Pathological concentration of zinc dramatically accelerates abnormal aggregation of full-length human Tau and thereby significantly increases Tau toxicity in neuronal cells

Abstract

A pathological hallmark of Alzheimer disease and other tauopathies is the formation of neurofibrillary tangles mainly composed of bundles of fibrils formed by microtubule-associated protein Tau. Here we study the effects of Zn on abnormal aggregation and cytotoxicity of a pathological mutant ΔK280 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 Zn dramatically accelerates the fibrillization of ΔK280 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 Zn remarkably enhances ΔK280 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 Zn on the fibrillization and the consequent cytotoxicity of ΔK280. Furthermore, Zn is co-localized with and highly enriched in amyloid fibrils formed by ΔK280 in SH-SY5Y cells. The results from isothermal titration calorimetry show that Zn binds to full-length human Tau by interacting with Cys-291 and Cys-322, forming a 1:1 Zn-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. Our findings could explain how pathological zinc regulates Tau aggregation and toxicity associated with Alzheimer disease.

Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau

“…Nevertheless, both studies were carried out in the presence of heparin, which induces Tau fibrillization into thioflavin T (ThT)positive PHFs. Recently, it was demonstrated that zinc not only accelerates aggregation of a pathological mutant ΔK280 of hTau40 induced by Congo red in vitro, but also significantly increases its toxicity in neuronal cells [25]. This study also demonstrated the importance of Cys-291 and Cys-322 residues both for zinc binding and Tau aggregation [23,25].…”

“…Here, we have investigated aggregation of fulllength human hTau40 isoform in the absence of heparin but in the presence of zinc ions under a wide range of temperature conditions using turbidimetry, isothermal titration calorimetry (ITC), dynamic light scattering (DLS) and transmission electron microscopy. We found that, contrary to its aggregation in the presence of heparin or with other inducers such as arachidonic acid or congo red [25,27,28], selfassembly of Tau is a reversible process that depends on temperature and is induced by zinc ions. Moreover, our findings point to an important role of low-affinity auxiliary zinc-binding sites in this process.…”

“…Recently, it was demonstrated that zinc not only accelerates aggregation of a pathological mutant ΔK280 of hTau40 induced by Congo red in vitro, but also significantly increases its toxicity in neuronal cells [25]. This study also demonstrated the importance of Cys-291 and Cys-322 residues both for zinc binding and Tau aggregation [23,25]. While using non-physiological specific inductors of Tau aggregation such as heparin or Congo red is the most common way to induce PHFs structure and test inhibitors, it might not be the most pertinent model to study PHF formation itself, especially in the early stages where the process has already been hypothesized to be reversible [26].…”

Zinc promotes liquid–liquid phase separation of tau protein

“…One possible mechanism by which zinc could exert this effect is by increasing the aggregation propensity of proteins such as Aβ and tau, and such effects have indeed been observed in experiments in vitro (24,(32)(33)(34)(35). However, accelerated tau fibrillization was observed only in the presence of non-physiological cofactors such as heparin (24) or Congo red (33). The present finding that zinc strongly promotes tau LLPS brings a new dimension to understanding the link between abnormal zinc homeostasis and the pathogenic process in AD and other tauopathies, especially since recent data indicate that the environment of liquid droplets strongly modulates the aggregation behavior of several proteins involved in neurodegenerative disorders (14,(18)(19)(20)28,36,37).…”

“…Thus, it has been widely postulated that zinc may play an important role in AD and other neurodegenerative diseases (9,12,31). One possible mechanism by which zinc could exert this effect is by increasing the aggregation propensity of proteins such as Aβ and tau, and such effects have indeed been observed in experiments in vitro (24,(32)(33)(34)(35). However, accelerated tau fibrillization was observed only in the presence of non-physiological cofactors such as heparin (24) or Congo red (33).…”