ATAD1 Overexpression Enhances Mitochondrial and Peroxisomal Function in Zellweger Syndrome Disorder Models

Abstract

Zellweger Spectrum Disorders (ZSDs) are caused by mutations in any of the different peroxin (PEX) genes, which are essential for peroxisome biogenesis and function. Clinical features of ZSDs include seizures, leukodystrophy, renal and liver dysfunction, skeletal abnormalities, and they usually result in death during infancy or early childhood. There are no treatments for ZSDs, and their rarity, the large size of the PEX genes, and the numerous different genes, has impaired therapeutic development. We previously demonstrated that ATAD1, a mitochondrial protein quality control chaperone, could correct both mitochondrial and peroxisomal phenotypes in PEX3 patient fibroblasts. In this study, we investigated whether overexpressing ATAD1 could provide similar benefits in PEX1 and PEX6 patient cell lines, which account for over 70% of ZSD cases. We used established PEX6-/- HEK293 cells, patient-derived fibroblasts with pathogenic PEX1 mutations, and newly created zebrafish mutants. Lipidomic profiling of the cell lines demonstrated widespread dysregulation, including accumulation of lysophosphatidylcholines with very-long-chain fatty acids, depletion of plasmalogens and cholesteryl esters containing polyunsaturated fatty acids, and a decrease in cardiolipins. Overexpressing ATAD1 partly corrected these imbalances, including normalizing VLCFA metabolism in PEX1 fibroblasts and restoring plasmalogens and cardiolipins in PEX6-deficient cells. Mitochondrial function analysis (Seahorse XF) showed that ATAD1 increased basal and ATP-linked respiration in both PEX1- and PEX6-deficient cells, sometimes surpassing the effects of PEX gene re-expression. ATAD1 increased peroxisome numbers in both PEX6 and PEX1 cells. Zebrafish Pex1 mutants exhibited impaired maximal respiration despite normal basal activity, confirming mitochondrial vulnerability in vivo. These findings further confirm a role for ATAD1 as a modifier that improves lipid metabolism, mitochondrial function, and peroxisome abundance that could function across multiple ZSDs.