NOX activation and mitochondrial ROS production are involved in redox state alteration of RTT cells

Rett syndrome is a rare neurodevelopmental disorder affecting almost exclusively females associated in up to 95% of cases to de novo loss-of-function mutations in the X-chromosome-linked gene encoding the methyl-CpG-binding protein 2 (MeCP2). In the last few years a strong correlation between the levels of Oxidative Stress (OS) and the clinical severity of RTT has been well documented although the source of oxidative stress and the specific effect of ROS production in this pathology has not been yet investigated. Using freshly isolated skin fibroblast from RTT patients and healthy subjects we have determined the source of oxidative stress and the activity of specific enzymes and proteins involved in the modulation of cellular redox balance. RTT fibroblast presented high intracellular levels of H2O2 and 4HNE protein adducts. This finding was correlated with the constitutive activation of NADPH oxidase (NOX) and was reverted by DPI (NOX inhibitor) or Desferal (Iron chelator) pre-treatment. In parallel, an increase in mithocondrial ROS production, as measured by superoxide ion levels (Mitosox stain) and H2O2 was also determined. To confirm the alteration of status redox in RTT cells, the activity of several enzymes involved in protecting the cell from OS was also evaluated. Surprisingly, gene expression and activity of Catalase was increased respect to control, whereas Glutathione peroxidase (GPx), Supeoxide dismutase and Glucose-6-phospho deidrogenase (G6PDH) activity were decreased respect to control. Taken all together, our findings show the possible source of OS in RTT, which is due not only to mitochondrial ROS production but also to the activation of the endogenous enzyme NOX