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

Abstract

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.

Fig. 1. Filament morphology

(a) Distribution of 27 amino acid residues affected by pathological missense tau mutations currently tabulated at http://www.molgen.ua.ac.be/FTDMutations (Rademakers et al. 2004) and depicted on isoform 2N4R. This isoform contains alternatively spliced exons 2 and 3 (E2 and E3), each of which encodes an acidic 29-residue segment, and exon 10 (E10), which encodes an additional microtubule binding repeat sequence. Mutants R5L, G272V, P301L, V337M, and R406W modeled herein span the tau molecule and are distinguished graphically with raised hollow symbols. (b–g) Full-length wild-type 2N4R tau (b) and mutants R5L (c), G272V (d), P301L (e), V337M (f), and R406W (g) were incubated (1 μM concentration) without agitation in the presence of 100 μM Thiazine red (24 h at 37°C), spotted onto copper Formvar-carbon mesh grids, stained with 2% uranyl acetate, and viewed by transmission electron microscopy. All tau species produced unbranched filaments ~16 nm in diameter with no obvious differences in morphology or length distribution. Scale bar = 200 nm.

Fig. 2. Effect of tau mutations on critical concentration

Wild-type 2N4R tau (●) and mutants R5L (○), G272V (▼), P301L (△), V337M (■), and R406W (□) were incubated at varying bulk concentrations in the presence of Thiazine red inducer for 24 h at 37°C, then assayed for filament formation by electron microscopy. (a) Plot of mean total filament length against bulk protein concentration, where each data point represents the mean ± SD of triplicate determinations and the solid lines represent best fit of the data points to linear regression. The abscissa intercept, which was obtained by extrapolation (dotted lines), was used to estimate critical concentration (K_crit). (b) Replot of data from Panel (a), where each bar represents the K_crit for examined tau species relative to wild-type 2N4R (dashed line). Both G272V and P301L tau mutants aggregated with significantly lower K_crit values than did wild-type 2N4R. **, p < 0.01 versus wild-type 2N4R tau.

Fig. 3. Tau mutants share a common sensitivity to aggregation inducer Thiazine red

Wild-type 2N4R (●) and mutants R5L (○), G272V (▼), P301L (▵), V337M (■), and R406W (□) were incubated (24 h at 37°C) at constant supersaturation (i.e., 0.5 μM above K_crit) in the presence of varying concentrations of Thiazine red, and then assayed for filament formation by electron microscopy. Each data point represents mean total filament lengths/field from triplicate determinations, whereas the solid curve is drawn solely to aid visualization. Under these conditions, the concentration effect relationship for Thiazine red was similar for all tau species.

Fig. 4. Estimation of extention association and dissociation rate constants

(a) Tau filaments prepared from wild-type 2N4R (●) and mutants R5L (○), G272V (▼), P301L (▵), V337M (■), and R406W (□) in the presence of 100 μM Thiazine red at 37°C were diluted 10-fold into assembly buffer containing Thiazine red, and the resultant disaggregation was followed as a function of time by electron microscopy. Each data point represents total filament length per field ± SD (n = 3 observations), whereas the solid lines represent best fit of the data points to linear regression. The first order decay constant k_app was estimated from each regression, and used in conjunction with measured K_crit values to estimate k_e− and k_e+ as described in the Experimental Procedures section. (b) Replot of data from Panel (a), where each bar represents k_e−and k_e+ values for each examined tau species relative to wild-type 2N4R (dashed line). Filaments composed of mutants G272V and P301L extended significantly faster than did 2N4R tau. **, p < 0.01 versus wild-type 2N4R tau.

Fig. 5. Effects of tau mutations on aggregation time course

(a) Wild-type 2N4R (●) and mutants R5L (○), G272V (▼), P301L (▵), V337M (■), and R406W (□) were incubated (37°C) at constant supersaturation (i.e., 0.2 μM above K_crit) in the presence of 100 μM Thiazine red, and then assayed for filament formation as a function of time. Each data point represents mean total filament lengths/field (expressed as % plateau length) calculated from triplicate electron microscopy images whereas each normalized curve represents best fit of the data points to a three parameter Gompertz growth function. The fits were used to calculate lag time as described in the Experimental Procedures section. (b) Replot of data from Panel (a), where each bar represents the lag time ± SE calculated from each Gompertz regression normalized to wild-type isoform 2N4R (dotted line). Mutants R5L, G272V, P301L, and V337M aggregated significantly faster than wild-type 2N4R when compared at constant supersaturation. *, p < 0.05; **, p < 0.01 versus wild-type 2N4R tau.

Fig. 6. Mutants R5L and V337M yield increased filament number concentration

(a) Mathematical simulation of reaction time course for R5L, V337M, and wild-type 2N4R tau at constant supersaturation (i.e., 0.2 μM above K_crit) using equations 3 – 5 and the kinetic parameters summarized in Table 1. Each curve represents the predicted evolution of filament number concentration (c_p) as function of time. The simulations predict that the decreases in K_n associated with R5L and V337M should yield increases in filament number concentration relative to 2N4R tau under these conditions. (b) R5L, V337M, and wild-type 2N4R were incubated (37°C) under identical conditions as in Panel (a) and then subjected to electron microscopy analysis at 24h. Each column represents mean total filament number/field ± S.D. (normalized to wild-type 2N4R tau) calculated from quadruplicate electron microscopy images. Consistent with mathematical simulation, R5L and V337M yielded significantly more tau filaments relative to wild-type 2N4R tau at reaction plateau. **, p < 0.01; n.s., p > 0.05.

Fig. 7. Effect of FTLD mutations on the fibrillization pathway

Normal tau binds tightly to microtubules, but dissociates upon phosphorylation to form free tau, which exists as a natively disordered, assembly incompetent monomer (U_x). A conformational change to an assembly competent state accelerates polymerization (U_c). Once assembly competent species form, the rate-limiting step in tau fibrillization is formation of dimer, which represents the thermodynamic nucleus (N). Following nucleation, extension occurs through further addition of assembly competent monomers to the filament (F) ends. Tau mutations promote fibrillization at multiple points in the pathway. Reactions characterized herein are shown in green lettering, whereas those reported in the literature are shown in red lettering. See text for details.