Elsevier

Neurobiology of Aging

Volume 27, Issue 8, August 2006, Pages 1129-1136
Neurobiology of Aging

Mitochondrial and nuclear DNA-repair capacity of various brain regions in mouse is altered in an age-dependent manner

https://doi.org/10.1016/j.neurobiolaging.2005.06.002Get rights and content

Abstract

Aging is associated with increased susceptibility to neuronal loss and disruption of cerebral function either as a component of senescence, or as a consequence of neurodegenerative disease or stroke. Here we report differential changes in the repair of oxidative DNA damage in various brain regions during aging. We evaluated mitochondrial and nuclear incision activities of oxoguanine DNA glycosylase (OGG1), uracil DNA glycosylase (UDG) and the endonuclease III homologue (NTH1) in the caudate nucleus (CN), frontal cortex (FC), hippocampus (Hip), cerebellum (CE) and brain stem (BS) of 6- and 18-month-old male C57Bl/6 mice. We observed a significant age-dependent decrease in incision activities of all three glycosylases in the mitochondria of all brain regions, whereas variable patterns of changes were seen in nuclei. No age- or region-specific changes were observed in the mitochondrial repair synthesis incorporation of uracil-initiated base-excision repair (BER). We did not observe any age or region dependent differences in levels of BER proteins among the five brain regions. In summary, our data suggest that a decreased efficiency of mitochondrial BER-glycosylases and increased oxidative damage to mitochondrial DNA might contribute to the normal aging process. These data provide a novel characterization of oxidative DNA damage processing in different brain regions implicated in various neurodegenerative disorders, and suggest that this process is regulated in an age-dependent manner. Manipulation of DNA repair mechanisms may provide a strategy to prevent neuronal loss during age-dependent neurodegenerative disorders.

Introduction

Aging is an inevitable biological process, which is characterized by a general decline in physiological function that leads to morbidity and mortality [20]. The detrimental effects of aging are best observed in post-mitotic tissues, where cells that are irreversibly damaged or lost cannot be replaced by mitosis of intact ones. Among such tissues the brain is most important, as it has the main role in homeostasis of the organism. The generation of various oxidants, both reactive oxygen (ROS) and nitrogen-species (RNS), leads to macromolecular damage, a characteristic of aging [28]. Aging enhances the generation of ROS and RNS in mouse models of dopaminergic damage [9], [29]. The hippocampus and cortex of aged rats have been reported to have increased neuronal nitric oxide synthase mRNA, resulting in an increased free radical production in these brain regions [15]. Antioxidant enzymes, such as manganese superoxide dismutase, were also found to be decreased in the brain of these rats [4]. Steady state levels of 8-oxoguanine (8-oxoG) have been reported to increase significantly in the striatum of aged rats [5]. Oxidative damage to DNA may play a role in both normal aging and neurodegenerative diseases [21], [22]. Recent studies suggest a role of free radicals in neuronal degeneration [19]. Oxidative damage is a well-recognized mechanism of injury in several neurodegenerative dementias including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), and is thought to contribute to the neuronal death associated with these disorders [26].

Free radical attack on nuclear and mitochondrial DNA is believed to be a major contributing factor to aging [23]. The levels of damaged DNA and the ability of the neurons to repair such damage may be an important predictor of the maximum life span of each species [24]. Oxidative damage to DNA may be specifically important because it has the potential to affect expression of wide variety of genes. It can also stimulate cell-cycle-related intrinsic DNA repair mechanisms that may be either reparative or deleterious [14]. The free radical theory of aging postulates that increased DNA damage and/or decreased DNA repair could lead to the accumulation of DNA lesions, ultimately resulting in cellular senescence and death [2].

The base-excision repair (BER) pathway repairs small base modifications, including lesions generated by reactive oxygen species. A DNA glycosylase, which catalyzes the hydrolysis of the N-glycosyl bond, initiates BER by releasing the base and generating an abasic site. The abasic site is cleaved by an AP lyase or AP endonuclease and the one base gap is filled in by a DNA polymerase and ligated by a DNA ligase. The specificity of BER is provided by the DNA glycosylases, which have precise substrate specificities. Mammalian cells have several substrate-specific DNA glycosylases: oxoguanine DNA glycosylase (OGG1), which primarily recognizes 8-oxo-dG but is active on other oxidized purines; uracil DNA glycosylase (UDG), which removes deoxyuracil from DNA; endonuclease III homologue (NTH1), which recognizes and cleaves oxidized pyrimidines such as thymine glycol and 5-hydroxycytosine (5-OH-dC); and 3-methyl adenine DNA glycosylase, which removes alkylated bases [27]. Although various reports have shown an accumulation of different oxidative DNA lesions in brain and possibly a decrease in DNA repair in various cell-types of brain, no study has been reported so far that examines the processing of DNA lesions in different brain regions during the normal aging process. In this study, we sought to evaluate the activity of three major DNA glycosylases, OGG1, UDG and NTH1, in different brain regions of young and old wild-type mice in order to understand the age-dependent status of their capacity to incise oxidative DNA lesions. We also evaluated regional changes in nuclear and mitochondrial protein levels of two DNA-repair proteins involved in different steps of BER, as well as uracil-initiated mitochondrial repair synthesis incorporation as a function of aging.

Section snippets

Materials

HEPES, benzamidine–HCl, dithiothreitol (DTT), bovine serum albumin (BSA), and acrylamide/bis-acrylamide (19:1) were from Sigma Chemicals. Leupeptin was from Roche. Isotopes were from NEN Life Science Products; G25 spin columns were from Amersham. T4 polynucleotide kinase was from Stratagene. All other reagents were of ACS grade from Sigma.

Animals

We used male C57Bl/6 mice (6 month and 18 month) obtained from the National Institute on Aging animal colony. The animals were fed regular Purina animal chow

Results

DNA glycosylases initiate BER by recognizing and removing the damaged base. In the present study, we examined DNA glycosylase activity as a measure of DNA repair in various mouse brain regions that are implicated in different varieties of neurodegeneration with respect to age. We compared the DNA incision activity of nuclear and mitochondrial extracts of five brain regions that include caudate nucleus (CN), frontal cortex (FC), hippocampus (Hip), cerebellum (CE) and brain stem (BS) of young (6

Discussion

Different tissue/organs have different metabolic profiles and are exposed to different levels of DNA damaging agents. It is therefore not surprising that DNA repair activities vary considerably from tissue to tissue. In a previous study we demonstrated that BER activities differ significantly in both mitochondria and nuclei from 6 mouse tissues, including brain [13]. However, while the other organs (liver, heart, kidney, skeletal muscle and testis) are relatively homogeneous as to cellular

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