Study On The Mechanism Of BMAL1 Regulating STX17 To Induce The Fusion Of Autophagosome And Lysosome In Hippocampal Neuron In The Abnormal Deposition Of Aβ

With the acceleration of the aging process of the population,the number of patients with Alzheimer’s disease(AD)is increasing year by year.By the end of 2021,3.75 of per 100 elderly people in China will suffer from Alzheimer’s disease.And the fatality rate of the disease is also rising,ranking fifth in the list of major causes of death in China in 2019.However,there is no effective treatment at present,so the research on its pathogenesis deeply is particularly important.The main pathological changes of AD are denatured protein aggregates of amyloid β(Aβ)and Tau in the cerebral cortex and hippocampus.At present,it is believed that the aggregation of Aβ plays a key role in the process of AD,and Aβ can induce progressive irreversible damage to neurons,resulting in cognitive dysfunction and memory loss.Among them,the imbalance between the production and clearance of Aβ is an important reason for the abnormal accumulation of Aβ.Studies have shown that the accumulation of extracellular Aβ is closely related to the abnormal deposition of intracellular Aβ,and the toxic effect of intracellular Aβ occurs even earlier than that of extracellular Aβ.Intracellular Aβ is formed by its amyloid precursor protein(APP)on the organelle membrane after cleavage by β-secretase and γ-secretase or endocytosis of extracellular Aβ.Intracellular Aβ mainly clean through autophagy,endosome/lysosome degradation and ubiquitin-proteasome system(UPS)pathway.However,in the brains of AD patients,the clearance rate of Aβ is significantly reduced,so how to increase the clearance rate of Aβhas become a key problem to be solved.Autophagy,as an important pathway for Aβ clearance,has an important neuroprotective function.After the formation of autophagosomes,it fuses with lysosomes to remove damaged and toxic proteins in cells,preventing neuron loss and delaying the onset of AD.Studies have found that there are a large number of abnormally accumulated autophagosomes at the axon terminals of AD patients,suggesting that autophagic flow is blocked in the process of AD.In the first part of our study,by detecting the autophagy indicators and Aβ in the hippocampus tissue of APP/PS1 double transgenic mice and the hippocampal nerve cells of APP-overexpressed virus,it was found that abnormal deposition of Aβ and autophagy block phenomenon existed in the AD model.In addition,electron microscope observation of the hippocampal tissue of APP/PS1 double transgenic mice found that there were a large number of autophagosomes and a small amount of autophagy lysosomes in neurons,suggesting that autophagy blockage may be related to the fusion disorder of autophagosomes and lysosomes.Whereas,the mechanism of autophagosome and lysosome fusion has remained poorly characterized.Therefore,in the second part of our study,through bioinformatics analysis,a significant differential factor syntaxin 17(STX17)that fuses with autophagosomes and lysosomes was found in the brains of AD patients.STX17 is a unique hairpin structure formed by two transmembrane domains,and each domain contains a glycine zipper structure,which can realize the localization of autophagosomes.STX17 combines with soluble NSF adhesion protein 29(SNAP29)and vesicle-associated membrane protein 8(VAMP8)to form the SNARE complex,which contribute autophagosomes and lysosomes fusion.It plays an important role in the fusion process of SNARE,among which STX17 is the initiating factor and key factor for the formation of SNARE complex.In the second part of our study,it was found that the expression of STX17 was reduced in AD models.After knockdown STX17,the autophagic flu was blocked;but after overexpression of STX17,it was found that the increased formation of SNARE complexes promoted the restoration of autophagic flu and reduced the abnormal deposition of Aβ.It was speculated that the transcriptional inhibition of STX17 in AD model neuronal cells was an important factor in autophagosomes and lysosomes fusion.However,the mechanism of downregulation of STX17 transcription in AD process is still unclear.Studies have found that circadian rhythm disturbance symptoms such as sleep-wake cycle disruption can appear in AD patients at an early stage.The circadian rhythm takes 24 hours as a cycle,and its production and maintenance are regulated by biological clock-related genes.Negative feedback regulatory pathways at the level of transcription and translation(Transcriptional-Translational Feedback Loop,TTFL).The autophagy rhythm is regulated by the circadian rhythm of the biological clock,among which STX17 belongs to the clock-controlled genes(CCGs,genes regulated by the circadian rhythm output signal downstream of the biological clock gene).BMAL1,as a positive regulator,can enter the nucleus to regulate the transcription of downstream factors,suggesting the possibility that BMAL1 protein can regulate the transcription of STX17.In the third part of this study,Motif was used to predict the binding site of BMAL1 and STX17,and the combination of BMAL1 and STX17 was observed by dual luciferase reporter technology.In summary,this results proposes that STX17 is a key factor regulating the fusion of autophagosomes and lysosomes,and BMAL1 protein can promote the transcription of STX17 and induce the formation of STX17-SNAP29-VAMP8 complex,thereby clearing Aβ through autophagy and delaying the pathogenesis of AD.This study intends to analyze the mechanism of Alzheimer’s disease from the perspective of autophagy and biological rhythm,aiming to provide a new strategy for the development of therapeutic drugs for AD..Part 1.Autophagosome and lysosome fusion disorders and abnormal deposition of Aβ in hippocampal neurons during ADObjective:To explore the fusion disorder of autophagosome and lysosome in hippocampal neurons and the accumulation of intracellular Aβ in the process of AD.Methods:(1)Grouping of experimental animals and collection of hippocampal tissue: male WT mice and APP/PS1 double transgenic mice of the same ages(4 months,8 months)were selected to obtain mouse hippocampus tissues.