dosificación , Dolor Crónico/tratamiento farmacológico , Preparaciones de Acción Retardada/metabolismo , Nanopartículas/metabolismo , Antagonistas del Receptor de Neuroquinina-1/administración & dosificación , Animales , Aprepitant/farmacocinética , Aprepitant/uso terapéutico , Línea Celular , Dolor Crónico/metabolismo , Sistemas de Liberación de Medicamentos , Endosomas/metabolismo , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Masculino , Ratones Endogámicos C57BL , Antagonistas del Receptor de Neuroquinina-1/farmacocinética , Antagonistas del Receptor de Neuroquinina-1/uso terapéutico , Ratas , Receptores de Neuroquinina-1/metabolismoRESUMEN
"Ryegrass staggers" is a neurological condition of unknown mechanism that impairs motor function in livestock. It is caused by infection of perennial ryegrass pastures by an endophytic fungus that produces neurotoxins, predominantly the indole-diterpenoid compound lolitrem B. Animals grazing on such pastures develop uncontrollable tremors and become uncoordinated in their movement. Lolitrem B and the structurally related tremor inducer paxilline both act as potent large conductance calcium-activated potassium (BK) channel inhibitors. Using patch clamping, we show that their different apparent affinities correlate with their toxicity in vivo. To investigate whether the motor function deficits produced by lolitrem B and paxilline are due to inhibition of BK ion channels, their ability to induce tremor and ataxia in mice deficient in this ion channel (Kcnma1(-/-)) was examined. Our results show that mice lacking Kcnma1 are unaffected by these neurotoxins. Furthermore, doses of these substances known to be lethal to wild-type mice had no effect on Kcnma1(-/-) mice. These studies reveal the BK channel as the molecular target for the major components of the motor impairments induced by ryegrass neurotoxins. Unexpectedly, when the response to lolitrem B was examined in mice lacking the beta4 BK channel accessory subunit (Kcnmb4(-/-)), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK beta4 subunit in motor control. The beta4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.
Asunto(s)
Enfermedades de los Bovinos/etiología , Indoles/toxicidad , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Micotoxinas/toxicidad , Enfermedades del Sistema Nervioso/etiología , Animales , Bovinos , Alcaloides Indólicos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Lolium/microbiología , Ratones , Ratones Noqueados , Enfermedades del Sistema Nervioso/veterinaria , Técnicas de Placa-ClampRESUMEN
BACKGROUND AND PURPOSE: Inhibitory neurotransmission plays an important role in controlling excitability within nociceptive circuits of the spinal cord dorsal horn. Loss of inhibitory signalling is thought to contribute to the development of pathological pain. Preclinical studies suggest that increasing inhibitory glycinergic signalling is a good therapeutic strategy for treating pain. One approach to increase synaptic glycine is to inhibit the activity of the glycine transporter 2 (GlyT2) on inhibitory nerve terminals. These transporters are involved in regulating glycine concentrations and recycling glycine into presynaptic terminals. Inhibiting activity of GlyT2 increases synaptic glycine, which decreases excitability in nociceptive circuits and provides analgesia in neuropathic and inflammatory pain models. EXPERIMENTAL APPROACH: We investigated the effects of reversible and irreversible GlyT2 inhibitors on inhibitory glycinergic and NMDA receptor-mediated excitatory neurotransmission in the rat dorsal horn. The effect of these drugs on synaptic signalling was determined using patch-clamp electrophysiology techniques to measure glycine- and NMDA-mediated postsynaptic currents in spinal cord slices in vitro. KEY RESULTS: We compared activity of four compounds that increase glycinergic tone with a corresponding increase in evoked glycinergic postsynaptic currents. These compounds did not deplete synaptic glycine release over time. Interestingly, none of these compounds increased glycine-mediated excitatory signalling through NMDA receptors. The results suggest that these compounds preferentially inhibit GlyT2 over G1yT1 with no potentiation of the glycine receptor and without inducing spillover from inhibitory to excitatory synapses. CONCLUSIONS AND IMPLICATIONS: GlyT2 inhibitors increase inhibitory neurotransmission in the dorsal horn and have potential as pain therapeutics. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.
