| Background and ObjectiveAs a neurodegenerative disease,Alzheimer’s disease(AD)is characterized by progressive memory and cognitive decline,behavioral and personality changes.The epidemiological survey results showed that the incidence of AD in the elderly increased year by year.AD has become a global public health problem that needs to be addressed urgently due to its heavy burden on patients and families.But so far,there is no definitive cure for the disease.As we all know,the pathogenesis of AD is complex,among these,Extracellular deposition of amyloid beta protein(Aβ)plays a central role in the pathogensis of AD.Therefore,further studies on the mechanism of the neurotoxicity of Aβ will help us understand the pathogenesis of AD and play an important role in the treatment of AD.As the power plants of cells,mitochondria play an important role in many age-related diseases.Numerous studies have showed that the dysfunction of mitochondria also present in AD.When large amounts of errors or unfolded proteins accumulate in the mitochondria,the unfolded protein response(UPRmt)is activated.Under stress,the UPRmt is activated and then activates the mitochondrial protective gens through the signal transduction between mitochondria and nucleus,thus rebuild the mitochondiral protein homeostasis.There are specific molecular chaperones in the mitochondrial matrix and intermembrane space that help maintain efficient mitochondrial protein folding and assembly.The Hsp60 chaperone protein is located in the mitochondrial matrix and promotes folding of relatively small soluble monomer proteins.In addition to molecular chaperones,mitochondria also contain several quality control proteases used to identify and degrade misfolded or assembled proteins.Among which CLPP and LONP1 are ATPases located in the mitochondrial matrix,mainly responsible for degrading misfolded proteins.The protease Htra2/OMI in the mitochondrial intermembrane space also has the function of identifying and degrading misfolded proteins.A large amount of studies have shown that the UPRmt is involved in the pathogenesis of various diseases.Studies have found that UPRmt related genes were up-regulated in the frontal cortex samples of postmortem patients with both familial and sporadic AD.Activation of UPRmt has also been reported in AD mice and C.legans models.However,there are still no relevant studies on UPRmt in AD cell model,and its specific changes are still unclear.In mammals,the mevalonate signaling pathway is responsible for catalyzing the synthesis of metabolites that play an important role in cell metabolism,growth,and differentiation,and plays an important role in maintaining normal cell function.Many studies have shown that the mitochondria can affect the mevalonate pathway through various pathways,including UPRmt.In the C.legans model,the mevalonate pathway plays an important role in the activation of UPRmt.However,in mammalian cells,especially in the AD cell model,there has been no study on the interaction between UPRmt and mevalonate pathway.Ceramide plays an important messenger role in regulating many biological processes such as cell growth,differentiation and apoptosis.A lot of evidence showed that there is a direct or indirect link between ceramide and Aβ.Ceramide levels are elevated in both neurons treated with Aβ and in the brain tissue of AD patients.However,studies on the interaction between ceramide pathway and UPRmt are still lacking.Only in the C.legans model is the ceramide signaling pathway considered necessary to activate UPRmt.However,the interaction between UPRmt and ceramide pathway has not been studied in mammalian cells,especially in the AD cell model.Therefore,in this study,we observed the role of UPRmt in the pathogenesis of AD by detecting the levels of UPRmt in SHSY5Y cells treated with Aβ and UPRmt in APP/PS1 transgenic mice.Small interfering RNA(siRNA)and chemical reagents were used to interfere with the mevalonate or ceramide signaling pathways,so as to observe the role of these two pathways in the UPRmt response induced by Aβ in SHSY5Y cells.Then we used HEK293 and 20E2 cells to detect the effect of endogenous overexpression of APPsw on UPRmt and the role of mevalonate pathway in UPRmtMethods1.SHSY5Y cells were cultured and given to Aβ25-35 with different concentrations.Collected cells 24h later and detected the expression of UPRmt related proteins by Western blot.SHSY5Y cells were cultured and treated with 20uM Aβ25-35 for 4h,then collected cells and detected the expression of UPRmt related proteins by Western blot.C57 control mice and APP/PS1 transgenic mice were bred to be 3 months and 9 months old,extracted the the protein of hippocampal tissue and detected the expression of UPRmt related proteins by Western blot.2.After treated cells with 20uM Aβ25-35 for 