Molecular Mechanisms For Tau Misfolding And Toxicity Modulated By Zn²⁺

Abnormal accumulation of misfolded hyperphosphorylated microtubule-associated protein Tau is a prominent feature of Alzheimer disease (AD), the most common neurodegenerative condition in the elderly. Zinc in amyloid plaques deposits of the AD brain tissue reaches an enormous concentration of 1000 ?M, suggesting that zinc dyshomeostasis is closely linked to the development and progression of this disease. Our previous studies have found that Zn2+accelerates the fibrillization of human Tau fragment Tau244-372 via bridging Cys-291 and Cys-322 in vitro. Here, by using a previously established inducible cell model for Tau aggregation and Tauopathy, we further investigated the effects of Zn2+on abnormal aggregation, post-translational modifications and cytotoxicity of a pathological mutant ?K280 of full-length human Tau. As revealed by Congo red binding assays, transmission electron microscopy, immunofluorescence, Western blot, and immunogold electron microscopy, pathological concentration of Zn2+dramatically accelerates the fibrillization of Tau-?K280 both in vitro and in SH-SY5Y neuroblastoma cells. As evidenced by annexin V-FITC apoptosis detection assay and MTT reduction assay, pathological concentration of Zn2+remarkably enhances Tau-?K280 fibrillization-induced apoptosis and toxicity in SH-SY5Y cells. Substitution of Cys-291 and Cys-322 with Ala, however, essentially eliminates such enhancing effects of Zn2+on the fibrillization and the consequent cytotoxicity of ?K280. Furthermore, Zn2+is co-localized with and highly enriched in amyloid fibrils formed by Tau-?K280 in SH-SY5Y cells. The results from isothermal titration calorimetry show that Zn2+ binds to full-length human Tau by interacting with Cys-291 and Cys-322, forming a 1:1 Zn2+-Tau complex. Our data demonstrate that zinc dramatically accelerates abnormal aggregation of human Tau and significantly increases Tau toxicity in neuronal cells mainly via bridging Cys-291 and Cys-322.Hyperphosphorylation of Tau, which is also closely linked to the aggregation of toxicity of Tau, is considered as another key process in AD pathology. In this study, exogenous aggregates formed by over expressed K18-?K280 fragment are found to significantly induce the abnormal phosphorylation and aggregation of endogenous Tau in SH-SY5Y cells. Moreover, Zn2+enhances the phosphorylation of Ser-396 of both Tau-?K280 and its Cys mutants, indicating that the enhancement effects of Zn2+ on Tau aggregation and phosphorylation could be independent. Taken together, we propose that Zn2+binds to Tau via tetrahedral coordination to Cys-291 and Cys-322 as well as His-330 and His-362 to promote the accumulation of Tau protein. The accumulation of over-expressed proteins could induce the abnormal aggregation and phosphorylation of endogenous Tau and further promotes Tau toxicity. Cys mutations destroy this kind of tetrahedral structure and thus Zn2+loses its ability to accelerate Tau aggregation and toxicity. Our study provides new clues to understanding the molecular mechanisms for Tau protein misfolding and toxicity regulated by Zn2+and the role of zinc dyshomeostasis in the etiology of neurodegenerative diseases.