ubgroups with ROS Exposure The reserve capacity at baseline and the change in response to increasing ROS was used to divide the AD LCLs into normal and abnormal subgroups. Reserve capacity was significantly elevated at baseline in the AD-A subgroup relative to both controls and to AD-N LCLs, which likely represents a compensatory adaptive response to the chronic elevations in ROS. Because we demonstrated that that mitochondrial copy numbers were not different in the two AD subgroups, the compensatory response that leads to increased reserve capacity in the AD-A subgroup was not likely due to differences in the number of mitochondria per cell but more likely due to either up-regulation of ETC complexes or to regulation of substrate supply and allosteric regulation of key metabolic enzymes. Despite the elevated reserve capacity at baseline, exposure to ROS resulted in a more precipitous decrease in reserve capacity in the AD-A LCLs as compared to the AD-N LCLs. This is significant since reduced reserve capacity is linked to several diseases such as aging, heart disease and neurodegenerative disorders. Reserve capacity is depleted when the mitochondria function at their maximal capacity, and depletion of reserve capacity renders the cell unable to meet any additional ATP demand. Reserve capacity depletion has been shown to result in cell death in several cell types under conditions of oxidative stress including cardiomyocytes and endothelial cells, and in neurons during glutamate toxicity or ETC inhibition. Overall proton leak respiration was 23428871 slightly but significantly higher in AD-N LCLs as compared to GW 5074 site control LCLs. This is not surprising as several indices of oxidative stress indicate that the AD-N LCLs have a more oxidized microenvironment as compared to control LCLs. As a result of this slight increase in proton leak respiration, the reserve capacity was slightly reduced in the AD-N LCLs as compared to control LCLs. In contrast to the mild differences in respiratory parameters between the AD-N and control LCLs, the differences in respiratory parameters between the AD-A LCL subgroup and both control and the AD-N LCL subgroup are particularly striking. First, the increased proton leak respiration in the AD-A LCLs compared to control LCLs was much more marked at baseline and became exaggerated as DMNQ increased. Second, the differences in reserve capacity were much more marked with a significantly 6099352 higher reserve capacity at baseline for the AD-A LCL subgroup with a significant decrease in reserve capacity with increasing DMNQ concentrations. Third, unlike the AD-N LCL subgroup, the AD-A LCL subgroup demonstrated significant elevations in ATP-linked respiration and maximal respiratory capacity at baseline with this difference diminishing as DMNQ increased. These differences were also seen when comparing the AD-A and AD-N subgroup, demonstrating that the AD-A LCLs represent a distinct subgroup of LCLs with an atypical mitochondrial response to chronic and acute increases in intracellular ROS. Molecular Mechanisms Associated with the Increase in ATP-linked Respiration Elevations in ATP-linked respiration in the AD-A LCL subgroup is consistent with clinical reports of electron transport chain over-activity in ASD children. Frye and Naviaux reported five ASD/MD children with complex IV over-activity and Graf et al reported a ASD/MD child with complex I over-activity. The fact that ATP-linked respiration is increased at baseline suggests that iubgroups with ROS Exposure The reserve capacity at baseline and the change in response to increasing ROS was used to divide the AD LCLs into normal and abnormal subgroups. Reserve capacity was significantly elevated at baseline in the AD-A subgroup relative to both controls and to AD-N LCLs, which likely represents a compensatory adaptive response to the chronic elevations in ROS. Because we demonstrated that that mitochondrial copy numbers were not different in the two AD subgroups, the compensatory response that leads to increased reserve capacity in the AD-A subgroup was not likely due to differences in 23570531 the number of mitochondria per cell but more likely due to either up-regulation of ETC complexes or to regulation of substrate supply and allosteric regulation of key metabolic enzymes. Despite the elevated reserve capacity at baseline, exposure to ROS resulted in a more precipitous decrease in reserve capacity in the AD-A LCLs as compared to the AD-N LCLs. This is significant since reduced reserve capacity is linked to several diseases such as aging, heart disease and neurodegenerative disorders. Reserve capacity is depleted when the mitochondria function at their maximal capacity, and depletion of reserve capacity renders the cell unable to meet any additional ATP demand. Reserve capacity depletion has been shown to result in cell death in several cell types under conditions of oxidative stress including cardiomyocytes and endothelial cells, and in neurons during glutamate toxicity or ETC inhibition. Overall proton leak respiration was slightly but significantly higher in AD-N LCLs as compared to control LCLs. This is not surprising as several indices of oxidative stress indicate that the AD-N LCLs have a more oxidized microenvironment as compared to control LCLs. As a result of this slight increase in proton leak respiration, the reserve capacity was slightly reduced in the AD-N LCLs as compared to control LCLs. In contrast to the mild differences in respiratory parameters between the AD-N and control LCLs, the differences in respiratory parameters between the AD-A LCL subgroup and both 
control and the AD-N LCL subgroup are particularly striking. First, the increased proton leak respiration in the AD-A LCLs compared to control LCLs was much more marked at baseline and became exaggerated as DMNQ increased. Second, the differences in reserve capacity were much more marked with a significantly higher reserve capacity at baseline for the AD-A LCL subgroup with a significant decrease in reserve capacity with increasing DMNQ concentrations. Third, unlike the AD-N LCL subgroup, the AD-A LCL subgroup demonstrated significant elevations in ATP-linked respiration and maximal respiratory capacity at baseline with this difference diminishing as DMNQ increased. These differences were also seen when comparing the AD-A and AD-N subgroup, demonstrating that the AD-A LCLs represent a distinct subgroup of LCLs with an atypical mitochondrial response to chronic and acute increases in intracellular ROS. Molecular Mechanisms Associated with the Increase in ATP-linked Respiration Elevations in ATP-linked respiration in the AD-A LCL subgroup is consistent 18753409 with clinical reports of electron transport chain over-activity in ASD children. Frye and Naviaux reported five ASD/MD children with complex IV over-activity and Graf et al reported a ASD/MD child with complex I over-activity. The fact that ATP-linked respiration is increased at baseline suggests that i