The defense mechanisms in mammalian cells against oxidative damage in nucleic acids and their involvement in the suppression of mutagenesis and cell death

To counteract oxidative damage in nucleic acids, mammalian cells are equipped with several defense mechanisms. We herein review that MTH1, MUTYH and OGG1 play important roles in mammalian cells avoiding an accumulation of oxidative DNA damage, both in the nuclear and mitochondrial genomes, thereby suppressing carcinogenesis and cell death. MTH1 efficiently hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP and 2-hydroxy (OH)-dATP, to the monophosphates, thus avoiding the incorporation of such oxidized nucleotides into the nuclear and mitochondrial genomes. OGG1 excises 8-oxoG in DNA as a DNA glycosylase and thus minimizes the accumulation of 8-oxoG in the cellular genomes. MUTYH excises adenine opposite 8-oxoG, and thus suppresses 8-oxoG-induced mutagenesis. MUTYH also possesses a 2-OH-A DNA glycosylase activity for excising 2-OH-A incorporated into the cellular genomes. Increased susceptibilities to spontaneous carcinogenesis of the liver, lung or intestine were observed in MTH1-, OGG1- and MUTYH-null mice, respectively. The increased occurrence of lung tumors in OGG1-null mice was abolished by the concomitant disruption of the Mth1 gene, indicating that an increased accumulation of 8-oxoG and/or 2-OH-A might cause cell death. Furthermore, these defense mechanisms also likely play an important role in neuroprotection.     

MTH1, an oxidized purine nucleoside triphosphatase, prevents the cytotoxicity and neurotoxicity of oxidized purine nucleotides

In human and rodent cells, MTH1, an oxidized purine nucleoside triphosphatase, efficiently hydrolyzes oxidized dGTP, GTP, dATP and ATP such as 2'-deoxy-8-oxoguanosine triphosphate (8-oxo-dGTP) and 2'-deoxy-2-hydroxyadenosine triphosphate (2-OH-dATP) in nucleotide pools, thus avoiding their incorporation into DNA or RNA. MTH1 is expressed in postmitotic neurons as well as in proliferative tissues, and it is localized both in the mitochondria and nucleus, thus suggesting that MTH1 plays an important role in the prevention of the mutagenicity and cytotoxicity of such oxidized purines as 8-oxoG which are known to accumulate in the cellular genome. Our recent studies with MTH1-deficient mice or cells revealed that MTH1 efficiently minimizes accumulation of 8-oxoG in both nuclear and mitochondrial DNA in the mouse brain as well as in cultured cells, thus contributing to the protection of the brain from oxidative stress.     

Oxidation of mitochondrial deoxynucleotide pools by exposure to sodium nitroprusside induces cell death

Human MutT homolog (hMTH1) hydrolyzes oxidized purine nucleoside triphosphates to monophosphates, thereby avoiding incorporation of such oxidized purines into DNA or RNA. We examined whether hMTH1 prevents cellular dysfunction induced by sodium nitroprusside, a spontaneous NO donor. Exposure to sodium nitroprusside caused an 8-oxoguanine (8-oxoG) buildup in DNA of proliferating MTH1-null cells which underwent mitochondrial degeneration and subsequently died. Quiescent MTH1-null cells also died with 8-oxoG buildup but only when the buildup affected mitochondrial and not nuclear DNA. In both proliferative and quiescent conditions, the accumulation of 8-oxoG in DNA and cell death was effectively prevented by hMTH1. Knockdown of MUTYH in quiescent MTH1-null cells significantly prevented the cell death, suggesting that 8-oxoG incorporated into mitochondrial DNA is a main cause of this form of cell death. To verify this possibility, an artificially modified hMTH1, namely mTP-EGFP-hMTH1, which localizes exclusively in mitochondria, was expressed in MTH1-null cells. mTP-EGFP-hMTH1 selectively prevented buildup of 8-oxoG in mitochondrial but not nuclear DNA after exposure of proliferating cells to sodium nitroprusside, and also efficiently prevented cell death. We thus concluded that exposure of cells to sodium nitroprusside causes oxidation of mitochondrial deoxynucleotide pools, and that buildup of oxidized bases in mitochondrial DNA initiates cell death.     

