In the present study, we demonstrated that 8-oxoG accumulated within mtDNA and nDNA under conditions of oxidative stress is differentially involved in neurodegeneration through activation of the calpain and PARP/AIF pathways. MTH1 and OGG1, which prevent accumulation of 8-oxoG in the respective DNAs, protect the brain, while MUTYH, which initiates excision repair of adenine opposite 8-oxoG, triggers neurodegeneration.
We thus propose that the neurotoxicity of oxidative stress in the mammalian brain predominantly results from MUTYH-initiated excision repair of adenine opposite 8-oxoG, which is mostly derived from 8-oxo–dGTP accumulated in the nucleotide pool under oxidative stress.
MTH1 and OGG1 suppress neurodegeneration as well as mutagenesis and carcinogenesis by preventing accumulation of 8-oxoG in cellular DNAs. It has been widely accepted that both degenerative diseases and cancers are tightly associated with DNA damage (1
). We previously showed that Mth1
-KO and Ogg1
-KO mice exhibit increased incidence of spontaneous tumors in liver and lung, respectively. This was accompanied by accumulation of 8-oxoG in their nDNAs, thus demonstrating that MTH1 and OGG1 prevent accumulation of 8-oxoG in nDNA, thereby suppressing mutagenesis and carcinogenesis (12
). However, we also found that mice lacking both MTH1 and OGG1 developed no lung tumors, although 8-oxoG was highly accumulated in nDNA (13
). This observation suggests that excess accumulation of 8-oxoG under combined deficiency of MTH1 and OGG1 may induce tumor cell death, thereby leading to diminished occurrence of lung tumors (41
). Furthermore, we have shown that increased accumulation of 8-oxoG in nDNA and/or mtDNA causes cell death and that MTH1 and OGG1 suppress cell death (34
We previously reported that Mth1
-KO mice exhibit increased accumulation of 8-oxoG in the mtDNA of striatal nerve terminals of dopamine neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, followed by more severe terminal degeneration of dopamine neurons in comparison to WT mice (31
). MTH1 also efficiently suppresses the accumulation of 8-oxoG in mtDNA in the hippocampus caused by kainate-induced excitotoxicity, especially in microglia (43
). Moreover, it has been shown that human MTH1 transgenic mice are resistant to 3-NP–induced striatal degeneration (27
), suggesting that increased accumulation of 8-oxoG in brain causes neurodegeneration.
In the present study, we showed that double deficiency of MTH1 and OGG1 renders the striatum highly vulnerable to 3-NP–induced neurodegeneration, accompanied by highly increased accumulation of 8-oxoG in both the mtDNA of MSNs and the nDNA of microglia in the striatum. This is the first experimental evidence that OGG1 and MTH1 cooperatively suppress 8-oxoG accumulation in brain DNAs, thus protecting the brain from neuronal loss and microgliosis, which result in severe neurodegeneration and behavioral impairments.
Mitochondria prepared from caudate nucleus as well as other regions of mouse brain possess substantial levels of OGG1 incision activity (44
). In the present study, we found that OGG1 was predominantly localized in the mitochondria of MSNs in normal striatum (Figure A) and that OGG1 deficiency significantly increased accumulation of mitochondrial 8-oxoG and ssDNA in MSNs after exposure to 3-NP. Considering these findings together, we concluded that mitochondrial OGG1 is crucial to protect striatal MSNs from oxidative stress.
MUTYH suppresses carcinogenesis but promotes neurodegeneration by inducing cell death. We have shown that Mutyh
-KO mice exhibit the most significant increase in spontaneous occurrence of tumors among Mth1
-, and Mutyh
-KO mice (14
). It has been reported that Ogg1/Mutyh
-DKO mice (15
) and Mth1
–triple-KO mice exhibit markedly increased spontaneous tumorigenesis in various tissues over a much shorter period (our unpublished observations). These observations indicate that MUTYH functions as the strongest suppressor of tumorigenesis among the 3, because Mth1/Ogg1
-DKO mice develop no lung tumors whereas Ogg1
-KO mice do (13
). Thus, we hypothesized that MUTYH induces cell death, thereby suppressing tumorigenesis in the absence of MTH1 and OGG1. We previously showed that MUTYH is required for generation of SSBs in DNA and induction of cell death when 8-oxoG is highly accumulated in either nDNA or mtDNA under conditions of oxidative stress (34
In the present study, we found that MUTYH protein is highly expressed in the striatum and is mostly present in the mitochondria of MSNs and that Ogg1/Mutyh-DKO mice exhibit markedly increased resistance to 3-NP–induced striatal degeneration in comparison with Ogg1-KO mice and even WT mice. These results clearly demonstrate that MUTYH enhances neurodegeneration through the induction of cell death once 8-oxoG becomes accumulated in neurons and/or microglia.