(2)Construction of AD cell model: the mouse hippocampal neuron HT22 was infected with lentivirus to overexpress APP in the cells.(3)Western Blot and RT-PCR were used to detect the expression changes of autophagy-related indicators LC3Ⅱ and P62 in the AD cell model and after 20 μmol/L chloroquine treatment for 24 hours.(4)The fusion of autophagosomes and lysosomes in mouse hippocampal tissue was observed by transmission electron microscopy.(5)HE staining was used to observe the morphological changes of hippocampal neurons in APP/PS1 double transgenic mice.(6)Congo red staining was used to observe the deposition state of Aβ in the AD model.Results:(1)The hippocampus of APP/PS1 double transgenic mice at different ages showed blockage of autophagic flow,which manifested as autophagosome and lysosome fusion adisorder.(2)The autophagic flow was blocked in HT22 cells with APP overexpression,which manifested as the fusion disorder of autophagosomes and lysosomes.(3)Neuronal necrosis and abnormal deposition of Aβ increased in the hippocampus of APP/PS1 double transgenic mice.(4)The HT22 cells with APP overexpression showed increased abnormal deposition of Aβ.Conclusion:In the process of AD,the fusion of autophagosomes and lysosomes in hippocampal nerve cells is impaired,and the abnormal deposition of Aβ increases.Part 2.Reduced expression of STX17 induces fusion of autophagosomes and lysosomes in hippocampal neurons and promotes abnormal accumulation of AβObjective:To clarify the important role of the decreased expression of STX17 in the fusion disorder of autophagosomes and lysosomes in hippocampal neurons.Methods:(1)Bioinformatics analysis: The GEO database of differential genes and the autophagy database were intersected to find key factors that fuse with autophagosomes and lysosomes in the brains of AD patients.(2)The expression changes of STX17 were observed by Western Blot and RT-PCR.(3)Immunofluorescence technique was used to observe the localization of STX17.(4)STX17 was knocked down by si RNA,the autophagic flow was observed by Western Blot and RT-PCR.(5)STX17 was overexpressed using lentivirus,and chloroquine was further used to inhibit the fusion of autophagosomes and lysosomes,and Western Blot and RT-PCR techniques were used to observe autophagic flow.(6)Co-immunoprecipitation was used to observe the combination of STX17 and SNAP29 in the SNARE complex in the AD cell model and after STX17 overexpression;and Immunofluorescence technique was used to observe the co-localization of STX17 and VAMP8.(7)Aβ deposition was observed by Congo red staining.Results:(1)Bioinformatics analysis suggested that STX17 was the significantly differential gene associated with fusion of autophagosomes and lysosomes in the brains of AD patients.(2)The expression of STX17 decreased in AD animals and cell models.(3)si RNA knockdown of STX17 further blocks autophagy in HT22 cells and increases the accumulation of Aβ.(4)Overexpression of STX17 by lentivirus partially restored the autophagic flow in HT22 cells,thereby reducing Aβ deposition.(5)STX17 promotes the fusion of autophagosomes and lysosomes in nerve cells by combining with SNAP29 and VAMP8 in the SNARE complex.Conclusion:The decreased expression of STX17 inhibits the combination of STX17,SNAP29 and VAMP8,then the decreased formation of SNARE complex leads to the fusion barrier of autophagosome and lysosome and the increase of Aβ deposition.Part 3.Bmal1 expression decreased to inhibit the transcription of STX17 to make hippocampal neuronal autophagosome and lysosome fusion disorderObjective:To explore the mechanism by which the decreased expression of Bmal1 inhibits the transcription of STX17 and induces fusion barriers between autophagosomes and lysosomes.Methods:(1)Place 4-month-old male WT mice and APP/PS1 double transgenic mice in LD(12h light/12 h dark)environment for one week,turn on the lights at 8:00,20:00 lights off,and DD(24h dark)environment for two weeks.Changes in free-running cycle and average daily activity of rats.(2)In the DD environment,the animal’s endogenous biological clock circadian time(Circadian time,CT)is used to describe.When the mouse starts to move,it is recorded as CT12,and after the running wheel is finished,it is recorded at four time points of CT0,CT6,CT12,and CT18.Observe the rhythmic changes of BMAL1 in mouse hippocampus.(3)Add 0.1 μmol/L dexamethasone to APP-overexpressed HT22 cells for 2 hours to synchronize the cells,collect samples at CT0,CT6,CT12,and CT18 respectively,and observe the rhythmic effect of Aβ on BMAL1.(4)Western Blot was used to observe the expression changes of BMAL1 in the AD model,and the expression rhythm was analyzed by JTK-CYCLE.(5)The combination of BMAL1 and STX17 was observed by dual luciferase reporter gene detection.(6)Bmal1 was overexpressed using a plasmid to detect autophagy flow and Aβ deposition.Results:(1)APP/PS1 double transgenic mice showed circadian rhythm disorder of the running wheel,prolonged free-running period,and increased daytime activity.(2)The expression of BMAL1 in the hippocampus of APP/PS1 double transgenic mice decreased and the expression of rhythm was abnormal.(3)The expression of BMAL1 in APP-overexpressed HT22 cells was reduced and the rhythm was disrupted.(4)BMAL1 can combine with STX17 promoter in hippocampal neurons to promote STX17 transcription.(5)After the overexpression of BMAL1,the increased expression of STX17 partially restored the autophagic flow and reduced the abnormal deposition of Aβ.Conclusion:The decreased expression of BMAL1 during AD can inhibit the expression of STX17 and interfere with the fusion of autophagosomes and lysosomes mediated by the SNARE complex.