Differential age-related changes in mitochondrial DNA repair activities in mouse brain regions

Abstract

Aging in the brain is characterized by increased susceptibility to neuronal loss and functional decline, and mitochondrial DNA (mtDNA) mutations are thought to play an important role in these processes. Due to the proximity of mtDNA to the main sites of mitochondrial free radical generation, oxidative stress is a major source of DNA mutations in mitochondria. The base excision repair (BER) pathway removes oxidative lesions from mtDNA, thereby constituting an important mechanism to avoid accumulation of mtDNA mutations. The complexity of the brain implies that exposure and defence against oxidative stress varies among brain regions and hence some regions may be particularly prone to accumulation of mtDNA damages. In the current study we investigated the efficiency of the BER pathway throughout the murine lifespan in mitochondria from cortex and hippocampus, regions that are central in mammalian cognition, and which are severely affected during aging and in neurodegenerative diseases. A regional specific regulation of mitochondrial DNA repair activities was observed with aging. In cortical mitochondria, DNA glycosylase activities peaked at middle-age followed by a significant drop at old age. However, only minor changes were observed in hippocampal mitochondria during the whole lifespan of the animals. Furthermore, DNA glycosylase activities were lower in hippocampal than in cortical mitochondria. Mitochondrial AP endonuclease activity increased in old animals in both brain regions. Our data suggest an important regional specific regulation of mitochondrial BER during aging.

Fig. 1

Western blot analysis of Lamin B and VDAC1 in whole brain extract and mitochondrial fractions isolated from cortex and hippocampus. 40 μg of mitochondrial protein from hippocampus (A) and cortex (B) were loaded and resolved on a SDS-PAGE gel as described in Materials and Methods. Representative blot images are shown. Lane 1: young group; lane 2: adult group; lane 3: middle-aged group; lane 4: old group; lane 5: whole brain extract.

Fig. 2

Changes in DNA glycosylase activities in mitochondria from cortex and hippocampus with aging. Top panels show quantification of DNA glycosylase activities and bottom panels show representative gels from cortical mitochondria. Incision activities were calculated from the amount of radioactivity in the products relative to the total radioactivity in the lane. Each lane in the gels represents one animal. Values presented are means ± S.E.M. of triplicate measurements from three to five mitochondrial preparations and are expressed as fmol per hour per 10 μg of mitochondrial protein for UNG1 and NTH1 and fmol per 3 h per 30 μg of mitochondrial protein for OGG1 activity. Filled bars represent cortical mitochondrial activities; Open bars represent hippocampal mitochondrial activities. (A) UNG1 activity; (B) NTH1 activity; (C) OGG1 activity. (a) Indicates significant difference versus adult group; (b) indicates significant difference versus middle-aged group; (c) indicates significant difference versus young group. Significant changes in cortical mitochondria: *p ≤ 0.06; **p ≤ 0.01; ***p ≤ 0.001; significant changes in hippocampal mitochondria: ^#p ≤ 0.06; ^##p ≤ 0.01.

Fig. 3

Changes in DNA glycosylase activities in mitochondria from cerebellum with aging. Quantification of DNA glycosylase activities as described for Fig. 2. Values presented are means ± S.E.M. of triplicate measurements from three to five mitochondrial preparations and are expressed as fmol per hour per 10 μg of mitochondrial protein for UNG1 and NTH1 and fmol per 3 h per 30 μg of mitochondrial protein for OGG1 activity. Asterisk (*) indicates significant difference versus middle-aged group; **p ≤ 0.01.

Fig. 4

NEIL activity in mitochondria from cortex and hippocampus in different age groups. Top panel shows quantification of NEIL activity and bottom panel shows representative gel. Quantification of NEIL activities as described for Fig. 2. Each lane in the gels represents one animal. Values presented are means ± S.E.M. of triplicate measurements from three to five mitochondrial preparations and are expressed as fmol per 3 h per 30 μg of mitochondrial protein. Filled bars represent cortical mitochondrial activities; Open bars represent hippocampal mitochondrial activities. (a) Indicates significant difference versus adult group; (b) indicates significant difference versus middle-aged group; *p ≤ 0.06.

Fig. 5

MtAPE activity in cortical and hippocampal mitochondria with aging. Quantification of mtAPE activity in cortical (A) and hippocampal (B) mitochondria. Values presented are means ± S.E.M. of triplicate measurements from three to four mitochondrial preparations and are expressed as fmol per hour per 10 μg of mitochondrial protein. (C and D) Representative gels of AP incision in cortical and hippocampal mitochondria respectively. Each lane in the gels represents one animal. (E) Western blot analyses of APE in cortical and hippocampal mitochondria from adult and old groups. Representative blot image is shown. (a) Denotes significant difference versus adult group; (b) indicates significant difference versus middle-aged group; *p ≤ 0.06; **p ≤ 0.01; ***p ≤ 0.001.