IP3 receptor depletion in a spontaneous canine model of Charcot-Marie-Tooth disease 1J with amelogenesis imperfecta

Abstract Inositol 1,4,5-trisphosphate receptors (IP 3 R) mediate Ca 2+ release from intracellular stores, contributing to complex regulation of numerous physiological responses. The involvement of the three IP 3 R genes ( ITPR1 , ITPR2 and ITPR3 ) in inherited human diseases has started to shed light on the essential roles of each receptor in different human tissues and cell types. Variants in the ITPR3 gene, which encodes IP 3 R3, have recently been found to cause demyelinating sensorimotor Charcot-Marie-Tooth neuropathy type 1J (CMT1J). In addition to peripheral neuropathy, immunodeficiency and tooth abnormalities are occasionally present. Here, we report the identification of a homozygous nonsense variant in the ITPR3 gene in Lancashire Heeler dogs, presenting with a severe developmental enamel defect and reduced nerve conduction velocity. We studied the primary skin fibroblasts of the affected dogs and observed that the nonsense variant in ITPR3 led to a complete absence of full-length IP 3 R3 protein. Unexpectedly, the protein levels of IP 3 R1 and IP 3 R2 were also markedly decreased, suggesting co-regulation. Functional Ca 2+ measurements revealed reduced IP 3 R-mediated Ca 2+ flux upon stimulation of G-protein-coupled-receptors in the affected dog fibroblasts. We were able to rescue the IP 3 R1 and IP 3 R2 depletion by proteasome inhibition but not the IP 3 R3 loss, which was facilitated by nonsense-mediated mRNA decay. These findings highlight the first spontaneous mammalian phenotype caused by a nonsense variant in ITPR3 , leading to the loss of IP 3 R3. The human and canine IP 3 R3 proteins are highly similar, and our study suggests that the tissue involvement resulting from the receptor’s dysfunction is also conserved. In summary, IP 3 R3 is critical for enamel formation and peripheral nerve maintenance. Author summary We investigated pet dogs, Lancashire Heelers, with impairments in tooth development and in the nerves that regulate limb muscles. Through genetic studies of the dog pedigree, we found that the phenotypes were caused by a recessively inherited mutation in the ITPR3 gene, which encodes one of three IP 3 receptors (IP 3 R) isoforms (IP 3 R3 isoform) that are needed for intracellular Ca 2+ signaling. Mutated IP 3 R3 has been recently linked to a human inherited neuropathy called Charcot-Marie-Tooth disease type 1J, which impairs peripheral nerve function and is accompanied by immunodeficiency and abnormal teeth in some individuals. We showed that in the skin cells of the affected dogs, the full-length IP 3 R3 protein was completely absent, and also the protein levels of the other two IP 3 R isoforms (IP 3 R1 and IP 3 R2) were severely lowered. This led to impaired agonist-induced Ca 2+ release and signaling. Our results demonstrate the high conservation between human and canine IP 3 receptors and their significance for different tissue systems. The genetic studies now highlight that IP 3 R3 is vital for peripheral nerve function and enamel development.

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