Haishan Yin

Tau aggregation and toxicity in a cell culture model of tauopathy.

Intracellular aggregation of the microtubule-associated protein tau into filamentous inclusions is a defining characteristic of Alzheimer disease. Because appearance of tau-aggregate bearing lesions correlates with both cognitive decline and neurodegeneration, it has been hypothesized that tau aggregation may be directly toxic to cells that harbor them. Testing this hypothesis in cell culture has been complicated by the resistance of full-length tau isoforms to aggregation over experimentally tractable time periods. To overcome this limitation, a small-molecule agonist of the tau aggregation reaction, Congo red, was used to drive aggregation within HEK-293 cells expressing full-length tau isoform htau40. Formation of detergent-insoluble aggregates was both time and agonist concentration dependent. At 10 μm Congo red, detergent-insoluble aggregates appeared with pseudo-first order kinetics and a half-life of approximately 5 days. By 7 days in culture, total tau levels increased 2-fold, with ∼30% of total tau converted into detergent-insoluble aggregates. Agonist addition also led to rapid losses in the tubulin binding activity of tau, although tau was not hyperphosphorylated as judged by occupancy of phosphorylation sites Ser396/Ser404. Tau aggregation was associated with decreased viability as detected by ToPro-3 uptake. The results, which establish a new approach for analysis of tau aggregation in cells independent of tau hyperphosphorylation, suggest that conformational changes associated with aggregation are incompatible with microtubule binding, and that toxicity associated with intracellular tau aggregation is not acute but develops over a period of days.

C-terminal truncation modulates both nucleation and extension phases of τ fibrillization

Proteolytic post‐translational modification has been proposed as an early stage event in the aggregation of τ protein and formation of neurofibrillary lesions in Alzheimers disease. Caspases and other proteases cleave τ in vivo at discrete locations including Asp421 and Glu391. Both cleavage products are prone to aggregation relative to wild‐type, full‐length τ protein. To determine the mechanism underlying this effect, the fibrillization of τ truncated after Asp421 and Glu391 residues was characterized in a full‐length four‐repeat τ background using quantitative electron microscopy methods under homogeneous nucleation conditions. Both C‐terminal truncations decreased critical concentration relative to full‐length τ, resulting in more filament mass at reaction plateau. Moreover, truncation directly augmented the efficiency of the nucleation reaction. The results suggest the mechanism through which C‐terminal proteolysis can modulate τ filament accumulation depending on whether it precedes or follows nucleation.

Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps

Mutations in the MAPT gene encoding tau protein lead to neurofibrillary lesion formation, neurodegeneration, and cognitive decline associated with frontotemporal lobar degeneration. While some pathogenic mutations affect MAPT introns, resulting in abnormal splicing patterns, the majority occur in the tau coding sequence leading to single amino acid changes in tau primary structure. Depending on their location within the polypeptide chain, tau missense mutations have been reported to augment aggregation propensity. To determine the mechanisms underlying mutation‐associated changes in aggregation behavior, the fibrillization of recombinant pathogenic mutants R5L, G272V, P301L, V337M, and R406W prepared in a full‐length four‐repeat human tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Results indicated that the mutants differ from each other and from wild‐type tau in their aggregation propensity. G272V and P301L mutations increased the rates of both filament nucleation and extension reactions, whereas R5L and V337M increased only the nucleation phase. R406W did not differ from wild‐type in any kinetic parameter. The results show that missense mutations can directly promote tau filament formation at different stages of the aggregation pathway.