Effects Of UCH-L1Inhibition On The Degradation Of Tau And The Underlying Mechanisms

Ubiquitin C-terminal hydrolase L1(UCH-L1) is a very important deubiquitinating enzymes (DUBs) in the ubiquitin-proteasome system(UPS), which participates in and maintains the normal morphology of neurons and its cognitive function in the brain. Tau is a microtubule-associated protein whose main biological function is to promote microtubule assembly and maintain its stability. The abnormal modification of tau can form the neurofibrillary tangles then in turn cause neurodegenerative diseases. And the UPS system can degrade the abnormal modification of tau to maintain the normal morphology of neurons. Studies have shown that the activity of UCH-L1is negatively correlated with the number of neurofibrillary tangles, suggesting that UCH-L1is closely related to the abnormal tau protein. Our study focuses on the effects of UCH-L1inhibition on the degradation and function of tau and the underlying mechanism in mouse N2a neuroblastoma cells (N2a cells).We respectively used various concentrations of the inhibitor LDN (2.5,5,10μM) to inhibit the activity of UCH-L1for1h in N2a cells. We firstly examined the changes of poly-and mono-ubiquitin in N2a cells by western blot. And then western blot and immunoprecipitation were used to detecte the level of ubiquitinated tau and phosphorylated tau. And we also analyzed the microtubules-binding ability of tau by western blot. Next, we used Thioflavine-S and AT8staining to observe whether the phosphorylation of tau protein formed in abnormal aggregates in N2a cells. Finally, we examed the levels of p-CREB and PSD-95which both have a close contact with the composition of memory related protein and the structure of neuronal synaptic. The results showed that:(1) By detecting the changes of poly and mono-ubiquitin with UCH-L1inhibition, poly-ubiquitination protein gradually increased while the mono-ubiquitin decreased in LDN dose-dependent manner (P<0.05).(2) UCH-L1inhibition changed the level of ubiquitinated tau which increased in a manner of LDN dose-dependent (P<0.05).(3) Compared with the control group, LDN treatment respectively increased the levels of total tau, phosphorylated tau in a dose-dependent manner (P<0.05).(4) Our study found that, throughout the taxol stabilized microtubules, the level of tau in supernantant increased in a dose-dependent (P<0.05), but the level of tau in precipitate was quite opposite. These results suggest that with LDN treatment, the microtubule-binding ability of tau was obviously decreased.(5) We used AT8(recognize tau-Ser202/Thr205) and Thioflavine-S staining (recognize the β-sheet secondary structure of protein) to observe the intracellular immunofluorescence colocalization. And the results showed that, AT8and Thioflavine-S staining both significantly strengthened with the LDN treatment, and they also make a co-localization in the cell, suggesting that LDN treatment induced the formation of the abnormal aggregates in the cell, and the hyperphorylation of tau protein directly related with the aggregates.(6) UCH-L1inhibition induced the levels of phosphorylated CREB and PSD-95decreased in a dose-dependent manner (P<0.05). These results suggest that the inhibition of UCH-L1may damage the structure and the function of tau protein by affect the recycling of mono-ubiquitin, then further affect the structure of synaptic and the synthesis of memory related protein.