Research On The Mechanism Of Pgc-1alpha Improving Alzheimer’s Disease By Rugulating Mitochondrial Anterograde Axonal Transport

Alzheimer’s disease（AD）is a common neurodegenerative disease that affects memory,cognition,and behavior.The typical pathological features areβ-amyloid deposition and neurofibrillary tangles formed by hyperphosphorylation of tau protein and massive loss of neurons.So far,the pathogenesis of AD has not been fully elucidated.It was recently found that the mitochondrial soma-axonal transport damage plays a crucial role in the pathogenesis of AD.Peroxisome proliferator-activated receptorγcoactivator-1alpha（Pgc-1alpha）,as a key factor in mitochondrial biosynthesis,has a protective effect on the nervous system.It has been reported that Pgc-1alpha can stimulate the activity of the MFN2 promotor and regulate the expression of MFN2.MFN2 not only plays a vital role in the fusion of mitochondria,but also plays an important role in the mitochondrial axonal transport.However,whether Pgc-1alpha can improve the occurrence of AD,its molecular mechanism of improving AD and its potential site of action need further study.Objectives（1）To verify the correlation between Pgc-1alpha and AD.（2）To explore the mechanism of improving AD by Pgc-1alpha.（3）To investigate the key sites in the improvement of AD by Pgc-1alpha.Methods（1）Verifing the correlation between the expression of Pgc-1alpha and AD.（1）Effectineness evaluation of AD models in vivo and in vitro:6-month-old APP/PS1 mice were selected as animal models,and the expression of Aβdeposition in the lateral parietal association cortex（LPt A）of the brain was detected by immunofluorescence to verify the effectiveness of the model;The APPswe plasmid was transfected in the N₂A cell line to construct an AD cell model,and the overexpression of the plasmid was verified by Western Blotting.（2）Immunohistochemical and Western Blotting were used to detect the expression of apoptosis-related proteins（BAX,Bcl-2）in AD models in vivo and in vitro.（3）Immunofluorescence and Western Blotting were used to detect the expression of PGC-1αin AD models in vivo and in vitro.（4）Overexpression of PGC-1αinduced by gene recombination in vivo and in vitro models of AD:the animal model was induced by microinjection technology to induce overexpression of PGC-1αin the LPt A of APP/PS1.The overexpression of PGC-1αwas verified by immunofluorescence and Western Blotting after three weeks of virus expression;the cell model was co-transfected with APPswe and p ECMV/Pgc-1alpha plasmids on the N₂A cell line.After 48 hour,Western Blotting was used to verify the overexpression of PGC-1α.（5）Immunofluorescence detection of the changes in the area and number of Aβ-positive plaques in APP/PS1 mice after overexpression of PGC-1α;Immunohistochemical and Western Blotting were used to detect the expression of apoptosis-related proteins（BAX,Bcl-2）after PGC-1αoverexpression,which can evaluate the improvement effect of PGC-1αon AD pathology.（2）Exploring the mechanism of improving AD by Pgc-1alpha.（1）Immunohistochemical and Western Blotting were used to detect the expressions of MFN2,KIF5A,and KIF5B in AD models in vivo and in vitro,which can evaluate the changes of anterograde axonal transport in AD.（2）In the AD model in vitro,Mito-Tracker probes were used to label mitochondria,and the changes in mitochondrial distribution were observed.Using the JC-1 fluorescent probe,observe the fluorescence intensity of JC-1 aggregates that produce red fluorescence and JC-1monomers that produce green fluorescence,and evaluate the change of mitochondrial membrane potential.（3）Western Blotting was used to detect the expressions of PSD95,SYP in AD cell model.（4）Immunohistochemical and Western Blotting were used to detect the effect of PGC-1αoverexpression on mitochondrial anterograde transport-related proteins MFN2,KIF5A,and KIF5B in AD,which can evaluate the role of PGC-1αin improving anterograde axonal transport.（5）In the cell model of PGC-1αoverexpression,Mito-Tracker probes were used to mark mitochondria,and the changes in mitochondrial distribution were observed.Using the JC-1 fluorescent probe,observe the fluorescence intensity of JC-1 aggregates that produce red fluorescence and JC-1 monomers that produce green fluorescence,and evaluate the effect of mitochondrial membrane potential by PGC-1α.（6）Western Blotting was used to detect the expressions of PSD95,SYP in the cell model of PGC-1αoverexpression.（3）Investigating the key sites in the improvement of AD by Pgc-1alpha.（1）Construction of Pgc-1alpha mutant plasmids,which are the LKKLL motif located at positions 145-149（m L2）and the LLKYL motif located at positions 209-213（m L3）of Pgc-1alpha,and verify whether the mutation is successfully induced.（2）In AD cell model,Western Blotting was used to detect the effect of Pgc-1alpha mutant plasmids on apoptosis-related proteins（BAX,Bcl-2）and synaptic associated proteins（PSD95,SYP）.（3）In the AD cell model,Western Blotting was used to detect the effect of Pgc-1alpha mutant plasmids on mitochondrial anterograde transport-related proteins MFN2,KIF5A,and KIF5B.（4）In the AD cell model,Mito-Tracker probes were used to label mitochondria,and the effect of Pgc-1alpha mutants on the distribution of mitochondria was observed.Using the JC-1 fluorescent probe,observe the fluorescence intensity of JC-1 aggregates that produce red fluorescence and JC-1monomers that produce green fluorescence,and evaluate the influence of the mitochondrial membrane potential of the Pgc-1alpha mutant.Results（1）6-month-old APP/PS1 mice had obvious Aβdeposition in LPt A,and the N₂A cell line was successfully transfected with the APPswe plasmid,which proved that the AD models in vivo and vitro were successfully constructed;In these models,it were found that:（1）BAX expression increased,Bcl-2 decreased expression and cell apoptosis increased;（2）PGC-1αexpression level decreased.After the APP/PS1 mice were injected with p AAV-MCS-Ppargc1α-m-FLAG-HA,the PGC-1αwas expressed successfully;The N₂A cell line was successfully transfected with APPswe and Pgc-1alpha plasmids;In these models of PGC-1αoverexpression,it were found that:（1）Aβ-positive plaque area and number decreased;（2）BAX expression decreased,Bcl-2 expression increased,and cell apoptosis decreased.（2）In the models of AD in vivo and vitro,it were found that:（1）MFN2,KIF5A,KIF5B protein expression decreased,and anterograde axonal transport was impaired;（2）Mitochondrial distribution was abnormal and membrane potential decreased;（3）PSD95,SYP protein expression decreased.In the models of PGC-1αoverexpression,it were found that:（1）MFN2,KIF5A,KIF5B protein expression increased,and anterograde axonal transport improved;（2）Mitochondria distribution improved,the membrane potential rises;（3）PSD95,SYP protein expression increased.（3）The plasmid tag was successfully expressed,which proved that the cell models were successfully constructed;in the cell models,it were found that:（1）The effect of PGC-1αon apoptosis and synaptic improvement after the L3 site mutation was weakened;（2）The effect of PGC-1αon MFN2,KIF5A,and KIF5B after the L3 site mutation Weakened;（3）The improvement effect of PGC-1αon mitochondrial distribution and mitochondrial membrane potential after L3 site mutation was weakened.ConclusionsIn AD,mitochondrial anterograde axonal transport damage is accompanied by decreased expression of PGC-1αprotein.Pgc-1alpha can improve the synaptic damage and apoptosis in AD by regulating the anterograde axonal transport of mitochondria,and its function site is the L3 motif.The regulation mechanism of Pgc-1alpha on mitochondrial anterograde axonal transport provides a new strategy for the treatment of AD.