Impaired enzymatic defensive activity, mitochondrial dysfunction and proteasome activation are involved in RTT cell oxidative damage

Abstract

A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment.

Increased Mitochondrial Mass and Cytosolic Redox Imbalance in Hippocampal Astrocytes of a Mouse Model of Rett Syndrome: Subcellular Changes Revealed by Ratiometric Imaging of JC‐1 and roGFP1 Fluorescence

“…Yet, the total mitochondrial content determined in parallel and labeling with the mitochondria-specific marker MitoTracker Red also confirm an increased mitochondrial mass in Mecp2 −/y astrocytes. It therefore seems that the increased mitochondrial content constitutes a cell-endogenous response to compensate for the mitochondria-related deficits and their limited metabolic/respiratory capacity in RTT [26, 27, 31, 40]. Interestingly, an increased mitochondrial mass was also reported for Mecp2 −/y microglia of the juvenile mouse brain [78].…”

“…In mouse models of RTT, symptomatic animals also showed reduced enzyme activities of respiratory chain complexes II, III, IV, and ANT1, as well as reduced glutathione levels in the brain and/or skeletal muscle [26–29]. Furthermore, the ANT1 gene encoding the mitochondrial adenine nucleotide translocase is highly upregulated in the Mecp2 −/y mouse brain and Rett patient fibroblasts [30], the latter of which also show clear signs of metabolic mitochondrial dysregulation, oxidative stress, and diminished redox-balancing capabilities [31]. Also, the elevated blood lactate and pyruvate levels suggest defects in the mitochondrial respiratory chain and the urea cycle [32].…”

Alterations of mitochondrial bioenergetics, dynamics, and morphology support the theory of oxidative damage involvement in autism spectrum disorder

“…Mitochondrial‐derived ROS were determined in cells grown onto coverslips and stained at 37°C for 30 minutes with 5 µM MitoSOX Red (cat. M36008, Thermo Fisher Scientific) as reported previously 9 . After staining, the cells were washed and analyzed by a confocal microscope.…”

“…The NOX activity was measured using the lucigenin‐enhanced chemiluminescence assay for the detection of superoxide anions production, as previously described 9 . Briefly, cell suspensions (2 × 10 5 cells) in Hanks' Balanced Salt solution (HBSS) were mixed with lucigenin (50 μM) (cat.…”

“…Metabolic status of fibroblasts was determined using a Seahorse Bioscience XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA), as previously reported 9 . A Seahorse XF Cell Mito Stress Test Kit (cat.…”