RESUMO
Aicardi-Goutières syndrome (AGS) is an autoinflammatory disease characterized by aberrant interferon (IFN)-α production. The major cause of morbidity in AGS is brain disease, yet the primary source and target of neurotoxic IFN-α remain unclear. Here, we demonstrated that the brain was the primary source of neurotoxic IFN-α in AGS and confirmed the neurotoxicity of intracerebral IFN-α using astrocyte-driven Ifna1 misexpression in mice. Using single-cell RNA sequencing, we demonstrated that intracerebral IFN-α-activated receptor (IFNAR) signaling within cerebral endothelial cells caused a distinctive cerebral small vessel disease similar to that observed in individuals with AGS. Magnetic resonance imaging (MRI) and single-molecule ELISA revealed that central and not peripheral IFN-α was the primary determinant of microvascular disease in humans. Ablation of endothelial Ifnar1 in mice rescued microvascular disease, stopped the development of diffuse brain disease, and prolonged lifespan. These results identify the cerebral microvasculature as a primary mediator of IFN-α neurotoxicity in AGS, representing an accessible target for therapeutic intervention.
Assuntos
Encéfalo , Interferon-alfa , Microvasos , Malformações do Sistema Nervoso , Receptor de Interferon alfa e beta , Animais , Humanos , Camundongos , Interferon-alfa/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/genética , Microvasos/patologia , Malformações do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Células Endoteliais/metabolismo , Camundongos Knockout , Masculino , Feminino , Transdução de Sinais , Camundongos Endogâmicos C57BL , Astrócitos/metabolismo , Modelos Animais de DoençasRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has become an integral part of the molecular biology toolkit. CRISPRa genetic screens are an exciting high-throughput means of identifying genes the upregulation of which is sufficient to elicit a given phenotype. Activation machinery is continually under development to achieve greater, more robust, and more consistent activation. In this review, we offer a succinct technological overview of available CRISPRa architectures and a comprehensive summary of pooled CRISPRa screens. Furthermore, we discuss contemporary applications of CRISPRa across broad fields of research, with the aim of presenting a view of exciting emerging applications for CRISPRa screening.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas/genética , Humanos , Edição de Genes/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos/métodos , AnimaisRESUMO
Alzheimer's disease (AD) is an age-related disease, with loss of integrity of the blood-brain barrier (BBB) being an early feature. Cellular senescence is one of the reported nine hallmarks of aging. Here, we show for the first time the presence of senescent cells in the vasculature in AD patients and mouse models of AD. Senescent endothelial cells and pericytes are present in APP/PS1 transgenic mice but not in wild-type littermates at the time of amyloid deposition. In vitro, senescent endothelial cells display altered VE-cadherin expression and loss of cell junction formation and increased permeability. Consistent with this, senescent endothelial cells in APP/PS1 mice are present at areas of vascular leak that have decreased claudin-5 and VE-cadherin expression confirming BBB breakdown. Furthermore, single cell sequencing of endothelial cells from APP/PS1 transgenic mice confirms that adhesion molecule pathways are among the most highly altered pathways in these cells. At the pre-plaque stage, the vasculature shows significant signs of breakdown, with a general loss of VE-cadherin, leakage within the microcirculation, and obvious pericyte perturbation. Although senescent vascular cells were not directly observed at sites of vascular leak, senescent cells were close to the leak area. Thus, we would suggest in AD that there is a progressive induction of senescence in constituents of the neurovascular unit contributing to an increasing loss of vascular integrity. Targeting the vasculature early in AD, either with senolytics or with drugs that improve the integrity of the BBB may be valid therapeutic strategies.
Assuntos
Doença de Alzheimer , Barreira Hematoencefálica , Humanos , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Doença de Alzheimer/metabolismo , Células Endoteliais , Camundongos Transgênicos , EnvelhecimentoRESUMO
The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.
