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

PLoS Genet. 2019 Jun 14;15(6):e1008226. doi: 10.1371/journal.pgen.1008226. eCollection 2019 Jun.

Abstract

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 CA8-204G transcript (G allele at rs6471859), CA8-204C (C allele at rs6471859 reversion mutation) and CA8-201 (full length transcript). CA8-204G transcript expression occurred predominantly in non-neuronal cells (HEK293), while CA8-204C expression was restricted to neuronal derived cells (NBL) in vitro. CA8-204G 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 CA8-204G 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-CA8-204G 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 CA8-204G 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / genetics
  • Animals
  • Biomarkers, Tumor / genetics*
  • Biomarkers, Tumor / pharmacology
  • Calcium Signaling / genetics*
  • Cerebellum / drug effects
  • Cerebellum / metabolism
  • Ganglia, Spinal / metabolism
  • Ganglia, Spinal / pathology
  • Gene Transfer Techniques
  • HEK293 Cells
  • Humans
  • Inositol 1,4,5-Trisphosphate Receptors / genetics*
  • Mice
  • Mutation / genetics
  • Neurons / metabolism*
  • Neurons / pathology
  • Organ Specificity
  • Pain / genetics*
  • Pain / pathology
  • Peptides / genetics
  • Peptides / pharmacology
  • Quantitative Trait Loci / genetics
  • RNA Splice Sites / genetics
  • Sciatic Nerve / drug effects
  • Sciatic Nerve / metabolism

Substances

  • Biomarkers, Tumor
  • CA8 protein, human
  • ITPR1 protein, human
  • Inositol 1,4,5-Trisphosphate Receptors
  • Peptides
  • RNA Splice Sites