Pathogenic mutations in ATAD3A cause dysregulation of RagC/D-TFEB axis and disrupt lysosomal homeostasis
We previously discovered that a de novo variant p.R528W in ATAD3A, encoding a mitochondrial membrane-anchored protein, causes a human neurological syndrome. While ATAD3A mutations induce aberrant lysosomal expansion accompanied by undigested material in the lysosomes, how mutant ATAD3A disrupts lysosomal homeostasis and whether this contributes to neurodevelopmental defects remain unknown. Here we show that pathogenic ATAD3A p.R528W expression disrupts the mTORC1-TFEB axis as revealed by dysregulation of mTORC1 substrate phosphorylation, TFEB nuclear localization, and CLEAR gene activation associated with lysosomal biogenesis. ATAD3A binds to lysosome-localized Rag C/D GTPases, which constitute a platform for TFEB recruitment, with pathogenic variants increasing this association and thereby decreasing lysosomal localization of Rag GTPases. Importantly, overexpression of RagC or RagD restores TFEB phosphorylation in human cells expressing p.R528W, and RagC-D overexpression or TFEB/Mitf knockdown rescues lysosomal expansion and neurodevelopmental defects in Drosophila. These data indicate that disrupted Rag GTPase recruitment to lysosomes and subsequent aberrant TFEB/Mitf activation contribute to neurodevelopmental and lysosomal phenotypes caused by pathogenic mutations in ATAD3A. Our work reveals a novel role for the mitochondrial resident protein ATAD3A in modulating lysosomal homeostasis through regulation of the mTORC1-TFEB axis, providing a mechanistic link between impaired mitochondrial and lysosomal homeostasis in a neurodevelopmental disorder.