Assuntos
Síndrome de Angelman , Transtorno Autístico , Animais , Camundongos , Transtorno Autístico/genética , Modelos Animais de Doenças , Mutação com Ganho de Função , Interneurônios/metabolismo , Herança Materna , Fenótipo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/genética , Antivirais/farmacologia , Enzima de Conversão de Angiotensina 2/metabolismo , Fibroblastos/metabolismo , Ligação Proteica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable and scalable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use this to examine eight public single-cell RNA-seq datasets (six from peripheral blood mononuclear cells, one from bronchoalveolar lavage and one from nasopharyngeal), with a total of 211 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients. Overall, this workflow can be generalized and scaled to combine multiple scRNA-seq datasets to uncover cell-cell interactions.
Assuntos
COVID-19 , Comunicação Celular , Humanos , Leucócitos Mononucleares , SARS-CoV-2 , Fluxo de TrabalhoRESUMO
Skeletal muscle weakness is linked to many adverse health outcomes. Current research to identify new drugs has often been inconclusive due to lack of adequate cellular models. We previously developed a scalable monolayer system to differentiate human embryonic stem cells (hESCs) into mature skeletal muscle cells (SkMCs) within 26 days without cell sorting or genetic manipulation. Here, building on our previous work, we show that differentiation and fusion of myotubes can be further enhanced using the anabolic factors testosterone (T) and follistatin (F) in combination with a cocktail of myokines (C). Importantly, combined TFC treatment significantly enhanced both the hESC-SkMC fusion index and the expression levels of various skeletal muscle markers, including the motor protein myosin heavy chain (MyHC). Transcriptomic and proteomic analysis revealed oxidative phosphorylation as the most up-regulated pathway, and a significantly higher level of ATP and increased mitochondrial mass were also observed in TFC-treated hESC-SkMCs, suggesting enhanced energy metabolism is coupled with improved muscle differentiation. This cellular model will be a powerful tool for studying in vitro myogenesis and for drug discovery pertaining to further enhancing muscle development or treating muscle diseases.
Assuntos
Células-Tronco Embrionárias Humanas , Diferenciação Celular/genética , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , ProteômicaRESUMO
Peripheral nerve injury induces long-term pro-inflammatory responses in spinal cord glial cells that facilitate neuropathic pain, but the identity of endogenous cells that resolve spinal inflammation has not been determined. Guided by single-cell RNA sequencing (scRNA-seq), we found that MRC1+ spinal cord macrophages proliferated and upregulated the anti-inflammatory mediator Cd163 in mice following superficial injury (SI; nerve intact), but this response was blunted in nerve-injured animals. Depleting spinal macrophages in SI animals promoted microgliosis and caused mechanical hypersensitivity to persist. Conversely, expressing Cd163 in spinal macrophages increased Interleukin 10 expression, attenuated micro- and astrogliosis, and enduringly alleviated mechanical and thermal hypersensitivity in nerve-injured animals. Our data indicate that MRC1+ spinal macrophages actively restrain glia to limit neuroinflammation and resolve mechanical pain following a superficial injury. Moreover, we show that spinal macrophages from nerve-injured animals mount a dampened anti-inflammatory response but can be therapeutically coaxed to promote long-lasting recovery of neuropathic pain.
Assuntos
Hiperalgesia/metabolismo , Macrófagos/fisiologia , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Medula Espinal/metabolismo , Animais , Modelos Animais de Doenças , Inflamação/metabolismo , Camundongos , Nociceptividade/fisiologia , Medição da DorRESUMO
Neuropathic pain causes severe suffering, and most patients are resistant to current therapies. A core element of neuropathic pain is the loss of inhibitory tone in the spinal cord. Previous studies have shown that foetal GABAergic neuron precursors can provide relief from pain. However, the source of these precursor cells and their multipotent status make them unsuitable for therapeutic use. Here, we extend these findings by showing, for the first time, that spinally transplanted, terminally differentiated human induced pluripotent stem cell-derived GABAergic (iGABAergic) neurons provide significant, long-term, and safe relief from neuropathic pain induced by peripheral nerve injury in mice. Furthermore, iGABAergic neuron transplants survive long term in the injured spinal cord and show evidence of synaptic integration. Together, this provides the proof in principle for the first viable GABAergic transplants to treat human neuropathic pain patients.
