IKKβ,Modulated By DJ-1/p-VHL,Reduces Phosphorylated Tau(p-Tau) Accumulation Via Autophagy In Alzheimer’s Disease Model

Objective:Alzheimer’s disease（AD）is a neurodegenerative disease characterized by progressive irreversible impairment of memory,cognition and other functions.It mainly occurs in the elderly with cognitive impairment,abnormal mental behavior and gradual decline of daily living ability.Amyloid plaques formed byβ-amyloid beta（Aβ）and neurofibrillary tangles by phosphorylated tau protein（p-Tau）are two typical pathological features of AD.It has been found that in AD model mice,the enhancement of autophagy reduces the accumulation of p-Tau in Tau-transgenic mice and improves their synaptic and cognitive functions.Appropriate low-level inflammatory stimulation is beneficial to maintain the autophagy ability of neurons and reduce the expression of p-Tau in Tau-transgenic mice,so as to improve the cognitive function of mice.Inhibitor kappaβ（IKKβ）,as a key inflammatory signaling molecule,is normally expressed in neurons,and neuroinflammation can enhance autophagy activity in the brain.Then,that whether increase IKKβcan promote autophagy mediated p-Tau clearance remains to be further studied.The studies have found that DJ-1 interacts with WSB-1 to negatively regulate the expression of p-VHL,which mediates IKKβubiquitination degradation.Therefore,does DJ-1 affect IKKβubiquitination degradation by regulating p-VHL to reduce the accumulation of p-Tau in AD patients is not clear.All in all,this study intends to explore DJ-1 and IKKβeffect on autophagy mediated p-Tau clearance by means of gene transfection,immunofluorescence,immunohistochemistry and so on.Methods:1.Firstly,we took mouse neuronal cell HT-22 and used 0,0.1,1,5 and 10μM concentration of Aβ_1-42 to construct AD in vitro cell model.MTT was used to detect cell survival,Hoechst staining and TUNEL staining were used to detect cell death,and DCFH-DA was used to detect the production of ROS.Then,we constructed GFP-LC3 adeno-associated virus,infected into the AD in vitro cell model,and observed the distribution and proportion of green fluorescence under fluorescence microscope to judge the form of autophagy flow.Finally,DJ-1,IKKβ,autophagy related proteins including Beclin1,LC3II/I and ATG5,and p-Tau were detected by Western blotting under treatment with 0,0.1,1,5 and 10μM concentration of Aβ_1-42.2.pc DNA-IKKβwere transfected into HT-22 cells to construct IKKβoverexpression HT-22 cells before cells were treated 5μM Aβ_1-42.The viability of HT-22 cells was evaluated by MTT assay,the cell death was detected by TUNEL staining,and the expression of autophagy related proteins（Beclin 1,LC3Ⅱ/I and ATG5）and p-Tau protein were detected by Western blotting.3.pc DNA-DJ-1 were transfected into HT-22 cells to construct DJ-1overexpression HT-22 cells before cells were treated 5μM Aβ_1-42.The viability of HT-22 cells was evaluated by MTT assay,the cell death was detected by TUNEL staining,and the expression of autophagy related proteins（Beclin 1,LC3Ⅱ/I and ATG5）and p-Tau protein were detected by Western blotting.4.pc DNA-DJ-1 or/and sh IKKβtransfected into HT-22 cells to construct DJ-1overexpression or/and IKKβknockdown HT-22 cells before cells were treated 5μM Aβ_1-42.MTT assay and TUNEL staining were used to detect the viability and death of HT-22 cells,and Western blotting was used to detect the expression of autophagy related proteins（Beclin 1,ATG5,LC3II/I）and p-Tau protein.Finally,we detected the degradation of p-tau protein in HT-22 by autophagy lysosome by immunofluorescence.5.pc DNA-DJ-1 were transfected into HT-22 cells to construct DJ-1overexpression HT-22 cells before cells were treated 5μM Aβ1-42.Western blotting was performed to study the expression level of DJ-1,WSB-1,p-VHL and IKKβ.Then,Co-IP analysis was used to evaluate the interaction between p-VHL and IKKβ,determine the effects of p-VHL overexpression on K63 linked IKKβpolyubiquitination.6.In order to further study the role of DJ-1 in the progression of AD,10 mg/kg of Aβ_1-42 treated mice and injected 10 m M sh-DJ-1 into mice.We performed water maze test to detect the cognitive behavior of mice,and immunohistochemistry and HE staining were used to detect Aβdeposition and tissue lesion in mouse hippocampus,respectively.Finally,the expression of autophagy related protein and p-Tau in hippocampus were