| Research BackgroundAlzheimer's disease(AD)is a neurodegenerative disease that occurs mostly in old age and is the most common cause of dementia.With the development of an aging society,the impact of AD on human health has become more and more concerned.Alzheimer's disease is more insidious and progressive,and its main clinical manifestations are memory loss,cognitive dysfunction,behavioral impairment,speech disorder,etc.,and ultimately loss of daily living ability.The incidence of AD is increasing.Epidemiological surveys in China show that the number of AD patients in the elderly population has exceeded 6 million.It is estimated that the number of people with AD will exceed 20 million by 2050,which is the largest number of AD patients and the fastest growing region in the world.But there is currently no effective diagnosis and treatment for AD.The classic pathological features of Alzheimer's disease are recognized as the formation of senile plaques caused by abnormal deposition of amyloid beta(A?)in the brain,the accumulation of highly phosphorylated tau in cells,the formation of neurofibrillary tangles,and the number of neuronal cells.Missing.However,its specific pathogenesis is still unclear.There are various hypotheses for the pathogenesis of AD,among which the A? over deposition hypothesis is recognized by most people,but with the failure of clinical trials to treat AD to clear A?,people hope to find new targets to treat AD.Mitochondria are an important part of cell productivity and are essential for maintaining the normal function of cells.Mitochondrial dysfunction can lead to cell damage or even death.Neuronal cells are cells that consume more energy,and because they cannot store energy,they are very dependent on the function of mitochondria to produce ATP.Therefore,it is speculated that mitochondrial dysfunction can lead to the death of neuronal cells and cause clinical symptoms.At present,mitochondrial dysfunction has become a research hotspot in the pathogenesis of AD.Mitochondrial dysfunction includes mitochondrial DNA damage,mitochondrial dynamics imbalance,mitochondrial energy metabolism disorders,and increased ROS production.Any of the factors that cause this may be involved in the mechanism of mitochondrial dysfunction.A key enzyme in the mitochondrial tricarboxylic acid cycle is succinate-CoA synthetase,which is composed of ap heterodimers,including a constant a subunit and a variable ? subunit,of which the SCS a subunit It is encoded by SCLUG1,and the SCS ? subunit has two forms,which are the ?-subunit formed by the ADP-encoded by SUCLA2 and the ?-subunit formed by the GDP-encoded by the SUCLG2.The enzyme is present in the mitochondrial matrix and catalyzes the conversion of succinyl-CoA and NTP(ATP/GTP)to a reversible reaction of succinyl,coenzyme A and NDP(ADP/GDP).Previously,we are concerned that the deletion of the SUCLA2 gene can lead to mitochondrial DNA depletion syndrome,which is characterized by a group of clinical syndromes characterized by high energy-consuming organ dysfunction,but the underlying pathological feature is the reduction of mitochondrial DNA copy number;and SUCLA2 may specifically highly expressed in neurons,so we speculate that due to the important relationship between SUCLA2 and mitochondrial DNA integrity and mitochondrial dynamics,it may be involved in the development of AD.Therefore,our research focused on the changes in SUCLA2 in A?-stimulated neurons and the mechanisms by which this change may lead to mitochondrial dysfunction,in the hope of finding a new target for the diagnosis and treatment of AD.Research Aims1.To investigate whether SUCLA2 is highly expressed in neurons and the expression of SUCLA2 is changed after treatment of neuronal cells with A?1-42 oligomer.2.To study the mechanism of mitochondrial dysfunction after SUCLA2 knock down.Research Methods1.Neonatal mice cerebral cortex primary neuron culture:the newborn mice were washed in 70%alcohol,then the whole brain was taken out and placed in DMEM for soaking;the cortex was separated under the microscope,the meninges were removed,and the peeled cortex was placed.In another dish containing DMEM;(Operation on the above process ice box)quickly remove the cortex of the meninges back into the ultra-clean table,place on the ice box,absorb excess DMEM,and use the sterile blade to brain tissue Thoroughly chopped,transferred to a 10 ml centrifuge tube;washed twice with cold HBSS,and allowed to aspirate the supernatant after standing;pipette 5.5 ml of HBSS into another centrifuge tube,add 2 ml of trypsin and mix at 37?The box was preheated;the brain tissue was placed in preheated trypsin,and the mixture was placed in a water bath at 37 ? for 15 minutes in a water bath for 5 minutes;the digestion was terminated by adding 1 ml of FBS.After mixing,the tissue is completely sunk to the bottom of the tube,the supernatant is discarded,the pellet is resuspended with NeurobasalA,and the digested tissue is blown off with a pipette 15 times;the mixed cells are blown with a 40 nm filter.Filtration,transfer the filtered liquid to another 10ml centrifuge tube,centrifuge at 