Ogg1 null mice exhibit age-associated loss of the nigrostriatal pathway and increased sensitivity to MPTP

Cumulative damage to cellular macromolecules via oxidative stress is a hallmark of aging and neurodegenerative disease. Whether such damage is a cause or a subsequent effect of neurodegeneration is still unknown. This paper describes the development of an age-associated mild parkinsonian model in mice that lack the DNA repair enzyme 8-oxoguanine glycosylase 1 (Ogg1). Aged OGG1 knock-out (OGG1 KO) mice show a decreased spontaneous locomotor behavior and evidence a decrease in striatal dopamine levels, a loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN), and an increase in ubiquitin-positive inclusions in their remaining SN neurons. In addition, young OGG1 KO mice are more susceptible to the dopaminergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) than their wild-type (WT) counterparts. Age-associated increases in 7,8-dihydro-2'-deoxyguanine (oxo(8)dG) have been reported in brain regions and neuronal populations affected in Parkinson's disease (PD), toxin-induced parkinsonian models, and mice harboring genetic abnormalities associated with PD. Because of these increased oxo(8)dG levels, the OGG1 KO mouse strain could shed light on molecular events leading to neuronal loss as a consequence of cumulative oxidative damage to DNA during aging and after toxicological challenge.     

Apoptosis signal-regulating kinase 1 mediates MPTP toxicity and regulates glial activation

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase 3 family, is activated by oxidative stress. The death-signaling pathway mediated by ASK1 is inhibited by DJ-1, which is linked to recessively inherited Parkinson''s disease (PD). Considering that DJ-1 deficiency exacerbates the toxicity of the mitochondrial complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we sought to investigate the direct role and mechanism of ASK1 in MPTP-induced dopamine neuron toxicity. In the present study, we found that MPTP administration to wild-type mice activates ASK1 in the midbrain. In ASK1 null mice, MPTP-induced motor impairment was less profound, and striatal dopamine content and nigral dopamine neuron counts were relatively preserved compared to wild-type littermates. Further, microglia and astrocyte activation seen in wild-type mice challenged with MPTP was markedly attenuated in ASK1^−/− mice. These data suggest that ASK1 is a key player in MPTP-induced glial activation linking oxidative stress with neuroinflammation, two well recognized pathogenetic factors in PD. These findings demonstrate that ASK1 is an important effector of MPTP-induced toxicity and suggest that inhibiting this kinase is a plausible therapeutic strategy for protecting dopamine neurons in PD.     

Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids

Genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are generated both as byproducts of oxygen respiration or molecular executors in the host defense, and by environmental exposure to ionizing radiation and chemicals. To counteract such oxidative damage in nucleic acids, mammalian cells are equipped with three distinct enzymes. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-2'-deoxyguanosine triphosphate and 2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP), to the corresponding monophosphates. We observed increased susceptibility to spontaneous carcinogenesis in MTH1-null mice, which exhibit an increased occurrence of A:T-->C:G and G:C-->T:A transversion mutations. 8-Oxoguanine (8-oxoG) DNA glycosylase, encoded by the OGG1 gene, and adenine DNA glycosylase, encoded by the MUTYH gene, are responsible for the suppression of G:C to T:A transversions caused by the accumulation of 8-oxoG in the genome. Deficiency of these enzymes leads to increased tumorigenesis in the lung and intestinal tract in mice, respectively. MUTYH deficiency may also increase G:C to T:A transversions through the misincorporation of 2-OH-dATP, especially in the intestinal tract, since MUTYH can excise 2-hydroxyadenine opposite guanine in genomic DNA and the repair activity is selectively impaired by a mutation found in patients with autosomal recessive colorectal adenomatous polyposis.     

