RESUMO
Allosteric modulation of metabotropic glutamate receptor 2 (mGlu2) has demonstrated efficacy in preclinical rodent models of several brain disorders, leading to industry and academic drug discovery efforts. Although the pharmacology and binding sites of some mGlu2 allosteric modulators have been characterized previously, questions remain about the nature of the allosteric mechanism of cooperativity with glutamate and whether structurally diverse allosteric modulators bind in an identical manner to specific allosteric sites. To further investigate the in vitro pharmacology of mGlu2 allosteric modulators, we developed and characterized a novel mGlu2 positive allosteric modulator (PAM) radioligand in parallel with functional studies of a structurally diverse set of mGlu2 PAMs and negative allosteric modulators (NAMs). Using an operational model of allosterism to analyze the functional data, we found that PAMs affect both the affinity and efficacy of glutamate at mGlu2, whereas NAMs predominantly affect the efficacy of glutamate in our assay system. More importantly, we found that binding of a novel mGlu2 PAM radioligand was inhibited by multiple structurally diverse PAMs and NAMs, indicating that they may bind to the mGlu2 allosteric site labeled with the novel mGlu2 PAM radioligand; however, further studies suggested that these allosteric modulators do not all interact with the radioligand in an identical manner. Together, these findings provide new insights into the binding sites and modes of efficacy of different structurally and functionally distinct mGlu2 allosteric modulators and suggest that different ligands either interact with distinct sites or adapt different binding poses to shared allosteric site(s).
Assuntos
Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Regulação Alostérica , Sítio Alostérico , Animais , Linhagem Celular , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Ligantes , Mutagênese , Ligação Proteica , Ensaio Radioligante , Ratos , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismoRESUMO
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are highly homologous yet distinct components of signal transduction pathways known to regulate cell survival and function. Recent evidence indicates an isoform-specific role for ERK2 in pain processing and peripheral sensitization. However, the function of ERK2 in primary sensory neurons has not been directly tested. To dissect the isoform-specific function of ERK2 in sensory neurons, we used mice with Cre-loxP-mediated deletion of ERK2 in Nav1.8(+) sensory neurons that are predominantly nociceptors. We find that ERK2, unlike ERK1, is required for peripheral sensitization and cold sensation. We also demonstrate that ERK2, but not ERK1, is required to preserve epidermal innervation in a subset of peptidergic neurons. Additionally, deletion of both ERK isoforms in Nav1.8(+) sensory neurons leads to neuron loss not observed with deletion of either isoform alone, demonstrating functional redundancy in the maintenance of sensory neuron survival. Thus, ERK1 and ERK2 exhibit both functionally distinct and redundant roles in sensory neurons. SIGNIFICANCE STATEMENT: ERK1/2 signaling affects sensory neuron function and survival. However, it was not clear whether ERK isoform-specific roles exist in these processes postnatally. Previous work from our laboratory suggested either functional redundancy of ERK isoforms or a predominant role for ERK2 in pain; however, the tools to discriminate between these possibilities were not available at the time. In the present study, we use new genetic knock-out lines to demonstrate that ERK2 in sensory neurons is necessary for development of inflammatory pain and for postnatal maintenance of peptidergic epidermal innervation. Interestingly, postnatal loss of both ERK isoforms leads to a profound loss of sensory neurons. Therefore, ERK1 and ERK2 display both functionally distinct and redundant roles in sensory neurons.
Assuntos
Hiperalgesia/metabolismo , Inflamação/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Allosteric modulation of metabotropic glutamate (mGlu) receptors offers a promising pharmacological approach to normalize neural circuit dysfunction associated with various psychiatric and neurological disorders. As mGlu receptor allosteric modulators progress through discovery and clinical development, both technical advances and novel tool compounds are providing opportunities to better understand mGlu receptor pharmacology and neurobiology. Recent advances in structural biology are elucidating the structural determinants of mGlu receptor-negative allosteric modulation and supplying the means to resolve active, allosteric modulator-bound mGlu receptors. The discovery and characterization of allosteric modulators with novel pharmacological profiles is uncovering the biological significance of their intrinsic agonist activity, biased mGlu receptor modulation, and novel mGlu receptor heterodimers. The development and exploitation of optogenetic and optopharmacological tools is permitting a refined spatial and temporal understanding of both mGlu receptor functions and their allosteric modulation in intact brain circuits. Together, these lines of research promise to provide a more refined understanding of mGlu receptors and their allosteric modulation that will inform the development of mGlu receptor allosteric modulators as neurotherapeutics in the years to come.
