Study On The Mechanism Of HIF-1 Mediating Tau Phosphorylation Under Chronic Hypoxia

BackgroundAlzheimer’s Disease（AD） is the most common neurodegenerative disease in the world.It is mainly manifested by aggravating cognitive impairment and mental behavior abnormality with memory impairment as the core.There were two typical pathological features in the cerebral cortex and hippocampus.One was the aggregation of pairs helical filaments（PHF）by abnormal hyperphosphorylation of microtubule-associated protein tau in neurons,and further formation of neurofibrillary tangles（NFTs）.The other is Senile plaques（SP）,which are deposited byβ-amyloid（Aβ）.Aβis an insoluble polypeptide produced by the hydrolysis of amyloid precursor protein（APP）byβ-secretase andγ-secretase.Tau is a microtubule-associated protein（MAP）,whose main biological function is to promote the assembly and stabilization of microtubules.Studies have shown that the content of NFTs is positively correlated with the degree of clinical dementia in AD,so the hyperphosphorylation and aggregation of tau protein play an important role in the pathogenesis and progression of AD.Tau hyperphosphorylation is caused by an imbalance of protein kinases and phosphatases in the brain.Two key enzymes responsible for tau hyperphosphorylation include protein kinases GSK3βand phosphatase PP2A.Brain tissue has high oxygen consumption and is extremely sensitive to hypoxia,so hypoxia is a risk factor for AD.Hypoxia-inducible factor HIF-1 is an important transcription factor involved in hypoxia response,consisting of oxygen-sensitiveαsubunit and constituentβsubunit.It has been found that the overexpression of HIF-1 can led to the increase of Aβ,and then cause the pathological changes of Alzheimer’s disease.The level of tau phosphorylation is increased during chronic hypoxia,but the mechanism of HIF-1 involvement in tau lesions remains unclear.The aim of this study was to elucidate the effect of HIF-1αon the expression and activity of key tau kinases and phosphatase,and to elucidate the molecular mechanism of HIF-1αmediating tau phosphorylation under chronic hypoxia.ObjectiveTo explore whether HIF-1 participate in tau lesions and cognitive dysfunction by regulating key kinase and/or phosphatase（such as GSK3β,PP2A）and promote the pathogenesis of AD in chronic hypoxia.This study provides new clues for the pathogenesis of chronic hypoxia.Methods1.By consulting literature,we chose a classical hypoxia model.A 2-month-old SD rat was placed in a 10%O₂ hypoxia incubator,and establishment of control group（ctr）,and chronic hypoxia model group（hypoxia）.Each group have 12 SD rats.The open field experiment,new object recognition and water maze experiment were used to evaluate the memory status of rats.Western blotting detected tau of different sites phosphorylation levels（p S199,p S262,p S396,p S404）,tau5 and HIF-1αexpression changes,and methylation levels of protein phosphates PP2A were also detected,and the activity of the PP2A was detected by the kit.2.In rat primary hippocampal neurons,we have control group（hypoxia 0h）and chronic hypoxia group（hypoxia 48h）.Western blotting detected tau of different sites phosphorylation levels（p S199,p S262,p S396,p S404）,tau5 and HIF-1αexpression changes.We also detected GSK3βand protein phosphatases PP2A levels,and the activity of the PP2A was detected by the kit.3.In rat primary hippocampal neurons,we transfected the cells transiently with no-load（vector）or knockdown HIF-1αlentivirus 72h.The cells were divided into three groups:transfection of no-load lentivirus（ctr group）;transfection of no-load lentivirus and hypoxia48h（hypoxia group）;transfection of knockdown HIF-1αlentivirus and hypoxia 48h（si HIF-1α+hypoxia group）.The HIF-1αknock efficiency of neurons was detected by Western blotting,and we also detected tau different sites（p S199,p S262,p S396,p S404）phosphorylation levels,and methylation levels of tau5 and PP2A.4.And in rat glioma cells（C6 cell）,we transfected cell with plasmid 48 h later,and put in a certain concentration of Co Cl₂ stimulation for 8 h.Western blotting detected tau of different sites phosphorylation levels（p S199,p S262,p S396,p S404）,tau5 and HIF-1αexpression changes.We also detected changes in PP2A methylation.Results1.In SD rats:（1）The results of open field,new object recognition and water maze test showed that the motor ability of chronic hypoxia rats were not affected,and the cognitive function was significantly reduced;（2）Western blotting assay showed that tau protein phosphorylation increased in chronic hypoxia group,but total Tau did not change.The methylation level and activity of PP2A were decreased.The expression of methyltransferase in PP2Ac was decreased.The expression of HIF-1αwas increased in rats in the experimental group.2.In the primary hippocampal neurons after chronic hypoxia for 48h,western blotting results showed that the expression of HIF-1αwas increased,and tau protein phosphorylation was increased,and the methylation level and activity of PP2A were decreased,and the expression of PP2Ac methyltransferase was decreased in the hypoxia group.3.In rat primary hippocampal neurons.We knock down HIF-1α.Western blotting results showed that the level of tau protein phosphorylation was decreased and PP2A methylation was increased in the down-knocked HIF-1αneurons compared with the hypoxia group.4.In astroglioma cells C6,after 8h of cobalt chloride administration,western blotting results showed that HIF-1αexpression was increased,and tau phosphorylation level was increased.PP2A methylation level was decreased,and PP2Ac methyltransferase expression was decreased in the experimental group compared with the control group.ConclusionChronic hypoxia may decrease LCMT1 by upregulating HIF-1α,resulting in down regulation of PP2Ac methylation level,and decreased activity and abnormal hyperphosphorylation of Tau.