Assuntos
Transplante de Células , Neurônios GABAérgicos/transplante , Células-Tronco Pluripotentes Induzidas/citologia , Interneurônios/transplante , Neuralgia/fisiopatologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Corno Dorsal da Medula Espinal , Animais , Comportamento Animal , Cálcio/metabolismo , Neurônios GABAérgicos/citologia , Humanos , Interneurônios/citologia , Camundongos , Inibição Neural , Neuralgia/terapia , Neurogênese , Imagem ÓpticaRESUMO
Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.
Assuntos
Neoplasias Cerebelares/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Hedgehog/antagonistas & inibidores , Meduloblastoma/genética , Transdução de Sinais/genética , Anilidas/farmacologia , Anilidas/uso terapêutico , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/patologia , Cerebelo/citologia , Cerebelo/patologia , Feminino , Mutação com Ganho de Função , Proteínas Hedgehog/genética , Humanos , Masculino , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Proteína MyoD/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , RNA-Seq , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Receptor Smoothened/genética , Fatores de Transcrição HES-1/genéticaRESUMO
Nerve injury leads to devastating and often untreatable neuropathic pain. While acute noxious sensation (nociception) is a crucial survival mechanism and is conserved across phyla, chronic neuropathic pain is considered a maladaptive response owing to its devastating impact on a patient's quality of life. We have recently shown that a neuropathic pain-like response occurs in adult Drosophila. However, the mechanisms underlying this phenomenon are largely unknown. Previous studies have shown that the α2δ peripheral calcium channel subunit straightjacket (stj) is a conserved factor required for thermal pain perception. We demonstrate here that stj is required in peripheral ppk+ sensory neurons for acute thermal responses and that it mediates nociceptive hypersensitivity in an adult Drosophila model of neuropathic pain-like disease. Given that calcium channels are the main targets of gabapentinoids (pregabalin and gabapentin), we assessed if these drugs can alleviate nociceptive hypersensitivity. Our findings suggest that gabapentinoids may prevent nociceptive hypersensitivity by preserving central inhibition after nerve injury. Together, our data further highlight the similarity of some mechanisms for pain-like conditions across phyla and validates the scientific use of Drosophila neuropathic sensitization models for analgesic drug discovery. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.
Assuntos
Canais de Cálcio/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Neuralgia/genética , Animais , Canais de Cálcio/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Expressão Gênica/fisiologia , Larva/genética , Larva/fisiologia , Neuralgia/fisiopatologiaRESUMO
The development of the mammalian cerebral cortex depends on careful orchestration of proliferation, maturation, and migration events, ultimately giving rise to a wide variety of neuronal and non-neuronal cell types. To better understand cellular and molecular processes that unfold during late corticogenesis, we perform single-cell RNA-seq on the mouse cerebral cortex at a progenitor driven phase (embryonic day 14.5) and at birth-after neurons from all six cortical layers are born. We identify numerous classes of neurons, progenitors, and glia, their proliferative, migratory, and activation states, and their relatedness within and across age. Using the cell-type-specific expression patterns of genes mutated in neurological and psychiatric diseases, we identify putative disease subtypes that associate with clinical phenotypes. Our study reveals the cellular template of a complex neurodevelopmental process, and provides a window into the cellular origins of brain diseases.
