Effects Of UCH-L1 Inhibition On The Clearance Of Tau Aggresome And The Underlying Mechanisms

Tau is a microtubule associated protein which can promote microtubule assembly and maintain microtubule stability.Abnormal aggregation of tau in neurons is one of the major neuropathological features of AD,which can cause neurdegeneration.Ubiquitin-proteasome system(UPS)and autophagy-lysosomal pathway(ALP)are the two major intracellular protein degradation systems,and the two degradation process are directly related to the ubiquitination of substrate proteins.Ubiquitin C-terminal hydrolase L1(UCH-L1)is a vital member of UCH family of deubiquitinating enzymes which plays an important role in the hydrolysis of polyubiquitin chains and the recycling of ubiquitin.Meanwhile,it is found that UCH-L1 also has ubiquitin ligase activity,which can produce unanchored K63-Ub chains.Targeted binding of K63 and substrate protein will facilitate the degradation of substrate protein by ALP,and this process is directly related to the HDAC6 activity.our previous studies found that UCH-L1 inhibiton could lead to tau abnormal phosphorylation and ubiquitination modification in cell and mouse hippocampal slices.However,up to now,there is no direct report about the relationship between UCH-L1 and tau protein aggregation clearance.Based on the above research background,we used the human embryonic kidney 293 cells stabling expressing human tau441(HEK293/tau441)as the research model.Study the effects of UCH-L1 inhibition on the clearance of tau aggresome and the associated mechanisms.Objective:Investigating the effects of UCH-L1 inhibition on the clearance of tau aggresome and the underlying mechanisms.Method:First,we treated cells with proteasome inhibitor MG132 for 24 hr;Then,we eluted MG 132 and added fresh medium for 24 hr;Finally,we added UCH-L1 inhibitor LDN during elution MG 132 for 24 hr.After the above treatment,we detect the relevant biochemical markers by immunofluorescence technology,immunoblotting technology,immunoprecipitation technology and HDAC6 activity assay kit.Results:(1)After treating cell with MG 132 for 24 hr,tau protein and vimentin(the marker protein of aggresome formation)aggregated into perinuclear and formed a distinct globular aggresome;After elution MG132 for 12 hr,tau protein and vimentin were scattered in the perinuclear and the globular aggresome was gradually disintegration;After elution MG132 for 24 hr,tau and vimentin were distributed evenly in the perinuclear and the globular aggresome is disappeared;After MG132 elution plus LDN(UCH-L1 inhibitor)treatment for 24 hr,the tau and vimentin formed spherical globular aggresome in the perinuclear and the two proteins demonstrated obvious co-localization.(2)Immunoblotting results showed that compared with DMSO group,the K63 content was increased in MG132 group(P<0.01);compared with MG132 group,the K63 content was decreased in elution MG132 group(P<0.01);compared with MG132 elution group,K63 content were significantly decreased in MG132 elution plus LDN treatment group(P<0.01).(3)Immunoprecipitation results showed compared with DMSO group,the affinity of HDAC6 and K63 were augmented in MG132 group(P<0.01);compared with MG132 group,the affinity of K63 and HDAC6 were decreased in MG132 elution group(P<0.05);compared with MG132 elution group,the affinity of K63 and HDAC6 were further decreased in MG132 elution plus LDN treatment group(P<0.05).(4)HDAC6 activity results showed that compared with DMSO group,the activity of HDAC6 was raised in MG132 group(P<0.01);compared with MG132 group,the activity of HDAC6 decreased in MG132 elution group(P<0.05);compared with MG132 elution group,the activity of HDAC6 was further decreased(P<0.01)in MG132 elution plus LDN treatment group.Meanwhile,we also detected the acetylation level of a-tubulin,a substrate protein of HDAC6,by Western blotting to reflect the change of HDAC6 activity.We found that the results were in accordance with the results of HDAC6 Activity assay.(5)Immunofluorescence results indicated that compared with MG132 elution for 12 hr or 24 hr alone,MG132 elution plus HDAC6 inhibitor TBSA treatment induced the clearance impairment of tau aggresome.(6)Immunoblotting results showed that compared with the DMSO group,the content of LC3-? was significantly increased in MG132 treatment group(P<0.01);compared with MG132 treatment group,the content of LC3-II was decreased in MG132 elution group(P<0.05);compared with MG132 elution group,and the content of LC3-II was significantly decreased in MG132 elution plus LDN treatment group(P<0.05).Conclusion:The above results suggested that UCH-L1 deficiency decreased the content of K63 and downregulated the activity of HDAC6 and autophagy,ultimately impaired the clearance of tau aggresome.