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Single cell RNAseq analysis of dorsal root ganglia from global and cLTMR-specific Nav1.7 knockout mice

ABSTRACT: We investigated changes in gene expression and cell type abundance using single cell RNA sequencing of dorsal root ganglia (DRG) isolated from global (pCAGG-CreER x Nav1.7 floxed) and C-low threshold mechanoreceptor (cLTMR)-specific (Slc17a8-Cre x Nav1.7 floxed) Nav1.7 knockout mice. We identified a reduction in the fraction of cLTMRs and a concomitant emergence of a distinct cell cluster located closely to cLTMRs in the UMAP projection in global Nav1.7 knockout DRGs. This novel cell cluster expressed previously described cLTMR markers with distinct gene expression changes such as a downregulation of Ceacam10 and an upregulation of Penk expression. We further found that cLTMR-specifc knockout of Nav1.7 induced the same shift in cLTMR cells to a knockout-specific cluster with upregulated Penk expression.

ORGANISM(S): Mus musculus

PROVIDER: GSE213825 | GEO | 2023/05/22

REPOSITORIES: GEO

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Bulk RNAseq analysis of dorsal root ganglia from global Nav1.7 knockout mice

Project description:We investigated changes in gene expression of dorsal root ganglia (DRG) isolated from global (pCAGG-CreER x Nav1.7 floxed) Nav1.7 knockout mice using bulk RNA sequencing. We found an upregulation of Penk and downregulation of Ceacam10 in Nav1.7 knockouts compared to controls, as well as other gene expression changes similar to those previously reported for Nav1.7 knockout mice (https://doi.org/10.1038/ncomms9967). We identified that genes selectively expressed in the C low-threshold mechanoreceptor (cLTMR) class of DRG neurons showed a general reduction in expression in Nav1.7 knockouts compared to littermate controls.

2023-05-22 | GSE213826 | GEO

The molecular basis of analgesia in congenital insensitivity to pain associated with loss of Nav1.7 function

Project description:Loss of function mutations in the SCN9a gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain (CIP) and anosmia in otherwise normal humans and mice, suggesting that this channel may be a good analgesic drug target. Surprisingly, potent selective antagonists of Nav1.7 are weak analgesics. We therefore investigated whether Nav1.7 , as well as contributing to electrical signalling may have an additional function. Here we report that Nav1.7 deletion has profound effects on the sensory neuron transcriptome, leading to dysregulation of a number of transcription factors as well as upregulation of enkephalin precursor PENK mRNA and down regulation of CEACAM10 mRNA, a protein involved in noxious thermosensation. PENK mRNA is transcriptionally upregulated in Nav1.7 null mutant female sensory neurons, resulting in increased enkephalin expression in the dorsal horn of the spinal cord. PENK expression is down-regulated by addition of the sodium ionophore monensin, suggesting that sodium may play a role as a second messenger. Application of the opioid antagonist naloxone strongly enhances noxious peripheral input into the spinal cord, and dramatically reduces analgesia in both male and female Nav1.7 null mutant mice, as well as in human Nav1.7 null mutants. These data show that loss of Nav1.7 expression increases opioid drive over the lifetime of mice and humans. They further suggest that Nav1.7 channel blockers alone may not replicate the phenotype of null mutant humans and mice, but should be potentiated with exogenous opioids. RNA was extracted from Dorsal Root Ganglia tissue from Nav1.7 Knock-Out, Nav1.8 KO and Nav1.9 KO mice (n = 3) and hybridised on Affymetrix Mouse Genome 430 2.0 Array (GPL1261)

2014-09-13 | E-GEOD-61373 | biostudies-arrayexpress

The molecular basis of analgesia in congenital insensitivity to pain associated with loss of Nav1.7 function

