Figure 1.
The extensive cortical tau tangle pathology present in 5.5 months old vehicle (veh) treated P301S mice (A) was widely attenuated in long-term rapamycin (rapa) treated mice (B).
The lowering in tangle formation was most pronounced in the motor cortex (C: left vehicle treated/right rapamycin) and associated with reduced pathological tau hyperphosphorylation at the AT8 and AT100 epitopes (D, E). In parallel, cortical astrogliosis was diminished following rapamycin treatment (F). While there was a trend towards a reduction of the sparse tangles in the hippocampus, the advanced tau pathology in the brain stem however was not significantly ameliorated by rapamycin (G, H). Short-term treatment at 3 months of age for 6 weeks again resulted in a marked reduction of cortical tangles (I) (A–H: 5MT group; I: 6WT. A–C, G–I: Gallyas silver stain; D: AT8 IHC; E: AT100 IHC; F: GFAP IHC. Bar in A equals 300 µm in A and B, 150 µm in C, G and I, 75 µm in D–F, 600 µm in H).
Figure 2.
After long-term rapamycin treatment (n = 6), unbiased stereology confirmed a significant reduction of cortical tau tangles to only 14% of the amount of tangles seen in vehicle treated (n = 5) P301S mice (vehicle = 100%).
The number of AT8 stained cells containing hyperphosphorylated tau was reduced to 30% in aged rapamycin treated mice compared to controls (100%) (5MT group). A significant attenuation of Gallyas-stained (to 39% of controls) and AT8-positive cells (to 46% of controls) was also achieved by late short-term rapamycin treatment (6WT, n = 6/6). The reduction of tangles in the hippocampus (to 33% of controls) and the brain stem (to 72% of controls) did not reach the level of significance adjusted for multiple testing. Pairwise reduction of Gallyas or AT8 positive counts was analysed using one-sample T-tests per brain region. Significant p-values, adjusted for the multiple comparisons of all 6 tested groups by the Holm-Bonferroni method, are outlined as follows: *p<0.05, **p<0.01, and ***p<0.001.
Figure 3.
Levels of sarkosyl extracted insoluble tau were significantly reduced in the forebrain of P301S mice after 5 months of long-term rapamycin treatment (R, n = 6) when compared to vehicle treated mice (V, n = 5) (A, 5MT group; Western blot using BR134 antibody).
A comparable lowering of insoluble tau was obtained by late short-term rapamycin administration over 6 weeks (B; 6WT group, n = 6/6). In parallel, the accumulation of tau hyperphosphorylated at the AT8 and AT100 epitopes was significantly lowered (C; 6WT group, n = 6/6). Quantification of tau Western blots was subjected to unpaired T-tests and T-tests adjusted for unequal variances (Welch-Test), yielding both similar results. *p<0.05 and **p<0.01.
Figure 4.
Consistent with cerebral mTOR inhibition, phosphorylation of S6 (S6P) was significantly reduced following rapamycin administration (A, 5MT group, n = 4/4).
Compatible with an induced autophagy pathway, LC3II levels were increased upon rapamycin treatment (B, 6WT group, n = 6/6). High levels of the autophagy associated proteins p62 and LC3 were measured in aged vehicle treated P301S transgenic mice (C; 5MT Vehicle, n = 4). This accumulation of p62 and LC3 was prevented by long-term rapamycin administration, pointing towards a restored autophagic flux (C; 5MT Rapa, n = 4). Forebrain tissue was used and data was analyzed by ANOVA. *p<0.05, **p<0.01, and ***p<0.001.