Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis-eQTL.

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. Author summary Carbonic anhydrase-8 (CA8) inhibits IP3 binding to the inositol trisphosphate receptor-1(ITPR1), which regulates intracellular calcium signaling critical to neuronal functions. Recessive CA8-null mutants are associated with spinocerebellar ataxia and neurodegenerative disorders. We have previously demonstrated that nociception and analgesic responses are associated with a DRG cis-eQTL that regulates murine expression of this gene. This study focuses on a human DRG exon-level cis-eQTL (rs6471859) that regulates CA8-204 alternative splicing producing a truncated 1,697bp transcript. Herein, we demonstrate the G allele at rs6471859 produces a cryptic 3'UTR splice site regulating tissue-specific CA8-204 expression. In vitro studies show the G allele (CA8-204(G)) produces a stable peptide that inhibits ITPR1 activation and Ca++ release in non-neuronal cells, but not in neuronal cells. However, using AAV8 gene transfer in vivo we show CA8-204(G) peptide is expressed in both glial and to a lesser extent in neuronal cells, producing profound analgesia and anti-hyperalgesia using inflammatory pain models, similar to the full length CA8-201 positive control. These data significantly extend our understanding of CA8 structure-function, demonstrating the truncated peptide may represent a novel therapeutic candidate.