Assuntos
Linhagem da Célula/genética , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Neocórtex/embriologia , Animais , Sequência de Bases , Linhagem Celular , Ciliopatias/genética , Feminino , Células HEK293 , Humanos , Masculino , Transtornos Mentais/genética , Camundongos , Camundongos Endogâmicos C57BL , Transtornos do Neurodesenvolvimento/genética , Neurônios/citologia , Neurônios/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Células-Tronco/citologia , Transcrição Gênica/genéticaRESUMO
Abstract: Phosphatidylinositol 4-phosphate 5-kinase type 1 gamma (Pip5k1c) generates phosphatidylinositol 4,5-bisphosphate, also known as PI(4,5)P2 or PIP2. Many pronociceptive signaling pathways and receptor tyrosine kinases signal via PIP2 hydrolysis. Previously, we found that pain signaling and pain sensitization were reduced in Pip5k1cþ/ global heterozygous knockout mice. Here, we sought to evaluate the extent to which dorsal root ganglia selective deletion of Pip5k1c affected nociception in mice. Initially, we crossed sensory neuron-selective Advillin-Cre mice with a conditional Pip5k1c knockout (cKO) allele (Pip5k1cfl/fl). However, these mice displayed an early onset proprioceptive deficit. To bypass this early onset phenotype, we used two different tamoxifen-inducible Cre lines (Brn3a-Cre-ERT2 and Advillin-Cre-ERT2) to conditionally delete Pip5k1c in adults. Tamoxifen induced high efficiency deletion of PIP5K1C in dorsal root ganglia and slightly reduced PIP5K1C in spinal cord and brain in Brn3a-Cre-ERT2 Pip5k1cfl/fl (Brn3a cKO) mice while PIP5K1C was selectively deleted in dorsal root ganglia with no changes in spinal cord or brain in Advillin-Cre-ERT2 Pip5k1cfl/fl (Advil cKO) mice. Acute thermosensation and mechanosensation were not altered in either line relative to wild-type mice. However, thermal hypersensitivity and mechanical allodynia recovered more rapidly in Brn3a cKO mice, but not Advil cKO mice, following hind paw inflammation. These data collectively suggest that PIP5K1C regulates nociceptive sensitization in more regions of the nervous system than dorsal root ganglia alone.
Assuntos
Deleção de Genes , Inflamação/genética , Nociceptividade/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Células Receptoras Sensoriais/metabolismo , Animais , Gânglios Sensitivos/fisiopatologia , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Hiperalgesia/genética , Hiperalgesia/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Medula Espinal/metabolismoRESUMO
The neurobiological mechanisms underlying chronic pain associated with cancers are not well understood. It has been hypothesized that factors specifically elevated in the tumor microenvironment sensitize adjacent nociceptive afferents. We show that parathyroid hormone-related peptide (PTHrP), which is found at elevated levels in the tumor microenvironment of advanced breast and prostate cancers, is a critical modulator of sensory neurons. Intraplantar injection of PTHrP led to the development of thermal and mechanical hypersensitivity in both male and female mice, which were absent in mice lacking functional transient receptor potential vanilloid-1 (TRPV1). The PTHrP treatment of cultured mouse sensory neurons enhanced action potential firing, and increased TRPV1 activation, which was dependent on protein kinase C (PKC) activity. Parathyroid hormone-related peptide induced robust potentiation of TRPV1 activation and enhancement of neuronal firing at mild acidic pH that is relevant to acidic tumor microenvironment. We also observed an increase in plasma membrane TRPV1 protein levels after exposure to PTHrP, leading to upregulation in the proportion of TRPV1-responsive neurons, which was dependent on the activity of PKC and Src kinases. Furthermore, co-injection of PKC or Src inhibitors attenuated PTHrP-induced thermal but not mechanical hypersensitivity. Altogether, our results suggest that PTHrP and mild acidic conditions could induce constitutive pathological activation of sensory neurons through upregulation of TRPV1 function and trafficking, which could serve as a mechanism for peripheral sensitization of nociceptive afferents in the tumor microenvironment.