MUTYH-initiated BER induces accumulation of SSBs in DNA, thereby triggering 2 distinct cell death pathways dependent on calpain or PARP-AIF. We previously established that MUTYH initiates the 2 distinct pathways of cell death while 8-oxoG accumulates selectively in mtDNA or nDNA in Ogg1
-KO cell lines (34
). In the present study, we found that 8-oxoG is significantly accumulated first in the mtDNA of MSNs (P
< 0.0001) and then in the nDNA of microglia during striatal degeneration caused by 3-NP in the absence of OGG1 and/or MTH1. This is followed by selective accumulation of SSBs in each type of DNA. Calpain activation was observed in MSNs, while nuclear accumulation of PAR-polymer and AIF was evident in microglia. Calpain activation, PARP-AIF activation, and accumulation of SSBs were observed in Ogg1
-KO mice, but were efficiently suppressed by a lack of MUTYH, which initiated BER of adenines inserted opposite 8-oxoG (41
). Moreover, administration of inhibitors of calpain or PARP to Mth1/ Ogg1
-DKO mice efficiently suppressed 3-NP–induced loss of MSNs and microgliosis in the striatum, and this was followed by improvement of motor impairment. Considering these observations together, we concluded that MUTYH-initiated BER induces accumulation of SSBs in DNA, thereby triggering 2 distinct cell death pathways dependent on calpain or PARP-AIF, as summarized in Figure . The anti-ssDNA antibody used in the present study also recognizes single-stranded regions of double-stranded breaks (DSBs) generated by resection of the ends (45
). It is thus possible that DSBs may also be generated through incision of the template strand if there are other types of damaged bases close to 8-oxoG in the template DNA generated under increased oxidative stress.
Molecular mechanisms underlying 8-oxoG–induced striatal degeneration.
Recently, Foti et al. reported that cytotoxicity of bactericidal antibiotics such as β-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxoG lesions and is efficiently suppressed by mutM/mutY
-double deficiency (5
). Considered together with our previous findings (28
), it is noteworthy that 8-oxoG accumulated in the genome causes cell death through a common mechanism from bacteria to mammals. In the mammalian brain, however, there are different types of cells, such as neurons and microglia, and each cell has 2 distinct genomes, 1 in the nucleus, and 1 in mitochondria. Our findings in the present study show for what we believe is the first time that neurodegeneration is a complex process caused by 8-oxoG accumulated in the 2 distinct genomes in the brain.
Neurodegeneration induced by 8-oxoG and MUTYH depends on both replication of mtDNA in neurons and nDNA in microglia. Overexpression of human MTH1 in mouse striatum efficiently abrogates 3-NP–induced striatal degeneration accompanied by effective suppression of 8-oxoG accumulation in the striatum (27
-DKO mice exhibited the highest susceptibility to 3-NP–induced striatal degeneration with the highest levels of 8-oxoG in striatal DNAs. These findings indicate that the major source of 8-oxoG accumulated in DNA are 8-oxo–dGTP generated in nucleotide pool; the former is repaired by OGG1 and the latter is hydrolyzed by MTH1.
DNA replication is essential for accumulation of 8-oxoG in the respective DNA; moreover, insertion of adenine opposite 8-oxoG also depends on replication. Neurons are postmitotic and only mtDNA but not nDNA is always replicated in neurons in order to provide the energy essential for maintenance of neuronal functions. 8-oxoG is therefore dominantly accumulated in mtDNA of neurons under conditions of oxidative stress, resulting in SSB accumulation in mtDNA during MUTYH-initiated BER of adenine inserted opposite 8-oxoG. In contrast, microglia are mitotic; thus, 8-oxoG and SSBs accumulated in nDNA dependent on their replication and MUTYH-initiated BER (Figure ). Thus, replication of the respective DNAs in neurons and microglia determines which of the 2 separate signaling pathways must be activated upon accumulation of 8-oxoG under conditions of oxidative stress.
Microgliosis is a delayed event in striatal degeneration induced by 3-NP (Figure D); therefore, it is likely that accumulation of 8-oxoG followed by ssDNA accumulation in nDNA of microglia (Figures H and 7B) is not due to a direct effect of mitochondrial ROS generated in MSNs immediately after exposure to 3-NP. Neurons damaged by mitochondrial ROS thereafter induce a delayed inflammatory response, resulting in microgliosis, which is known to be accompanied by secondary generation of ROS in brain (46
). Since inhibition of PARP activity, which is activated by SSBs accumulated in the nDNA of microglia resulting in AIF activation (Figure , A and D), efficiently suppressed neuronal loss as well as microgliosis, ROS generated in microglia may in turn attack neurons to promote the degenerative process as suggested by Brouillet et al. (36
), thus initiating a vicious cycle of neurodegeneration.
Growing evidence suggests that mitochondrial dysfunction is generally associated with neurodegenerative diseases (47
) and that microglial activation is also involved in the pathogenesis of most forms of neurodegeneration (48
). It is known that activation of calpain, PARP, and AIF is involved in various neurodegenerative diseases such as AD, PD, and HD (38
), in which 8-oxoG is a common molecular marker (50
), together with altered expression of MTH1, OGG1, and MUTYH (16
). Inhibition of calpain and PARP has been considered to be a potential therapeutic approach for neurologic disorders (38
Our findings in the present study show that 2 distinct molecular pathways are separately activated in the mitochondria of neurons and the nuclei of microglia through oxidative DNA damage and its repair, thus providing insights into the molecular mechanisms underlying neurodegeneration under conditions of oxidative stress as well as providing therapeutic targets for neurodegenerative diseases. Suppression of MUTYH may thus be an efficient strategy for protecting the brain under conditions of oxidative stress. However, it will be important not to increase the risk of carcinogenesis under MUTYH suppression; therefore, further experimental evaluation is essential.