Cell Sorting Experiments Link Persistent Mitochondrial DNA Damage with Loss of Mitochondrial Membrane Potential and Apoptotic Cell Death

Abstract

In order to understand the molecular events following oxidative stress, which lead to persistence of lesions in the mtDNA, experiments were performed on normal human fibroblast (NHF) expressing human telomerase reverse transcriptase (hTERT). The formation and repair of H 2 O 2 -induced DNA lesions were examined using quantitative PCR. It was found that NHF hTERTs show extensive mtDNA damage (ϳ4 lesions/10 kb) after exposure to 200 M H 2 O 2 , which is partially repaired during a recovery period of 6 h. At the same time, the nDNA seemed to be completely resistant to damage. Cell sorting experiments revealed persistent mtDNA damage at 24 h only in the fraction of cells with low mitochondrial membrane potential (⌬⌿m). Further analysis also showed increased production of H 2 O 2 by these cells, which subsequently undergo apoptosis. This work supports a hypothesis for a feed-forward cascade of reactive oxygen species generation and mtDNA damage and also suggested a possible mechanism for persistence of lesions in the mtDNA involving a drop in ⌬⌿m, compromised protein import, secondary reactive oxygen species generation, and loss of repair capacity.

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

“…The preferential effects of oxidative stress on mtDNA noted herein concur with others (4,5). Furthermore, the protective effects of mt-hOgg1-WT we show are in accordance with studies from several groups as well as our own (7)(8)(9)(10)(11)(12). Although unclear with AEC, a causal link between mtDNA damage and apoptosis has been established in various cell types (7)(8)(9)(10)12).…”

Calcium Homeostasis and Mitochondrial Dysfunction in Striatal Neurons of Huntington Disease

“…ROS have long been known to directly damage mitochondria: they dissipate the ⌬ m , impairing the production of ATP, and preferentially damage mitochondrial DNA (18,(45)(46)(47). The mitochondrial dysfunction mediated by ROS has received new impulse from recent work showing that in HD striata, and even in the model cells used in this work, a transcriptional coactivator of the genes of ROS-scavenging enzymes (PGC-1␣) is transcriptionally down-regulated (21).…”

Dysregulation of Mitochondrial Calcium Signaling and Superoxide Flashes Cause Mitochondrial Genomic DNA Damage in Huntington Disease

“…Overproduction of ROS in HD neurons causes oxidative damage to mitochondrial nucleic acids, proteins, and lipids, which in turn impair the function of mitochondria. Naked mitochondrial genomic DNA (part of which encodes subunits of complex I, III, IV, and V) and the iron-sulfur cluster-containing proteins, including the succinate dehydrogenase B subunit of complex II and the Rieske protein of complex III, are particularly vulnerable to oxidative stress (30,78,79). Postmortem studies of symptomatic HD patients revealed a drastic deficiency of complexes II and III and a lesser deficiency of complex IV in the caudate or putamen, with relatively normal levels in the frontal cortex or cerebellum (31,32).…”

“…Interestingly, oxidative damage in mitochondrial genomic DNA (mtDNA) preferentially occurs in the parietal region of the human HD brain, but not frontal cortex or cerebellum (26), and striatal MSN particularly accumulates more mtDNA mutations than any other brain cells (27), indicating the brain regional specificity of oxidative damage in HD. Deficiency of respiratory chain complexes has been suggested in enhanced reactive oxygen species generation in the HD yeast model (30), and complex II and III defects have been detected in HD brain (31)(32)(33), suggesting that defects of respiratory chain complexes may cause mitochondrial dysfunction and ROS overproduction. However, no significant deficiency of respiratory chain complexes has been dem-* This work was supported National Basic Research Program of China Grants onstrated in presymptomatic patients or in HD model mice expressing full-length HTT exp , suggesting that respiratory chain defects are a secondary feature in HD pathogenesis (34,35).…”