| Alzheimer’s disease(AD),commonly known as Alzheimer’s disease,is a common neurodegenerative disease that is mainly manifested by reduced cognitive and memory abilities.As society ages,the prevalence of AD is gradually increasing.In China,about70%-80% of patients have varying degrees of intellectual disability and mental symptoms,which seriously affect people’s quality of life.Although drugs such as cholinesterase inhibitors and NMDA receptor antagonists are currently on the market,the effect is still not satisfactory,and there is currently no effective treatment.Therefore,further exploration of the pathogenesis of AD is an urgent problem to be solved.The main pathological manifestations of AD are Amyloid-β protein(Aβ)deposition,neurofibrillary tangles and nerve cell death,of which Aβ toxicity is particularly important.In the brain of AD patients,Aβ plaques may damage nerve cells through autotoxicity or upregulation of inflammatory levels.The traditional view is that Aβ precursor protein APP(Amyloid precursor protein)through β secretase and γsecretase cleavage to form Aβ toxic fragments located outside the cell,age spots mainly gathered in the hippocampus and cerebral cortex and formed,in recent years researchers have gradually confirmed that the abnormal accumulation of Aβ in cells causes toxic effects,and the formation of Aβ in cells is even earlier than extracellular.Aβ in cells is generated by cutting organelle membranes such as Golgi apparatus,endoplasmic reticulum,lysosomes and mitochondria by APP,and can be secreted to the outside of cells by secreting vesicles or autophagosomes to form age spots;Extracellular Aβ also binds to receptors in the cell membrane and endocytosis.Therefore,it is very important to prevent the production of Aβ or speed up its clearance.In studies to reduce Aβoverproduction,researchers using(R)-flubiprofen to regulate γ secretase to reduce Aβproduction has been used in clinical trials,but the desired therapeutic effect has not been achieved;At present,it is believed that the most critical factor responsible for the large-scale aggregation of Aβ is the serious destruction of Aβ clearance.However,the mechanism leading to the occurrence of Aβ clearance disorder still needs to be further studied and explored.In the body’s normal metabolism,cells that abnormally produce aggregated proteins or damaged organelles that can be excreted normally by the body to maintain normal homeostasis.The abnormally aggregated protein component of Aβ can be cleared in cells mainly through the following three pathways,namely autophagy,endosomal/lysosomal degradation and ubiquitin-proteasome system(UPS),among which the autophagy clearance pathway is particularly important.Autophagy,that is,"eating oneself",is a lysosomal degradation pathway,after the formation of autophagysomes,through the fusion between the two and lysosomes,it can eventually degrade the abnormal folding and deposited proteins in the cell,as well as damaged organelles,and then maintain cellular homeostasis.Abnormal accumulation of autophagosomes containing large amounts of Aβ was detected in the brains of AD patients and the brains of APP/PS1 double-transgenic AD mice,and baflumycin A1 inhibited autophagosome and lysosomal fusion,suggesting that autophagic dysfunction in AD may be caused by impaired autophagosome and lysosomal fusion.At the same time,if abnormal aggregation of Aβ is found in the cell,it is often accompanied by a large accumulation of autophagies in the cell,and the autophagic body membrane can fuse with the cell membrane.Further development is that intracellular Aβ is gradually released from inside nerve cells to reach outside the cell,and cytotoxic Aβ will also aggregate outside the cell,that is,the accumulation of intracellular Aβ aggravates the toxicity of extracellular Aβ.It can be seen that the fusion disorder of autophagosomes and lysosomes in nerve cells can simultaneously cause abnormal accumulation of Aβinside and outside cells.However,the key mediators that cause autophagosome and lysosomal fusion disorders in the AD process are not fully understood.Autophagosome-lysosomal fusion requires the participation of a large number of proteins that promote membrane fusion,among which the role of the SNAREs membrane fusion protein family is particularly important.We performed biotrust GEO analysis in the early stage,retrieved the AD dataset GSE48350,used AD patients and normal people to intersect with autophagy database,and then intersected with SNAREs protein complex,and the top 7 genes in the obtained intersection results because the same gene can have multiple different ID numbers,gene BNIP1 accounted for the highest proportion.However,the specific mechanism of action of BNIP1 in AD is unclear.Therefore,the content of this study is to demonstrate: In Alzheimer’s disease,does the content of the protein BNIP1 in mouse hippocampal tissues and nerve cells change?Whether it affects the fusion of autophagosomes and lysosomes when it changes,and the mechanism of action by which this protein ultimately inhibits autophagosomelysosomal fusion leading to intracellular Aβ accumulation.Part I: Alzheimer’s disease hippocampal neurocell autophagosome-lysosomal fusion disorder involved in Aβ depositionObjective:To investigate the possible mechanisms in AD that affect changes in autophagosome-lysosomal fusion disorders in hippocampal nerve cells.Methods:At the body level:APP/PS1 double-transgenic mice were selected as 6-month-old and littermates and month-old control