Figure 1

Redox biology pathways in premutation carriers. (A) Increases in mitochondria-derived superoxide anion production can lead to the formation of the highly reactive species resulting in biomolecule damage. In parallel, hyperactivation of the polyol pathway—usually associated with mitochondrial dysfunction—leads to excessive accumulation of sorbitol and fructose contributing to increases in cytosolic [NADH]/[NAD⁺] ratios reducing the glucose flux via glycolysis and pentose phosphate pathway. This scenario impacts the mitochondrial (NADH)/(NAD⁺) leading to the generation of “reactive aldehydes” and oxidatively modified proteins or advanced glycation end-products. (B) Electron paramagnetic detection of free radicals in fibroblasts. Aliquots of cells from control and two premutation donors supplemented with deferoxamine, DMSO and DMPO were taken within 10–15 min after the addition of the spin trap. A weak EPR background signal was detected with cell culture media alone (a), which was higher upon addition of control cells (b). This latter signal was constituted by a quartet with an intensity ratio of 1:2:2:1 and hyperfine splitting constants a^N = a^Hβ = 14.9 G for N and H^β consistent with the detection of DMPO-hydroxyl radical adduct, further confirmed by spectra simulation. Cells from two premutation carriers exhibited EPR spectra similar to that of controls constituted solely by hydroxyl radical (c) or by a combination of hydroxyl and a second adduct at a ratio of 19 to 1 (d). This secondary adduct (a^N = 16.4 G and a^Hβ = 23.3 G) indicated the presence of DMPO-methyl adduct which resulted from the quenching of methyl radical (formed from the reaction between hydroxyl radical and DMSO) by DMPO. Lower panel: Representative EPR spectra of a cell line from a carrier with and without the addition of FCCP. (C) Representative Western blot image of 3-nitrotyrosine content in tubulin and actin of fibroblasts from controls, asymptomatic (PA) and FXTAS-affected or symptomatic (PS) premutation carriers. Intensity of nitrated actin and tubulin (expressed as arbitrary units of densitometry, AUD) were normalized by their respective total protein. ANOVA followed by Bonferroni was performed when comparing controls, PA and PS. *p < 0.05 versus controls. (D) Representative Western blot image and correspondent densitometry of MDA-protein adducts from controls and FXTAS-affected (PS) and unaffected (PA) carriers. ANOVA followed by Bonferroni’s post hoc test was used for the statistical analysis. *p < 0.005 versus controls. (E) Mitochondrial DNA copy number and deletions in control and premutation fibroblasts and correlations between these outcomes. P values were obtained with ANOVA followed by Bonferroni’s post hoc test. For correlations: Pearson’s r value = −0.500 and −0.244 (p = 0.034 and 0.230) for controls and premutation respectively.