Car8 dorsal root ganglion expression and genetic regulation of analgesic responses are associated with a cis-eQTL in mice

Abstract

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 this report, we show that DRG Car8 expression is variable across 25 naïve-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; V5-Car8 ^WT 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 ITPR1-dependent calcium release in mice.

Figure 1. Relative DRG expression, and eQTL analysis of Car8, as a function of inbred strain

(A) The relative expression of Car8 is plotted (grey bars) for each inbred strain along with haplotypes in the region covering Car8. (B) The haplotypes for thirteen variable single nucleotide polymorphisms (SNPs) are indicated by their allele, SNP ID, chromosomal location, and colored to indicate haplotype groupings. The Car8 locus is shown in yellow. (C) Genome wide association analysis of DRG mRNA gene expression patterns (e-QTL analysis) reveals a highly significant locus on Chr4 (−logP value=11.5) in the Manhattan plot. The locus marked by SNP rs27660559, delineates a strong association between gene expression pattern and the genomic locus for Car8 indicating cis-regulation of gene expression. Alternating black and grey bands denote each chromosome number.

Figure 2. Morphine-induced calcium release in NBL cells is attenuated by Car8 overexpression

Morphine induces increased intracellular free calcium in NBL cells. Intracellular free calcium was analyzed using Fura 2 imaging. (A) Morphine induces increase of intracellular free calcium denoted on the Y-axis as % difference from baseline in a dose-response manner denoted on the X-axis as morphine dose (micromolar). The EC₅₀ to morphine was approximately 9micromolar. (B) The response to 10 micromolar morphine was attenuated after NBL cells were infected with AAV8-V5-Car8^WT, but not after infection with AAV8-V5-Car8^MT or empty vector. N=6 from 2 independent cultures. ***P<0.001; ANOVA.

Figure 3. Immunocytochemistry and western blotting confirm Car8 and mu opioid receptor expression in NBL cells

Immunocytochemistry demonstrates levels of V5-tagged Car8 after infection with AAV8-V5-Car8^WT (A–C), and AAV8-V5-Car8^MT (D–F). Western blotting shows endogenous mu opioid receptor expression in NBL cells that are responsive to morphine (G). Scale bar = 100 μm.