RESUMO
Strong human genetic evidence points to an essential contribution of the voltage-gated sodium channel Nav1.7 to pain sensation: loss of Nav1.7 function leads to congenital insensitivity to pain, whereas gain-of-function mutations in the SCN9A gene that encodes Nav1.7 cause painful neuropathies, such as inherited erythromelalgia, a syndrome characterized by episodic spontaneous pain. Selective Nav1.7 channel blockers thus hold promise as potential painkillers with improved safety and reduced unwanted side effects compared with existing therapeutics. To determine the maximum effect of a theoretically perfectly selective Nav1.7 inhibitor, we generated a tamoxifen-inducible KO mouse model enabling genetic deletion of Nav1.7 from adult mice. Electrophysiological recordings of sensory neurons from these mice following tamoxifen injection demonstrated the loss of Nav1.7 channel current and the resulting decrease in neuronal excitability of small-diameter neurons. We found that behavioral responses to most, but surprisingly not all, modalities of noxious stimulus are abolished following adult deletion of Nav1.7, pointing toward indications where Nav1.7 blockade should be efficacious. Furthermore, we demonstrate that isoform-selective acylsulfonamide Nav1.7 inhibitors show robust analgesic and antinociceptive activity acutely after a single dose in mouse pain models shown to be Nav1.7-dependent. All experiments were done with both male and female mice. Collectively, these data expand the depth of knowledge surrounding Nav1.7 biology as it relates to pain, and provide preclinical proof of efficacy that lays a clear path toward translation for the therapeutic use of Nav1.7-selective inhibitors in humans.SIGNIFICANCE STATEMENT Loss-of-function mutations in the sodium channel Nav1.7 cause congenital insensitivity to pain in humans, making Nav1.7 a top target for novel pain drugs. Targeting Nav1.7 selectively has been challenging, however, in part due to uncertainties in which rodent pain models are dependent on Nav1.7. We have developed and characterized an adult-onset Nav1.7 KO mouse model that allows us to determine the expected effects of a theoretically perfect Nav1.7 blocker. Importantly, many commonly used pain models, such as mechanical allodynia after nerve injury, appear to not be dependent on Nav1.7 in the adult. By defining which models are Nav1.7 dependent, we demonstrate that selective Nav1.7 inhibitors can approximate the effects of genetic loss of function, which previously has not been directly established.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/deficiência , Insensibilidade Congênita à Dor/metabolismo , Percepção da Dor/fisiologia , Dor/metabolismo , Bloqueadores dos Canais de Sódio/uso terapêutico , Animais , Células Cultivadas , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor/tratamento farmacológico , Dor/genética , Insensibilidade Congênita à Dor/tratamento farmacológico , Insensibilidade Congênita à Dor/genética , Percepção da Dor/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metaboliteâgenerated by aldehyde oxidase (AO)âpossessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.
Assuntos
Doenças Respiratórias , Canais de Potencial de Receptor Transitório , Humanos , Canais de Potencial de Receptor Transitório/metabolismo , Canal de Cátion TRPA1 , Aldeído Oxidase/metabolismo , Oxirredutases/metabolismo , Proteínas do Citoesqueleto/metabolismoRESUMO
Marked hypersensitivity to heat and mechanical (pressure) stimuli develop after a burn injury, but the neural mechanisms underlying these effects are poorly understood. In this study, we establish a new mouse model of focal second-degree burn injury to investigate the molecular and cellular basis for burn injury-induced pain. This model features robust injury-induced behavioral effects and tissue-specific altered cytokine profile, but absence of glial activation in spinal dorsal horn. Three voltage-gated sodium channels, Na(v)1.7, Na(v)1.8, and Na(v)1.9, are preferentially expressed in peripheral somatosensory neurons of the dorsal root ganglia (DRGs) and have been implicated in injury-induced neuronal hyperexcitability. Using knock-out mice, we provide evidence that Na(v)1.7 selectively contributes to burn-induced hypersensitivity to heat, but not mechanical, stimuli. After burn model injury, wild-type mice display increased sensitivity to heat stimuli, and a normally non-noxious warm stimulus induces activity-dependent Fos expression in spinal dorsal horn neurons. Strikingly, both effects are absent in Na(v)1.7 conditional knock-out (cKO) mice. Furthermore, burn injury increases density and shifts activation of tetrodotoxin-sensitive currents in a hyperpolarized direction, both pro-excitatory properties, in DRG neurons from wild-type but not Na(v)1.7 cKO mice. We propose that, in sensory neurons damaged by burn injury to the hindpaw, Na(v)1.7 currents contribute to the hyperexcitability of sensory neurons, their communication with postsynaptic spinal pain pathways, and behavioral thresholds to heat stimuli. Our results offer insights into the molecular and cellular mechanisms of modality-specific pain signaling, and suggest Na(v)1.7-blocking drugs may be effective in burn patients.
