Oxidative Stress Induces Mitochondrial Dysfunction in a Subset of Autism Lymphoblastoid Cell Lines

Thursday, May 15, 2014
Atrium Ballroom (Marriott Marquis Atlanta)
S. Rose1, R. E. Frye2, J. C. Slattery3, R. A. Wynne4, M. Tippett5, S. Melnyk6 and S. J. James1, (1)University of Arkansas for Medical Sciences, Little Rock, AR, (2)Arkansas Children's Hospital Research Institute, Little Rock, AR, (3)Pediatric Neurology, Arkansas Children's Hospital Research Institute, Little Rock, AR, (4)Arkansas Children's Hospital, Little Rock, AR, (5)ACHRI, Little Rock, AR, (6)Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR
Background:  Mitochondrial dysfunction (MD) is associated with autism spectrum disorders; yet little is known about the etiology of MD or how it might interact with other physiological abnormalities associated with autism, such as oxidative stress. Primary lymphocytes and lymphoblastoid cell lines (LCLs) derived from children with AD exhibit decreased glutathione-mediated redox capacity and higher reactive oxygen species (ROS) compared to controls. This decreased ability to counter endogenous ROS production in AD immune cells may result in increased vulnerability to oxidative damage and mitochondrial dysfunction during pro-oxidant exposures.

Objectives:  We sought to determine whether mitochondrial respiration in AD and age and gender-matched control LCLs differed at baseline and in response to ROS. We hypothesized that upon increasing ROS, a subgroup of AD LCLs will demonstrate abnormal reserve capacity, a measure the mitochondrial ability to respond to physiological stress. To further investigate whether mechanisms to compensate for increased ROS differed between control and AD LCLs, we measured glycolysis as an intracellular compensatory mechanism and uncoupling protein 2 (UCP2) content as an intramitochondrial compensatory mechanism. UCP2, a major control mechanism for reducing ROS at the inner mitochondrial membrane, is up-regulated in many cell types under conditions of chronic mitochondrial oxidative stress.

Methods:  Mitochondrial oxygen consumption and glycolysis were measured using Seahorse extracellular flux (XF) technology. We compared bioenergetic profiles from 25 AD and control LCL pairs, matched by age and gender, before and after 1 h exposure to 5-15μM DMNQ (2,3-dimethoxy-1,4-napthoquinone), a ROS generator. Intracellular glutathione was measured by HPLC, intracellular ROS by CellRox green fluorescence and flow cytometry, UCP2 content by western blot, and mitochondrial DNA copy number by real-time PCR.

Results:  Compared to controls, AD LCLs exhibited abnormally elevated reserve at baseline that was severely depleted with increasing ROS. The changes in reserve capacity in AD LCLs resulted from higher ATP-linked and maximal respiratory capacity at baseline and a marked increase in proton leak with increasing ROS. These abnormalities were driven by a subgroup of eight (32%) of 25 AD LCLs, and further investigation revealed that this subgroup produced increased ROS and demonstrated a greater reliance on glycolysis and on UCP2 in attempt to regulate oxidative stress at the inner mitochondria membrane. Mitochondrial copy number was not different between the two AD subgroups. Glutathione redox capacity was reduced in the AD LCLs compared to controls but was not different between the two AD subgroups.

Conclusions:  This study suggests that a significant subgroup of AD children may have altered mitochondrial function rendering them more vulnerable to a pro-oxidant microenvironment derived from intrinsic and extrinsic sources of ROS such as immune activation and pro-oxidant environmental toxins. These findings are consistent with the notion that AD is caused by a combination of genetic and environmental factors.  An underlying defect in mitochondrial function could be a key deficit in AD affecting high energy demanding organs, particularly the brain and immune system, and could account for the commonly reported systemic abnormalities associated with ASD, such as immune dysfunction.