Molecular Mechanism Of Disorders Of Mitochondrial Morphology And Function Induced By ApoE4(?272-299)triggering Endoplasmic Reticulum Stress In Neurons

Background:Alzheimer's disease(AD)is a common age-related neurodegenerative disease.There are various hypotheses that attempt to reveal the pathogenesis of AD.One of the most widely accepted is the theory of genetics.One of the most widely recognized susceptibility genes in the academia is the apolipoprotein E4(apoE4)gene.There is a specific apoE protease in neurons,which can cleave apoE4 at Met 272 to produce apoE4(?272-299).Previous studies have shown that apoE4(?272-299)in neurons may be the main effector molecule of apoE4-induced AD lesions.In recent years,a large number of studies have confirmed that endoplasmic reticulum stress(ERS)plays a crucial pathogenic role in AD.Under the influence of external stimuli,the folding modification or assembly of nascent proteins in the endoplasmic reticulum is abnormal,causing unfolded or misfolded proteins to accumulate in the ER,which causes cells to produce ERS.Is there a link between apoE4(?272-299)abnormally retained in neurons and ERS,and what is the mechanism of action? These scientific issues need to be further clarified.Objective: Our study established two different models from animals to cells to investigate the effect of apoE4(?272-299)on ERS in neurons,and in apoE4(?272-299)transgenic mice,we further observed the effects of neuron-specific expression of apoE4(?272-299)on the learning and memory and spatial exploration capabilities of model mice.Methods: First,we used mouse neuroblastoma(Neuro-2a,N2a)cells as experimental objects,and recombinant plasmids p IRES-EGFP-apoE4(p-EGFP-apoE4)and p IRES-EGFP-apoE4(?272-299)(p-EGFP-apoE4(?272-299)constructed and stored in our laboratory were used to establish in vitro cell intervention models.Reverse transcription real-time quantitative PCR technology was used to detect the expressionchanges of ERS marker proteins GRP78,GRP75 and CHOPm RNA.Western-blot technology was used to detect the expressionchanges of ERS marker proteins GRP78,GRP75 and CHOP protein.Cellular immunofluorescence technology is used to detect the changes in cell levels of GRP78 and GRP75.Secondly,the ERS protective agent PBA was used for intervention treatment to detect the m RNA level and protein level changes of the ERS marker protein in the above cell model to further verifythe activation of ERS by apoE4(?272-299).Subsequently,in order to further verify the results of the above-mentioned cellular experiments,we used neuron-specific human apoE4(?272-299)transgenic mice as animal models to detect m RNA and protein expression changes of ERS marker proteins GRP78,GRP75 and CHOP.In addition,we further used the experiment of Morris water maze to test the learning or memory and space exploration capabilities of the transgenic model mice.Results: First,in the N2 a cell model,compared with the control group,we found that the m RNA expression levels of ERS marker proteins GRP78,GRP75,and CHOP were significantly increased after N2 a cells were transfected with p-EGFP-apoE4(?272-299),and the protein expression levels had also increased significantly.However,overexpression of full-length apoE4 did not result in changes in the m RNA and protein levels of the above ERS marker molecules.The results of cell immunofluorescence detection also showed that the expression levels of GRP78 and GRP75 in the cells increased.In addition,after transfection of N2 a cells with p-EGFP-apoE4(?272-299),using ERS protector PBA,we found that GRP78,GRP75,and CHOP m RNA and protein expression levels were significantly down-regulated.Secondly,in apoE4(?272-299)transgenic model mice,we found that compared with wild-type control mice,the expression changes of ERS marker proteins GRP78,GRP75,and CHOP m RNA and protein in hippocampus tissue of model mice were consistent with the results of the above cell model.Finally,in order to further observe whether apoE4(?272-299)can cause learning and memory impairment and spatial exploration dysfunction in transgenic model mice,we used morris water maze experiment to conduct behavioral experiments.The results showed that,compared with the normal control group,transgenic model mice took longer time to find the platform.At the same time,the apoE4(?272-299)transgenic model mouse stayed in the target quadrant significantly shorter than the normal control group.The above results indicated that neuron-specific apoE4(?272-299)can cause behavioral disorders of learning and memory in mice.Conclusion: Neuron-specific expression of apoE4(?272-299)can trigger ERS,cause learning and memory impairment,and have neurotoxic