Accumulation of pathological tau species and memory loss in a conditional model of tauopathy

Abstract

Neurofibrillary tangles (NFTs) are a pathological hallmark of Alzheimer's disease and other tauopathies, but recent studies in a conditional mouse model of tauopathy (rTg4510) have suggested that NFT formation can be dissociated from memory loss and neurodegeneration. This suggests that NFTs are not the major neurotoxic tau species, at least during the early stages of pathogenesis. To identify other neurotoxic tau protein species, we performed biochemical analyses on brain tissues from the rTg4510 mouse model and then correlated the levels of these tau proteins with memory loss. We describe the identification and characterization of two forms of tau multimers (140 and 170 kDa), whose molecular weight suggests an oligomeric aggregate, that accumulate early in the pathogenic cascade in this mouse model. Similar tau multimers were detected in a second mouse model of tauopathy (JNPL3) and in tissue from patients with Alzheimer's disease and FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17). Moreover, levels of the tau multimers correlated consistently with memory loss at various ages in the rTg4510 mouse model. Our findings suggest that accumulation of early-stage aggregated tau species, before the formation of NFT, is associated with the development of functional deficits during the pathogenic progression of tauopathy.

Figure 1.

Tau multimers in rTg4510 mice. A, Western blot of total extracts from 3.5-month-old rTg4510 and age-matched tTa animals (without mutant P301L tau transgene). Tau migrating at ∼55 kDa can be detected with the human-specific E1 antibody in rTg4510 but not in tTa animals. Tau multimers migrating at ∼170 (called tau170) and ∼140 kDa (called tau140) can be seen in rTg4510 but not in age-matched tTa animals, when film is exposed for a longer time. B, Schematic location of epitopes on tau that are recognized by phosphorylation-independent antibodies tau12, E1, TauC3, and tau46. Tau12 and E1 are N-terminal, whereas tau46 is C-terminal. TauC3 (which does not detect tau multimers) is selective for tau cleaved at Asp421. C, Tau multimers are detected in total brain extracts from 3.5-month-old rTg4510 with a variety of tau antibodies (E1, CP13, PHF1, PS422, AT8, and T46). Age-matched tTa animals (without mutant P301L tau transgene) were used as a control. Tau170 and tau140 are seen by E1, CP13, PHF1, and T46, whereas PS422 and AT8 selectively detects tau170. D, Dephosphorylation leads to disappearance of tau170, although tau140 is still present.

Figure 2.

Biochemical properties of tau species. A, Schematic diagram of the tau extraction procedure that is based on previously published methology (Greenberg and Davies, 1990). For details, see Material and Methods. B, Brain extracts from 6.5-month-old rTg4510 were processed as described in A. Tau ∼55 kDa and tau 64 kDa are present in total extracts at 6.5 months of age (assessed by E1 antibody). Tau ∼55 kDa is found in soluble (S1) fraction, whereas hyperphosphorylated tau 64 kDa is found in the sarkosyl-insoluble (P3) fraction. Tau170 and tau140 are present in total lysate from 6.5-month-old rTg4510. Tau140 is mostly found in soluble (S1) fraction, whereas tau170 is mostly in the sarkosyl-insoluble (P3) fraction, as assessed by E1 antibody. Tau170 kDa, present in the sarkosyl-insoluble (P3) fraction, is strongly immunoreactive with AT8, whereas tau140, present in soluble (S1) fraction, exhibits very little immunoreactivity with AT8 antibody. C, Summary of how the various tau species are extracted into the different fractions. D, Analysis of the size of the different tau species in vivo. Size-exclusion chromatography was performed with total extracts from 4.5-month-old rTg4510. Tau140 and tau 55 kDa elute at a similar size and appear relatively small, whereas tau170 and tau 64 kDa are part of larger aggregates. Tau140 and tau170 have distinct sizes that do not overlap at this time point. Molecular weight scale corresponds to standards (see Materials and Methods) eluting at respective fractions shown on the graph (∼67 kDa eluted in fraction 34). E, Size-exclusion chromatography analysis using extracts of older 6.5-month-old rTg4510. Hyperphosphorylated species (64 kDa, tau170) are characterized by two distinct elution peaks, suggesting they are derived from two distinct populations of small and large aggregated species. Tau140 and tau170 have overlapping size ranges at 6.5 months of age.

