Effects Of Zinc In HDAC6Mediated Tau Degradation By Autophagy-lysosome Pathway

Abnormal protein aggregation is an important pathologic sign of neurodegenerative diseases, and it has important clinical significance to explore the mechanism of the degradation of abnormal proteins in the cell aggregation. In tauopathies (such as Alzheimer’s diseases), it is often shown abnormal aggregation and post-transcriptional modification of tau protein, which caused by the dysfunction of degradation system, such as the functional impairment of proteasome. Tau is the degradation substrate of both ubiquitin proteasome system and autophagy-lysosome pathway. When the avtivity of proteasome is inhibited, Histone deacetylase6(HDAC6) has the capacity to bind both the polyubiquitinated misfolded protein and the dynein motor to form a complex, then transport the aggregate proteins to microtubule-organizing center (MTOC) where the aggresomal particles are sequestered into a single large aggresome, and subsequently MTOC transports the aggregate proteins to lysosomes to be degraded by autophagy. It has been confirmed that the HDAC6ZnF-UBP domain contains three Zn atoms, which is critical for ubiquitin binding, and the presence of zinc is indispensable in the normal physiological function of HDAC6. Biological functions of zinc in neurogenesis, neuronal migration, synaptogenesis and neurotransmission make it closely related to learning and memory function. Malfunction in any of these processes induced by zinc deficiency can contribute to defects in cognitive development and function with some typical behavioural paradigms. But the molecular mechanism of znic in neurodegenerative disease is unclear. In this study, we aim to explore the HDAC6mediated effects of dysregulated znic on the degradation of tau by autophagy-lysosome pathway in HEK293/tau441cells.Experiments were divided into four groups with corresponding drug treatments respectively. DMSO group was the control group; MG132group was treated with MG132for8hr; MG132+TPEN group was first treated with MG132for2hr, then exposed by TPEN for6hr; MG132+TPEN+ZnSO4group was first with MG132treatment for2hr, then added TPEN and ZnSO4for6hr. The final concentration of MG132, TPEN and ZnSO4are2μM,2μM and5μM respectively. After drug treatments, using Western blot to detect the level of cellular tau-5, LC3and HDAC6, with activity kit to detect the activity change of intracellular HDAC6, with immunofluorescence technology to detect tau aggresome formation, with immunoprecipitation technology to detect the binding ability between HDAC6and tau. The results show that:(1) compared with control, proteasome activity inhibition induce the level of total tau and LC3increase (p<0.01); compared with MG132group, zinc deficiency induce the level of total tau increase (p<0.01), while the level of LC3decrease (p<0.01); compared with MG132+TPEN group, adding exogenous zinc could reverse the changes of tau protein level and autophagy activity which induced by TPEN;(2) compared with control, proteasome activity inhibition induce the level (p<0.01) and activity (p<0.01) of HDAC6increase; compared with MG132group, zinc deficiency lower HDAC6activity (p<0.001), but there is no significant change in the level of HDAC6; adding exogenous zinc on the basis of TPEN, the activity of HDAC6increased compared with MG132+TPEN group (p<0.05), but still lower than MG132group (p<0.01), while the level of HDAC6has no obvious change compared with MG132group and MG132+TPEN group;(3) compared with control, proteasome activity inhibition induce tau aggresome formation; compared with MG132group, zinc deficiency block tau aggresome formation; compared with MG132+TPEN group, added exogenous zinc could promote the formation of tau aggresome;(4) compared with control, proteasome activity inhibition enhance the binding force between HDAC6and tau; compared with MG132group, zinc deficiency block the binding force between HDAC6and tau; compared with MG132+TPEN group, adding exogenous zinc promote HDAC6combined with tau.Above results show that when proteasome activity is inhibited, zinc deficiency caused by TPEN suppresses the autophagy-lysosome degradation of tau protein, makes intracellular tau level increase. The invovled mechanism may be due to the decreased activity of HDAC6and the binding ability between HDAC6and tau protein following zinc deficiency, thus blocks the process of tau aggresome formation which is the key step in autophagy-lysosome degradation. This experiment first explore the molecular mechanism of zinc deficiency in neurodegenerative diseases related to tau aggregation, and provide some basis experimental evidence for the establishment of HDAC6as the therapeutic target of tau related neurodegenerative diseases.