Rna Oxidation On The Pathogenesis Of Alzheimer's Disease, The Role Of Mechanism Research

Alzheimer’s disease (AD) is a kind of neuron degenerative disorders which are characterized by progressive and irreversible loss of brain neurons (especially the cortex and hippocampus). Cognitive disorders, memory impairment are the main clinical features. Senile plaques (SP), neurofibrillary tangles (NFT) and a large loss of neurons constitute the pathological hallmarks of AD. The aging of the population gives a sharp rise in the number of AD patients, Therefore, clarifying the pathogenesis of AD is crucial to solve a series of problems caused by the aging population.The main constituent of SP is the β-amyloid protein (Aβ), which is formed after sequential cleavage of the amyloid precursor protein by β secretase and y secretase. Aβ mainly exists in two forms:Aβ1-40and Aβ1-42, while the latter one is more toxic than the former.Five steps are needed for Aβ to exhibit neurotoxicity:The APP gene�'APP mRNA�'APP protein�'AP polypeptide�'Aβ oligomers. Studies suggest that the mutation of the APP gene at the DNA level is the main cause of increased Aβ production, but this can only use to explain a small number of the pathogenesis of AD, since familial AD (FAD) only account for1%of the total AD. No mutation of the APP gene was found in the vast majority of sporadic AD patients,Our laboratory has long been engaged in the research of the molecular mechanism of RNA oxidation with aging and aging-related diseases. The most important product of RNA oxidation is8-oxidized guanosine (8-oxoG),8-oxoG can either pair with cytosine or adenine on the DNA template, cause transcription error, then translate to sequence varied protein. Therefore, the genomic DNA information cannot be passed accurately to the protein.In1999, researchers found that RNA oxidation product8-oxoG was significantly higher in the patients with AD, and this increasing is clearly correlated with the progress of the disease. To verify this result,2008, we used immunohistochemical methods to detect the DNA and RNA oxidation in the hippocampi of the senescence-accelerated mouse8(SAMP8) and its normal control SAMR1mouse. Results showed that8-oxodG and8-oxoG increased with aging in the2strains of the mice, and P8showed higher oxidation level than its age-matched control. Subsequently, we detected DNA and RNA oxidation in the hippocampus of AD patients by using autopsy specimen.8-oxodG in the nucleus and8-oxoG in the cytoplasm were increased compared with age-matched normal control subjects.The results of immunohistochemistry are influenced by the operator’s personal judgment. It’s unable to accurately assess the level of RNA oxidation according to the gray scale. In this study, we established an accurate and sensitive high performance liquid chromatography-triple quadruple mass spectrometry (LC-MS/MS) detection system, by using this system, we detected DNA and RNA oxidation level in various tissues of SAMP8and SAMR1mice, including the brain, lungs, heart, liver, kidneys and testes, we also detected the concentration of DNA and RNA oxidative metabolites in plasma and urine. Our results confirmed the nucleic acid oxidation, especially RNA oxidation was correlated with decreased learning and memory abilities in SAMP8mice. We also found that8-oxoG content in the urine was much higher than the plasma8-oxodG, plasma8-oxoG and urine8-oxodG. Therefore,8-oxoG in urine may be a better biological marker for the evaluation of the oxidative damage.Based on these, we hypothesize that oxidized APP mRNA cause transcription and translation errors of the APP gene, change of the amino acid sequence of the APP protein. This may make APP protein turn from its normal a, y proteolytic pathways to the β, γ proteolytic pathways which produce more kinds and amount of the Aβ fragment and aggregate to form senile plaques, then leading to AD.To test this hypothesis, we stably transfected human APP gene into CHO cells, and knocked down the RNA oxidation suppressor gene MTH1to build CHO_APP_iMTHl monoclonal cell lines. We added8-oxoGTP to the cell culture medium, since8-oxoGTP can be incorporated into RNA chain competitively with GTP during transcription, thus cause an increase in RNA oxidation. We observed the change of the kinds and amounts of Aβby ELISA, we found the amount of Aβ1-x、Aβ40and Aβ42were significantly increased(P<0.05). By using LC-Q-TOF technology, we discovered69and108kinds of Aβ wt fragments in con and oxo groups, respectively. And a majority of the Aβ fragments were increases in oxo group compared with the con group.Meanwhile, we injected nanoparticles packed8-oxoGTP through the lateral ventricles and the tail vein into8-month old SAMR1and SAMP8mouse, which could cause an increase in RNA oxidation in various organs of the body, especially the brain.2months later, we investigated the behavior change indicators (including the Morris water maze, object recognition, balance beam test, and open field test). In R1strain, the latency of the oxo group was much longer than the saline group(P<0.05), while in P8strain, there’s no difference in the2groups. The other3behavior tests, however, didn’t show any changes.In conclusion, we found increased RNA oxidation lead to accumulation of AP protein at the cellular level, increased RNA oxidation lead to learning and memory decline at the animal level, indicating that the the RNA oxidation may be one of the pathogenesis of Alzheimer’s disease. We planned to examine the senile plaques by PET-CT, and Aβ1-x、 Aβ40and Aβ42in the brains by immunohistochemistry when8-oxoGTP are injected for4months which could further enhance our previous research from imaging and pathology aspects.