Figure 3.

Changes in the levels of tau species between 1 and 4.5 months. A, Tau species present in total extracts in 1- and 4.5-month-old rTg4510. Abundant ∼55 kDa tau is detected at both time points, and the levels of this species appear to increase over this time period. Hyperphosphorylated tau 64 kDa can be detected at 4.5 months with the AT8 antibody (arrow, the top band in the doublet). Levels of the tau140 multimers are increased at 4.5 months, relative to 1 month, and tau170, also detected by the AT8 antibody, is detectable only at 4.5 months. B, Tau species present in the soluble (S1) fraction in 1- and 4.5-month-old rTg4510. Only tau140 is present in the soluble (S1) fraction, whereas tau170 is absent. Tau140 levels increase between 1 and 4.5 months similar to that observed in total fraction. C, Tau ∼55 kDa positively correlates with the levels of the multimers (tau140) in the soluble (S1) fraction. Data from 1-month-old animals are depicted in squares, whereas data from 4.5-month-old animals are in triangles; the same applies to D. D, Levels of tau140 multimers positively correlate with levels of tau170 in total extracts.

Figure 4.

Analysis of tau species present at 5.5-month-old rTg4510. A, Tau species present in total extracts in 5.5-month-old rTg4510. The abundant ∼55 kDa tau is observed with the E1 antibody without obvious variability between mice. Hyperphosphorylated 64 kDa tau is detected both by E1 and the phospho-epitope-specific AT8 antibody. Both tau170 and tau140 are seen with the E1 antibody, whereas only tau170 is recognized specifically by AT8. Levels of the tau multimers (tau170 and tau140) appear to be variable between different animals. B, Soluble (S1) fraction contains tau ∼55 kDa and tau140, but tau 64 kDa and tau170 are absent. C, Levels of multimers in total extracts exhibit a higher extent of variability across the group (SD of 0.33; 48% of the mean) compared with the levels of multimers in the soluble (S1) fraction (SD of 0.06; 21% of the mean).

Figure 5.

Relationship of tau species and memory impairment (memory index) in 5.5-month-old rTg4510. A, Levels of multimers (tau170 and tau140) in total extracts showed a significant negative correlation with memory index, whereas multimers in the soluble (S1) fraction did not. B, Levels of ∼55 kDa tau in total extracts or in the soluble (S1) fraction did not significantly correlate with memory index. C, Levels of hyperphosphorylated tau ∼64 kDa in total extracts did not exhibit significant correlation with memory index. However, the subpopulation of this species that was extracted into the sarkosyl-insoluble (P3) fraction correlated significantly and negatively with memory index.

Figure 6.

Changes in tau species after suppression of transgene expression with doxycycline between 6.5 and 8 months. A, Transgene suppression with doxycycline led to decreased levels of ∼55 kDa tau, 64 kDa tau, and tau multimers (tau170 and tau140) in total extracts. B, Transgene suppression with doxycycline also led to decreased levels of tau species in the soluble (S1) fraction. C, Levels of sarkosyl-insoluble 64 kDa tau and tau multimers in the P3 fraction were variable and did not show a consistent decrease with transgene suppression. D, Quantification of changes of different tau species from experiment in Figure 6A–C. The values of tau species in animals not treated with doxycycline was set as 100% (n = 10 for animals without doxycycline; n = 11 for animals treated with doxycycline). ***p < 0.001; NS p > 0.05. Two-sample two-tailed t test was used.