detected by Western blotting.Result:1.With the increase of Aβ_1-42 concentration,cell activity was decreased,while cell death showed an increasing trend,accompanied by the increase of ROS.The points of LC3 autophagy flow were distributed around the nucleus and increased with Aβ_1-42.The green fluorescence emitted by LC3 were decreased with the increase of Aβ_1-42 concentration.With Aβ_1-42 concentration increased,DJ-1,IKKβ,and autophagy related proteins Beclin1 and ATG5 was decreased,the ratio of LC3II/I was reduced,while the expression of p-Tau was increased.2.IKKβand Aβ_1-42 inhibited the viability of HT-22 cells,but in AD cell model,IKKβoverexpression reduced Aβ_1-42-causing suppression of the viability of HT-22cells.Similarly,IKKβand Aβ_1-42 accelerated HT-22 cell death,while IKKβoverexpression alleviated Aβ_1-42exerting promotiving effect on HT-22 cell death.Further study also found that Aβ_1-42 downregulated IKKβ,Beclin 1,LC3Ⅱ/Ⅰand ATG5 protein levels,while up-regulated the expression of p-Tau.However,IKKβoverexpression promoted the expression of autophagy related proteins in HT-22 cells,but inhibited the expression of p-Tau protein.In addition,IKKβoverexpression reversed the inhibition of autophagy and up-regulation of p-Pau protein induced by Aβ_1-42.3.DJ-1 overexpression and Aβ_1-42 inhibited the viability of HT-22 cells and accelerated the death of HT-22 cells,while DJ-1 overexpression reduced effects on the viability and death of HT-22 cells induced by Aβ_1-42.DJ-1 overexpression promotes the expression level of IKKβand autophagy related protein,but inhibited the expression of p-Tau protein.In addition,DJ-1 overexpression upregulated the decreasing IKKβand autophagy related protein and downregulated increasing p-Tau protein induced by Aβ_1-42.4.DJ-1 overexpression accelerated the viability of HT-22 cells and inhibited the death of HT-22 cells,while IKKβknockdown inhibited the viability of HT-22 cells and promoted death of HT-22 cells after Aβ_1-42 treatment.Overexpression of DJ-1promoted the expression of IKKβprotein,while IKKβknockdown had no effect on the expression of DJ-1.DJ-1 overexpression promoted autophagy related protein expression,but downregulated p-Tau protein,while IKKβknockdown shows the opposite trend.Notably,overexpression of DJ-1 reversed IKKβknockdown induced decrease of autophagy related protein and increase of p-Tau protein.Compared with lysosomal protein LAMP-1,IKKβoverexpression promoted the expression of p-Tau in lysosomes,indicating that it promoted its degradation,while IKKβknockdown inhibited the expression of lysosomal p-Tau,indicating that it inhibited its degradation.5.Overexpression of DJ-1 promoted WSB-1 and IKKβ,but inhibited the expression of p-VHL protein.p-VHL and IKKβcould combine with each other.Further studies showed that overexpression of Ub（K63）promoted IKKβubiquitination,and p-VHL overexpression enhanced K63 linked IKKβpolyubiquitination.6.Aβ_1-42 treatment led to behavioral retardation and regression of learning,cognition and memory ability of mice,while knockdown of DJ-1 could also lead to behavioral retardation and reduced escape potential of mice,and exacerbated AD symptom,indicating that the learning and memory ability of mice was more significantly weakened.Aβ_1-42 treatment resulted in Aβaggregation and pathological injury in mouse hippocampus while knockdown of DJ-1 aggravates Aβ_1-42 induced Aβaggregation and pathological injury.Besides,knocking down DJ-1 can downregulate IKKβand autophagy related proteins,and up-regulated the expression of p-Tau protein,which is similar with Aβ_1-42 treatment.Thus,knockdown of DJ-1exacerbates the reduction of autophagy and the accumulation of p-Tau in AD disease models.Conclusion:1.With the increase of Aβ_1-42 concentration,autophagy was decreased and the expression of p-Tau was increased;2.Overexpression of IKKβcaused the increase of autophagy and the decrease of p-Tau expression in AD cell model in vitro;3.Overexpression of DJ-1 caused the increase of autophagy and the decrease of p-Tau expression in AD cell model in vitro;4.DJ-1 regulates the increase of autophagy and the decrease of p-Tau expression in AD cell model in vitro caused by IKKβ;5.DJ-1 regulated IKKβubiquitination degradation by p-VHL;6.Knockdown of DJ-1 slowed behavior,inhibited autophagy and promoted the accumulation of p-Tau in AD disease model mice.