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

• Published in: PLoS genetics Vol. 15; no. 6; p. e1008226
• Main Authors: Upadhyay, Udita, Zhuang, Gerald Z, Diatchenko, Luda, Parisien, Marc, Kang, Yuan, Sarantopoulos, Konstantinos D, Martin, Eden R, Smith, Shad B, Maixner, William, Levitt, Roy C
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14-06-2019; Public Library of Science (PLoS)
• Subjects: 3' Untranslated regions; Alleles; Allosteric properties; Alternative splicing; Alternative Splicing - genetics; Amino acids; Analgesia; Analgesics; Anesthesiology; Animals; Ataxia; Biology and life sciences; Biomarkers, Tumor - genetics; Biomarkers, Tumor - pharmacology; Calcium (intracellular); Calcium Signaling - genetics; Calcium signalling; Carbonic anhydrases; Cerebellar ataxia; Cerebellum; Cerebellum - drug effects; Cerebellum - metabolism; Dorsal root ganglia; Excitability; Exons (Molecular genetics); Ganglia, Spinal - metabolism; Ganglia, Spinal - pathology; Ganglion cysts; Gender differences; Gene expression; Gene therapy; Gene transfer; Gene Transfer Techniques; Genes; Genetic aspects; Genetic regulation; Genomics; Glial cells; Health aspects; HEK293 Cells; Homozygotes; Humans; Immunohistochemistry; Inflammation; Inositol; Inositol 1,4,5-trisphosphate receptors; Inositol 1,4,5-Trisphosphate Receptors - genetics; Intracellular; Kinases; Laboratory animals; Medicine; Medicine and Health Sciences; Mice; Mutation; Mutation - genetics; Neuronal-glial interactions; Neurons; Neurons - metabolism; Neurons - pathology; Neurophysiology; Organ Specificity; Pain - genetics; Pain - pathology; Pain management; Pain perception; Peptides; Peptides - genetics; Peptides - pharmacology; Phenotypes; Physiological aspects; Quantitative trait loci; Quantitative Trait Loci - genetics; Research and Analysis Methods; RNA Splice Sites - genetics; Sciatic Nerve - drug effects; Sciatic Nerve - metabolism; Software; Transcription; United States; North Carolina; Canada; Montreal Quebec Canada; Florida; United States > US; Quebec Canada
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1008226

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.