Effects Of UCH-L1 On The Formation Of Tau Aggresome And The Associated Mechanism

The ubiquitin-proteasome system (UPS) is the most noticeable system in charge of degrading of proteins, which is involved in the degradation of abnormal proteins. When abnormal proteins clearance is failure by ubiquitin-proteasome pathway due to proteasome impairment, the autophagy-lysosomal system (ALS), also named as aggresome pathway, will be activated. Polyubiquitinated protein aggregates are recruited and transported towards microtubule organizing center (MTOC) to form the aggresome via the microtubule network by a protein complex consisting of histone deacetylase 6 (HDAC6) and the dynein motor complex. The formation of aggresome is a vital process in protein aggregates cleanrance through autophagy-lysosomal pathway. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), an important member of UCH family of deubiquitinating enzymes, is responsible for neurons to survive and to maintain their proper function. Several functions of UCH-L1 have been proposed, other than as an ubiquitin hydrolase, it also serves as an ubiquitin ligase activity. Dysfunction of UCH-L1 has been identified in neurodegenerative diseases such as Parkinson’s Disease and Alzheimer’s Disease, which have a difficulty in protein clearance. A recent study has shown that UCH-L1 inhibition leads to abnormal aggregation of tau. However, little is known about the underlying pathogenic mechanism of UCH-L1 involved in aggresome clearance by autophagy-lysosomal pathway. It has been reported that several deubiquitinating enzymes have been identified in regulation of aggresome formation or autophagic activiation by producing unanchored K63-linked ubiquitin chains (K63-Ub), which can bind and activate HDAC6 activity. Therefore, we speculate that K63-linked ubiquitin chains produced by UCH-L1 may bind and activate HDAC6 activity, consequently promoting aggresome formation. When UCH-L1 activity is inhibited, loss of K63-Ub leads to aggresome formation unsuccessfully.Objective:Exploring the effects and mechanism of UCH-L1 in tau aggresome formation when UCH-L1 activity is inhibited during proteasome impairment.Methods:For drug treatment, proteasome inhibitor MG132 was added at a final concentration of 3 μM. After 2 hr incubation in the presence of MG132, HEK293/tau441 cells were treated or untreated with UCH-L1 inhibitor LDN for 6 hr at a final concentration of 5 μM. The cells were treated with Dimethyl Sulfoxide (DMSO) as control. The total time of exposure to drug treatment was 8 hr and then cells were analyzed by western blot, immunofluorescence and immunoprecipitation.Results:(1) Polyubiquitin protein levels were found significantly increased (P<0.05) while monomeric ubiquitin decreased (P<0.01) in HEK293/tau441 cells incubated with MG132 treatment and MG132-LDN treatment compared to control cells. Meanwhile, compared with MG132 treatment group, the level of monomeric ubiquitin was surprisingly decreased after MG132 exposure and LDN treatment (P<0.05). (2) Treatment of cells with MG132 promoted tau protein gathering into a large spherical peri-nuclear structure aggresome and caused a dramatic co-localization between tau and ubiquitin as well as tau and vimentin in aggresome. In stark contrast, in cells treated with MG132 in combination with UCH-L1 inhibitor LDN, tau aggresome formation was largely blocked and ubiquitin failed to aggregation. (3) The immunoprecipitation assays showed a stronger interaction between HDAC6 and tau when cells were treated with MG132 compared with control group (P<0.001). However, compared with MG132 treatment group, the interaction was markedly weakened after LDN exposure during proteasome failure (P<0.01). (4) HDAC6 protein levels were both increased in MG132 treated cells incubated with or without LDN compared to control cells. Strikingly, the level of acetylated a-tubulin was found significantly decreased as expected when cells were treated with MG132 (P<0.05) and significantly increased after MG132 and LDN exposure (P<0.01). (5) Compared with control group, the level of K63-Ub was significantly increased in MG132 treated cells (P<0.01). Meanwhile, the level of K63-Ub was surprisingly decreased after MG132 and LDN exposure (P<0.05) and further reduced compared with MG132 treatment group (P<0.01). (6) The immunoprecipitation assays showed that the binding ability between HDAC6 and K63-Ub increased in MG132 treatment group compared with control group (P<0.01). On the contrary, the interaction was markedly weakened after LDN exposure (P<0.05) and further reduced compared with MG132 treatment group (P<0.01).Conclusions:Altogether, these findings indicate that HDAC6 could promote aggresome formation when proteasome impairment by activating HDAC6 deacetylases activity due to the stronger interaction with K63-Ub and strengthening the binding ability between HDAC6 and tau. However, inhibition of UCH-L1 activity when proteasome impairment leads to decrease in K63-Ub and weak interaction between HDAC6 and K63-Ub, which downregulates HDAC6 deacetylases activity, then caused the weak binding ability between HDAC6 and tau, finally blocking formation of tau aggresome. This research primary investigated the underlying pathogenic mechanism of UCH-L1 involved in tau aggresome formation. It provided a novel experimental evidence to further illustrate the function and mechanism of UCH-L1 in neurodegenerative diseases.