Assuntos
Queimaduras/complicações , Temperatura Alta/efeitos adversos , Hiperalgesia/etiologia , Limiar da Dor/fisiologia , Canais de Sódio/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Análise de Variância , Animais , Biofísica , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Edema/etiologia , Estimulação Elétrica , Lateralidade Funcional , Gânglios Espinais/citologia , Glicoproteínas/metabolismo , Hiperalgesia/patologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Canal de Sódio Disparado por Voltagem NAV1.7 , Canal de Sódio Disparado por Voltagem NAV1.8 , Canal de Sódio Disparado por Voltagem NAV1.9 , Neuralgia/etiologia , Neuralgia/patologia , Neuroglia/patologia , Técnicas de Patch-Clamp , Proteínas/genética , RNA Mensageiro/metabolismo , RNA não Traduzido , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/deficiência , Tetrodotoxina/farmacologia , TransfecçãoRESUMO
OBJECTIVE: Cerebellar dysfunction in multiple sclerosis (MS) contributes significantly to disability, is relatively refractory to symptomatic therapy, and often progresses despite treatment with disease-modifying agents. We previously observed that sodium channel Nav1.8, whose expression is normally restricted to the peripheral nervous system, is present in cerebellar Purkinje neurons in a mouse model of MS (experimental autoimmune encephalomyelitis [EAE]) and in humans with MS. Here, we tested the hypothesis that upregulation of Nav1.8 in cerebellum in MS and EAE has functional consequences contributing to symptom burden. METHODS: Electrophysiology and behavioral assessment were performed in a new transgenic mouse model overexpressing Nav1.8 in Purkinje neurons. We also measured EAE symptom progression in mice lacking Nav1.8 compared to wild-type littermates. Finally, we administered the Nav1.8-selective blocker A803467 in the context of previously established EAE to determine reversibility of MS-like deficits. RESULTS: We report that, in the context of an otherwise healthy nervous system, ectopic expression of Nav1.8 in Purkinje neurons alters their electrophysiological properties, and disrupts coordinated motor behaviors. Additionally, we show that Nav1.8 expression contributes to symptom development in EAE. Finally, we demonstrate that abnormal patterns of Purkinje neuron firing and MS-like deficits in EAE can be partially reversed by pharmacotherapy using a Nav1.8-selective blocker. INTERPRETATION: Our results add to the evidence that a channelopathy contributes to cerebellar dysfunction in MS. Our data suggest that Nav1.8-specific blockers, when available for humans, merit study in MS.
Assuntos
Doenças Cerebelares/fisiopatologia , Canalopatias/fisiopatologia , Encefalomielite Autoimune Experimental/fisiopatologia , Esclerose Múltipla/fisiopatologia , Compostos de Anilina/uso terapêutico , Animais , Doenças Cerebelares/genética , Cerebelo/citologia , Cerebelo/metabolismo , Cerebelo/patologia , Canalopatias/genética , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Furanos/uso terapêutico , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/genética , Canal de Sódio Disparado por Voltagem NAV1.8 , Células de Purkinje/patologia , Células de Purkinje/fisiologia , Bloqueadores dos Canais de Sódio/uso terapêutico , Canais de Sódio/biossíntese , Canais de Sódio/genética , Canais de Sódio/metabolismo , Regulação para Cima/genéticaRESUMO
Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.