Preventive role of PD‐1 on MPTP‐induced dopamine depletion in mice

Many current studies of Parkinson's disease (PD) suggest that inflammation is involved in the neurodegenerative process. PD‐1, a traditional Korean medicine, used to treat various brain diseases in Korea. This study was designed to investigate the effect of PD‐1 extract in the Parkinson's model of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) lesioned mice. The MPTP administration caused the dopamine neuron loss in the striatum and substantia nigra pars compacta (SNpc), which was demonstrated by a depletion of tyrosine hydroxylase (TH). In addition, a reduction of bcl‐2 expression with elevation of bax expression, caspase‐3 activation, and release of cytochrome c into cytosol in dopaminergic neurons of SNpc were noted. Oral administration of PD‐1 extract (50 and 100 mg kg^?1) attenuated the MPTP‐induced depletion of TH proteins in the striatum and SNpc and prevented the apoptotic effects. These results indicate that PD‐1 extract is able to protect dopaminergic neurons from MPTP‐induced neuronal death, with important implications for the treatment of PD. Copyright © 2010 John Wiley & Sons, Ltd.     

Iron-sulfur enzyme mediated mitochondrial superoxide toxicity in experimental Parkinson's disease

Mitochondrial oxidative stress is thought to be an important pathological mediator of neuronal death in Parkinson's disease. However, the precise mechanism by which mitochondrial oxidative stress mediates the death of dopaminergic neurons of the substantia nigra remains unclear. We tested the idea that neuronal damage in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease results, in part, from superoxide radical toxicity via inactivation of an iron-sulfur (Fe-S) protein, mitochondrial aconitase. Administration of MPTP in mice resulted in inactivation of mitochondrial aconitase, but not fumarase in the substantia nigra. MPTP treatment mobilized an early mitochondrial pool of iron detectable by bleomycin chelation that coincided with mitochondrial aconitase inactivation. MPTP-induced mitochondrial aconitase inactivation, iron accumulation and dopamine depletion were significantly attenuated in transgenic mice overexpressing mitochondrial Sod2 and exacerbated in partial deficient Sod2 mice. These results suggest that mitochondrial aconitase may be an important early source of mitochondrial iron accumulation in experimental Parkinson's disease, and that superoxide radical toxicity manifested by oxidative inactivation of mitochondrial aconitase may play a pathogenic role in Parkinson's disease.     

Increased vulnerability of dopaminergic neurons in MPTP-lesioned interleukin-6 deficient mice

To test the hypothesis that neuroinflammation contributes to dopaminergic neuron death in the MPTP-lesioned mouse, we compared nigrostriatal degeneration in interleukin (IL)-6 (+/+) with IL-6 (-/-) mice. In the absence of IL-6, a single injection of MPTP (30 mg/kg) resulted in significantly greater striatal dopamine depletion than that measured in IL-6 (+/+) mice. The observed dopamine depletion was MPTP dose dependent. This loss of striatal dopamine and a significantly greater loss of TH+ cells in the substantia nigra pars compacta in IL-6 (-/-) mice as compared with control IL-6 (+/+) mice, suggest that IL-6 is neuroprotective in the MPTP-lesioned nigrostriatal system. Co-localization experiments identified striatal astrocytes as the source of IL-6 in IL-6 (+/+) mice at 1 and 7 days postinjection of MPTP. The increased sensitivity of dopaminergic neurons to neurotoxicant in the absence of IL-6, is compatible with a neuroprotective activity of IL-6 in the injured nigrostriatal system.     

Effects of mitochondrial antioxidant SkQ1 on biochemical and behavioral parameters in a Parkinsonism model in mice

According to one hypothesis, Parkinson’s disease pathogenesis is largely caused by dopamine catabolism that is catalyzed on mitochondrial membranes by monoamine oxidase. Reactive oxygen species are formed as a byproduct of these reactions, which can lead to mitochondrial damage followed by cell degeneration and death. In this study, we investigated the effects of administration of the mitochondrial antioxidant SkQ1 on biochemical, immunohistochemical, and behavioral parameters in a Parkinson-like condition caused by protoxin MPTP injections in C57BL/6 mice. SkQ1 administration increased dopamine quantity and decreased signs of sensory-motor deficiency as well as destruction of dopaminergic neurons in the substantia nigra and ventral tegmental area in mice with the Parkinson-like condition.     

Inactivation of Pink1 gene in vivo sensitizes dopamine-producing neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and can be rescued by autosomal recessive Parkinson disease genes, Parkin or DJ-1

Mutations in the mitochondrial PTEN-induced kinase 1 (Pink1) gene have been linked to Parkinson disease (PD). Recent reports including our own indicated that ectopic Pink1 expression is protective against toxic insult in vitro, suggesting a potential role for endogenous Pink1 in mediating survival. However, the role of endogenous Pink1 in survival, particularly in vivo, is unclear. To address this critical question, we examined whether down-regulation of Pink1 affects dopaminergic neuron loss following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the adult mouse. Two model systems were utilized: virally delivered shRNA-mediated knockdown of Pink1 and germ line-deficient mice. In both instances, loss of Pink1 generated significant sensitivity to damage induced by systemic MPTP treatment. This sensitivity was associated with greater loss of dopaminergic neurons in the Substantia Nigra pars compacta and terminal dopamine fiber density in the striatum region. Importantly, we also show that viral mediated expression of two other recessive PD-linked familial genes, DJ-1 and Parkin, can protect dopaminergic neurons even in the absence of Pink1. This evidence not only provides strong evidence for the role of endogenous Pink1 in neuronal survival, but also supports a role of DJ-1 and Parkin acting parallel or downstream of endogenous Pink1 to mediate survival in a mammalian in vivo context.     

Therapeutic Effects of Rapamycin on MPTP-Induced Parkinsonism in Mice

In neurodegenerative disorders such as Parkinson’s disease (PD), autophagy is implicated in the process of dopaminergic neuron cell death. The α-synuclein protein is a major component of Lewy bodies and Lewy neurites, and mutations in α-synuclein have been implicated in the etiology of familial PD. The current work investigates the mechanisms underlying the therapeutic effects of the autophagy-stimulating antibiotic rapamycin in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Male C57BL/6 mice were treated with intravenous rapamycin or saline control for 7 days following MPTP administration. Immunohistochemistry and western blotting were used to detect alterations in the expression of PD biomarkers, including tyrosine hydroxylase (TH), and the level of autophagy was evaluated by the detection of both microtubule-associated protein light chain 3 (LC3) and α-Synuclein cleavage. In addition, levels of monoamine neurotransmitters were measured in the striatum using high performance liquid chromatography (HPLC). Immunohistochemistry using antibodies against TH indicated that the number of dopaminergic neurons in the substantia nigra following MPTP treatment was significantly higher in rapamycin-treated mice compared with saline-treated controls (p < 0.01). Levels of TH expression in the striatum were similar between the groups. α-synuclein Immunoreactivity was significantly decreased in rapamycin-treated mice compared with controls (p < 0.01). Immunoreactivity for LC3, however, was significantly higher in the rapamycin-treated animals than controls (p < 0.01). The concentrations of both striatal dopamine, and the dopamine metabolite DOPAC, were significantly decreased in both MPTP-treated groups compared with untreated controls. The loss of DOPAC was less severe in rapamycin-treated mice compared with saline-treated mice (p < 0.01) following MPTP treatment. These results demonstrate that treatment with rapamycin is able to prevent the loss of TH-positive neurons and to ameliorate the loss of DOPAC following MPTP treatment, likely via activation of autophagy/lysosome pathways. Thus, further investigation into the effectiveness of rapamycin administration in the treatment of PD is warranted.     

Hydrogen in Drinking Water Reduces Dopaminergic Neuronal Loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mouse Model of Parkinson's Disease

It has been shown that molecular hydrogen (H₂) acts as a therapeutic antioxidant and suppresses brain injury by buffering the effects of oxidative stress. Chronic oxidative stress causes neurodegenerative diseases such as Parkinson''s disease (PD). Here, we show that drinking H₂-containing water significantly reduced the loss of dopaminergic neurons in PD model mice using both acute and chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The concentration-dependency of H₂ showed that H₂ as low as 0.08 ppm had almost the same effect as saturated H₂ water (1.5 ppm). MPTP-induced accumulation of cellular 8-oxoguanine (8-oxoG), a marker of DNA damage, and 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation were significantly decreased in the nigro-striatal dopaminergic pathway in mice drinking H₂-containing water, whereas production of superoxide (O₂•⁻) detected by intravascular injection of dihydroethidium (DHE) was not reduced significantly. Our results indicated that low concentration of H₂ in drinking water can reduce oxidative stress in the brain. Thus, drinking H₂-containing water may be useful in daily life to prevent or minimize the risk of life style-related oxidative stress and neurodegeneration.     

Ventricular injection of nerve growth factor increases dopamine content in the striata of MPTP-treated mice

Experimental depletion of dopaminergic striatal neurons was induced in mice with the neurotoxin MPTP. To investigate a possible effect of nerve growth factor on the damaged neurons, we injected 4 g into the right cerebral ventricle of mice three days after the last administration of MPTP. We found a significant increase of dopamine and homovanillic acid in the striatum of MPTP treated mice after NGF administration when compared with dopamine and HVA levels in MPTP-treated control mice (p<0.001). The increase of dopamine and homovanillic acid seems to be related to a partial restorative effect of NGF on the damaged dopaminergic cells, since ventricular administration of NGF to normal mice did not increase dopamine or homovanillic acid contents above the levels measured in untreated controls. It appears that administration of nerve growth factor prcduces a beneficial effect on damaged dopaminergic neurons; this effect could be due to stimulation of neuron sprouting from neurons that survived the toxic effect of MPTP. The increase of dopamine levels was seen 8 days after injection of nerve growth factor and was maintained at least until day 25, showing a lasting persistence of the restorative effect.     

Effects of Systemic Administration of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine to Mice on Tyrosine Hydroxylase, l-3,4-Dihydroxyphenylalanine Decarboxylase, Dopamine β-Hydroxylase, and Monoamine Oxidase Activities in the Striatum and Hypothalamus

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg subcutaneously per day for 8 days) to C57BL/6N mice were studied on tyrosine hydroxylase (TH), L-3,4-dihydroxyphenylalanine decarboxylase (DDC), and monoamine oxidase (MAO) activities in the striatum, and TH, DDC, dopamine-beta-hydroxylase (DBH), and MAO activities in the hypothalamus. Treatment with MPTP led to a large decrease in TH activity and a parallel decrease in DDC activity in the striatum, as compared with the saline controls. In contrast, MPTP administration did not cause a decrease of the activities of TH, DDC, and DBH in the hypothalamus. There was also no reduction in MAO activities of striatum and hypothalamus. These data indicate that MPTP administration to mice results in specific degeneration of the dopaminergic nigrostriatal pathway and that DDC in the mouse striatum may mainly be localized in the dopaminergic neurons with TH.     

Combination therapy with Coenzyme Q10 and creatine produces additive neuroprotective effects in models of Parkinson's and Huntington's Diseases

Coenzyme Q₁₀ (CoQ₁₀) and creatine are promising agents for neuroprotection in neurodegenerative diseases via their effects on improving mitochondrial function and cellular bioenergetics and their properties as antioxidants. We examined whether a combination of CoQ₁₀ with creatine can exert additive neuroprotective effects in a MPTP mouse model of Parkinson's disease, a 3-NP rat model of Huntington's disease (HD) and the R6/2 transgenic mouse model of HD. The combination of the two agents produced additive neuroprotective effects against dopamine depletion in the striatum and loss of tyrosine hydroxylase neurons in the substantia nigra pars compacta (SNpc) following chronic subcutaneous administration of MPTP. The combination treatment resulted in significant reduction in lipid peroxidation and pathologic α-synuclein accumulation in the SNpc neurons of the MPTP-treated mice. We also observed additive neuroprotective effects in reducing striatal lesion volumes produced by chronic subcutaneous administration of 3-NP to rats. The combination treatment showed significant effects on blocking 3-NP-induced impairment of glutathione homeostasis and reducing lipid peroxidation and DNA oxidative damage in the striatum. Lastly, the combination of CoQ₁₀ and creatine produced additive neuroprotective effects on improving motor performance and extending survival in the transgenic R6/2 HD mice. These findings suggest that combination therapy using CoQ₁₀ and creatine may be useful in the treatment of neurodegenerative diseases such as Parkinson's disease and HD.     

