Dual phases of respiration chain defect-augmented mROS-mediated mCa ²⁺ stress during oxidative insult in normal and ρ ⁰ RBA1 astrocytes

Mitochondrial respiratory chain (RC) deficits, resulting in augmented mitochondrial ROS (mROS) generation, underlie pathogenesis of astrocytes. However, mtDNA-depleted cells (ρ ⁰) lacking RC have been reported to be either sensitive or resistant to apoptosis. In this study, we sought to determine the effects of RC-enhanced mitochondrial stress following oxidative insult. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy, the ability to resist oxidative stress and levels of mROS formation and mitochondrial calcium (mCa²⁺) were compared between two different astrocyte cell lines, control and ρ ⁰ astrocytes, over time upon oxidative stress. Our results showed that the cytoplasmic membrane becomes permeated with YO-PRO-1 dye at 150 and 130 minutes in RBA-1 and ρ ⁰ astrocytes, respectively. In contrast to RBA-1, 30 minutes after 20 mM Hexposure, ρ ⁰ astrocytes formed marked plasma membrane blebs, lost the ability to retain Mito-R, and showed condensation of nuclei. Importantly, Hinduced ROS and accompanied mCa²⁺ elevation in control showed higher levels than ρ ⁰ at early time point but vice versa at late time point. Our findings underscore dual phase of RC-defective cells harboring less mitochondrial stress due to low RC activity during short-term oxidative stress but augmented mROS-mediated mCa²⁺ stress during severe oxidative insult.