200g for 5 minutes at room temperature;discard the supernatant after centrifugation,resuspend the cells in neuron medium,count under the microscope,then press about 4-6 x 106/ml was seeded into wells coated with polylysine in advance and placed in a 37 ?,5%C02 incubator.Primary neuronal cells take about 3-4 days to change medium.2.Glial cell culture of newborn mice:The newborn mice were washed in 70%alcohol,then the whole brain was taken out and placed in DMEM to soak;the whole dish containing the whole brain was placed on a microscope ice box under a microscope.The cortex is separated,the meninges are removed,and the peeled cortex is placed in another petri dish containing DMEM(on the ice box);the cortex peeling off the meninges is quickly taken back into the ultra-clean table,placed on the ice box,and the excess is taken.The brain tissue was completely chopped with a sterile blade,transferred to a 10 ml centrifuge tube;washed twice with cold HBSS,left to remove the supernatant;pipet 5.5 ml HBSS into another centrifuge tube,add 2 ml trypsin was mixed and placed in a 37 ? incubator for preheating;the brain tissue was placed in preheated trypsin,and the mixture was placed in a water bath at 37 ? for 15 minutes,mixed every 5 minutes;1 ml of FBS was added to terminate digestion.After mixing,the tissue was completely sunk to the bottom of the tube and the supernatant was discarded.The pellet was resuspended in DMEM(+10%FBS)and gently pipetted 25 times with a pipette;the cells mixed with the blow were filtered through a 40 nm filter.The filtered liquid was transferred to another 10 ml centrifuge tube,and centrifuged at 200 g for 5 minutes at room temperature.After centrifugation,the supernatant was discarded,the cells were resuspended in glial cell culture medium,counted under a microscope,and seeded into a cell culture flask with medium,then placed in a 37 ?,5%C02 incubator.The complete medium is replaced as appropriate according to the density,state,and color of the cells in the culture dish,and is usually replaced once every 1-2 days.The medium was preheated in 37 ? water bath before use.When changing the solution,discard the old medium,wash it twice with PBS,and add new medium.When the cells were passaged,the culture flask/well plate was first coated with PDL,and washed and dried with ddH20 before passage;when the cells were over 90%passaged.The medium was first aspirated,washed 3 times with sterile PBS pre-warmed at 37 ?,and 1 ml of pre-warmed 0.25%trypsin was added.After the cells were lightly tapped,they were detached,and 2 ml of complete medium was added to terminate the digestion.The digested cells were collected into a 15 ml centrifuge tube,centrifuged at 1000 rpm for 5 minutes,the supernatant was discarded,the cells were resuspended in the medium,inoculated according to the desired density,and cultured at 37 ? in a 5%CO 2 incubator.3.Western blot analysis:The sample was extracted with 1×loading buffer,collected,and then boiled for 10 minutes in boiling water.The prepared SDS-PAGE gel was placed in a vertical electrophoresis tank,and 1×running buffer,70 V,was used for 30 minutes pre-electrophoresis.The Marker protein and the target protein were added to the wells as required and electrophoresed.The electrophoresis is divided into two stages:one stage 30-40V,and the second stage 150V.The electrophoresis time is determined according to the position of the protein Marker,and the electrophoresis is turned off after the protein to be detected is separated.Prepare the PVDF membrane and filter paper as needed,soak the PVDF membrane in methanol before use.There are 4 layers of filter paper on the upper and lower sides of the transfer clamp.Add the cold transfer membrane to the tray and cut the glue according to the marker instructions.The film was transferred at low temperature.Using a constant current of 180 mA,the film transfer time was adjusted according to the molecular weight of the protein.After the protein is transferred,the PVDF film was taken out,naturally dried,soaked in methanol for 1 minute,soaked in deionized water for 1 minute,and dyed on a 0.5%PS horizontal shaker.After photographing,the background color was washed with deionized water.According to Marker's molecular weight labeling,the required strips were cut and blocked with 1 x TBST of 5%milk or 5%BSA(according to different protein requirements)at room temperature on a horizontal shaker for 1 hour.After the milk was drained,it was washed with 1 xTBST,and the primary antibody in an appropriate ratio was added,and shaken at 4 0 C horizontally overnight.The primary antibody was recovered the next day,and the membrane was washed 3 times with TBST,each time on a horizontal shaker for 10 minutes;the corresponding secondary antibody was added and placed on a shaker at room temperature for 1 hour.The secondary antibody was recovered,and the membrane was washed three times with a TBST horizontal shaker for 10 minutes each time;after the membrane was washed,the membrane was washed with a filter paper,and the developer was evenly applied at the position corresponding to the target protein,and developed by an Alpha Flurochem Q imaging analysis system.4.Preparation and cell treatment of A? oligomer:In the fume hood,resuspend the freeze-dried powder A?1-42(1mgA?