The Abnormal Distribution And Expression Of BAG3 In The Brain Of Alzheimer's Disease Transgenic Mice And Its Involvement In Pathogenesis Of AD

Objectives: Alzheimer's disease(AD)is the most common chronic and progressive degenerative disease of the central nervous system.It is mainly characterized by dementia and manifests as memory impairment,cognitive function,and language impairment.The main pathological features of AD are the deposition of extracellular amyloid plaques,the hyperphosphorylation of intracellular tau protein leads to neurofibrillary tangles and the loss of neurons in specific brain regions.Amyloid precursor protein(APP)is cleaved by ?-secretase and ?-secretase to produce soluble APP?(s APP?),insoluble beta amyloid protein(A?)and APP intracellular Structural domain(AICD).In contrast,?-secretase and ?-secretase could cleave APP to produce nutrient peptides,soluble APP?(s APP?),P3 peptides and AICD.The regulation of?-secretase that protects nerves and ?-secretase and ?-secretase related to neurotoxicity are thought to play a vital role in the etiology and pathogenesis of AD.SAPP? is considered to have neurotrophic and neuroprotective properties,and may play a role in eliminating A? neurotoxicity.BAG(Bcl2-associated athanogene)is an anti-apoptotic protein family,currently consists of 6 members(BAG1-6),all members share the BAG domain.In addition to its anti-apoptotic activity,BAG family proteins have unique functions in health and diseases.BAG3 has become one of the hot spots in many researches.BAG3(also known as CAIR-1 or Bis)is a member of the BAG family.In addition to the BAG domain,BAG3 also has a WW domain,two IPV motifs,and a proline-rich(PXXP)motif.Through these motifs,BAG3 interacts with other binding proteins such as Hsp22 and phospholipase C(PLC),and participates in a variety of cellular processes,including apoptosis,proliferation,migration,and autophagy.Various functions of BAG3 are related to diseases such as cancer,myopathy,and neurodegeneration.BAG3 is related to tau phosphorylation and s APP?.BAG3 could not only play a selective autophagy effect to clear the phosphorylated tau protein of misfolded proteins,but also interact with s APP? to participate in cell regulation.BAG3-mediated selective macroautophagy could be used as an adaptive mechanism to maintain cell homeostasis,stress,and aging,making BAG3 an important target for future drug intervention.In this study,we systematically analyzed the expression changes,localization,and distribution of BAG3 in the cerebral cortex and hippocampus of 5 × FAD transgenic mice,APP/PS1 transgenic mice and in APPsw cells,as well as the correlation between BAG3 and A? co-expression and hyperphosphorylation of Tau protein.It is of great significance to further explore the relationship between BAG3 and the pathophysiological mechanism of AD to investigate whether the overexpression of BAG3 inhibitions A? secretion and its effect on autophagy.Methods:1.7-month-old 5 × FAD transgenic mice were used.(1).The co-expression of BAG3 and A? in the cerebral cortex and hippocampus of mice was detected by immunofluorescence.(2).The localization of BAG3,Neun and GFAP in the cerebral cortex and hippocampus of mice was detected by immunofluorescence.2.3,6,9,12 months old APP/PS1 transgenic mice were used.(1).Western blot was used to detect the change of protein level of BAG3.(2).RT-PCR was used to detect the change of total m RNA expression of BAG3.(3).The protein levels of ADAM10,?-catenin and phosphorylated ?-catenin(serine 33 and serine 37)were detected by Western blot.3.Mouse derived neuroblastocyte N2 a cells and N2 a cells stably transfected with APP695 swe gene were used as cell lines.(1).The expression of BAG3 was detected by immunofluorescence technique.(2).Western blot was used to detect the change of total protein level of BAG3.(3).RT-PCR was used to detect the expression changes of BAG3 total m RNA.4.8-month-old Rosa 26-BAG3 KI transgenic mice were used.The protein expression changes of A? and APP during hydrolysis were detected by Western blot.5.The cell line uses N2 a cells stably transfected with APP695 swe gene and N2 a cells stably transfected with human APP695 swe gene to infect BAG3(BAG3-APPsw cells)with lentivirus.(1).CCK8 technology was used to detect the protective effect of BAG3 on cells.(2).The expression of A? was detected by immunofluorescence technique.(3).The protein expression levels of A? and APP during hydrolysis were detected by Western blot.(4).The m RNA expression level of APP hydrolase was detected by RT-PCR.(5).After the addition of ?-catenin inhibitor XAV939,the changes of ?-catenin and APP enzyme digestion protein were detected by Western blot.(6).The protein expression levels of P62,LC3?/?,Tau and phosphorylated Tau(site of threonine 231,serine 396,serine 404),GSK3? and phosphorylated GSK3?were detected by Western blot.Results:1.BAG3 in the cerebral cortex and hippocampus of non-Tg mice was expressed in the cytoplasm of neurons;BAG3 in the cerebral cortex and hippocampus of 5 ×FAD transgenic mice were positively expressed in garland shape in senile plaques.The BAG3 protein and m RNA levels of APP/PS1 transgenic mice increased in the juvenile period(3 months of age),and decreased in the middle age(9 months of age).The expression of ADAM10 and ?-catenin in the cerebral cortex and hippocampus of APP/PS1 transgenic mice decreased,and the expression of phosphorylated ?-catenin(serine 37)increased.In APPsw cells,the change trend of BAG3 protein level and m RNA level was consistent with the in vivo experiment.2.The level of ADAM10 protein in the cerebral cortex of BAG3 KI transgenic mice increased,and the level of A? protein decreased.The proliferation level of BAG3-APPsw cells was higher than that of control group.A? protein expression decreased and the fluorescence intensity decreased.ADAM10 protein and m RNA levels in BAG3-APPsw cells increased;?-catenin protein level increased.After adding the ?-catenin inhibitor XAV939,the ADAM10 protein level decreased.3.The P62 and LC3?/? protein levels of BAG3-APPsw cells increased,the phosphorylated tau protein(threonine 231,serine 396,serine 404)protein level decreased.Phosphorylated GSK3?(serine 9 position)level increased and total GSK3? protein decreased.Conclusion:1.BAG3 and A? were co-localize in the senile plaques in the brain of AD mice,and BAG3 expression was reduced in the brain neurons of AD mice.2.BAG3 expression increased in the cerebral cortex and hippocampus of young AD mice,but decreased in the brains of middle-aged AD mice.3.BAG3 overexpression could promote the expression of ADAM10.4.BAG3 increased the expression of ADAM10 by up-regulating ?-catenin,thereby promoting APP non-amyloid proteolysis.5.BAG3 overexpression could promote the process of AD autophagy.6.BAG3 overexpression could reduce the expression of phosphorylated tau protein.