2014-09-13 | GSE61373 | GEO

Long-lasting Analgesia via Targeted in situ Repression of NaV1.7

Project description:Current treatments for chronic pain rely largely on opioids despite their significant side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Towards this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells, and then by the lumbar intrathecal route delivered both epigenome-engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain and BzATP-induced pain. Our results demonstrate: i) effective repression of NaV1.7 in lumbar dorsal root ganglia; ii) reduced thermal hyperalgesia in the inflammatory state; iii) decreased tactile allodynia in the neuropathic state; and iv) no changes in normal motor function. We anticipate this genomically scarless and non-addictive pain prevention and amelioration approach enabling Long-lasting Analgesia via Targeted in vivo Epigenetic Repression of NaV1.7, a methodology we dub pain LATER, will have significant therapeutic potential in management of persistent pain states.

2022-01-01 | GSE159818 | GEO

Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene targeted mice

Project description:The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. As well as action potential propagation, NaV1.7 regulates neurotransmitter release, integrates depolarizing inputs over long periods and regulates transcription. In order to better understand these functions, we generated an epitope-tagged NaV1.7 mouse that showed normal NaV1.7 channel activity and normal pain behavior. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 NaV1.7 associated proteins including known and novel interactors such as sodium channel β3 subunit (Scn3b) and collapsin response mediator protein (Crmp2). Selected NaV1.7 protein interactors, such as Crmp2, membrane-trafficking protein synapototagmin-2 (Syt2), G protein-regulated inducer of neurite outgrowth 1 (Gprin1), L-type amino acid transporter 1 (Lat1) and transmembrane P24 trafficking protein 10 (Tmed10) were validated using co-immunoprecipitation and functional assays. The information provided with this physiologically normal epitope-tagged mouse should provide useful insights into the downstream mechanisms associated with NaV1.7 channel function.

2018-01-15 | PXD005155 | Pride

Long-lasting Analgesia via Targeted in situ Repression of NaV1.7

Project description:Long-lasting Analgesia via Targeted in situ Repression of NaV1.7

| PRJNA670519 | ENA

MicroRNA-96 is required to prevent allodynia by repressing voltage-gated sodium channels in spinal cord

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.

2021-07-06 | GSE144832 | GEO

Transcription profiling of Drosophila mechanoreceptor-rich tissue compared mechanoreceptor-poor tissue shows DCX-EMAPis required for mechanoreception in sensory cilia

Project description:Determining which genes are expressed in mechanoreceptor-rich tissue (pedicel) compared mechanoreceptor-poor tissue (capitellum) and a neuronal subtraction control (thoracic ganglion) in Drosophila melanogaster

2009-05-01 | E-MEXP-1921 | biostudies-arrayexpress

Development of Photocrosslinking Probes Based on Huwentoxin-IV to Map the Site of Interaction on Nav1.7

Project description:This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of Nav1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-2 and helix S3 of VSD2 in a chimeric channel system. Sample preparation of purified chimeric channels crosslinked with the photoprobes was based on the FASP protocol. Various digestion enzymes were evaluated in order to maximise the sequence coverage of this membrane protein. Finally, MeroX was employed in order to identify the sites of crosslinking. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on Nav channels.

2019-11-18 | PXD015037 | Pride

Mouse retina from Msi1/Msi2 double knockout in photoreceptor cells

Project description:This quantitative protepomics study (TMT10 isobaric labeling) of the protein expression in the retina of Msi1/Msi2 double knockout mouse compared to floxed controls. The Msi1 and Msi2 genes were knocked out in photoreceptor cells using tamoxifen inducible Cre (Cre-ERT2) under the control of Pde6g promoter. Tamoxifen was administered by intrapertoneal injection for three consecutive day starting at postnatald day 30, and the retina was collected at postnatal day 51. Retinas from floxed animals lacking the Cre recombinase and treated with tamoxifen were used as controls. Five biological replicates for each knockout (three femailes, two males) and control (two femaes and three males) group were used.

2022-03-10 | PXD030748 | Pride

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