+300ulHIFP)with HIFP,pre-cool the ultrasonic water bath with ice pack,mix well and place in the ultrasonic water bath dissolved for 30 minutes.The solution was centrifuged at 10,000 rpm for 5 minutes,and the supernatant was transferred and centrifuged again.The conditions were the same,and the supernatant was retained,and the collected supernatants were mixed twice and loaded into EP tubes as required.The HIFP in the A? solution was completely evaporated by drying in a sterile hood overnight,and the bottom of the tube was an A? peptide membrane.Store it in a-80? refrigerator for later use.The peptide membrane was taken out before the cells were treated,and the peptide membrane was dissolved by repeated soaking in sterile DMSO 60 ?l,and then dissolved in 900 ?l of PBS(pH=8.0)in a 10 ? ultrasonic water bath for 5 minutes,centrifuged at 10 ? for 10 minutes at 10000 rcf,and the supernatant was aspirated.The concentration of A? was determined by the BCA method.Incubation of the A?solution at 4 0 C overnight can be considered as the A? oligomer state,validation by Western blotting.The cells were stimulated by adding A? to the cell culture medium at the desired concentration.Care should be taken when performing cell processing to adjust the medium of each well to an equal amount.5.Plasmid preparation and cell transfection:The filter paper labeled with the plasmid was cut along the mark with a sterile scissors and placed in the EP.Add 40 ?l of sterile deionized water to each EP,soak the filter paper,seal with the sealing film,mark it,and store it in a refrigerator at-20?.Prepare LB medium,prepare LA culture dish,prepare competent cells(Stbl3 according to the instructions),apply the plate in the bacteria clean bench,and after influencing the bacterial solution,put it into the 37? incubator overnight.The colonies cultured overnight were observed,and single colonies were selected for shaking amplification.Plasmid extraction was performed using a small plasmid cassette(NucleoSpin Plamsid).The extracted plasmid cells were subjected to cell transfection.After the virus is prepared,the label is dispensed,stored in a-80 ? refrigerator,and melted at 4 ? when used.The virus was directly added to the neurons,4 ?l was added to each well,and the cells were collected after 3-6 days of incubation.The TRC was added only once,which was consistent with the virus for the longest time.Western blot detection of protein was performed after 6 days.6.Mitochondrial membrane potential,dendritic mitochondrial content and ATP measurement:Primary cultured neurons were incubated with 200 nM TMRM and 400 nM mitochondrial tracer green dye in a cell culture incubator(5%CO 2,37 0 C)for 30 minutes,pre-warmed the neuron medium,and removed the neurons.The neuron was washed by pre-warmed medium and then incubated in the incubator for 15 minutes.Image acquisition was performed using an incubator with a Nikon inverted microscope.The ATP content was determined by the Luminescent ATP Detection Assay Kit.7.Data processing and statistical analysis:Western blot images were processed by the US Department of Health ImageJ software for gray scale processing and analysis.The gray value of the target protein was adjusted by the corresponding mitochondrial internal reference protein or total protein internal reference protein.The group value is set to 1,and then statistical analysis is performed.Data analysis was processed by ImageJ software.Statistical analysis SPSS software(IBM software)was used for one-way analysis of variance,Bonferroni post hoc test or t test for repeated analysis and statistical analysis.The distribution and variation between the groups were basically the same.p<0.05 was considered statistically significant.All data are expressed as mean ± standard deviation.Research Results1.A? oligomer can induce apoptosis of neuronWe used normal wild-type C57/BL mice(within 24 hours of birth)to culture the primary cells of the neurons,and selected cells with good growth conditions to add the prepared A? oligomers and incubate for 24-48 hours to observe the survival of the cells.In this state,it was found that most of the neuronal cells collapsed into cell debris(Figl-2).At the same time,we analyzed the effect of DMSO and A?concentration on the activity of experimental cells(Fig 4-5),and confirmed that the apoptosis of neuron was caused by AP under the experimental conditions.2.SUCLA2 is specifically expressed in neuronWe performed primary neuron culture and glial cell culture of normal cerebral cortical cells through normal wild-type C57/BL newborn mice,and selected cells with good growth state to compare the expression levels of SUCLA2,SUCLG1 and SUCLG2 by Western blot.Changes in these two cells suggest that SUCLA2 is specifically expressed in neurons and is expressed more in mature neurons(Fig 6,P<0.05).3.Changes in the expression of SUCLA2:the expression of SUCLA2 was significantly decreased in A?