Succinate supplementation ameliorates musculoskeletal defects caused by PLOD3 mutations in a BCARD syndrome model

BCARD syndrome is a rare complex connective tissue disorder associated with variants in the PLOD3 gene, presenting with musculoskeletal, vascular, and sensory deficits. The role of PLOD3 in post-translational modifications of collagens has been established. However, limited treatment options exist to correct connective tissue deficits linked to PLOD3, largely due to sparse knowledge of cellular and molecular mechanisms driving phenotypic changes. To explain the mechanisms of PLOD3 genotype-phenotype associations, we have used clinical data, molecular assays in patient-derived fibroblasts, perturbation experiments in zebrafish models, cellular and molecular experiments, and unbiased genome- and transcriptome-wide approaches. We show that wild-type human PLOD3 mRNA partially rescued musculoskeletal, vascular, and brain phenotypes in zebrafish plod3 mutants, while clinically identified variants had only a limited effect, validating the pathogenicity of the variants and the high conservation of PLOD3 function across vertebrates. We found that, at the molecular level, organ systems selectively upregulated the PERK pathway of the Unfolded Protein Response and subsequently activated autophagy as an adaptive response to an extracellular matrix (ECM) protein backlog; however, autophagy inhibitors did not rescue the plod3 mutant phenotypes. Bulk RNA-seq analysis of plod3 mutants revealed downregulation of genes in metabolic pathways, including the electron transport chain and the tricarboxylic acid (TCA) cycle, consistent with structural defects in electron micrographs of mitochondria. Search of Drug Repurposing Data Portals identified a dietary supplement, succinate, to be associated with PLOD3 and 25 additional genes, involved in the TCA cycle and collagen synthetic pathways. We showed that treatment with succinate ameliorated BCARD features, i.e., musculoskeletal defects, and restored reduced expression of TCA cycle genes in the zebrafish model. Our data indicate that the interaction between ECM synthesis and mitochondrial energy metabolism offers an entry point for novel therapies to prevent complex connective tissue decline in BCARD and, potentially, in other rare and common musculoskeletal disorders and conditions such as aging, cancer, or injury. Moreover, the genetic models developed here, and succinate, should be valuable tools in future studies of the underlying mechanisms of the BCARD extensive medical phenome.