Despite being one of the most prevalent metabolic abnormalities affecting children with autism spectrum disorders (ASD), the significance of mitochondrial dysfunction in ASD remains poorly understood. We and others have previously demonstrated that many children with ASD exhibit elevated oxidative stress markers. In addition, we have shown a reduction in glutathione-mediated redox capacity in plasma, primary immune cells, as well as lymphoblastoid cell lines (LCLs) and mitochondria isolated from LCLs derived from children with ASD. Autism LCLs produce significantly more reactive oxygen species (ROS) and exhibit an increased susceptibility to mitochondrial membrane depolarization following acute nitric oxide exposure relative to control LCLs. This evidence suggests that glutathione-mediated redox capacity is insufficient to counter endogenous ROS production in ASD LCLs resulting in increased vulnerability to oxidative damage and mitochondrial dysfunction during pro-oxidant exposures.
We sought to determine whether decreased glutathione-mediated redox capacity in ASD LCLs renders them more susceptible than control LCLs to mitochondrial dysfunction following acute oxidant exposure and whether pretreatment to increase the intracellular glutathione concentration would confer protection from mitochondrial dysfunction.
Mitochondrial function was measured in intact LCLs in real-time using Seahorse extracellular flux (XF) technology. The oxygen consumption rate (OCR) at baseline (Basal OCR) and upon the sequential addition of mitochondrial inhibitors was measured and a bioenergetic profile was derived from these measurements. We compared bioenergetic profiles from 15 ASD and control LCLs before and after a 1 h exposure to 5-15 μM DMNQ (2,3-dimethoxy-1,4-napthoquinone), a ROS generator. To determine whether changes in mitochondrial bioenergetics after DMNQ exposure could be prevented by NAC-induced increases in the intracellular glutathione redox capacity, a third comparison group consisted of the ASD LCLs pretreated for 48 h with 1 mM N-acetyl-cysteine (NAC). In addition, the intracellular glutathione redox capacity in ASD and control LCLs was measured by HPLC, and the mitochondrial to nuclear DNA ratios (mtDNA/nDNA) were determined.
At baseline (i.e. no DMNQ challenge), ASD LCLs exhibited a significantly increased mitochondrial Reserve Capacity (Maximal OCR- Basal OCR) relative to controls (p=0.04). Challenge with DMNQ resulted in significantly higher Basal OCR (10-15 μM; p≤0.04), Proton Leak (5-10 μM; p≤0.03), and a greater depletion of Reserve Capacity (10-15 μM; p≤0.03) in ASD LCLs relative to control LCLs. NAC pretreatment of the ASD LCLs successfully increased the intracellular glutathione-mediated redox capacity (p<0.001) and attenuated the abnormal depletion of Reserve Capacity observed with DMNQ challenge relative to control LCLs. The average mtDNA to nDNA ratio was significantly lower in the ASD LCLs relative to control LCLs (p<0.01).
We demonstrate that acute exposure to a ROS-producing agent results in a detrimental effect on mitochondrial bioenergetics that is greater for ASD LCLs as compared to control LCLs. Further, targeted treatment to restore intracellular glutathione redox capacity improves the ability of the ASD LCLs to withstand excessive ROS exposure. The increased mitochondrial Reserve Capacity of the ASD LCLs is likely an adaptive response to chronic oxidative stress and initial studies indicate that increase Reserve Capacity is not due to increased numbers of mitochondria.
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