-toxic neurons,and it decreased more with the increase of A? concentration and the prolongation of action timeWe cultured primary C57/BL newborn mice with primary neurons of cerebral cortex cells,and prepared A? oligomers.The concentration was determined by BCA method,and 0.1umol/1 and 1.Oumol/I were added to neurons.After 24 and 48 hours of incubation,protein was collected,and the protein expression was detected by Western blot.It was found that the expression of SUCLA2 was significantly decreased in A?-toxic neurons,and decreased with the increase of Ap concentration and time of action.(Fig 7,P<0.05).4.The study of mitochondrial dysfunction with SUCLA2 expression down-regulates cell model4.1 Decreased expression of SUCLA2 simultaneous down-regulation of SUCLG1 expressionIn order to determine the effect of decreased SUCLA2 expression on neurons,we used C57/BL newborn mice to culture primary neurons of cerebral cortical cells,and exposed the developed neuronal cells to specific SUCLA2-targeted shRNA,control group.It is a shRNA exposed to a non-targeting gene,and the expression level of SUCLA2 in the neuron is detected by the WB method.We first detected the most significant transfected virus that down-regulated SUCLA2(Fig 8,P<0.05)and treated it with cells.The results showed that the expression of SUCLA2 in SUCLA2 shRNA-induced neurons was significantly decreased(Fig 9,P<0.05).At the same time,it was observed that there was an increase in SUCLG2 in SUCLA2 down-regulated neurons(Fig9,P<0.05).At the same time,we also found that the expression level of SUCLG1 was also significantly decreased with the down-regulation of SUCLA2(Fig9,P<0.05).Based on this result,we hypothesized that when SUCLA2 targets shRNA to down-regulate the expression level of SUCLA2,the overall function of SCS is decreased.4.2 Decreased expression of SUCLA2 inhibit neuronal mitochondria bioenergeticsWe all know that the physiological activities of neurons depend on ATP produced by mitochondrial OXPHOS,while SCS catalyzes the phosphorylation of substrate levels in TCA.The substrate is involved in the electron transport of OXPHOS,so the maintenance of SCS activity is essential for the respiratory chain.In order to determine the effect of SUCLA2 deficiency on mitochondrial energy production,the ATP content was determined by chemiluminescence method,and the amount of ATP produced by SUCLA2-targeted shRNA-treated neurons was significantly decreased(Fig 8 P<0.01).We know that mitochondrial membrane potential is both the basis of electron transport chain activity and its product.Therefore,we also performed mitochondrial membrane potential detection.The results showed that SUCLA2 down-regulated neuronal dendritic cell mitochondria at TMRM density compared with control neurons.The reduction was 36%(Fig 8 al and a3.P<0.001);there was no significant difference in total dendritic cell mitochondria content(Fig 8 a2 and a3).These results indicate that the mitochondrial membrane potential collapse of neurons caused by SUCLA2 deficiency may be a manifestation of impaired mitochondrial oxidative phosphorylation efficiency.Based on this result,we speculate that the oxidative phosphorylation metabolism of neuronal mitochondria is significantly reduced by down-regulating the expression level of SUCLA2.4.2 Decreased expression of SUCLA2 afects mitochondrial dynamics proteinsMitochondrial dynamics is controlled by mitochondrial fusion and fission proteins,and its dynamic balance is essential for maintaining mitochondrial function.To determine whether SUCLA2 deficiency affects mitochondrial fusion and fission proteins,we collected non-targeted genes and SUCLA2 shRNA-treated neurons for immunoblotting to detect mitochondrial fusion proteins including mitofusin 2(Mfn2)and Optic atrophyl(Opal)and mitochondrial fission protein fssion 1(Fis 1),dynamin-like protein1(Dlp1)and phosphorylated Dlp1(pDlpl S616).As shown in Fig.11-13,the expression levels of Mfn2 and Opal caused by SUCLA2 deficiency were significantly decreased.Interestingly,we found that the loss of SUCLA2 also inhibited the expression of mitochondrial fission proteins.SUCLA2 down-regulation neurons underwent dramatically decreased the levels of Fisl and DLP1.The results indicate that the dcreased expression of SUCLA2 can inhibit mitochondrial fusion and fission protein expression,resulting in imbalance of mitochondrial dynamics.Research Conclusion1.SUCLA2 is highly expressed in neurons,so the changes of its expression have a greater effect on neurons.2.The expression of SUCLA2 in A?-toxic neurons decreased significantly,and the expression of SUCLA2 decreased further with the increase of A? concentration and the prolongation of action time.3.Decreased expression of SUCLA2 mediates mitochondrial dysfunction,possibly due to the following mechanisms:the scs dysfunction?impaired OXPHOS efficiency and disruption of mitochondrial fission and fusion balance.4.Our data further demonstrate that SUCLA2 plays an essential role in the maintenance of mitochondrial physiology in neurons,suggesting that SUCLA2 may be a potential new target for the treatment of AD in the future. |
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