Assuntos
Hiperalgesia/induzido quimicamente , Proteína Relacionada ao Hormônio Paratireóideo/toxicidade , Transporte Proteico/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Animais , Capsaicina/farmacologia , Células Cultivadas , AMP Cíclico/metabolismo , Feminino , Gânglios Espinais/citologia , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Proteína Quinase C-épsilon/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Canais de Cátion TRPV/genética , Regulação para Cima/genéticaRESUMO
Existing analgesics are not efficacious in treating all patients with chronic pain and have harmful side effects when used long term. A deeper understanding of pain signaling and sensitization could lead to the development of more efficacious analgesics. Nociceptor sensitization occurs under conditions of inflammation and nerve injury where diverse chemicals are released and signal through receptors to reduce the activation threshold of ion channels, leading to an overall increase in neuronal excitability. Drugs that inhibit specific receptors have so far been unsuccessful in alleviating pain, possibly because they do not simultaneously target the diverse receptors that contribute to nociceptor sensitization. Hence, the focus has shifted toward targeting downstream convergence points of nociceptive signaling. Lipid mediators, including phosphatidylinositol 4,5-bisphosphate (PIP2), are attractive targets, as these molecules are required for signaling downstream of G-protein-coupled receptors and receptor tyrosine kinases. Furthermore, PIP2 regulates the activity of various ion channels. Thus, PIP2 sits at a critical convergence point for multiple receptors, ion channels, and signaling pathways that promote and maintain chronic pain. Decreasing the amount of PIP2 in neurons was recently shown to attenuate pronociceptive signaling and could provide a novel approach for treating pain. Here, we review the lipid kinases that are known to regulate pain signaling and sensitization and speculate on which additional lipid kinases might regulate signaling in nociceptive neurons.
Assuntos
Dor Crônica/enzimologia , Dor Crônica/terapia , Metabolismo dos Lipídeos/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Fosfotransferases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , HumanosRESUMO
Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c(+/-) mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2-dependent nociceptive signaling and suggest that PIP5K1C is a therapeutic target for chronic pain.
Assuntos
Hiperalgesia/metabolismo , Limiar da Dor/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/genética , Gânglios Espinais/citologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medição da Dor/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfopiruvato Hidratase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Tempo de Reação/genética , Células Receptoras Sensoriais/fisiologia , Medula Espinal/citologiaRESUMO
Infection with human immunodeficiency virus-1 (HIV-1) within the brain has long been known to be associated with neurodegeneration and neurocognitive disorder (referred as HAND), a condition characterized in its early stages by declining cognitive function and behavioral disturbances. Mechanistically, the HIV-1 coat glycoprotein 120 (gp120) has been suggested to be a critical factor inducing apoptotic cell death in neurons via the activation of p38 mitogen-activated protein kinase (MAPK), upon chronic exposure to the virus. Here we show that acute exposure of neurons to HIV-1 gp120 elicits a homeostatic response, which provides protection against non-apoptotic cell death, involving the major somatodendritic voltage-gated K⺠(Kv) channel Kv2.1 as the key mediator. The Kv2.1 channel has recently been shown to provide homeostatic control of neuronal excitability under conditions of seizures, ischemia and neuromodulation/neuroinflammation. Following acute exposure to gp120, cultured rat hippocampal neurons show rapid dephosphorylation of the Kv2.1 protein, which ultimately leads to changes in specific sub-cellular localization and voltage-dependent channel activation properties of Kv2.1. Such modifications in Kv2.1 are dependent on the activation of the chemokine co-receptors CCR5 and CXCR4, and subsequent activation of the protein phosphatase calcineurin. This leads to the overall suppression of neuronal excitability and provides neurons with a homeostatic protective mechanism. Specific blockade of calcineurin and Kv2.1 channel activity led to significant enhancement of non-apoptotic neuronal death upon acute gp120 treatment. These observations shed new light on the intrinsic homeostatic mechanisms of neuronal resilience during the acute stages of neuro-HIV infections.
