Effects Of HDAC6 On Abnormal Aggregated Tau Protein Degradation And The Associated Mechanism

Abnormal pathological aggregation of tau protein is one of the most significant pathological features of neurodegenerative diseases. Investigating the mechanism of abnormal tau protein degradation is vital important in treating neurodegenerative disease such as Alzheimer’s disease (AD). The ubiquitin-proteasome system (UPS) is an important pathway to degrade intracellular short-lived and soluble low molecular weight proteins. However, in tauopathies, dysfunction is often occurred in proteasome which leads to the result that misfolded tau protein cannot be effectively cleaned by UPS. Histone deacetylase 6 (HDAC6) can bind ubiquitinated substrate protein into a combination. Then it can be transported towards microtubule organizing center (MTOC) through the microtubule with the participation of dynein and form the functional protein aggresome and activate autophagy-lysosomal pathway. Finally aggresomes with substrate protein are degraded through autophagy-lysosomal pathway. The autophagy-lysosomal pathway plays an important compensatory role in maintaining protein content in cells when the activities of proteasome are inhibited. The binder of ubiquitin zinc finger domain (BUZ) of HDAC6 has the capacity to bind with ubiquitin and polyubiquitin chain. And precious studies have found that HDAC6 could mediate the formatin of tau aggresome, but the mechanism is still unclear. Therefore, we speculate that when the activities of proteasome are inhibited, the ubiquitinated tau aggregate content will increase. And HDAC6 could bind with ubiquitinated tau protein through BUZ, mediate the formation of tau aggregate and finally enchance in tau protein degradation through autophagy-lysosomal system.Objective:Exploring the effect and mechanism of HDAC6 in mediating tau degradation when ubiquitinated tau aggregate in mouse neuroblastoma N2a cells (Neuro-2a) which is treated by the proteasome inhibitor MG132.Methods:(1) The HDAC6 expression in N2a cells was disturbed by siRNA and evaluated the influence of HDAC6 siRNA treatment on HDAC6 contents by western-blotting technique. (2) After being disturbed by siRNA for 6 hr, the N2a cells were transfected by FLAG-HDAC6 and FLAG-HDAC6 ABUZ plasmids for 36 hr respectively. Also the western-blotting technique was used to detect transfection efficacies. (3) After suppressing the expression of HDAC6 for 6 hr by siRNA, we transferred FLAG-HDAC6 and FLAG-HDAC6 ABUZ plasmids into N2a cells for 36 hr. Then the cells were treated with MG132 (5μM) for 24 hr in order to cause proteasome dysfunction. The changes of tau protein expression level were assessed by western-blotting. (4) Under the same condition as in (3), the autophagy active marker LC3 expression level was inspected by western-blotting method. (5) The FLAG-HDAC6/EGFP-tau and FLAG-HDAC6 ABUZ/EGFP-tau plasmids were co-transfected into N2a cells for 36 hr, and then the cells were exposed to MG132 (5μM) for 12 hr or 24 hr. The locations of HDAC6 and tau protein in the cell and the situation of tau protein functional aggregation were observed using immunofluorescence technique. (6) Under the same condition as (5), we observed the binding force between HDAC6 and tau protein by immunoprecipitation.Results:(1) the HDAC6 expression level in N2a cell decreased significantly with 80% when treated with 20 nM HDAC6 siRNA for 6 hr. (2) FLAG expression increased in FLAG-HDAC6 and FLAG-HDAC6 ABUZ plasmid transfection groups based on FLAG antibody detection. (3) The tau protein level increased significantly compared with control group (P<0.05); compared with MG132 treating group, tau protein level in cells transfected by FLAG-HDAC6 plasmid decreased significantly (P<0.05); compared with FLAG-HDAC6 plasmid transfection, tau protein level increase significantly in cells transfected by FLAG-HDAC6 ABUZ plasmid with P<0.05. (4) Compared with control group, autophagy active marker LC3 expression level upregulated significantly in MG132 treating group; Compared with MG132 treating group, LC3 expression level in cells transfected by FLAG-HDAC6 plasmid upregulated (P<0.05); compared with FLAG-HDAC6 plasmid transfection, LC3 expression level decrease significantly in cells transfected by FLAG-HDAC6 ABUZ plasmid with P<0.05. (5) Compared with control group, HDAC6 and tau protein in MG132 treating group aggregated around nuclear areas, and tau aggresome was formed, which indicated the colocalization between HDAC6 and tau protein. But tau protein in FLAG-HDAC6 ABUZ and EGFP-tau plasmid co-transfection groups dispersed and did not form tau aggresome compared with FLAG-HDAC6 and EGFP-tau co-transfection group, and the cololization phenomenon dispeared. (6) Compared with control group, the binding ability between HDAC6 and tau increased in MG132 treating group (P<0.05). On the contrary, that binding ability in FLAG-HDAC6 ABUZ and EGFP-tau co-transfection group decreased compared with FLAG-HDAC6 and EGFP-tau co-transfection group (P<0.05).Conclusions:These results shown that HDAC6 could activate autophagy-lysosomal pathway to efficiently enhance degradation by strengthening the binding ability with tau and participating tau aggresome formation while proteasome activity is suppressed. This research preliminary discussed the approach and mechanism of HDAC6 participating in tau protein degration based on N2a cell model. It provided good experimental bases to further explicit the function and mechanism of HDAC6 regulated tau degration in autophagy-lysosome system.