Assuntos
Analgésicos Opioides , Nociceptores , Camundongos , Humanos , Animais , Analgésicos Opioides/farmacologia , Potenciais de Ação , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor/genética , Células Receptoras Sensoriais , Peptídeos Opioides , Encefalinas , Gânglios EspinaisRESUMO
Behavioral responses to painful stimuli require peripheral sensory neurons called nociceptors. Electrophysiological studies show that most C-fiber nociceptors are polymodal (i.e., respond to multiple noxious stimulus modalities, such as mechanical and thermal); nevertheless, these stimuli are perceived as distinct. Therefore, it is believed that discrimination among these modalities only occurs at spinal or supraspinal levels of processing. Here, we provide evidence to the contrary. Genetic ablation in adulthood of unmyelinated sensory neurons expressing the G protein-coupled receptor Mrgprd reduces behavioral sensitivity to noxious mechanical stimuli but not to heat or cold stimuli. Conversely, pharmacological ablation of the central branches of TRPV1(+) nociceptors, which constitute a nonoverlapping population, selectively abolishes noxious heat pain sensitivity. Combined elimination of both populations yielded an additive phenotype with no additional behavioral deficits, ruling out a redundant contribution of these populations to heat and mechanical pain sensitivity. This double-dissociation suggests that the brain can distinguish different noxious stimulus modalities from the earliest stages of sensory processing.
Assuntos
Encéfalo/fisiologia , Fibras Nervosas Amielínicas/fisiologia , Dor/fisiopatologia , Animais , Comportamento Animal , Temperatura Baixa , Toxina Diftérica/farmacologia , Temperatura Alta , Camundongos , Camundongos Mutantes , Receptores Acoplados a Proteínas G/genética , Estresse Mecânico , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
Transient receptor potential ankyrin 1 (TRPA1) antagonists have generated broad interest in the pharmaceutical industry for the treatment of both pain and asthma. Over the past decade, multiple antagonist classes have been reported in the literature with a wide range of structural diversity. Our own work has focused on the development of proline sulfonamide and hypoxanthine-based antagonists, two antagonist classes with distinct physicochemical properties and pharmacokinetic (PK) trends. Late in our discovery program, cryogenic electron microscopy (cryoEM) studies revealed two different antagonist binding sites: a membrane-exposed proline sulfonamide transmembrane site and an intracellular hypoxanthine site near the membrane interface. A retrospective look at the discovery program reveals how the different binding sites, and their location relative to the cell membrane, influenced the optimization trajectories and overall drug profiles of each antagonist class.
RESUMO
The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.
Assuntos
Medição da Dor/métodos , Canal de Cátion TRPA1/agonistas , Canal de Cátion TRPA1/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Células HEK293 , Humanos , Ligantes , Masculino , Medição da Dor/efeitos dos fármacos , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Canal de Cátion TRPA1/químicaRESUMO
Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.
Assuntos
Asma/tratamento farmacológico , Inflamação Neurogênica/tratamento farmacológico , Dor/tratamento farmacológico , Prurido/tratamento farmacológico , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Canal de Cátion TRPA1/antagonistas & inibidores , Adolescente , Adulto , Animais , Estudos de Coortes , Modelos Animais de Doenças , Cães , Método Duplo-Cego , Feminino , Cobaias , Voluntários Saudáveis , Humanos , Isotiocianatos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Dor/induzido quimicamente , Prurido/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Canal de Cátion TRPA1/deficiência , Resultado do Tratamento , Adulto JovemRESUMO
Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.
Assuntos
Asma/tratamento farmacológico , Furanos/uso terapêutico , Purinas/uso terapêutico , Canal de Cátion TRPA1/antagonistas & inibidores , Animais , Asma/induzido quimicamente , Asma/complicações , Células CHO , Cricetulus , Furanos/síntese química , Furanos/metabolismo , Cobaias , Humanos , Inflamação/tratamento farmacológico , Inflamação/etiologia , Ligantes , Masculino , Estrutura Molecular , Ovalbumina , Oxidiazóis/síntese química , Oxidiazóis/metabolismo , Oxidiazóis/uso terapêutico , Ligação Proteica , Purinas/síntese química , Purinas/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Canal de Cátion TRPA1/metabolismoRESUMO
Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.
Assuntos
Azetidinas/farmacologia , Benzamidas/farmacologia , Descoberta de Drogas , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Azetidinas/química , Azetidinas/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Células Cultivadas , Células HEK293 , Humanos , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/farmacologia , Ratos Sprague-Dawley , Sulfonamidas/química , Sulfonamidas/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinéticaRESUMO
The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1 KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.
Assuntos
Asma/genética , Comportamento Animal/fisiologia , Dor/genética , Prurido/genética , Canal de Cátion TRPA1/genética , Animais , Asma/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dor/metabolismo , Fenótipo , Prurido/metabolismo , Ratos , Ratos Transgênicos , Canal de Cátion TRPA1/metabolismoRESUMO
Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/química , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Analgésicos/química , Analgésicos/metabolismo , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dor Crônica/tratamento farmacológico , Dor Crônica/patologia , Cães , Meia-Vida , Humanos , Ligantes , Masculino , Camundongos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Relação Estrutura-Atividade , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.
