The Mechanism Of MCU-knockdown In Hippocampal Neurons Improves Memory Performance In APP/PS1/tau Transgenic Mice

Objective:Alzheimer’s disease is the most typical senile neurodegenerative disease and the primary cause of senile dementia.Mitochondrial dysfunction is an important early cause of the development of AD and has attracted more and more attention.In recent years,it has been found that unidirectional mitochondrial calcium transporters,key channels for mitochondrial Ca2+uptake,are highly expressed in animal models of AD.Our objective was to observe whether knocking down the expression of Hippocampal nerve cell MCU can improve the cognitive function and pathological features of APP/PS1/tau mice in alzheimer’s disease model by optimizing mitochondrial function.Methods:1.Cytological experimentThe well-grown HT22 cells were infected with control lentiviruses with GFP and purinomycin resistance gene markers and three Lentiviruses with MCU knockdown sequence.The optimal knockdown sequence experiment of MCU was grouped as follows:Control group,sh MCU1,sh MCU 2 and sh MCU 3,the best sequence of MCU knockdown was selected by fluorescence observation and Western-blotting experiment.Two major pathological manifestations of AD,Aβplaque and phosphorylated Tau protein,were simulated in vitro by Aβ_1-42 and Okadaic acid（OA）,A specific inhibitor of protein phosphatase PP2A.Then MTS cell activity test was performed on the cells.Aβmodel experiment was divided into Control group,Aβ_1-42 group,Aβ_1-42+sh Con group and Aβ_1-42+sh MCU group.Tau model experiment was divided into Control group,OA group,OA+sh Con group and OA+sh MCU group.2.In vivo experiments on animals（1）Eight-month-old female APP/PS1/tau transgenic mice and control（WT）mice were selected and divided into WT group,APP/PS1/tau group,APP/PS1/tau+sh Con group and APP/PS1/tau+sh MCU group.Control viruses sh Con and sh MCU were injected into the hippocampus of mice by stereotaxic injection.Four weeks later,frozen brain sections of mice were taken for fluorescence observation,and proteins were extracted for Western-blotting test to identify the Knock-down of MCU.（2）Behavioral experimentOpen field test:This test is used to eliminate movement disorders and simply assess the anxiety of animals.Mice were placed into the central area for free exploration for5min,and the movement track,total movement distance and activity time of each mouse in the central area were analyzed.New object recognition:The new object recognition experiment evaluated the context recognition memory of mice by observing the exploration of new objects.In the experimental exploration phase,mice were put into an open field containing two identical objects to explore freely.After 6 hours,the mice entered the test period.One object was removed and replaced with a new object,and the mice were active for 10 minutes.The time of each mouse exploring new and old objects was analyzed,and the Novel object recognition Index（NOI）of each group was calculated.Y maze:THE Y maze experiment was used to assess short-term spatial working memory in mice.Mice in each group were placed separately into the three-arm intersection for free exploration for 8min,and the arm entry sequence of mice was recorded.The total number of arm entry and the number of correct arm entry of each mouse were counted,and the correct rate of spontaneous alternation was calculated..Morris Water Maze:This experiment tested the ability of long-term spatial reference memory in mice.The first stage was a five-day navigation experiment in which the mice were induced to rely on and remember the platform.The second stage was a space exploration experiment.After removing the platform,the mice were observed to remember the location of the previous platform.The visual platform experiment was conducted to exclude the influence of visual acuity on the whole experiment.（3）Western-blotting experiment:After protein extraction,RIPA lysate was added for protein extraction.Concentration was determined by BCA method,and denaturation was carried out by boiling at 95℃.Sds-page gel electrophoresis,membrane transfer,closed with skim milk powder,incubated primary antibody overnight,incubated secondary antibody after cleaning,and finally added exposure solution for exposure and imaging.（4）Transmission electron microscopy experiment:CA1 was extracted from the hippocampus of mice and placed in glutaric acid before fixation,1%osmium acid fixation solution after fixation,the tissues were placed in alcohol and acetone for gradient dehydration and infiltration,epoxy resin was immersed,dried and polymerized.The slicer stained the slices and dried them for observation.（5）Golgi staining experiment:After the brain tissues were collected,they were soaked in pre-prepared immersion solution to avoid light for 14 days.After the slices were sliced by the slicer,the working solution was stained,alcohol gradient dehydration,resin sealing and microscope observation.Results:1.Cytological experimentLow EXPRESSION of MCU can ameliorate the decreased activity of hippocampal neurons induced by amyloid beta protein（Aβ）and Okadaic acid（OA）（P<0.05）.2.In vivo experiments on animals（1）In the open field experiment,there was no significant difference between the total distance of the four groups of mice in the open field and the percentage of the time of each mouse in the central area（P<0.05）.（2）In the new object recognition experiment,there was no significant difference between the two same objects（P<0.05）in the exploration time;New object recognition index（NOI）in APP/PS1/tau group was lower than that in normal WT group,and NOI value increased after low expression of MCU（P<0.05）,sh MCU can improve the impaired object recognition and memory ability of APP/PS1/tau mice to a certain extent.（3）There was no significant difference in the total number of arm entry in Y maze test（P<0.05）;The correct rate of arm entry in APP/PS1/tau group was lower than that in normal WT group,and increased to a certain extent after low expression of MCU（P<0.05）.（4）In the Morris Water Maze test,the incubation period of the four groups of mice finding the platform decreased with the prolongation of training.Compared with WT group,the incubation period of the APP/PS1/tau group was longer,and significant differences began to appear on the fourth day（P<0.01）,the difference was more obvious on the fifth day（P<0.001）;Compared with APP/PS1/tau mice,the MCU virus group had less time to find the platform（P<0.05）,the percentage of sh MCU group staying in the target quadrant in the space exploration experiment without platform（P<0.05）and wear frequency（P<0.05）,indicating that the spatial memory ability of mice injected with sh MCU was recovered compared with that of APP/PS1/tau group.（5）Western-blotting results showed that the expression levels of synaptic proteins PSD95 and SYP and mitochondrial autophagy level proteins PINK1 and PARKIN in APP/PS1/Tau group were significantly lower than those in WT group（P<0.05）,low expression of hippocampal MCU increased the expression of PSD95,SYP,PINK1 and PARKIN proteins in APP/PS1/Tau mice（P<0.05）.（6）Transmission electron microscopy showed that APP/PS1/Tau group had less hippocampal protrusion（P<0.05）,the number and morphology of mitochondria were abnormal compared with the control group.The low expression of hippocampus MCU normalized the number of synapses and optimized the number and morphology of mitochondria.（7）Golgi staining showed that the density of dendritic spins per unit length in APP/PS1/Tau group was significantly lower than that in WT control group（P<0.05）,while the density of dendritic spines recovered after MCU was knocked down.Conclusion:Low expression of MCU ameliorates Aβ1-42 and Okada-induced decline in hippocampal neuron activity;Hippocampal neurons on low MCU can improve the APP/PS1/tau mice scene recognition memory ability,relieve the APP/PS1/tau mice in the short term and long term spatial working memory and reference memory ability;By optimizing the levels of PINK1 and Parkin in the mitochondrial autophagy pathways of PP/PS1/Tau mice,the morphology and number of mitochondria were standardized,and the levels of synaptic proteins PSD95 and SYP were increased,and the number of synaptic and dendritic spines tended to be normalized.This study suggests that the low expression of hippocampal neuron MCU has a certain anti-AD effect,which is expected to be a new strategy for the prevention and treatment of AD.