Effects Of Poh1 On The Clearance Of Tau Aggregates

There are three distinct fates for the misfolded and aggregated protein in eukaryotic cells.They may be refolded by the molecular chaperones or degraded by the ubiquitin-proteasome system (UPS) or the autophagosome-lysosome pathway (ALP). Under normal conditions, the UPS is the main system to degrade abnormal protein. The autophagosome-lysosome pathway will undertake this task unless the UPS is either inactivating or deficiencies in its function. Pohl, a Padl homologue of fission yeast in human, has various biological functions, such as maintaining the activity of cells, regulating embryonic stem cell differentiation, promoting the formation of tumor and so on. In addition, Pohl possesses a deubiquitinating enzyme activity, which is known as a subunit of 26S proteasome. Tauopathies, as a result of abnormal tau, belongs to the family of neurodegenerative disease, which is characterized of disorder of cytoskeleton and tau deposits in neuron and glia. Moreover, it is deficient in functions of UPS. Meanwhile, the ALP becomes the leader for degradation in the patients’bodies. Recently, a number of evidences indicate that Pohl is relative to the abnormal protein degradation. With cells treated by MG132, aggresomes were arrested in Pohl KD cells always. However, it was reversed when microinjected with K63 ubiquitin chains. So we assume that Pohl may participate in the clearance of the abnormal tau aggregates by K63-Ub chains mediating.This paper is based on the experiment with HEK293/tau441 cells. We first treated the cells with Pohl siRNA in order to reduce the level of endogenous Pohl, followed by 5 μM MG132 for 24 hr so that tau-contained aggregates formed. Then wash out MG132, and abserve the degradation of tau aggregates. As a result, we can learn more about the role and molecule mechanism of Pohl playing in the tau degradation. The results are as follows. (1) Compared with control group, endogenous Pohl decreased by 60% until Pohl siRNA was 80 nM. (2) In control groups, tau and vimentin were throughout the cells. However, both of them were gathered into aggresome closing to nuclear because of MG132. After MG132 washout, the tau and vimentin positive aggresome broke into small parts. (3) Similarly, Pohl and tau zoomed into a great peri-nuclear inclusion body when cells were treated with MG132. Amazingly, the structure became invisible instead of numerous and small aggregates when we removed MG132. (4) Compared with DMSO group, tau showed a increase because of MG132 (p<0.01) and a decrease because of MG132 washout (p<0.001). Knockdown of Pohl together with MG132 exposure induced a huge increase level of tau (p<0.001), while it was reversed on account of MG132 washout(p<0.001). Nevertheless, the ratio of MG132 washout/MG132 in PohlKD group was bigger than that in control group. (5) The western blot assay showed a moderate increase of LC3-Ⅱ with MG132 exposure (p<0.01), which was different from its behavior in both Pohl and proteasome deficient group. Because there was no apparent change with this treatment. (6) This result showed the variation of K63 ubiquitin chains. On condition that Pohl was well present, the level of K63 ubiquitin chains declined by 50% when MG132 was removed compared to MG132 treatment. Once Pohl was knockdown, the increase of K63 ubiquitin chains could be ignored. Whereas Poh1 deficient has an ability to make K63 ubiquitin chains decrease when cells were treated with MG132. (7) There was no influence on the stability of HDAC6 for Pohl. But HDAC6 activity was under the control of Pohl. Compared with DMSO group, the level of ac-tubulin declined greatly because of proteasome inhibitor MG132 (p<0.001). If suppressed Pohl at the same time, ac-tubulin showed a great decrease, too(p<0.001). Unfortunately, there is no different between them. However, ac-tubulin expression enhanced for Pohl KD in MG132 washout group(p<0.01). From the above, we consider that Pohl is a regular of ALP which can degrade tau finally. Pohl binds to HDAC6 by producing free K63 ubiqintin chains and then makes HDAC6 active so that HDAC6 can promote the ALP initiating. In the case of UPS deficient, the decrease of endogenous Pohl lead to a drop of K63 ubiquitin chains directly.Then both HDAC6 activity and APL activity will be inhibited resulting in tau accumulation in cells. The outcomes we have got explore the role and the mechanism of Pohl during the tau degradation by the ALP, and we also hope that these achievements can contribute to tauopathies clinic.