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Review
. 2007 Apr 14;145(4):1249-59.
doi: 10.1016/j.neuroscience.2006.10.002. Epub 2006 Nov 13.

Mitochondrial DNA repair: a critical player in the response of cells of the CNS to genotoxic insults

S P LeDoux  1 , N M Druzhyna, S B Hollensworth, J F Harrison, G L Wilson
Affiliations
Review

Mitochondrial DNA repair: a critical player in the response of cells of the CNS to genotoxic insults

S P LeDoux et al. Neuroscience. .

Abstract

Cells of the CNS are constantly exposed to agents which damage DNA. Although much attention has been paid to the effects of this damage on nuclear DNA, the nucleus is not the only organelle containing DNA. Within each cell, there are hundreds to thousands of mitochondria. Within each mitochondrion are multiple copies of the mitochondrial genome. These genomes are extremely vulnerable to insult and mutations in mitochondrial DNA (mtDNA) have been linked to several neurodegenerative diseases, as well as the normal process of aging. The principal mechanism utilized by cells to avoid DNA mutations is DNA repair. Multiple pathways of DNA repair have been elucidated for nuclear DNA. However, it appears that only base excision repair is functioning in mitochondria. This repair pathway is responsible for the removal of most endogenous damage including alkylation damage, depurination reactions and oxidative damage. Within the rat CNS, there are cell-specific differences mtDNA repair. Astrocytes exhibit efficient repair, whereas, other glial cell types and neuronal cells exhibit a reduced ability to remove lesions from mtDNA. Additionally, a correlation was observed between those cells with reduced mtDNA repair and an increase in the induction of apoptosis. To demonstrate a causative relationship, a strategy of targeting DNA repair proteins to mitochondria to enhance mtDNA repair capacity was employed. Enhancement of mtDNA repair in oligodendrocytes provided protection from reactive oxygen species- and cytokine-induced apoptosis. These experiments provide a novel strategy for protecting sensitive CNS cells from genotoxic insults and thus provide new treatment options for neurodegenerative diseases.

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Figures

FIGURE 1
FIGURE 1. Analysis of the formation of oxidative damage to mtDNA of CNS cells
Cells were exposed to concentrations of menadione ranging from 50μM to 200μM for one hour and lysed immediately. Control cultures were incubated in drug diluent only (HBSS). High molecular weight DNA was isolated and digested to completion with Bam H1. Samples were exposed to 0.1N NaOH prior to Southern blot analysis and hybridized with a PCR generated mitochondrial probe. Quantitation of the hybridization was performed using a Bio-Rad phosphoimager. The fraction of fragments free of damage (zero class) was determined by dividing the band intensity of the treated sample by the intensity of the nontreated sample. The number of lesions per fragment was determined using the Poisson expression. The values represent the mean for at least three separate experiments.
FIGURE 2
FIGURE 2. Comparison of mtDNA repair in CNS cells
Using quantitative Southern Blot analysis, autoradiographic bands representing a 10.8 kb DNA fragment were densitometrically scanned. The break frequency per fragment was determined using the Poisson expression (s=-lnP0; where s is the number of breaks per fragment and P0 is the fraction of fragments free of breaks). Repair is determined by BF0-BFt/BF0, where BF0 is the break frequency at 0 hours of repair and BFt is the break frequency at the time of interest. The values represent the mean ± SEM for at least three separate experiments.
FIGURE 3
FIGURE 3. Viability of CNS cells after treatment with 100μM menadione
Cells were plated into 24-well plates and exposed to 100μM menadione for a one-hour period. Following this incubation, cells were rinsed with HBSS and normal media was replaced for 2, 4, 6, or 24 hours. For control and 0-hour samples, a 10% trypan blue solution was added to the wells and viable cells counted using a light microscope.
FIGURE 4
FIGURE 4. Quantitation of apoptotic CNS cells following menadione exposure
In order to quantitate apoptosis, two commercially available kits were employed. The ApoTag kit from Oncor detects the 3′-OH groups generated during apoptosis while the Annexin-V-FITC kit from Trevigen (Gaithersburg, MD, USA) labels the phosphatidylserines that have been flipped to the outside of the cell membrane. Cells were plated into Labtech slides and treated with 100μM menadione for one hour. The cells were rinsed with HBSS and culture media replaced for 6 hours. At this time, cells were rinsed with cold 1XPBS and the ApoTag/Annexin V assays performed. Using an immunofluorescent microscope, cells in five random fields were counted and the number of positive-staining cells was divided by the total number of cells in the field to give a % of apoptotic cells.
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