Assuntos
Proteína gp120 do Envelope de HIV/farmacologia , HIV-1 , Hipocampo/citologia , Neurônios/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Canais de Potássio Shab/metabolismo , Animais , Apoptose/efeitos dos fármacos , Calcineurina/metabolismo , Cálcio/metabolismo , Feminino , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
The chemokine stromal cell-derived factor-1α (SDF-1α) has multiple effects on neuronal activity, survival, and death under conditions that generate a proinflammatory microenvironment within the brain, via signaling through C-X-C-type chemokine receptor 4 (CXCR4), although the underlying cellular/molecular mechanisms are unclear. Using rat hippocampal neurons, we investigated distinct modifications in the voltage-gated K⺠(Kv) channel Kv2.1 in response to short- and long-term SDF-1α/CXCR4-mediated signaling as an underlying mechanism for CXCR4-dependent regulation of neuronal survival and death. Acute exposure of neurons to SDF-1α led to dynamic dephosphorylation and altered localization of Kv2.1 channel, resulting in enhanced voltage-dependent activation of Kv2.1-based delayed-rectifier Kv currents (I(DR)). These changes were dependent on CXCR4- and/or NMDA receptor-mediated activation of calcineurin and provide neuroprotection. However, prolonged SDF-1α treatment leads to CXCR4-mediated activation of p38 mitogen-activated protein kinase, resulting in phosphorylation of Kv2.1 at S800 and enhanced surface trafficking of the channel protein, resulting in increased I(DR)/Kv2.1 current density. This, in combination with sustained dephosphorylation-induced enhancement of the voltage-dependent activation of I(DR)/Kv2.1, predisposed neurons to excessive K⺠efflux, a vital step for the neuronal apoptotic program. Such apoptotic death was dependent on CXCR4 and Kv2.1 function and was absent in cells expressing the Kv2.1-S800A mutant channel. Furthermore, similar modifications in Kv2.1 and CXCR4/Kv2.1-dependent apoptosis were observed following treatment of neurons with the human immunodeficiency virus-1 (HIV-1) glycoprotein gp120. Therefore, distinct modifications in Kv2.1 in response to short- and long-term CXCR4-mediated signaling could provide a basis for neuroprotection or apoptosis in neuropathologies, such as neuroinflammation, stroke, brain tumors, and HIV-associated neurodegeneration.
Assuntos
Apoptose/fisiologia , Quimiocina CXCL12/fisiologia , Receptores CXCR4/fisiologia , Canais de Potássio Shab/metabolismo , Animais , Apoptose/efeitos dos fármacos , Calcineurina/fisiologia , Inibidores de Calcineurina , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Quimiocina CXCL12/farmacologia , Interações Medicamentosas , Feminino , Células HEK293 , Proteína gp120 do Envelope de HIV/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas Mutantes/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/agonistas , Canais de Potássio Shab/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tacrolimo/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain essentially unknown. Many biological effects of NPs are mediated by guanylate cyclase (GC)-coupled NP receptors, NPR-A and NPR-B, whereas the third NP receptor, NPR-C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR-C can couple to specific Gα(i)-Gßγ-mediated intracellular signaling cascades in numerous cell types. We found that NPR-C is coexpressed in transient receptor potential vanilloid-1 (TRPV1)-expressing mouse dorsal root ganglia (DRG) neurons. NPR-C can be coimmunoprecipitated with Gα(i), and C-type natriuretic peptide (CNP) treatment induced translocation of protein kinase Cε (PKCε) to the plasma membrane of these neurons, which was inhibited by pertussis toxin pretreatment. Application of CNP potentiated capsaicin- and proton-activated TRPV1 currents in cultured mouse DRG neurons and increased their firing frequency, an effect that was absent in DRG neurons from TRPV1(-/-) mice. CNP-induced sensitization of TRPV1 activity was attenuated by pretreatment of DRG neurons with the specific inhibitors of Gßγ, phospholipase C-ß (PLCß), or PKC, but not of protein kinase A, and was abolished by mutations at two PKC phosphorylation sites in TRPV1. Furthermore, CNP injection into mouse hindpaw led to the development of thermal hyperalgesia that was attenuated by administration of specific inhibitors of Gßγ or TRPV1 and was also absent in TRPV1(-/-) mice. Thus, our work identifies the Gßγ-PLCß-PKC-dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity.