Therapeutic Effects of Hydrogen in Animal Models of Parkinson's Disease

Since the first description of Parkinson's disease (PD) nearly two centuries ago, a number of studies have revealed the clinical symptoms, pathology, and therapeutic approaches to overcome this intractable neurodegenerative disease. 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) are neurotoxins which produce Parkinsonian pathology. From the animal studies using these neurotoxins, it has become well established that oxidative stress is a primary cause of, and essential for, cellular apoptosis in dopaminergic neurons. Here, we describe the mechanism whereby oxidative stress evokes irreversible cell death, and propose a novel therapeutic strategy for PD using molecular hydrogen. Hydrogen has an ability to reduce oxidative damage and ameliorate the loss of nigrostriatal dopaminergic neuronal pathway in two experimental animal models. Thus, it is strongly suggested that hydrogen might provide a great advantage to prevent or minimize the onset and progression of PD

Cellular Levels of 8-Oxoguanine in either DNA or the Nucleotide Pool Play Pivotal Roles in Carcinogenesis and Survival of Cancer Cells

8-Oxoguanine, a major oxidized base lesion formed by reactive oxygen species, causes G to T transversion mutations or leads to cell death in mammals if it accumulates in DNA. 8-Oxoguanine can originate as 8-oxo-dGTP, formed in the nucleotide pool, or by direct oxidation of the DNA guanine base. MTH1, also known as NUDT1, with 8-oxo-dGTP hydrolyzing activity, 8-oxoguanine DNA glycosylase (OGG1) an 8-oxoG DNA glycosylase, and MutY homolog (MUTYH) with adenine DNA glycosylase activity, minimize the accumulation of 8-oxoG in DNA; deficiencies in these enzymes increase spontaneous and induced tumorigenesis susceptibility. However, different tissue types have different tumorigenesis susceptibilities. These can be reversed by combined deficiencies in the defense systems, because cell death induced by accumulation of 8-oxoG in DNA is dependent on MUTYH, which can be suppressed by MTH1 and OGG1. In cancer cells encountering high oxidative stress levels, a high level of 8-oxo-dGTP accumulates in the nucleotide pool, and cells therefore express increased levels of MTH1 in order to eliminate 8-oxo-dGTP. Suppression of MTH1 may be an efficient strategy for killing cancer cells; however, because MTH1 and OGG1 protect normal tissues from oxidative-stress-induced cell death, it is important that MTH1 inhibition does not increase the risk of healthy tissue degeneration

Nucleotides function as endogenous chemical sensors for oxidative stress signaling

Oxidized and nitrated nucleotides including 8-oxogunanine and 8-nitroguanine derivatives such as 8-nitroguanosine 3',5'-cyclic monophosphate were generated by reactive nitrogen oxides and reactive oxygen species in cultured cells and in tissues. 8-oxoguanine and 8-nitroguanine in DNA and RNA are potentially mutagenic, and the former also induces cell death. Some derivative, 8-nitroguanosine 3',5'-cyclic monophosphate a major nitrated guanine nucleotide, was identified as a novel second messenger. Surprisingly, the amount of 8-nitroguanosine 3',5'-cyclic monophosphate generated was found to be higher than that of guanosine 3',5'-cyclic monophosphate in cells expressing inducible nitric oxide synthase. More important, 8-nitroguanosine 3',5'-cyclic monophosphate is electrophilic and reacted efficiently with sulfhydryls of proteins to produce a novel posttranslational modification (named S-guanylation) via guanosine 3',5'-cyclic monophosphate adduction. For example, 8-nitroguanosine 3',5'-cyclic monophosphate-induced S-guanylation of Kelch-like ECH-associated protein 1 led to NF-E2-related factor activation and induction of antioxidant enzymes. 8-nitroguanosine 3',5'-cyclic monophosphate may thus protect cells against oxidative stress-related cytotoxicity. Therefore, although chemically modified nucleotides produced via oxidative and nitrative stress are regarded simply as endogenous mutagens, the endogenous nucleotides stored in cells per se may serve functionally as a sensing mechanism for reactive nitrogen oxides and oxygen species to induce cellular adaptive responses to oxidative stress

A functional analysis of the DNA glycosylase activity of mouse MUTYH protein excising 2-hydroxyadenine opposite guanine in DNA

2-Hydroxy-2-deoxyadenosine triphosphate (2-OH-dATP), generated by the oxidation of dATP, can be misincorporated by DNA polymerases opposite guanine in template DNA during DNA replication, thus causing spontaneous mutagenesis. We demonstrated that mouse MUTYH (mMUTYH) has a DNA glycosylase activity excising not only adenine opposite 8-oxoguanine (8-oxoG) but also 2-hydroxyadenine (2-OH-A) opposite guanine, using purified recombinant thioredoxin-mMUTYH fusion protein. mMUTYH formed a stable complex with duplex oligonucleotides containing an adenine:8-oxoG pair, but the binding of mMUTYH to oligonucleotides containing a 2-OH-A:guanine pair was barely detectable, thus suggesting that mMUTYH recognizes and interacts with these two substrates in a different manner which may reflect the difference in the base excision repair process for each substrate. Mutant mMUTYH with G365D amino acid substitution, corresponding to a G382D germline mutation of human MUTYH found in familial adenomatous polyposis patients, almost completely retained its DNA glycosylase activity excising adenine opposite 8-oxoG; however, it possessed 1.5% of the wild-type activity excising 2-OH-A opposite guanine. Our results imply that the reduced repair capacity of the mutant hMUTYH(G382D), which inefficiently excises 2-OH-A opposite guanine, results in an increased occurrence of somatic G:C to T:A transversion mutations in the APC gene as well as tumorigenesis in the colon

NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa− mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa− embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa− primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa− MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa− embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa− embryos. However, immortalized Itpa− MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa− MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals

APE1- and APE2-dependent DNA breaks in immunoglobulin class switch recombination

Antibody class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded breaks (DSBs) in switch-region DNA. The initial steps in DSB formation have been elucidated, involving cytosine deamination by activation-induced cytidine deaminase and generation of abasic sites by uracil DNA glycosylase. However, it is not known how abasic sites are converted into single-stranded breaks and, subsequently, DSBs. Apurinic/apyrimidinic endonuclease (APE) efficiently nicks DNA at abasic sites, but it is unknown whether APE participates in CSR. We address the roles of the two major mammalian APEs, APE1 and APE2, in CSR. APE1 deficiency causes embryonic lethality in mice; we therefore examined CSR and DSBs in mice deficient in APE2 and haploinsufficient for APE1. We show that both APE1 and APE2 function in CSR, resulting in the DSBs necessary for CSR and thereby describing a novel in vivo function for APE2

Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons

A molecular basis of survival from neuronal injury is essential for the development of therapeutic strategy to remedy neurodegenerative disorders. In this study, we demonstrate that an EF-hand Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1), one of the key proteins for various neuronal functions, also acts as an important survival factor. Overexpression of NCS-1 rendered cultured neurons more tolerant to cell death caused by several kinds of stressors, whereas the dominant-negative mutant (E120Q) accelerated it. In addition, NCS-1 proteins increased upon treatment with glial cell line–derived neurotrophic factor (GDNF) and mediated GDNF survival signal in an Akt (but not MAPK)-dependent manner. Furthermore, NCS-1 is significantly up-regulated in response to axotomy-induced injury in the dorsal motor nucleus of the vagus neurons of adult rats in vivo, and adenoviral overexpression of E120Q resulted in a significant loss of surviving neurons, suggesting that NCS-1 is involved in an antiapoptotic mechanism in adult motor neurons. We propose that NCS-1 is a novel survival-promoting factor up-regulated in injured neurons that mediates the GDNF survival signal via the phosphatidylinositol 3-kinase–Akt pathway

NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest kcat/Km values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP

MUTYH, an adenine DNA glycosylase, mediates p53 tumor suppression via PARP-dependent cell death

p53-regulated caspase-independent cell death has been implicated in suppression of tumorigenesis, however, the regulating mechanisms are poorly understood. We previously reported that 8-oxoguanine (8-oxoG) accumulation in nuclear DNA (nDNA) and mitochondrial DNA triggers two distinct caspase-independent cell death through buildup of single-strand DNA breaks by MutY homolog (MUTYH), an adenine DNA glycosylase. One pathway depends on poly-ADP-ribose polymerase (PARP) and the other depends on calpains. Deficiency of MUTYH causes MUTYH-associated familial adenomatous polyposis. MUTYH thereby suppresses tumorigenesis not only by avoiding mutagenesis, but also by inducing cell death. Here, we identified the functional p53-binding site in the human MUTYH gene and demonstrated that MUTYH is transcriptionally regulated by p53, especially in the p53/DNA mismatch repair enzyme, MLH1-proficient colorectal cancer-derived HCT116+Chr3 cells. MUTYH-small interfering RNA, an inhibitor for p53 or PARP suppressed cell death without an additive effect, thus revealing that MUTYH is a potential mediator of p53 tumor suppression, which is known to be upregulated by MLH1. Moreover, we found that the p53-proficient, mismatch repair protein, MLH1-proficient colorectal cancer cell line express substantial levels of MUTYH in nuclei but not in mitochondria, suggesting that 8-oxoG accumulation in nDNA triggers MLH1/PARP-dependent cell death. These results provide new insights on the molecular mechanism of tumorigenesis and potential new strategies for cancer therapies

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