Structural and Functional Characterization of Neurotoxic Oligomers of Pro-aggregant Tau Repeat Domain

Abstract

Tau protein is a microtubule associated protein present abundantly in the neurons of the central nervous system where it stabilizes the axonal microtubules thereby providing structural architecture for the axons of neurons. Aggregation of Tau occurs in many neurodegenerative diseases collectively termed tauopathies including Alzheimer disease (AD) and frontotemporal dementia (FTD). The mutation ΔK280 of Tau was originally discovered in cases of FTD (Rizzu et al., 1999). In vitro it leads to a pronounced propensity of the protein to aggregate (Barghorn et al., 2000). The repeat domain of Tau protein with this pro-aggregant mutation (Tau^RDΔ) induces toxicity in transgenic mice and organotypic hippocampal slice culture models (Sydow et al., 2011, Messing et al., 2013). One current concept of Tau-mediated toxicity in Alzheimer disease and related tau dependent pathologies is that it is based on low-n oligomeric species, rather than higher aggregated forms (fibers and neurofibrillary tangles). To test this we characterized oligomers from Tau^RDΔ protein assembled and purified in vitro. Since Tau oligomers are in dynamic equilibrium during aggregation, we tried to capture and stabilize only the oligomeric forms of Tau using EGCG (Epigallocatechin gallate). EGCG reduces the formation of fibrils and increases the SDS stable oligomers. However, the oligomers are not separable by gel filtration chromatography. Therefore we stabilized the tau oligomers using a low concentration of glutaraldehyde as a cross-linking reagent. This yielded SDS stable low-n oligomers predominantly in the form of dimers, trimers, tetramers with very low amounts of higher order species. The cross-linked Tau^RDΔ oligomers can be purified by hydrophobic interaction chromatography with ~95% purity. They exhibit enhanced fluorescence with the dye ANS, arguing for an altered conformation (compared with monomers) and possibly exposed hydrophobic surface patches. However, they do not contain substantial ß-sheet structure, as analyzed by thioflavin S fluorescence and circular dichroism. Atomic force microscopy (AFM) of Tau^RDΔ oligomers reveals that the particles are roughly globular in shape, with diameters in the range 1.6-5.4 nm (AFM height values). The hydrodynamic radius of Tau^RDΔ oligomers (~5.2 nm) is dominated by that of tetramers, as measured by dynamic light scattering. The size of Tau^RDΔ oligomers reveals that they contain up to 4-5 molecules of Tau, consistent with the SDS gel analysis. The Tau^RDΔ oligomers do not exhibit global toxicity towards rat primary neurons when applied to the extracellular medium, as judged by MTT and LDH assays. However, functional impairment can be deduced from a pronounced (up to 50%) decrease of dendritic spines and a shift from mushroom-shaped to stubby spines. Consistent with this, the expression of cytoskeletal proteins which are necessary to maintain the mushroom spines is reduced. The neurons also show an increase in reactive oxygen species and influx of calcium. In summary, low-n oligomers of Tau^RDΔ do not cause gross changes in viability, but induce subtle functional defects, leading to an increase in Ca++ and ROS, and consequently to loss of spines and associated shape changes.<br /> Since Tau is an intracellular protein and the formation of oligomers occurs inside the cells, we introduced low-n Tau oligomers by protein transfection into SH-SY5Y cells and primary rat hippocampal neurons and analyzed them by flow cytometry and western blot analysis. This showed that only the cells transfected with Tau^RDΔ oligomers (but not monomers) induce the intracellular aggregation of Tau and recruitment of endogenous Tau into the aggregates. This is accompanied by the hyperphosphorylation of aggregated Tau. Although Tau^RDΔ oligomer transfected cells do not undergo cell death within 15 h of transfection, we found the presence of annexin V positive cells. When compared to monomers and fibrils, the oligomer transfected cells show a 5 fold increase in annexin V positive cells suggesting enhanced apoptosis. We conclude that Tau^RDΔ oligomers applied extracellularly cause degeneration of spines without affecting cell viability, whereas introducing oligomers intracellularly leads to Tau aggregation and apoptosis.