Figure 7.

Relationship of tau species and memory index during transgene suppression by doxycycline between 6.5–8 months. A, Levels of multimers in total extracts correlated significantly and negatively with memory index, whereas levels of multimers in the soluble (S1) fraction do not. B, Levels of ∼55 kDa tau in total extracts or in soluble (S1) fraction did not exhibit significant correlation with memory index. C, Levels of hyperphosphorylated tau 64 kDa in total extracts did not significantly correlate with memory, whereas sarkosyl-insoluble 64 kDa tau in P3 fraction does exhibit significant and negative correlation with memory index.

Figure 8.

Tau multimers in 3-month-old rTg4510. A, Levels of multimers in total extracts did not correlate significantly with memory index at 3.5 months. However, removal of an outlier animal with memory index of 8.5 (square) resulted in a significant and negative correlation between levels of tau140 multimers in total extracts and memory index (R² = 0.45; p = 0.01). B, Levels of multimers in soluble (S1) fraction did not correlate significantly with memory index at 3.5 months, and this analysis is not affected by removing the outlier animal (R² = 0.11; p = 0.16).

Figure 9.

NFT counts and memory index in 5.5- and 8-month-old rTg4510. A, The number of neurofibrillary tangles in individual rTg4510 mice and memory index displayed a borderline significant negative correlation (R² = 0.33; p = 0.05) in 5.5-month-old rTg4510. B, In 8-month-old rTg4510 mice, the number of neurofibrillary tangles correlated negatively with memory index only in the no-dox cohort (R² = 0.47; p = 0.02), whereas this correlation was completely lost after transgene suppression in the dox cohort (R² = 0.03; p = 0.33). Dox treatment was for 6 weeks from 6.5 to 8 months.

Figure 10.

Relationship of tau species to motor deficits in JNPL3 mice. A, Multimers were detected in spinal cord extracts from 2-month-old JNPL3 mice (1–3) but were absent in nontransgenic littermates (NT). Asterisk marks nonspecific band also present in nontransgenic littermates, which is attributable to nonspecific binding of the secondary antibody. B, Spinal cord and brain extracts from JNPL3 female mice at the age of 12 (1) and 14 months (2). Tau multimers (tau170 and tau140) and 64 kDa tau species are present in spinal cord but are not clearly visible in brain. C, Tau multimers (tau170 and tau140) and hyperphosphorylated 64 kDa are detected in JNPL3 mice severely affected with motor dysfunction, but neither of these species is seen in unaffected JNPL3 mice of similar age. D, Tau multimers are not present in mice overexpressing wild-type human tau. wt tau, Spinal cord extracts from mice overexpressing wild-type human tau (JN25 line) at 14-months of age; JNPL3, spinal cord extracts from 14-month-old JNPL3 mice. Note that there is no evidence of tau multimers in mice expressing human wild-type tau, despite significantly higher levels of total tau being loaded onto the blot, compared with JNPL3 mice. The apparent low level of tau 55/64 kDa in the JNPL3 mice seen on this Western blot is only attributable to this adjustment (∼3 times more protein was loaded for wild-type tau).

Figure 11.

Species similar to tau multimers are found in extracts from patients with AD and FTDP-17. A, Species similar to tau multimers are present in total extracts from the temporal cortex of FTDP-17 patients (D1, D2) with the N279K mutation but not in extracts from the cerebellum. Note that the low-intensity band present in control individuals (C) or in cerebellum is attributable to nonspecific binding of the secondary antibody (data not shown). B, Comparison of the molecular weight of the multimers in total extracts from the spinal cords of JNPL3 mice (affected, 12 and 14 months of age), brain extracts from 6.5-month-old rTg4510, an AD patient (Braak stage 5 and 6), and two FTDP-17 cases with the N279K mutation. In both panels, the human-specific E1 antibody was used for detection of tau, and GAPDH was used as a loading control.