Assuntos
Encéfalo/efeitos dos fármacos , Desenho de Fármacos , Descoberta de Drogas/métodos , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Animais , Sítios de Ligação , Encéfalo/metabolismo , Agonistas de Aminoácidos Excitatórios/química , Agonistas de Aminoácidos Excitatórios/metabolismo , Humanos , Optogenética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Relação Estrutura-Atividade , Transmissão Sináptica/efeitos dos fármacosRESUMO
Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that regulate various aspects of central nervous system processing in normal physiology and in disease. They are thought to function as disulfide-linked homodimers, but studies have suggested that mGluRs can form functional heterodimers in cell lines. Using selective allosteric ligands, ex vivo brain slice electrophysiology, and optogenetic approaches, we found that two mGluR subtypes-mGluR2 and mGluR4 (or mGlu2 and mGlu4)-exist as functional heterodimers that regulate excitatory transmission in a synapse-specific manner within the rodent medial prefrontal cortex (mPFC). Activation of mGlu2/mGlu4 heterodimers inhibited glutamatergic signaling at thalamo-mPFC synapses but not at hippocampus-mPFC or amygdala-mPFC synapses. These findings raise the possibility that selectively targeting these heterodimers could be a therapeutic strategy for some neurologic and neuropsychiatric disorders involving specific brain circuits.
Assuntos
Córtex Pré-Frontal , Transmissão Sináptica , SinapsesRESUMO
The loss of nigral dopaminergic (DA) neurons in idiopathic Parkinson's disease (PD) is believed to result from interactions between genetic susceptibility and environmental factors. Evidence that inflammatory processes modulate PD risk comes from prospective studies that suggest that higher plasma concentrations of a number of proinflammatory cytokines correlate with an increased risk of developing PD and chronic nonsteroidal anti-inflammatory drug regimens reduce the incidence of PD. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, Parkin-deficient (parkin-/-) mice do not display nigrostriatal pathway degeneration, suggesting that a genetic factor is not sufficient, and an environmental trigger may be needed to cause nigral DA neuron loss. To test the hypothesis that parkin-/- mice require an inflammatory stimulus to develop nigral DA neuron loss, low-dose lipopolysaccaride (LPS) was administered intraperitoneally for prolonged periods. Quantitative real-time PCR and immunofluorescence labeling of inflammatory markers indicated that this systemic LPS treatment regimen triggered persistent neuroinflammation in wild-type and parkin-/- mice. Although inflammatory and oxidative stress responses to the inflammation regimen did not differ significantly between the two genotypes, only parkin-/- mice displayed subtle fine-motor deficits and selective loss of DA neurons in substantia nigra. Therefore, our studies suggest that loss of Parkin function increases the vulnerability of nigral DA neurons to inflammation-related degeneration. This new model of nigral DA neuron loss may enable identification of early biomarkers of degeneration and aid in preclinical screening efforts to identify compounds that can halt or delay the progressive degeneration of the nigrostriatal pathway.