4h,the expression of the enzyme HMGCS-1 in the mevalonate pathway were detected.Transfected cells with scramble siRNA or HMGCS-1 siRNAs for 48h,then treated with or without 20uM Aβ25-35 for another 4h,collected cells and detected the expression of UPRmt related proteins by Western blot.After treated SHSHY5Y cells with different concentrations of simvastatin for different times,each group was treated with 20uM Aβ25-35 for another 4h.Collected cells and detected the changes of UPRmt related proteins by Western blot.Observed the morphological of cells by microscopy.Observed the morphological changes of mitochondria by transmission electron microscopy.Fluorescence and quantitative detection of ROS level changes and CCK8 assays cell activity.3.After treated cells with 20uM Aβ25-35 for 4h,the expression of the enzyme SPTLC-1 were detected.Transfected cells with scramble siRNA or SPTLC-1 siRNAs for 48h,then treated with or without 20uM Aβ25-35 for another 4h,collected cells and detected the expression of UPRmt related proteins by Western blot.Observed the morphological changes of mitochondria by transmission electron microscopy.Fluorescence and quantitative detection of ROS level changes and CCK8 assays cell activity.4.Cultured HEK293 and 20E2 cells,and detectded the expression of UPRmt related proteins and HMGCS-1 by Western blot.After transfection with control or HMGCS-1shRNA for 48h,the expression of HMGCS-1 and UPRmt related proteins in HEK293 and 20E2 cells were detected by Western blot.After transfection with the control or HMGCS-1shRNA for 48h,then treated with 100uM mevalonate or sterilized water for 4h,collected cells and analysis the expression of HMGCS-1 and UPRmt related proteins in 20E2 cells by Western blot.Results1.UPRmt related proteins were upregulated in SHSY5Y cells treated with AP25-35 and in APP/PS1 transgenic mice1.1 SHSY5Y cells were treated with Aβ25-35 for 24h,and the concentration of A(3 was 2.5,5,10,and 20uM respectively.The results showed that exogenous Aβtreatment increased the expression of UPRmt related proteins in SHSY5Y cells,so Aβ25-35 treatment could activate the UPRmt response in SHSY5Y cells.1.2 Compared with the age-matched WT mice,the expression of Hsp60,HtrA2/Omi and CLPP proteins were increased in the hippocampus of APPsw/PS1dE9 transgenic mice at 3 months old,and the expression of Hsp60 and LONP1 proteins were increased in the hippocampus of APPsw/PS1dE9 transgenic mices at 9 months old.This indicated that UPRmt was also activated in AD mouse model.2.Blocking the mevalonate pathway by HMGCS-1 siRNAs or simvastatin inhibits the UPRmt activation induced by Aβ and exacerbates the cytotoxic effect of Aβ in SHSY5Y cellsIn this part of the study,we used HMGCS-1 siRNA and simvastatin to block the mevalonate pathway,respectively.2.1 First we detected that after treated with 20uM Aβ25-35 for 4h,HMGCS-1 protein level was increased in SHSY5Y cells.Without intervention with Aβ,there was no significant statistical difference in the expression of UPRmt related proteins between cells transfected with scramble and HMGCS-1 siRNAs.We also found that after transfection with different sequences of HMGCS-1 siRNAs,the expression of UPRmt related proteins HSP60,LONP1,HtrA2/Omi and CLPP were decreased in SHSY5Y cells treated with Aβ.2.2 Cells were treated with luM and lOuM simvastatin for 2,6 and 24h,and each group was treated with 20uM A 25-35 for another 4h before protein collection.Western blot results showed that UPRmt related proteins HSP60,LONP1,HtrA2/Omi and CLPP were decreased in all groups except the group that treated with luM simvastatin for 24h.Therefore,inhibition of mevalonate signaling pathway by HMGCS-1 siRNA or simvastatin can reduce UPRmt response caused by Aβ.2.3 In order to study the effect of inhibiting mevalonate signaling pathway on cells,we detected the changes in cell morphology,mitochondrial morphology,intracellular ROS levels and cellular activity after simvastatin pretreatment.The results showed that after lOuM simvastatin pretreatment for 24h,the cell morphology was significantly changed,and the mitochondrial morphological abnormalities caused by Aβ25-35 were aggravated in SHSY5Y cells,such as mitochondrial swelling,increased vacuolization and mitochondrial ridge deletion.The ROS detection results showed that after lOuM simvastatin pretreatment,the intracellular reactive oxygen species level was increased.CCK8 results also showed that the simvastatin pretreatment exacerbated the decrease of cell viability induced by Aβ25-35.Therefore,inhibition of the mevalonate pathway by simvastatin aggravate the cytotoxic effect of Aβ25-35 in SHSY5Y cells.3.Blocking the ceramide signaling pathway by SPTLC-1 siRNAs inhibits the UPRmt activation induced by Aβ and exacerbates the cytotoxic effect of Aβ in SHSY5Y cells3.1 It has