mice,and with the western blot detection of expression changes associated with autophagy in LC3 and P62 proteins.At the same time,tissue electron microscopy samples were prepared,and the number of autophagies and lysosomes and fusion were observed by electron microscopy.Furthermore,amyloid staining solution(modified Highman Congo red method)was used to detect the accumulation of amyloid sediment in the hippocampus of mice,and the morphological structure changes and utilization of the hippocampus of mice were observed by HE staining.In vivo level:First,an experimental cell model was constructed in mouse hippocampal neurons HT22 using APP overexpression vector,and with the western blot detection of expression changes associated with autophagy in LC3 and P62 proteins.Immunofluorescence staining was used to detect the expression and localization of autophagy-tagged protein LC3 and lysosomal labeled protein LAMP1.The accumulation of amyloid sediment in the hippocampus of mice was further detected by amyloid staining solution(modified Highman Congo red method).Results:(1)APP/PS1 disease model screening,AD hippocampal nerve cell construction;(2)AD hippocampal neuronal cell autophagosomal lysosomal fusion disorder;(3)abnormal deposition of amyloid in AD hippocampal tissue and hippocampal nerve cells;(4)The hippocampal area of APP/PS1 mice was abnormal.Conclusion:In AD,the fusion of autophagosomes and lysosomes is obstructed,there is abnormal amyloid deposition,and the cell structure is abnormal.Part II: Abnormal expression of BNIP1 in AD hippocampal tissue and hippocampal nerve cells,improvement/obstruction of autophagosome-lysosomal fusion disorder after overexpression/knockdown of BNIP1 in AD hippocampal nerve cellsObjectives:To investigate the changes of BNIP1 expression in AD hippocampal tissues and hippocampal nerve cells,as well as the effects of BNIP1 content changes in AD hippocampal nerve cells on autophagosome-lysosomal fusion disorders and their possible mechanisms.Method:The difference gene between AD and normal people was queried in the GEO database by bioinformatics method,and the intersection gene and autophagy gene bank were used to obtain the intersection gene BNIP1 by intersecting with the SNAREs protein family.Western Blot and immunofluorescence assays were used in AD to detect protein expression of BNIP1.After knocking down BNIP1 with BNIP1 overexpression plasmid or small interfering RNA,LC3 and P62 protein levels were detected by western blot experimentResults:(1)Decreased expression of BNIP1 in AD hippocampal tissues and hippocampal nerve cells: GEO database screened the differential genes between AD and normal people and prepared multi-data Wayne diagram to find intersection genes: Biotrust GEO analysis,retrieved AD dataset GSE48350,intersected with the autophagy database between AD patients and normal people,and then intersected with SNAREs protein complex,and the gene with the highest proportion in the obtained intersection results was BNIP1;(2)Autophagosomal lysosomal fusion was improved after overexpression of BNIP1 in AD hippocampal nerve cells;(3)Autophagosomal lysosomal fusion is blocked after knocking down BNIP1 in AD hippocampal nerve cells.Conclusion:The expression content of BNIP1 in AD hippocampal tissues and hippocampal nerve cells decreased,and the autophagosome-lysosomal fusion disorder improved/blocked after overexpression/knockdown of BNIP1 in AD hippocampal nerve cells.Part III: In AD hippocampal nerve cells,BNIP1 recruits Rab7 by binding to Rab5 b to promote autophagosomelysosomal fusionObjectives:Rab5b was detected by mass spectrometry in AD hippocampal nerve cells that the protein with the most obvious change in binding content to BNIP1 after its binding content was reduced,and the role of BNIP1 in recruiting Rab7 by binding to Rab5 b and its possible mechanism was explored.Method:The IP solution of mass spectrometry was prepared by AD hippocampal nerve cells,and after obtaining the mass spectrometry results,it was obtained that the protein with the most obvious change in binding content was screened after the content of BNIP1 in AD decreased.The Co-IP experimental method and Western Blot experimental method were used to detect the binding of BNIP1 to Rab5 b and the binding of Rab5 b to Rab7 in AD.After plasmid overexpression of BNIP1,the binding of BNIP1 to Rab5 b and the binding of Rab5 b to Rab7 in AD were detected by Co-IP experimental method and Western Blot experimental method.The binding of BNIP1 to Rab5 b and the binding of Rab5 b and Rab5 b and Rab5 b to Rab7 were detected by using Co-IP experimental method and Western Blot experimental method after knocking down BNIP1 by small interfering RNA.In AD hippocampal nerve cells,BNIP1 is overexpressed,then Rab5 b is knocked down under small interference,and LC3 and P62 expression levels are detected by western blot experiments.Results:(1)Increased binding of BNIP1 to Rab5 b in AD hippocampal nerve cells(2)After overexpression of BNIP1 by plasmids in AD hippocampal nerve cells,the binding of Rab5 b to Rab7 increased(3)After small interference knockdown BNIP1 in AD hippocampal nerve cells,the binding of Rab5 b to Rab7 is reduced(4)After the plasmid overexpressed BNIP1 in AD hippocampal nerve cells and then knocked down Rab5 b,the autophagosome and lysosomal fusion disorders were blockedConclusion:In AD hippocampal nerve cells,BNIP1 recruits Rab7 by binding to Rab5 b to promote autophagosome-lysosomal fusion. |
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