Assuntos
Inflamação/complicações , Degeneração Neural/etiologia , Substância Negra/patologia , Ubiquitina-Proteína Ligases/deficiência , Animais , Comportamento Animal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Dopamina/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/genética , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Inflamação/induzido quimicamente , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Análise Multivariada , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Degeneração Neural/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Polissacarídeos , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Teste de Desempenho do Rota-Rod , Substância Negra/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Tirosina 3-Mono-OxigenaseRESUMO
The exercise pressor reflex (EPR) is an important neural mechanism that controls blood pressure and heart rate during static muscle contraction. It has been previously demonstrated that the EPR is exaggerated in cardiomyopathy. Both mechanically (group III) and metabolically (group IV) sensitive afferent neurons are important to this reflex in normal humans and animals. In cardiomyopathy, however, the metabolically sensitive afferents are less responsive to activation whereas the mechanically sensitive fibres are overactive. We have demonstrated that this overactivity is responsible for the exaggeration in the EPR. Of importance, we have also demonstrated that the reduced responsiveness in the group IV afferent neuron is an initiating factor in the development of the exaggerated EPR. To date, the mechanism mediating this reduced group IV responsiveness remains unclear. Given that group IV afferent neurons are activated via chemically sensitive receptors, it is logical to suggest that changes in receptor function are responsible for the blunted behaviour of group IV neurons in cardiomyopathy. In order to test this postulate, however, potential receptor candidates must first be identified. The transient receptor potential vanilloid 1 (TRPv1) receptor is a non-selective cation channel that serves as a marker of the group IV afferent neurons in the periphery. We have demonstrated that the TRPv1 is abnormal in cardiomyopathy. It has been shown that the TRPv1 receptor is colocalized with the cannabinoid 1 (CB(1)) receptor on group IV afferent neurons. Therefore, we hypothesized that the function of CB(1) receptors is abnormal in cardiomyopathy. We explored this possibility by using anandamide (AEA), an endogenously produced cannabinoid that has been shown to control blood pressure via activation of the CB(1) receptor. In these studies, we evaluated the cardiovascular responses to intra-arterial injection of AEA into the hindlimb of normal, cardiomyopathic and neonatally capsaicin-treated (NNCAP) rats (rats that lack group IV afferent neurons) to determine whether administration of AEA results in abnormal responses of group IV afferent neurons in cardiomyopathic rats. We determined that AEA controls changes in blood pressure, predominately via activation of the CB(1) receptor in this preparation. We further observed that the blood pressure response to AEA is blunted in cardiomyopathic rats when compared to normal rats. We also observed a reduced blood pressure response to AEA in NNCAP animals, indicating that AEA is acting on group IV afferent neurons in this preparation. To determine whether programmed cell death could account for the decreased responsiveness that we observed during activation of the CB(1) and TRPv1 receptors on group IV afferent neurons in heart failure, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay. We observed no evidence of cell death within the dorsal root ganglia in rats with cardiomyopathy. The data suggest that the responsiveness of CB(1) receptors on group IV afferent neurons is blunted in cardiomyopathy. Importantly, these data indicate that group IV primary afferent neurons express multiple receptor defects in cardiomyopathy that may contribute to the decreased CB(1) receptor sensitivity in this disease.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Neurônios Aferentes/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Apoptose/fisiologia , Ácidos Araquidônicos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Moduladores de Receptores de Canabinoides/farmacologia , Capsaicina/farmacologia , Cardiomiopatia Dilatada/patologia , Modelos Animais de Doenças , Endocanabinoides , Gânglios Espinais/patologia , Masculino , Neurônios Aferentes/patologia , Alcamidas Poli-Insaturadas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/efeitos dos fármacos , Fármacos do Sistema Sensorial/farmacologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pronociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here, we demonstrate the different contributions of genetically defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception.
