(33) Investigation of combined oxidative phosphorylation deficiency 25 (COXPD25) in Mouse and hiPSCs Models

Background: Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) disorders are a rare class of mitochondrial disease with poorly understood pathophysiology. These enzymes are crucial for mitochondrial protein synthesis, catalyzing the attachment of amino acids to their cognate mitochondrial tRNA molecules, essential for energy production via oxidative phosphorylation. Pathogenic, biallelic single nucleotide variants in MARS2 cause COXPD25, characterized by intellectual disability, growth failure, and sensorineural hearing loss. Affected patients exhibit decreased oxidative phosphorylation enzyme activities of Complex I (CI) and Complex IV (CIV) in fibroblasts, and reduced protein expression of NDUFB8 (CI) and COXII (CIV) in fibroblasts and lymphoblasts.

Objectives: This study aims to define MARS2-regulated pathways in highly energetic tissues to elucidate COXPD25 pathophysiology using mouse models and human induced pluripotent stem cell (hiPSC) models to explore MARS2 mutation effects.

Design/Methods: We developed mouse and human iPSC models of COXPD25. The mouse model was generated using CRISPR-Cas9 to introduce the loss-of-function p.R135W variant, homologous to the p.R142W pathogenic mutation in patients. MARS2R135W/R135W mouse liver and heart were collected for immunoblotting using a mitochondrial complex antibody cocktail and RNA-seq. Differentially expressed genes (DEGs) were identified and gene ontology (GO) enrichment analysis was performed. Additionally, MARS2R135W/R135W hiPSCs were differentiated into immature (day 7) and mature (day 28) cortical neurons for RNA-seq to identify differentially expressed and coregulated genes.

Results: Immunoblots showed decreased expression of NDUFB8, representative of CI, in MARS2R135W/R135W liver. RNA-seq analysis revealed decreased expression of Ndufa11, Ndufb7, Ndufs8, Ndufv3, and Ndufa9 (CI subunits); Uqcr11, Uqcrc1, Cyc1 (CIII subunits); Cox6b1 (CIV subunit); and Atp5g1 (CV subunit) in MARS2R135W/R135W heart. GO enrichment analysis of DEGs in day 7 and day 28 cortical neurons indicated significant dysregulation of neurogenesis, nervous system development, and axonogenesis.

Conclusion: Our findings demonstrate abnormalities in OXPHOS in mouse liver and heart, implicating MARS2 in energy deficiencies leading to COXPD25. RNA-seq analysis confirms the critical role of MARS2 in neurogenesis. Utilizing both mouse and hiPSC models of COXPD25 enables investigation of mt-ARS disease pathophysiology in vivo and in vitro.