Asunto(s)
Glicinérgicos/farmacología , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Glicinérgicos/química , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Masculino , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Sinapsis/metabolismoRESUMEN
Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and ß-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.
Asunto(s)
Endosomas/metabolismo , Terapia Molecular Dirigida , Nocicepción , Dolor/tratamiento farmacológico , Receptores de Neuroquinina-1/metabolismo , Transducción de Señal , Animales , Compartimento Celular , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Lípidos/química , Modelos Biológicos , Antagonistas del Receptor de Neuroquinina-1/farmacología , Antagonistas del Receptor de Neuroquinina-1/uso terapéutico , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Nocicepción/efectos de los fármacos , Dolor/patología , Unión Proteica/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Médula Espinal/patología , Fracciones Subcelulares/metabolismo , Sustancia P/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Human genetic studies have implicated the voltage-gated sodium channel NaV1.7 as a therapeutic target for the treatment of pain. A novel peptide, µ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits NaV1.7 (IC50 0.9 nM) with at least 40-1000-fold selectivity over all other NaV subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by NaV1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective NaV1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective NaV1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted NaV1.7 inhibitors can only produce analgesia when administered in combination with an opioid.
RESUMEN
The development of neuropathic pain involves persistent changes in signalling within pain pathways. Reduced inhibitory signalling in the spinal cord following nerve-injury has been used to explain sensory signs of neuropathic pain but specific circuits that lose inhibitory input have not been identified. This study shows a specific population of spinal cord interneurons, radial neurons, lose glycinergic inhibitory input in a rat partial sciatic nerve ligation (PNL) model of neuropathic pain. Radial neurons are excitatory neurons located in lamina II of the dorsal horn, and are readily identified by their morphology. The amplitude of electrically-evoked glycinergic inhibitory post-synaptic currents (eIPSCs) was greatly reduced in radial neurons following nerve-injury associated with increased paired-pulse ratio. There was also a reduction in frequency of spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSC) in radial neurons without significantly affecting mIPSC amplitude. A subtype selective receptor antagonist and western blots established reversion to expression of the immature glycine receptor subunit GlyRα2 in radial neurons after PNL, consistent with slowed decay times of IPSCs. This study has important implications as it identifies a glycinergic synaptic connection in a specific population of dorsal horn neurons where loss of inhibitory signalling may contribute to signs of neuropathic pain.
Asunto(s)
Neuralgia/metabolismo , Células del Asta Posterior/metabolismo , Receptores de Glicina/metabolismo , Nervio Ciático/metabolismo , Transmisión Sináptica , Animales , Modelos Animales de Enfermedad , Masculino , Neuralgia/patología , Células del Asta Posterior/patología , Ratas , Ratas Sprague-Dawley , Nervio Ciático/patologíaRESUMEN
Orf virus causes pustular skin lesions (orf) in sheep, goats and humans. The virus encodes an interleukin-10 (orfvIL-10) that is identical in amino acid composition to ovine IL-10 (ovIL-10) over the C terminal two-thirds of the polypeptide, but not in the N terminal third. The immuno-suppressive and immuno-stimulatory activities of orfvIL-10 and ovIL-10 were compared. Both orfvIL-10 and ovIL-10 inhibited TNF-alpha and IL-8 cytokine production from stimulated ovine macrophages and keratinocytes and IFN-gamma and GM-CSF production from peripheral blood lymphocytes. OrfvIL-10 and ovIL-10 co-stimulated both ovine and murine mast cell proliferation in conjunction with IL-3 (ovine) or IL-4 (murine). Isoleucine at position 87 (Ile(87)) of the mature human IL-10 (huIL-10) has been reported as essential for the immuno-stimulatory activity of huIL-10. In spite of the differences in amino acids within the N-terminal third of orfvIL-10 compared with ovIL-10 and substitution of Ile(87) with Ala(87) in ovIL-10, these variants of ovIL-10 and orfvIL-10 all co-stimulated mast cell proliferation and inhibited macrophage IL-8 production. As ovIL-10 and orfvIL-10 have a similar structure to huIL-10 and conserved receptor-binding residues, it was concluded that Ile(87) is not essential for IL-10 immuno-stimulatory activity. Finally, ovine keratinocytes do not express ovIL-10. This might explain why orf virus has evolved a viral IL-10.