The Molecular Mechanisms Of Tau Misfolding And Liquid-Liquid Phase Separation Affected By Cd²⁺

Nerve fiber tangles（NFTs）formed by the misfolding of Tau protein is one of the main pathological features of Alzheimer’s disease（AD）.Tau protein is a highly soluble natural unfolded protein under normal physiological conditions,but under pathological conditions,it will misfold and aggregate.In recent years,many studies have found that Tau protein can undergo liquid-liquid phase separation（LLPS）,and it is generally believed that the Tau droplets formed by LLPS are the intermediate state of soluble Tau protein to insoluble Tau aggregates.LLPS can trigger Tau aggregation.Therefore,it is important to understand the molecular mechanisms of Tau protein misfolding and liquid-liquid phase separation for the pathogenesis of AD.At present,the environmental problems of cadmium pollution are becoming more and more serious.Studies have found the cadmium content in blood and cerebrospinal fluid of AD patients is significantly higher compared with healthy people.Cadmium may be one of the risk factors for AD,but its specific toxicity mechanism is still unclear.As belonging to the same family of elements,cadmium is very similar to zinc in chemical properties,and can competitively replace zinc in many physiological functions.Our previous studies have found that,in vitro and at the cellular level,Zn²⁺bind to Tau protein through Cys-291 and Cys-322 to promote Tau protein aggregation.We further explored whether Cd²⁺can also promote Tau aggregation by combining Cys-291 and Cys-322.Firstly,we extracted and purified the core fragment of Tau（K18-ΔK280）and its cysteine mutant in vitro,and using a series of experimental methods including Th T fluorescence spectroscopy,circular dichroism（CD）spectroscopy,transmission electron microscopy（TEM）,fluorescence microscopy,isothermal titration calorimetry（ITC）and fluorescence photobleaching recovery（FRAP）,we found that Cd²⁺interact with K18-ΔK280,and dramatically accelerates the liquid-liquid phase separation and aggregation of K18-ΔK280.However,substitution of Cys-291 and Cys-322 with Ala essentially eliminates such enhancing effects of Cd²⁺on the LLPS and fibrillization.At the same time,we constructed two SH-SY5Y cell lines that overexpress Tau-ΔK280 and its cysteine mutants.We have found that Cd²⁺can increase Tau-ΔK280 protein phosphorylation levels but have no obvious effect on Tau-ΔK280-C291/322A.Furthermore,our results revealed that the interaction of Cd²⁺with Tau protein may enhance the toxicity to nerve cells,mid-late cell apoptosis and oxidative stress on cells,and caused damage to the mitochondrial structure.Taken together,we propose that Cd²⁺could dramatically accelerate Tau protein misfolding and liquid-liquid separation via bridging cys-291 and Cys-322,thus inducing Tau protein aggregation in vitro and promoting Tau protein phosphorylation in cells.In addition,Cd²⁺may further enhance cytotoxicity by interacting with Tau protein.These results provide a new way to study the mechanisms for Tau protein accumulation and cytotoxicity regulated by Cd²⁺and the pathology of AD.