| Alzheimer's disease (AD) is the most common neurodegenerative disease,characterized by progressive loss of memory, cognitive and motor deficits, loss of thinkingability and the emergence of neuropsychiatric abnormalities.The etiology of AD is not clear, brought great difficulties for clinical diagnosis andtreatment.The drug therapy can alleviate the symptoms,but can not stop or reverse thedisease occurrence and development. The main pathologic changes of AD are reduction ofneurons and synapses, senile plaques (Sp) and neurofibrillary tangles (NFT) in thecerebral cortex and several local brain tissue. SP was mainly distributed in extraneuronal,formed by Aβ(amyloid-beta peptide) with surrounding materials. Aβ is consists of39-43amino acids, for enzymatic production of APP (amyloid precursor protein) hydrolysate.APP is a transmembrane protein of neurons, play a key role in neuronal growth, survivaland repair processes. NFT was mainly distributed in intraneuronal, formed by microtubuleassociated protein tau. Tau assists the formation of neuronal microtubules, as a trackinvolved in the transport of nutrients and signaling molecules. tau is hyperphosphorylatedto form NFT In AD brain. Then NFT dissociated from microtubules, leading to microtubuledepolymerization and disturbs material transport.Recent studies have shown that oxidativestress may cause tau phosphorylation, although several factors can cause tauphosphorylation, However, the reason is unclear.Autophagy can selectively degrade prone-aggregation or misfolded proteins. P62/SQSTM1(sequestosome1) as a receptor bind to ubiquitinated proteins, associated withautophagic degradation of mitochondria, peroxisomes, microorganisms and othersubstances and cell organelles. Studies found that p62exists in NFT, shown that p62mightbe related to the onset of AD, but whether participated in tau phosphorylation is not clear.p62also regulates oxidative stress, apoptosis and other functions. p62influencesKeap1(Kelch-like ECH-associated protein effect1) hydrolysis to change its half-life. Invitro experiments indicated that elevated expression of p62decrease the effet ofCul3-Rbx1-E3ubiquitin ligase by perturbing Keap1, and mutant p62didn`t affect thefunction of Keap1. Presumably, autophagy and p62may affect the ability of oxidative stress tolerance through impact the effect of Keap1-Nrf2-ARE system.This research copied AD rat model and studied the effect of autophagy and p62onKeap1-Nrf2-ARE system and the further effect on tau hyperphosphorylation wereinvestigated. In addition, the influence of tau hyperphosphorylation on structure ofneuronal and the memory of rats were studied. Our main target is to reveal the pathogenesisof AD, provides the theoretical basis for diagnosis and treatment.Methods(1)EstablishedAD rat model.Aβ25-35was injected to bilateral hippocampus and D-galactose injected intraperitoneal pathway.(2)Tested spacial memory of rats by using the Morris water maze.(3)Detected the neuron size, shape and number of rat brain cortex and hippocampus byHE staining method.(4) Detected the amount and structure of neurons, synapses, blood vessels and glialcells in rat cerebral cortex and hippocampal CA3area by transmission electron microscopy.(5) Detected the expression of autophagy related protein Atg12-Atg5, LC3andBeclin1, p62, Keap1, Nrf2, phosphorylated-tau in rat brain cortex and hippocampus byimmunohistochemical staining assay.(6) Detected the expression of autophagy related protein Atg12-Atg5, LC3andBeclin1, p62, tatal tau, phosphorylated tau in rat brain cortex and hippocampusus bywestern blotting assay.(7)Reverse transcription PCR assay detected GCLC/GCLM mRNAexpression in ratbrain cortex and hippocampus.Results(1)Rats with the application of Aβ25-35hippocampal injection and D-galactoseintraperitoneal injection, need longer time in finding the platform compared to normal ratsand saline group.These results indicated that AD model rats have significant injury inmemory function.(2)In AD model rats brain, the neuron number decreased in the hippocampus CA3region and cortex of the AD model group, and the cells were irregular and dark, sphericalmitochondria with broken cristae were observed. The neuropil of nerve fibers wassignificantly reduced, cavitated, the synapses were reduced. Brain capillary endothelial cell nuclear matrix appeared mild cavitation, cytoplasmic and membrane structure is not clear.Perivascular appeared bigger gap, have macrophages and secondary lysosomes. Glialnuclei were oval or round, nuclear matrix cavitated, heterochromatin reduced, cytoplasmwere cavitated, and organelles disappeared. These results indicated that AD model rat braintissue appeaed structural damage.(3)The results showed that the expression of autophagy-related protein Atg12-Atg5,LC3and beclin1increased in the cortex of AD model group, the level of Beclin1alsoincreased in the cotex of saline group. In hippocampus of AD model group, Atg12-Atg5,LC3and beclin1were also found to be increased, especially for Atg12-Atg5. Beclin1andLC3-Ⅱ also increased in saline group.(4) The expression of p62in the cortex and hippocampus of AD model group wasdecreased,especialy in the cortex. p62expression of saline group did not changed, themachanism is unknown.(5) Keap1increased in the hippocampus and cortex of the AD model group. Itdemonstrates that due to decreased p62, Keap1increased.(6) Nrf2expression reduced in cerebral cortex of AD model group, but not changedin hippocampus, the level of Nrf2presented increased trend in the AD model group. Thedecreased expression of GCLC, GCLM mRNA corresponding with Nrf2expression in thecortex, and the expression of GCLC, GCLM mRNA not changed in hippocampus. Theseresults indicated that the antioxidant capacity of AD model rat brain is insufficient.(7)The expression of total tau protein in the hippocampus and cortex of AD modelwas reduced, and phosphorylated tau was increased. These results indicated that theinsufficient of antioxidant capacity promoted up regulated tau phosphorylation and reducednomal tau.ConclusionsIn this research, we copied AD rat model by Aβprotein hippocampul injection andperitoneal injection of D-galactose. The results demonstated that AD model rats need longertime in finding the platform compared to normal rats.These results indicated that AD modelrats have significant injury in memory functions. Morphological studies showed that theneurons, neuropils, cytoplasm and organelles,capillaries,glialcells are changed in CA3 region and cortex of the AD model group.These results indicated that AD model rat brainappeared significant structural damage.The investigation of autophagy-related proteins and p62showed that the expression ofautophagy-related protein Atg12-Atg5, LC3and beclin1increased in the brain tissue of theAD model group. The expression of p62in the brain tissue of the AD model group wasdecreased. It can be confirmed from the above results that elevated autophagy in the braintissue can dicrease p62.Fuether investigation of Keap1-Nrf2-ARE related proteins and mRNAdemonstrated,Keap1increased in the hippocampus and cortex of AD model group.Nrf2dicreased in the cortex of AD model group,not changed in the hippocampus. Thetranscription of mRNA in GCLC,GCLM was reduced in the cortex, not changed in thehippocampus. These results indicate that the decrease of p62can weaken the anti-oxidativecapability.To investigate the expression of tau hyperphosphorylation showed that the expressionof total tau protein in the hippocampus and cortex of AD model was reduced, andphosphorylated tau was increased. Indicated decreased anti-oxidative capacity promoted tauhyperphosphorylation.This work mainly use the AD rat model, studied the effect of autophagy and p62on tauphosphorylation in animal brain. It demonstrated that increased autophagy degradedexcessive p62,further increased tau hyperphosphorylation by influencing antioxidantcapacity. Our result showed that, autophagy through excessive removal of multifunctionalprotein p62causes the dysfunction of Keap1-Nrf2-ARE pathway is one of reasons in tauphosphorylation in AD. |
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