The basal levels of 8-oxoG and other oxidative modifications in intact mitochondrial DNA are low even in repair-deficient (Ogg1(-/-)/Csb(-/-)) mice

Mitochondrial DNA (mtDNA) is assumed to be highly prone to damage by reactive oxygen species (ROS) because of its location in close proximity to the mitochondrial electron transport chain. Accordingly, mitochondrial oxidative DNA damage has been hypothesized to be responsible for various neurological diseases, ageing and cancer. Since 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most frequent oxidative base modifications, is removed from the mitochondrial genome by the glycosylase OGG1, the basal levels of this lesion are expected to be highly elevated in Ogg1^−/− mice. To investigate this hypothesis, we have used a mtDNA relaxation assay in combination with various repair enzymes (Fpg, MutY, endonuclease III, endonuclease IV) to determine the average steady-state number of oxidative DNA modifications within intact (supercoiled) mtDNA from the livers of wild-type mice and those deficient in OGG1 and/or the Cockayne syndrome B (CSB) protein for mice aged up to 23 months. The levels of all types of oxidative modifications were found to be less than 12 per million base pairs, and the difference between wild-type and repair-deficient (Ogg1^−/−/Csb^−/−) mice was not significant. Thus, the increase of 8-oxoG caused by the repair deficiency in intact mtDNA is not much higher than in the nuclear DNA, i.e., not more than a few modifications per million base pairs. Based on these data, it is hypothesized that the load of oxidative base modifications in mtDNA is efficiently reduced during replication even in the absence of excision repair.     

Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.     

Analysis of MTH1 gene function in mice with targeted mutagenesis

Oxidative DNA damage is thought to contribute to carcinogenesis, ageing, and neurological degeneration. Further, the cumulative risk of cancer increases dramatically with age in humans. In general terms, cancer can be regarded as a degenerative disease of ageing. There is evidence for the accumulation of oxidative DNA damage with age based on studies mainly measuring an increase in 8-oxoguanine. 8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is formed in the nucleotide pool of a cell during normal cellular metabolism. When 8-oxoguanine is incorporated into DNA causes mutation. Organisms possess 8-oxo-dGTPase, an enzyme that specifically degrades 8-oxo-dGTP to 8-oxo-dGMP. To analyze the function of MTH1 with 8-oxo-dGTPase activity in vivo, we generated a mouse line carrying a mutant MTH1 allele created by targeted gene disruption. MTH1 homozygous mutant mice were found to have a physically normal appearance, but seemed to have lost 8-oxo-dGTPase activity in liver extracts. When we examined the susceptibility of the mutant mice to spontaneous tumorigenesis, no significant difference was observed in survival rate of MTH1+/+ and MTH1-/- mice. However, pathological examination revealed a statistically significant difference in the incidence of tumors. More tumors were formed in lungs, livers, and stomachs of MTH1-/- mice than in those of the wild type mice. These studies with MTH1-null mutant mice provided an important insight into the role of this nucleotide sanitization enzyme in terms of the spontaneous tumorigenesis as well as mutagenesis caused by the oxygen-induced DNA damage.     

Overexpression of Parkinson's disease-associated alpha-synucleinA53T by recombinant adeno-associated virus in mice does not increase the vulnerability of dopaminergic neurons to MPTP

Mutations in the alpha-synuclein gene are linked to a rare dominant form of familial Parkinson's disease, and alpha-synuclein is aggregated in Lewy bodies of both sporadic and dominant Parkinson's disease. It has been proposed that mutated alpha-synuclein causes dopaminergic neuron loss by enhancing the vulnerability of these neurons to a variety of insults, including oxidative stress, apoptotic stimuli, and selective dopaminergic neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test this hypothesis in vivo, we overexpressed human alpha-synuclein(A53T) in the substantia nigra of normal and MPTP-treated mice by rAAV-mediated gene transfer. Determination of dopaminergic neuron survival, striatal tyrosine hydroxylase fiber density, and striatal content of dopamine and its metabolites in rAAV-injected and uninjected hemispheres demonstrated that alpha-synuclein(A53T) does not increase the susceptibility of dopaminergic neurons to MPTP. Our findings argue against a direct detrimental role for (mutant) alpha-synuclein in oxidative stress and/or apoptotic pathways triggered by MPTP, but do not rule out the possibility that alpha-synuclein aggregation in neurons exposed to oxidative stress for long periods of time may be neurotoxic.