Mitochondrial DNA Toxicity in Forebrain Neurons Causes Apoptosis,Neurodegeneration, and Impaired Behavior

Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIα-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration.A variety of both exogenous and endogenous reactive compounds present a constant threat to the integrity of DNA in living cells. DNA damage introduced by such compounds can lead to high and deleterious mutation rates as well as DNA cytotoxicity, both to the nuclear and the mitochondrial genome. This has triggered the evolution of several different DNA repair pathways (28). One is the base excision repair (BER) pathway, which repairs small base alterations that do not distort the DNA helix. Repair of such highly abundant lesions by BER is performed by a multistep process that is initiated by a damage-specific DNA glycosylase, which removes the damaged base. One of these glycosylases is uracil-DNA glycosylase (UDG), which acts to preserve the genome by removing mutagenic uracil residues from the DNA. This glycosylase, as well as the OGG1 glycosylase that is specialized for the removal of oxidized bases, exists in a nuclear and mitochondrial splice form (1, 11, 37, 45). Accordingly, BER of a variety of lesions has been observed in mitochondria (26, 31).Damage to the mitochondrial DNA (mtDNA) can cause respiratory chain deficiency and lead to disorders that have varied phenotypes (35, 41). Many involve neurological features that are often associated with cell loss within specific brain regions. These pathologies, along with the increasing evidence of a decline in mitochondrial function with aging, have raised speculation that key changes in mitochondrial DNA sequences and functions could have a vital role in age-related neurodegenerative diseases (41). This has also been studied in several model organisms. Mouse models with respiratory chain deficient dopamine neurons have demonstrated adult onset Parkinsonism phenotype (16), and cell death induced by mitochondrial toxicity is likely to underlie Alzheimer disease (32). Mitochondrial oxidative stress and accumulation of mtDNA damage are believed to be particularly devastating to postmitotic differentiated tissue, including neurons (30). The mtDNA contains genetic information for 13 polypeptides that are a part of the electron transport chain and for rRNAs and tRNAs that are necessary for mitochondrial protein synthesis. Thus, damage to the mtDNA genome will affect the energetic capacities of the mitochondria and also influence the level of reactive oxygen species (ROS) and ultimately the susceptibility to apoptosis (30, 35).Some recent influential studies have assessed the effect of mtDNA mutagenesis, including small base-pair substitutions and larger mtDNA deletions, on the life span of mice. It was concluded that a massive increase in the frequency of mtDNA base-pair substitutions are required for inducing premature aging, whereas the number of mtDNA deletions coincides better with natural aging (25, 47-49).In the present study, we have combined two novel transgenic mouse models, which allow the induction of a high number of apyrimidinic (AP) sites specifically to the mitochondrial genome in adults simply by the addition of doxycycline to the diet. Such AP sites are created by the expression of a mutated version of mitochondrion-targeted human UDG (abbreviated here as mutUNG1), whereby an amino acid substitution results in an enzyme that removes thymine, in addition to uracil, from DNA (23). The CaMKIIα promoter restricts expression of the mutUNG1 to forebrain neurons (34). We demonstrate that a continuous generation of AP sites leads to apoptosis, accelerated neurodegeneration, and impaired behavior.