Assuntos
Pró-Fármacos/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , Sulfonamidas/farmacologia , Canal de Cátion TRPA1/antagonistas & inibidores , Animais , Cães , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Ligantes , Células Madin Darby de Rim Canino , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Prolina/síntese química , Prolina/farmacocinética , Ratos , Solubilidade , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Sulfonamidas/farmacocinética , Canal de Cátion TRPA1/químicaRESUMO
Assessing BACE1 (ß-site APP cleaving enzyme 1) knockout mice for general health and neurological function may be useful in predicting risks associated with prolonged pharmacological BACE1 inhibition, a treatment approach currently being developed for Alzheimer's disease. To determine whether BACE1 deletion-associated effects in mice generalize to another species, we developed a novel Bace1-/- rat line using zinc-finger nuclease technology and compared Bace1-/- mice and rats with their Bace1+/+ counterparts. Lack of BACE1 was confirmed in Bace1-/- animals from both species. Removal of BACE1 affected startle magnitude, balance beam performance, pain response, and nerve myelination in both species. While both mice and rats lacking BACE1 have shown increased mortality, the increase was smaller and restricted to early developmental stages for rats. Bace1-/- mice and rats further differed in body weight, spontaneous locomotor activity, and prepulse inhibition of startle. While the effects of species and genetic background on these phenotypes remain difficult to distinguish, our findings suggest that BACE1's role in myelination and some sensorimotor functions is consistent between mice and rats and may be conserved in other species. Other phenotypes differ between these models, suggesting that some effects of BACE1 inhibition vary with the biological context (e.g. species or background strain).
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Deleção de Genes , Reflexo de Sobressalto/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Peso Corporal/genética , Peso Corporal/fisiologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Atividade Motora/fisiologia , Inibição Pré-Pulso/genética , Inibição Pré-Pulso/fisiologia , Ratos , Reflexo de Sobressalto/fisiologia , Especificidade da EspécieRESUMO
Cerebellar symptoms significantly diminish quality of life in patients with multiple sclerosis (MS). We previously showed that sodium channel Nav1.8, although normally restricted to peripheral somatosensory neurons, is upregulated in the cerebellum in MS, and that Nav1.8 expression is linked to ataxia and MS-like symptoms in mice. Furthermore, intracerebroventricular administration of the Nav1.8 blocker A-803467 temporarily reversed electrophysiological and behavioral manifestations of disease in a mouse MS model; unfortunately A-803467 is not orally bioavailable, diminishing the potential for translation to human patients. In the present study, we assessed the effect of per os (p.o.) dosing of a new orally bioavailable Nav1.8-selective blocker, PF-01247324, in transgenic mice expressing Nav1.8 in Purkinje neurons, and in wildtype mice in the experimental autoimmune encephalomyelitis (EAE) model. PF-01247324 was administered by oral gavage at 1000 mg/kg; control groups received an equal volume of vehicle. Behavioral assays of motor coordination, grip strength, and ataxia were performed. We observed significant improvements in motor coordination and cerebellar-like symptoms in mice that received PF-01247324 compared to control littermates that received vehicle. These preclinical proof-of-concept data suggest that PF-01247324, its derivatives, or other Nav1.8-selective blockers merit further study for providing symptomatic therapy for cerebellar dysfunction in MS and related disorders.
Assuntos
Esclerose Múltipla/tratamento farmacológico , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Células de Purkinje/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Administração Oral , Animais , Humanos , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Células de Purkinje/patologiaRESUMO
Mouse genetics has contributed significantly to our understanding of molecular mechanisms underlying tissue and nerve injury-induced persistent pain. To create a highly reproducible, relatively noninvasive model of neuropathic pain in the mouse, we examined the behavioral consequences of sparing each of the 3 distal branches of the sciatic nerve in wild-type mice after a model originally described in the rat. Sparing the tibial branch but sparing neither of the other branches produced robust mechanical allodynia while leaving heat sensibility intact. To assess the topographic organization of the IB4 population of afferents from each branch and to compare anatomic consistency across injury models, we examined loss of thiamine monophosphatase staining in the superficial dorsal horn after peripheral nerve injury. We found that each of the sciatic branches targets a distinct mediolateral location in inner lamina II and that each of the spared nerve injury models produced a more reproducible pattern of thiamine monophosphatase staining loss than did partial tight ligation. These results improve on previous nerve injury models in mouse, demonstrate similar behavioral changes as in rat, and provide novel information on the topographic organization of small diameter peripheral afferents in the mouse spinal cord.
