Molecular Mechanism For Tau Filament Formation Induced By Heparin And Accelerated By Exposure To Lead And Crowded Cell-like Environment

Neurofibrillary tangles, principally composed of bundles of filaments formed by microtubule associate protein Tau, is a hallmark in a group of neurodegenerative diseases such as Alzheimer’s disease. Polyanionic cofactors such as heparin could induce Tau filament formation. Here we quantitatively characterize the interaction between recombinant human Tau fragment Tau244-372and heparin (average MW=7kDa) as well as heparin-induced fibril formation by using static light scattering, isothermal titration calorimetry, turbidity assays and transmission electron microscopy. Our data clearly show that at physiological pH, heparin and human Tau244-372form a tight1:1complex with an equilibrium association constant exceeding106M"1under reducing conditions, triggering Tau fibrillization. Heparin shows a moderate binding affinity (105M’1) to Tau244-372in the absence of dithiolthreitol, triggering Tau fibrillization too. Further fibrillization kinetics analyses show that the lag time appears to be approximately invariant up to a molar ratio of2:1, and then gets longer at larger ratios of heparin/Tau. The maximum slope representing the apparent rate constant for fibril growth increases sharply with substoichiometric ratios of heparin/Tau, and then decreases to some extent with ratios>1:1. The retarding effect of heparin in excess is due to the large increase in ionic strength of the medium arising from free heparin. Together, these results suggest that the formation of1:1complex of Tau and heparin plays an important role in the inducer-mediated Tau filament formation, providing clues to understanding the pathogenesis of neurodegenerative diseases.Lead is a potent neurotoxin for human being especially for the developing children, and Pb2+at high concentrations is found in the brains of patients with Alzheimer disease. However, it has not been reported so far whether Pb2+plays a role in the pathology of Alzheimer disease through interaction with human Tau protein and thereby mediates Tau filament formation. In this study, we have investigated the effect of Pb2+on fibril formation of recombinant human Tau fragment Tau244-372and its mutants at physiological pH. As revealed by thioflavin T and8-anilino-1-naphthalene sulfonic acid fluorescence, the addition of5-40μM Pb2+significantly accelerates the exposure of hydrophobic region and filament formation of wild-type Tau244-372on the investigated time scale. As evidenced by circular dichroism and Fourier transform infrared spectroscopy, fibrils formed by wild-type Tau244-372in the presence of5-40μM Pb2+contain more β-sheet structure than those formed by the protein in the absence of Pb2+. However, unlike wild-type Tau244-372, the presence of5-40μM Pb2+has no obvious effects on fibrillization kinetics of single mutants H330A and H362A and double mutant H330A/H362A, and fibrils formed by such mutants in the absence and in the presence of Pb2+contain similar amounts of β-sheet structure. The results from isothermal titration calorimetry show that one Pb+binds to one Tau monomer via interaction with His-330and His-362, with sub-micromolar affinity. We demonstrate for the first time that the fibrillization of human Tau protein is accelerated by exposure to lead via interaction with His-330and His-362. Our results suggest the possible involvement of Pb+in the pathogenesis of Alzheimer disease and provide critical insights into the mechanism of lead toxicity.The role of crowded intracellular environments on Tau protein misfolding is not clearly understood. The addition of Ficoll70and dextran70at50-150g/1significantly accelerates filament formation of Tau244-372on the investigated time scale, and the enhancing effects increase with the increase of concentrations of crowding agents. Our data demonstrate that macromolecular crowding enhances the formation of inter-molecular disulfide bond between C291and C322of Tau244-372.Such an enhancing effect is also observed for pathological Tau mutants. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of Alzheimer disease by enhancing the formation of inter-molecular disulfide bond of Tau protein.