Abnormal redox balance at membrane contact sites causes axonopathy in GDAP1-related Charcot-Marie-Tooth disease

Pathogenic variants of GDAP1 cause Charcot-Marie-Tooth disease (CMT), an inherited neuropathy characterized by axonal degeneration. GDAP1, an atypical glutathione S-transferase, localizes to the outer mitochondrial membrane (OMM), regulating this organelle's dynamics, transport, and membrane contact sites (MCSs). It has been proposed that GDAP1 functions as a cellular redox sensor. However, its precise contribution to redox homeostasis remains poorly understood, as does the possible redox regulation at mitochondrial MCSs. Given the relationship between the peroxisomal redox state and overall cellular redox balance, we investigated the role of GDAP1 in peroxisomal function and mitochondrial MCSs maintenance by using high-resolution microscopy, live cell imaging with pH-sensitive fluorescent probes, and transcriptomic and lipidomic analyses in the Gdap1 ^-/- mice and patient-derived fibroblasts. We demonstrate that GDAP1 deficiency disrupts mitochondria-peroxisome MCSs and leads to peroxisomal abnormalities, which are reversible upon pharmacological activation of PPARγ or glutathione supplementation. These results identify GDAP1 as a new tether of mitochondria-peroxisome MCSs that maintain peroxisomal number and integrity. The supply of glutathione (GSH-MEE) or GDAP1 overexpression suffices to rescue these MCSs. Furthermore, GDAP1 may regulate the redox state within the microdomain of mitochondrial MCSs, as suggested by decreased pH at mitochondria-lysosome contacts in patient-derived fibroblasts, highlighting the relationship between GDAP1 and redox-sensitive targets. Finally, in vivo analysis of sciatic nerve tissue in Gdap1 ^-/- mice revealed significant axonal structural abnormalities, including nodes of Ranvier disruption and defects in the distribution and morphology of mitochondria, lysosomes, and peroxisomes, emphasizing the importance of GDAP1 in sustaining axon integrity in the peripheral nervous system. Taken together, this study positions GDAP1 as a multifunctional protein that mediates mitochondrial interaction with cellular organelles of diverse functions, contributes to redox state sensing, and helps maintain axonal homeostasis. In addition, we identify PPAR as a novel therapeutic target, based on knowledge of the underlying pathogenetic mechanisms.