Assuntos
Dor/patologia , Dor/psicologia , Traumatismos dos Nervos Periféricos , Doenças do Sistema Nervoso Periférico/patologia , Animais , Comportamento Animal/fisiologia , Densitometria , Modelos Animais de Doenças , Temperatura Alta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Aferentes/fisiologia , Limiar da Dor/fisiologia , Nervos Periféricos/patologia , Nervo Fibular/patologia , Estimulação Física , Reprodutibilidade dos Testes , Nervo Sural/patologia , Nervo Tibial/patologiaRESUMO
The Wnt family of proteins plays key roles during central nervous system development and has been involved in several neuropathologies during adulthood, including spinal cord injury (SCI). However, Wnts expression knowledge is relatively limited during adult stages. Here, we sought to define the Wnt family expression pattern after SCI in adult mice by using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Under physiological conditions, the messenger RNAs (mRNAs) of most Wnt ligands, inhibitors, receptors, and coreceptors are constitutively expressed in healthy adult mice. After dorsal hemisection, we found significant time-dependent variations, with a prominent up-regulation of Wnt inhibitory factor 1 (Wif1). IHC against Frizzled (Fz) 1 and Fz4, as representatives of late and acute up-regulated receptors, showed a differential expression in the uninjured spinal cord of Fz1 by neurons and oligodendrocytes and Fz4 by astrocytes. After injury, both receptors were maintained in the same type of cells. Finally, by using BATgal reporter mice, our results revealed active ß-catenin signaling in neurons of the dorsal horn and cells of the central canal of uninjured spinal cords, besides a lack of additional SCI-induced activation. In conclusion, we demonstrate Wnt expression in the adult spinal cord of mice that is modulated by SCI, which differs from that previously described in rats. Further, Fz receptors are differentially expressed by neurons and glial cells, suggestive for cell-specific patterns and thus diverse physiological roles. Further studies will help toward in-depth characterization of the role of all Wnt factors and receptors described and eventually allow for the design of novel therapies.
Assuntos
Astrócitos/metabolismo , Receptores Frizzled/metabolismo , Neurônios/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Proteínas Wnt/metabolismo , Animais , Feminino , Receptores Frizzled/genética , Camundongos , Camundongos Endogâmicos C57BL , Traumatismos da Medula Espinal/genética , Regulação para Cima , Proteínas Wnt/genéticaRESUMO
A vast diversity of salient cues is sensed by numerous classes of primary sensory neurons, defined by specific neuropeptides, ion channels, or cytoskeletal proteins. Recent evidence has demonstrated a correlation between the expression of some of these molecular markers and transmission of signals related to distinct sensory modalities (eg, heat, cold, pressure). Voltage-gated sodium channel Na(v)1.8 has been reported to be preferentially expressed in small-diameter unmyelinated sensory afferents specialized for the detection of noxious stimuli (nociceptors), and Na(v)1.8-Cre mice have been widely used to investigate gene function in nociceptors. However, the identity of neurons in which Cre-mediated recombination occurs in these animals has not been resolved, and whether expression of Na(v)1.8 in these neurons is dynamic during development is not known, rendering interpretation of conditional knockout mouse phenotypes problematic. Here, we used genetics, immunohistochemistry, electrophysiology, and calcium imaging to precisely characterize the expression of Na(v)1.8 in the peripheral nervous system. We demonstrate that 75% of dorsal root ganglion (DRG) neurons express Na(v)1.8-Cre, including >90% of neurons expressing markers of nociceptors and, unexpectedly, a large population (â¼40%) of neurons with myelinated A fibers. Furthermore, analysis of DRG neurons' central and peripheral projections revealed that Na(v)1.8-Cre is not restricted to nociceptors but is also expressed by at least 2 types of low-threshold mechanoreceptors essential for touch sensation, including those with C and Aß fibers. Our results indicate that Na(v)1.8 underlies electrical activity of sensory neurons subserving multiple functional modalities, and call for cautious interpretation of the phenotypes of Na(v)1.8-Cre-driven conditional knockout mice.