effects.Background: Mitochondria are cells' metabolic centers and energy factories.They provide cells with substrates required for energy and biosynthesis,which determine the cell's fate.Impaired mitochondrial morphology and function is an early pathological feature of AD.The mechanism of apoE4 leading to mitochondrial dysfunction in the pathogenesis of AD is still unclear.Previous studies have found that apoE4 can be cleaved into apoE4(?272-299)fragments with neurotoxicity by proteolytic enzymes in neuronal cells.Studies have found that this toxic fragment,after being overexpressed in neurons,damages mitochondrial respiratory chain-related complexes in neurons more severely than pure full-length apoE4.We speculate that this full-length apoE4 may exert toxic effects on mitochondria in neuronal cells by producing apoE4(?272-299)toxic effector molecules.Previous studies have paid more attention to the effect of apoE4(?272-299)fragment on mitochondrial respiration,but the effect on neuron mitochondrial morphology and function is still unknown.Objective: To investigate the effects of apoE4(?272-299)fragments on the morphology and function of neuron mitochondria through neuron-specific apoE4(?272-299)transgenic mouse models and cell models.Methods: First,we used a high-resolution transmission electron microscope to observe the morphological changes of mitochondria in the hippocampus of apoE4(?272-299)transgenic model mice.We used Western-blot technology to detect changes in protein expression levels of mitochondrial fusion and division-associated marker proteins in the hippocampus of transgenic model mice.Second,in order to verify the above results,we used N2 a cells as experimental objects,and transiently transfected the empty vectorp IRES and p IRES-EGFP-apoE4(?272-299)eukaryotic expression plasmids respectively,to observe the morphological changes of mitochondria by using the laser confocal microscopy.We used Western-blot technology to detect changes in protein expression of mitochondrial fusion and division-associated marker proteins in N2 a cells.Subsequently,in order to explore the effect of apoE4(?272-299)fragment on mitochondrial function,we used the JC-1 method to detect the mitochondrial membrane potential(MMP)of the cell model;the chemiluminescence method was used to detect the mitochondrial ATP generation;Spectrophotometer was used to detect the level of ROS in cells.Finally,we treated the above cell model with ERS protective agent PBA to further verify whether the effect of apoE4(?272-299)toxic fragments on mitochondrial morphology and function is related to triggering ERS.Results: First,in the hippocampus of transgenic model mice,transmission electron microscopy results showed that mitochondrial ridges in hippocampal neurons were broken or disappeared,and mitochondrial fragmentation increased significantly.Further detection of its mitochondrial fusion and division-related proteins revealed that mitochondrial fusion-related molecules MFN1,MFN2,and OPA1 protein expression level decreased significantly,while mitochondrial fission-associated marker molecule MFF protein expression level was significantly increased,and the phosphorylation level of p-DRP1 Ser616 was also increased significantly.Secondly,in the cell model,we found that,compared with the normal control group,the average length of mitochondria in the apoE4(?272-299)transfected group was significantly reduced,and the changes in the expression of mitochondrial fusion and division-associated marker proteins of the cells are consistent with the results of the above-mentioned transgenic model mice.We further detected mitochondrial function and found that apoE4(?272-299)transfection group significantly reduced mitochondrial MMP,significantly reduced ATP production,and significantly increased intracellular ROS levels.Finally,after further treating the above cell model with ERS protective agent PBA,we found that the PBA treatment group could significantly alleviate mitochondrial fragmentation caused by apoE4(?272-299)overexpression.The average length of mitochondria increased significantly compared with the transfection group,the expression level of mitochondrial fusion-related proteins increased significantly,and the expression level of mitochondrial division-related proteins decreased.At the same time,PBA treatment can significantly increase mitochondrial membrane potential and ATP synthesis level,and significantly reduce the production of ROS in cells.Conclusion: Neuron-specific expression of apoE4(?272-299)fragment can cause morphological and functional disorders of neuron mitochondria by triggering ERS.Background: The endoplasmic reticulum(ER)is the organelle with