Severe oligomeric tau toxicity can be reversed without long-term sequelae

Abstract

Tau is a microtubule stabilizing protein that forms abnormal aggregates in many neurodegenerative disorders, including Alzheimer's disease. We have previously shown that co-expression of fragmented and full-length tau in P301SxTAU62on tau transgenic mice results in the formation of oligomeric tau species and causes severe paralysis. This paralysis is fully reversible once expression of the tau fragment is halted, even though P301S tau expression is maintained. Whereas various strategies to target tau aggregation have been developed, little is known about the long-term consequences of reverted tau toxicity. Therefore, we studied the long-term motor fitness of recovered, formerly paralysed P301SxTAU62on-off mice. To assess the seeding competence of oligomeric toxic tau species, we also inoculated ALZ17 mice with brainstem homogenates from paralysed P301SxTAU62on mice. Counter-intuitively, after recovery from paralysis due to oligomeric tau species expression, ageing P301SxTAU62on-off mice did not develop more motor impairment or tau pathology when compared to heterozygous P301S tau transgenic littermates. Thus, toxic tau species causing extensive neuronal dysfunction can be cleared without inducing seeding effects. Moreover, these toxic tau species also lack long-term tau seeding effects upon intrahippocampal inoculation into ALZ17 mice. In conclusion, tau species can be neurotoxic in the absence of seeding-competent tau aggregates, and mice can clear these tau forms permanently without tau seeding or spreading effects. These observations suggest that early targeting of non-fibrillar tau species may represent a therapeutically effective intervention in tauopathies. On the other hand, the absent seeding competence of early toxic tau species also warrants caution when using seeding-based tests for preclinical tauopathy diagnostics.

Keywords: Alzheimer’s disease; neurodegeneration; oligomers; seeding; tau.

Figure 1

Early neurotoxic stress depends on full-length and Δtau co-expression and is reversible. (A) P301S heterozygous mice (P301Shet; n = 5) at 21 days of age show no signs of paralysis upon tail suspension, and histological tests with AT8 antibody did not detect hyperphosphorylated tau or Gallyas-positive tau fibrils in the tegmental reticular nucleus. (B) Hindlimbs of P301SxTAU62^on mice (n = 5) the same age as their heterozygous littermates are paralysed; upon histological characterization, paralysed P301SxTAU62^on mice show hyperphosphorylated tau but no Gallyas-positive tau fibrils in the same region. (C) At 3 weeks after halting doxycycline administration, P301SxTAU62^on-off mice (n = 5) show recovered motor functions and reverted tau hyperphosphorylation. Scale bar = 75 µm (A–C).

Figure 2

Absence of tau filaments in paralysed P301SxTAU62^on mice. (A) Immunoelectron microscopy shows HT7-positive tau filaments in total tau and sarkosyl extracts of brainstem tissue from a 6-month-old homozygous P301S mouse. (B) Total tau extract collected from a 3-week-old paralysed P301SxTAU62^on mouse reveals non-filamentous, oligomeric HT7 antibody positive tau structures while tau filaments are absent in its sarkosyl extract. (C) Absence of tau aggregates in a 3-week-old BL6 control mouse. The findings confirm that the severe neurotoxicity in P301SxTAU62^on mice does not depend on filamentous tau pathology, but is linked to hyperphosphorylated tau oligomers. Scale bar = 100 nm (A–C).

Figure 3

P301SxTAU62^on-off mice do not develop an impaired motor phenotype upon ageing. (A) Tail suspension revealed normal hindlimb spreading for BL6 mice, and pathological hindlimb spreading predominantly in heterozygous P301S transgenic littermates at 16 months of age, while this was less pronounced in age-matched P301SxTAU62^on-off (abbreviated P62^on-off) mice. (B) Rotarod test comparing BL6 (n = 10), P301Shet (n = 15), and P301SxTAU62^on-off mice (n = 13) at 16 months of age revealed that P301SxTAU62^on-off animals were not significantly more impaired than P301Shet mice that did not undergo early neurotoxic stress (P-value = 0.14). (C) Grid climbing of BL6 (n = 10), P301Shet (n = 17), and P301SxTAU62^on-off mice (n = 11) at 16 months also showed that P301SxTAU62^on-off mice were not experiencing heavier motor impairment when compared to their heterozygous littermates (P-value = 0.09). n.s. = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. See also Supplementary Table 1.

Figure 4

Aged P301SxTAU62^on-off mice show decreased hyperphosphorylated and fibrillar tau pathology compared to their heterozygous littermates. Histological tests comparing AT8 stained brainstem sections (A) of 16-month-old P301Shet (n = 7) and P301SxTAU62^on-off (abbreviated P62^on-off) mice (n = 7) revealed that the different performance in the behavioural tests was paralleled by different degrees of hyperphoshorylated tau pathology (B, P = 3.39 × 10⁻⁶). These results were also mirrored by Gallyas-stained brainstem sections (C) of 16-month-old P301Shet (n = 7), and P301SxTAU62^on-off mice (n = 7), which revealed different degrees of fibrillar tau pathology between the two groups (D, P = 7.77 × 10⁻⁵). ***P < 0.001. Scale bar = 200 µm (A and C). See also Supplementary Table 2.

Figure 5

Aged P301SxTAU62^on-off mice show lower total and soluble tau levels than their P301Shet littermates. Western blot analysis of total tau (HT7 antibody) revealed that P301Shet mice (n = 5) have a significantly higher level of total tau when compared to P301SxTAU62^on-off (abbreviated P62^on-off) mice (n = 5) at 16 months of age (A), which was confirmed by HT7/GAPDH ratio quantification (B, P = 1.32 × 10⁻⁶). Soluble tau levels (C) in P301Shet mice (n = 5) were also significantly higher than in P301SxTAU62^on-off mice (n = 5), as reflected by HT7/GAPDH ratio quantification (D, P = 0.0001). ***P < 0.001. See also Supplementary Table 2.

Figure 6

Total tau levels in young P301SxTAU62^on-off mice do not decrease compared to their heterozygous littermates. Western blot analysis (HT7; A) and HT7/GAPDH ratio (B) for both P301Shet (n = 6) and P301SxTAU62^on-off (abbreviated P62^on-off) mice (n = 6). Tau levels were not significantly different between the two groups (P = 0.35). n.s. = P > 0.05. Box plots with hinges, whiskers and outliers. See also Supplementary Table 2.

Figure 7

Seeding with high molecular weight tau results in the absence of fibrils in ALZ17 mice. ALZ17 mice seeded with brainstem (BS) homogenates from paralysed P301S homozygous (P301Shomo) mice (n = 5) revealed distinct granular tau pathology in CA1 (A) and the fornix (B), which was not detected in ALZ17 mice seeded with P301SxTAU62^on brainstem homogenate (n = 4). Scale bar = 37.5 µm (A and B).