Assuntos
Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Nociceptividade/fisiologia , Percepção da Dor/fisiologia , Substância Cinzenta Periaquedutal/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Nociceptividade/efeitos dos fármacos , Percepção da Dor/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Técnicas de Cultura de TecidosRESUMO
UNLABELLED: Neurofibromatosis type 1 (NF1) is characterized primarily by tumor formation in the nervous system, but patients report other neurological complications including pain and itch. Individuals with NF1 harbor 1 mutated NF1 allele causing heterozygous expression in all of their cells. In mice, Nf1 heterozygosity leads to hyperexcitability of sensory neurons and hyperproliferation of mast cells, both of which could lead to increased hypersensitivity and scratching in response to noxious and pruritic stimuli. To determine whether Nf1 heterozygosity may increase pain and itch behaviors independent of secondary effects of tumor formation, we used mice with a targeted, heterozygous Nf1 gene deletion (Nf1±) that lack tumors. Nf1± mice exhibited normal baseline responses to thermal and mechanical stimuli. Moreover, similar to wild-type littermates, Nf1± mice developed inflammation-induced heat and mechanical hypersensitivity, capsaicin-induced nocifensive behavior, histamine-dependent or -independent scratching, and chronic constriction injury-induced cold allodynia. However, Nf1± mice exhibited an attenuated first phase of formalin-induced spontaneous behavior and expedited resolution of formalin-induced heat hypersensitivity. These results are not consistent with the hypothesis that Nf1 heterozygosity alone is sufficient to increase pain and itch sensation in mice, and they suggest that additional mechanisms may underlie reports of increased pain and itch in NF1 patients. PERSPECTIVE: This study assessed whether Nf1 heterozygosity in mice increased hypersensitivity and scratching following noxious and pruritic stimuli. Using Nf1± mice lacking tumors, this study finds no increases in pain or itch behavior, suggesting that there is no predisposition for either clinical symptom solely due to Nf1 heterozygosity.
Assuntos
Neurofibromatose 1/complicações , Dor/diagnóstico , Dor/etiologia , Prurido/diagnóstico , Prurido/etiologia , Animais , Capsaicina/efeitos adversos , Cloroquina/toxicidade , Constrição , Modelos Animais de Doenças , Lateralidade Funcional , Histamina/toxicidade , Hiperalgesia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurofibromatose 1/genética , Neurofibromatose 1/metabolismo , Dor/induzido quimicamente , Medição da Dor , Limiar da Dor/fisiologia , Prurido/induzido quimicamente , p-Metoxi-N-metilfenetilamina/toxicidadeRESUMO
BACKGROUND: Mutations in the PLA2G6 gene have been identified in autosomal recessive neurodegenerative diseases classified as infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accumulation (NBIA), and dystonia-parkinsonism. These clinical syndromes display two significantly different disease phenotypes. NBIA and INAD are very similar, involving widespread neurodegeneration that begins within the first 1-2 years of life. In contrast, patients with dystonia-parkinsonism present with a parkinsonian movement disorder beginning at 15 to 30 years of age. The PLA2G6 gene encodes the PLA2G6 enzyme, also known as group VIA calcium-independent phospholipase A(2), which has previously been shown to hydrolyze the sn-2 acyl chain of phospholipids, generating free fatty acids and lysophospholipids. METHODOLOGY/PRINCIPAL FINDINGS: We produced purified recombinant wildtype (WT) and mutant human PLA2G6 proteins and examined their catalytic function using in vitro assays with radiolabeled lipid substrates. We find that human PLA2G6 enzyme hydrolyzes both phospholipids and lysophospholipids, releasing free fatty acids. Mutations associated with different disease phenotypes have different effects on catalytic activity. Mutations associated with INAD/NBIA cause loss of enzyme activity, with mutant proteins exhibiting less than 20% of the specific activity of WT protein in both lysophospholipase and phospholipase assays. In contrast, mutations associated with dystonia-parkinsonism do not impair catalytic activity, and two mutations produce a significant increase in specific activity for phospholipid but not lysophospholipid substrates. CONCLUSIONS/SIGNIFICANCE: These results indicate that different alterations in PLA2G6 function produce the different disease phenotypes of NBIA/INAD and dystonia-parkinsonism. INAD/NBIA is caused by loss of the ability of PLA2G6 to catalyze fatty acid release from phospholipids, which predicts accumulation of PLA2G6 phospholipid substrates and provides a mechanistic explanation for the accumulation of membranes in neuroaxonal spheroids previously observed in histopathological studies of INAD/NBIA. In contrast, dystonia-parkinsonism mutations do not appear to directly impair catalytic function, but may modify substrate preferences or regulatory mechanisms for PLA2G6.