Mitochondrial dysfunction and impaired oxidative stress defense as potential trigger of cerebral X-linked...

X-linked adrenoleukodystrophy (X-ALD) is caused by pathogenic ABCD1 variants, leading to a dysfunctional peroxisomal ABCD1 transporter, crucial for β-oxidation of very long chain fatty acids (VLCFA). The clinical manifestation ranges from asymptomatic carriers to severe childhood cerebral ALD (CALD). The underlying pathophysiology remains unclear, and while elevated oxidative stress and signs of mitochondrial dysfunction have been observed in X-ALD cells and tissues, their precise roles are still uncertain. This study aims to elucidate the interplay among excess VLCFA, mitochondrial function and oxidative stress in fibroblasts derived from CALD and non-CALD patients. Therefore, we measured reactive oxygen species (ROS) using the 2',7'-dichlorofluorescein diacetate assay, mitochondrial function with the Seahorse XFe24 flux analyzer and assessed the regulation of stress homeostasis on the genetic level by qPCR of NRF2-dependent genes NQO1, AR1B10 and AKR1C1. Additional stress was induced by exposure to tert-butyl hydroperoxide (TBHP) and hexacosanoic acid (C26:0). Scanning confocal microscopy and STED super-resolution microscopy was implemented for evaluation of mitochondrial structure and peroxisomal-mitochondrial crosstalk. Our findings indicate that non-CALD cell lines exhibit an overall compromised oxidative status under basal conditions, characterized by significantly reduced oxygen consumption rates (OCR) relative to both CALD and healthy controls, along with diminished expression of NRF2-regulated genes. Notably, ROS levels in non-CALD cells are comparable to those observed in CALD cells. However, when exposed to additional stress, these non-CALD cells show greater potential of defense mechanisms and compensation compared to CALD cells. These findings significantly improve our understanding of metabolic changes in X-ALD, focusing on the ability of different X-ALD phenotypes to cope with oxidative stress. They pave the way for further investigations to understand the different phenotypes and their disease progression, to find reliant biomarkers, and to develop therapeutic approaches and preventive measures for individual patients.