been reported that the sphingolipid biosynthetic signaling pathway is necessary for the activation of UPRmt in nematodes.In this part of the study,we used SPTLC-1 siRNA to block the sphingolipid pathway.First we detected that after treated with 20uM Aβ25-35 for 4h,SPTLC-1 protein level was increased in SHSY5Y cells.Without intervention with Aβ,there was no significant statistical difference in the expression of UPRmt related proteins between cells transfected with scramble and SPTLC-1 siRNAs.We also found that after transfection with different sequences of SPTLC-1 siRNAs,the expression of UPRmt related proteins HSP60,LONP1,HtrA2/Omi and CLPP were decreased in SHSY5Y cells treated with Aβ.Thus,inhibition of the ceramide signaling pathway can reduce intracellular UPRmt activation caused by Aβ.3.2 To further investigate the effects of inhibiting the ceramides signaling pathway on cells,we detected changes in mitochondrial morphology,intracellular ROS levels and cellular activity after transfection with SPTLC-1 siRNAs.The results showed that after transfection with SPTLC-1 siRNAs,the mitochondrial morphological abnormalities caused by Aβ25-35 were aggravated in SHSY5Y cells,such as increased mitochondrial vacuolization and mitochondrial ridge deletion.Both fluorescence and ROS quantitative test results showed that after transfection with SPTLC-1 siRNAs,the level of intracellular reactive oxygen species was higher than that of the group with Aβ25-35 treatment alone.CCK8 results also showed that the cell viability of SHSY5Y cells was decreased after transfected with SPTLC-1 siRNAs.Therefore,inhibition of the ceramide signaling pathway by SPTLC-1 siRNA aggravate the cytotoxic effect of Aβ25-35 in SHSY5Y cells.4.Stable overexpression of APPsw gene cause changes of UPRmt in HEK293 cells,and mevalonate pathway plays an important role in UPRmt4.1 To determine whether endogenous overexpression of APPsw can cause the above UPRmt reactions,we detected the UPRmt related proteins levels in HEK293 and 20E2 cells.The results showed that the UPRmt related protein Hsp60 was upregulated in 20E2 cell compared with HEK293 cell.However,the expression levels of the other two UPRmt related proteins HtrA2/Omi and CLPP in 20E2 cells were significantly lower than those in HEK293 cells.So our results suggest that the UPRmt related proteins level were different in 20E2 cells compared with HEK293 cells.4.2 In previous studies,we found that manipulate the mevalonate pathway through HMGCS-1 siRNAs inhibited UPRmt activation in SHSY5Y cells induced by Aβ.In this study,we used HMGCS-1 shRNAs to manipulate the mevalonate pathway.First,we found that the HMGCS-1 protein level was decreased in 20E2 cells compared with HEK293 cells.As shown in Figure 2B,there was no significant difference in UPRmt related protein levels between HEK293 cells transfection with HMGCS-1shRNA and HEK293 cells transfection with control shRNA.However,the protein levels of Hsp60、HtrA2/Omi and CLPP were both decreased in 20E2 cells after transfected with HMGCS-1 shRNA for 48h.Thus,we found that inhibition of the mevalonate pathw-ay by HMGCS-1 shRNA decreases the UPRmt related proteins level in 20E2 cells,so the mevalonate pathway is involved in the UPRmt changes in 20E2 cells.4.3 To further verify the expression of UPRmt related proteins in 20E2 cells mediated by the mevalonate pathway,we treated 20E2 cells with 100uM mevalonate for 4h after transfection with shRNAs,and then detected the expression of UPRmt related proteins by Western blot.The results showed that there was no significant change in UPRmt related protein levels in the cells transfected with control shRNA after mevalonate supplementation.But after treatment with 100uM mevalonate,the protein levels of Hsp60、HtrA2/Omi and CLPP in 20E2 cells were upregulated than those in the HMGCS-1 shRNA transfection group alone.Thus we found that supplemented with mevalonate restored the decreased UPRmt in 20E2 cells induced by HMGCS-1 shRNA,which further verify the important role of mevalonate pathway in mediating the UPRmt activation in 20E2 cells.Conclusion1.UPRmt was activated in SHSY5Y cells treated with Aβ25-35 and in APP/PS1 transgenic mice.2.In SHSY5Y cells,the activation of UPRmt induced by Aβ depends on the regulation of two pathways,mevalonate and ceramide signaling pathways.3.Inhibition of the above two pathways aggravate the cytotoxic effect of Aβ in SHSY5Y cells,suggesting that inhibition of UPRmt aggravate the cytotoxic effect of Aβ,which providing evidence for the protective effect of UPRmt during AD process.4.Compared with HEK293 cells,overexpression of APPsw gene cause the decrease of UPRmt,and the decrease of UPRmt may be related to the decrease of HMGCS-1 protein level in the mevalonate pathway... |
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