the largest membrane area in eukaryotic cells.In eukaryotic cells,mitochondria and ER,as two important organelles in the cell,do not exist completely independently.Earlier studies have found areas where mitochondria and ER are in contact with each other in parallel,but have not realized what role these closely-contacted areas play in the cell.Studies in recent years have shown that these structural coupling sites that are closely connected between ER and mitochondria,namely the mitochondria-associated ER membrane(MAM),play a critical role in the information exchange and material transfer between ER and mitochondria.MAM structural abnormalities can be widely involved in the occurrence of various diseases.The role of MAM in neurodegenerative diseases,especially AD,has become one of the hot topics in this field.We have found in previous studies that in the cell model and transgenic mouse hippocampus of apoE4(?272-299)overexpression,the ERS marker molecule GRP75 was significantly increased and mitochondrial morphological and functional disorders existed.Our previous studies have shown that mitochondrial morphology and dysfunction occur after ERS,but the mechanism is unknown.How neurons cells trigger ERS triggered by the toxic effects of apoE4(?272-299)fragments affects mitochondrial morphology and function,which is our concern.Given that MAM serves as an important "communication platform" between mitochondria and ER,MAM has an important role in regulating mitochondria.We speculate that MAM may be involved in the transmission of this ERS signal.Objective: To explore whether apoE4(?272-299)mediates mitochondrial morphology and dysfunction through MAM after ERS is triggered,and further explore its possible mechanism.Methods: First,we used transmission electron microscopy to observe changes in MAM between mitochondria and endoplasmic reticulum in the hippocampus of transgenic mice.Secondly,in a cell model,laser confocal microscopy was used to observe the changes in MAM area between the endoplasmic reticulum and mitochondria after transfection of the p-EGFP-apoE4(?272-299)expression plasmid.In addition,with different calcium ion probes,laser confocal microscopy was used to detect changes in mitochondrial calcium ions after transfection of the p-EGFP-apoE4(?272-299)expression plasmid.The N2 a cell model after transfection with p-EGFP-apoE4(?272-299)expression plasmid was further treated with GRP75 inhibitor combined intervention.The changes of mitochondrial fusion and division-related protein expression were detected by Western-blot technology.The mitochondrial function was also further detected.Finally,the GRP75 si RNA was used to verify the results of the above GRP75 inhibitor experiments,to detect the changes in cell mitochondrial fusion and division-related proteins,and to further detect changes in mitochondrial function.Results: Firstly,in the transgenic model mouse hippocampus,the results of transmission electron microscopy showed that the MAM distance between the mitochondria and the endoplasmic reticulum was significantly narrowed,and the area of the MAM region was significantly increased.Secondly,we found that,compared with the control group,the area of the endoplasmic reticulum and mitochondrial MAM area increased after N2 a cells were transfected with p-EGFP-apoE4(?272-299).However,after using GRP75 inhibitor,compared with the transfection group,its MAM area decreased.Secondly,compared with the control group,the mitochondrial Ca2+ concentration in the cells of the transfection group increased significantly,and mitochondrial Ca2+ overload occurred.In addition,at the level of mitochondrial division and fusion protein expression,the expression level of mitochondrial division protein MFF and DRP1 phosphorylation increased significantly in the transfected cells,and the expression levels of fusion proteins MFN1,MFN2,and OPA1 decreased significantly.In terms of mitochondrial function,apoE4(?272-299)overexpression can lead to a significant decrease in mitochondrial membrane potential,a significant decrease in ATP production,and a significant increase in ROS in N2 a cells.After treatment with GRP75 inhibitors,the imbalance between mitochondrial fusion and division was significantly alleviated,the mitochondrial membrane potential increased,the production of ATP increased,and the production of ROS also reduced.After treatment with GRP75 si RNA,the experimental results were consistent with the GRP75 inhibitors.Conclusion: apoE4(?272-299)can participate in the MAM transport of Ca2+ to mitochondria through GRP75 bridge molecule,which causes mitochondrial calcium overload and results in mitochondrial morphological and functional disorders.