MicroRNA-96 is required to prevent allodynia by repressing voltage-gated sodium channels in spinal cord
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PROVIDER: PRJNA605099 | ENA |
REPOSITORIES: ENA
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Project description:Voltage-gated sodium channels (Navs) 1.7, 1.8, and 1.9 are predominately expressed in peripheral sensory neurons and are critical for action potential propagation in nociceptors. Unexpectedly, we found that expression of SCN9A, SCN10A, SCN11A, and SCN2A, the alpha subunit of Nav1.7, Nav1.8, Nav1.9 and Nav1.2, respectively, are up-regulated in spinal dorsal horn (SDH) neurons of miR-96 knockout mice. These mice also have de-repression of CACNA2d1/2 in DRG and display heat and mechanical allodynia that could be attenuated by intrathecal or intraperitoneal injection of Nav1.7 or Nav1.8 inhibitors or Gabapentin. Moreover, Gad2::CreERT2 conditional miR-96 knockout mice phenocopied global knockout mice, implicating inhibitory neurons; nerve injury induced significant loss of miR-96 in SDH GABAergic and Glutamatergic neurons in mice which negative correlated to up-regulation of Nav1.7, Nav1.8, Nav1.9 and Scn2a, this dis-regulation of miR-96 and Navs in SDH neurons contributed to neuropathic pain which can be alleviated by intrathecal injection of Nav1.7 or Nav1.8 blockers. In conclusion, miR-96 is required to avoid allodynia through limiting the expression of VGCCs and Navs in DRG and Navs in SDH in naïve and nerve injury induced neuropathic pain mice. Our findings suggest that central nervous system penetrating Nav1.7 and Nav1.8 inhibitors may be efficacious for pain relief.
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Project description:The targeting of membrane proteins that are activated in cancer stem cells (CSCs) represents one of the key strategies in novel cancer therapy. The present study investigated ion channel expression profiles and functions in pancreatic CSCs (PCSCs). Cells strongly expressing aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were separated from PK59 cells, a human pancreatic cancer cell line, using fluorescence-activated cell sorting (FACS), and PCSCs were identified based on tumorsphere formation. A microarray analysis was performed to investigate gene expression profiles in PCSCs. ALDH1A1 messenger RNA levels were higher in PCSCs. PCSCs were resistant to 5-fluorouracil (5-FU) and capable of re-differentiation. The microarray analysis revealed that gene expression related to ion channels, including voltage-gated potassium channels (Kv), was up-regulated. 4-Aminopyridine (4-AP), a potent Kv inhibitor, exhibited greater cytotoxicity in PCSCs. In a xenograft model in nude mice, tumor volumes were significantly smaller in mice injected with PK59 cells treated with 4-AP than in those injected with non-treated cells. The present results implicate Kv in the persistence of PCSCs, and the Kv inhibitor, 4-AP, has potential as a therapeutic agent for pancreatic carcinoma.
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Project description:Scn2a encodes voltage-gated sodium channel NaV1.2, a main mediator of neuronal action potential firing. The current paradigm suggests that NaV1.2 gain-of-function variants enhance neuronal excitability resulting in epilepsy, whereas NaV1.2 deficiency impairs excitability contributing to autism. This paradigm, however, does not explain why 20~30% of patients with NaV1.2 deficiency still develop seizures. Here we report a counterintuitive finding that severe NaV1.2 deficiency results in increased neuronal excitability. Using a unique NaV1.2-deficient mouse model, we found enhanced intrinsic excitabilities of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in Scn2a-related seizures. This increased excitability is autonomous, and is reversible by the genetic restoration of Scn2a expression in adult mice. RNA-sequencing revealed that the downregulation of multiple potassium channels including KV1.1, and KV channel openers alleviated hyperexcitability of NaV1.2-deficient neurons. This unexpected neuronal hyperexcitability may serve as a cellular basis underlying NaV1.2 deficiency-related seizures.
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Project description:In multipolar vertebrate neurons, action potentials (AP) initiate close to the soma, at the axonal initial segment (AIS). Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the AIS in vertebrates. We used an intronic MiMIC to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para. Despite being the only NaV channel in the fly, we show that only 23 ±1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole 3rd instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore only 23 ±1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the Distal Axonal Segment (DAS). The DAS is present in multiple neuron classes in both the 3rd instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.
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Project description:In cardiomyocytes, Ca2+ influx through L-type voltage-gated calcium channels (LTCCs) following membrane depolarization regulates crucial Ca2+-dependent processes including duration and amplitude of the action potentials and excitation-contraction coupling. LTCCs are heteromultimeric proteins composed of the Cav1, Cav, Cav2 and Cav subunits. Here, using ascorbate peroxidase (APEX)-mediated proximity labeling and quantitative proteomics, we identified 61 proteins in the nano-environments of Cav2 in cardiomyocytes. These proteins are involved in diverse cellular functions such as cellular trafficking, muscular contraction, sarcomere organization and excitation-contraction coupling. Moreover, pull-down assays, co-immunoprecipitation analyses and super-resolution imaging using direct stochastic optical reconstruction microscopy (dSTORM) revealed that Cav2 interacts with the ryanodine receptor 2 (RyR2) in adult cardiomyocytes, probably coupling LTCCs and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src-homology 3 domain of Cav2 and is necessary for an effective pacing frequency‐dependent increase of the Ca2+-induced Ca2+ release mechanism in cardiomyocytes.
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Project description:Estrogen-related receptor γ (ERRγ) serves a critical O2-dependent regulatory role in differentiation of human cytotrophoblasts to syncytiotrophoblast. In this study, we investigated expression of genes encoding tissue kallikreins (KLK1) and voltage-gated K+ channels (KV7) during differentiation of human trophoblasts in culture and the roles of ERRγ and O2 tension in their regulation. Expression of KLK1 and the KV7 channel subunits, KCNQ1, KCNE1, KCNE3, KCNE5, increased during differentiation of cultured human trophoblast cells in a 20% O2 environment. Notably, together with ERRγ, expression of KLK1, KCNQ1, KCNE1, KCNE3 and KCNE5 was markedly reduced when cells were cultured in a hypoxic environment (2% O2). Moreover, upon transduction of trophoblast cells with shRNAs for endogenous ERRγ, KLK1, KCNQ1, KCNE1 and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRγ response elements (ERREs) within the KLK1 and KCNE1 5'-flanking regions required for ERRγ -stimulated transcriptional activity. Binding of endogenous ERRγ to these ERREs increased during trophoblast differentiation in culture and was inhibited by hypoxia. The KV7 blocker linopirdine reduced hCG secretion and aggregation of cultured human trophoblasts, suggesting a possible role of KV7 channels in cell fusion and differentiation. Illumina gene expression arrays of cultured human trophoblast cells revealed several genes upregulated during syncytiotrophoblast differentiation and downregulated upon ERRγ knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRγ mediates O2-dependent expression of genes involved in human trophoblast differentiation, function and vascular homeostasis.
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