Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wildtype Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wildtype ortholog of Car8 (CA8WT), but not the reported CA8 S100P loss-of-function mutation (CA8MT); inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high affinity receptor); and ITPR1-mediated cytoplasmic free calcium release in vitro. Additionally, we show that gene-transfer using AAV8-V5-CA8WT viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8WT expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8WT mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1.

Fission yeast have adapted to retrotransposon invasion by RNAi-mediated silencing, which has coevolved into a mechanism involving CENPB-mediated heterochromatinization together with ablation of RNAi components via accumulation of recombinogenic repeats in recently diverged species of Schizosaccharomyces. Similar trends are seen in the metazoans.

Carbonic anhydrase-8 (Car8 mouse gene symbol) is devoid of enzymatic activity, but instead functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) to regulate this intracellular calcium release channel important in synaptic functions and neuronal excitability. Causative mutations in ITPR1 and carbonic anhydrase-8 in mice and humans are associated with certain subtypes of spinal cerebellar ataxia (SCA). SCA mice are genetically deficient in dorsal root ganglia (DRG) Car8 expression and display mechanical and thermal hypersensitivity and susceptibility to subacute and chronic inflammatory pain behaviors in mice. In this report we show that DRG Car8 expression is variable across 25 naive-inbred strains of mice, and this cis-regulated eQTL (association between rs27660559, rs27706398, and rs27688767 and DRG Car8 expression; P<1×10−11) is correlated with nociceptive responses in mice. Next, we hypothesized that increasing DRG Car8 gene expression would inhibit intracellular calcium release required for morphine antinociception and might correlate with antinociceptive sensitivity of morphine and perhaps other analgesic agents. We show that mean DRG Car8 gene expression is directly related to the dose of morphine or clonidine needed to provide a half-maximal analgesic response (r=0.93, P<0.00002; r=0.83, P<0.0008, respectively), suggesting that greater DRG Car8 expression increases analgesic requirements. Finally, we show that morphine induces intracellular free calcium release using Fura 2 calcium imaging in a dose-dependent manner; and V5-Car8WT overexpression in NBL cells inhibits morphine-induced calcium increase. These findings highlight the ‘morphine paradox’ whereby morphine provides antinociception by increasing intracellular free calcium, while Car8 and other antinociceptive agents work by decreasing intracellular free calcium. This is the first study demonstrating that biologic variability associated with this cis-eQTL may contribute to differing analgesic responses through altered regulation of ITPRI-dependent calcium release in mice.

Recently, we showed murine DRG Car8 expression is a cis-regulated eQTL that determines analgesic responses. In this report, we show transduction via SN injection of DRG with human wildtype CA8 using AAV viral particles (AAV8-V5-CA8WT) produces analgesia in naïve male C57BL/6J mice and anti-hyperalgesia after carrageenan treatment. A peak mean increase of about 4 seconds in thermal hindpaw withdrawal latency equaled increases in thermal withdrawal latency produced by 10 mg/kg IP morphine in these mice. Allometric conversion of this IP morphine dose in mice equals an oral morphine dose of about 146 mg in a 60 kg adult. Our work quantifies for the first time analgesia and anti-hyperalgesia in an inflammatory pain model after DRG transduction by CA8 gene therapy.

Carbonic anhydrase-8 (CA8) is an intracellular protein that functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) critical to intracellular Ca ++ release, synaptic functions and neuronal excitability. We showed previously that murine nociception and analgesic responses are regulated by the expression of this gene in dorsal root ganglion (DRG) associated with a cis -eQTL. In this report, we identify an exon-level cis -eQTL (rs6471859) that regulates human DRG CA8 alternative splicing, producing a truncated 1,697bp transcript (e.g., CA8-204). Our functional genomic studies show the “G” allele at rs6471859 produces a cryptic 3’UTR splice site regulating expression of CA8-204. We developed constructs to study the expression and function of the naturally occurring CA8-204 G transcript (G allele at rs6471859), CA8-204 C (C allele at rs6471859 reversion mutation) and CA8-201 (full length transcript). CA8-204 G transcript expression occurred predominantly in non-neuronal cells (HEK293), while CA8-204 C expression was restricted to neuronal derived cells (NBL) in vitro . CA8-204 G produced a stable truncated transcript in HEK293 cells that was barely detectable in NBL cells. We also show CA8-204 produces a stable peptide that inhibits pITPR1 and Ca ++ release in HEK293 cells. These results imply homozygous G/G individuals at rs6471859, which are common in the general population, produce exclusively CA8-204 G that is barely detectable in neuronal cells. CA8 null mutations that greatly impact neuronal functions are associated with severe forms of spinal cerebellar ataxia, and our data suggest G/G homozygotes should display a similar phenotype. To address this question, we show in vivo using AAV8-FLAG-CA8-204 G and AAV8-V5-CA8-201 gene transfer delivered via intra-neural sciatic nerve injection (SN), that these viral constructs are able to transduce DRG cells and produce similar analgesic and anti-hyperalgesic responses to inflammatory pain. Immunohistochemistry (IHC) examinations of DRG tissues further show CA8-204 G peptide is expressed in advillin expressing neuronal cells, but to a lesser extent compared to glial cells. These findings explain why G/G homozygotes that exclusively produce this truncated functional peptide in DRG evade a severe phenotype. These genomic studies significantly advance the literature regarding structure-function studies on CA8-ITPR1 critical to calcium signaling pathways, synaptic functioning, neuronal excitability and analgesic responses.