The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of Huntington's disease

Abstract

Background: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD). In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting drug everolimus in the R6/2 mouse model of HD.

Results: Everolimus decreased phosphorylation of the mTOR target protein S6 kinase indicating brain penetration. However, everolimus did not activate brain macroautophagy as measured by LC3B Western blot analysis. Everolimus protected against early declines in motor performance; however, we found no evidence for neuroprotection as determined by brain pathology. In muscle but not brain, everolimus significantly decreased soluble mutant huntingtin levels.

Conclusions: Our data suggests that beneficial behavioral effects of everolimus in R6/2 mice result primarily from effects on muscle. Even though everolimus significantly modulated its target brain S6 kinase, this did not decrease mutant huntingtin levels or provide neuroprotection.

Figure 1

Everolimus penetrates the blood-brain barrier in R6/2 HD transgenic mice. Plasma (A) and brain (B) everolimus concentrations were measured 4 and 24 hours after the last dose following two weeks of treatment (black-bar = 10 μmol/kg, cross-hatched bar = 30 μmol/kg). Brain/plasma ratios (calculated as the ratio of brain to plasma concentration × 100) are higher than the reported [29] level of blood contamination (~6%) at 24, but not at 4 hours (C). Shown are mean ± SE. n = 3.

Figure 2

Mutant huntingtin and everolimus decrease S6 kinase phosphorylation in mouse HD brain. High (20 μmol/kg) but not low (10 μmol/kg) dose everolimus significantly decreases phosphorylation of S6 kinase at the S235-236 epitopes in cortex (A) and striatum (B). High-dose everolimus also significantly decreases phosphorylation of S6-kinase at the S240-244 epitopes in cortex (C) and striatum (D). S6 kinase phosphorylation at S235-236 is decreased in vehicle-treated HD compared to wild-type mice (A). Mice were treated from 4-12 weeks and sacrificed 4 hours after the last dose. X-axes show genotype-dose combinations. TG = transgenic, WT = wild-type. n = 10. p-values: * = < 0.05, ** = < 0.01

Figure 3

Everolimus provides early benefit against motor decline in R6/2 HD mice. A-B. Bars: solid black (a) = HD vehicle: cross-hatched; light gray (b) = low dose, dark gray (c) = high dose, white (d) = wild-type vehicle treated. WT to HD vehicle comparison significance bars are omitted for clarity. A. Low and high dose everolimus (10 and 20 μmol/kg, respectively) provide early, but not late, benefit against the decline in Rota-rod endurance in R6/2 HD mice. B. High dose everolimus has a small negative effect on body weight. Everolimus treatment does not improve measures of survival (C), brain weight (D), striatal volume (E), cell body volume (F) and nuclear aggregate density (G). Mice were dosed from 4-weeks of age. Shown are means + SE. X-axes show genotype-dose combinations (D-F). TG = transgenic, WT = wild type. n = 15-20 for behavior and survival (A-C), n = 12 for quantitative pathology (D-G). p-values: * = < 0.05

Figure 4

Everolimus does not affect LC3BII levels in mouse HD brain. There is no significant effect of everolimus on normalized LC3BII levels in striatum and cortex (A). There is a trend towards increased normalized LC3BII levels in muscle of everolimus treated R6/2 mice (global p-value = 0.12) (B). Mice were treated from 4-12 weeks and sacrificed 4-hours after the last dose. X-axes show genotype-dose combinations. TG = transgenic, WT = wild-type. n = 10.

Figure 5

Everolimus decreases LAMP1 expression in muscle, but not brain. A-B. Lysosome-associated membrane protein 1 (LAMP1) is increased in mouse HD striatal and cortical neurons (green) as measured by quantitative immunofluorescence. Ethidium dimer DNA/RNA counter stain (red). n = 5. C-D. There is no effect of everolimus on LAMP1 levels in striatum or cortex as measured by Western blot analysis. E-F. LAMP1 is increased in quadriceps femoris muscle; levels are significantly reduced by high-dose everolimus. Shown are means and standard errors. X-axes show genotype-dose combinations (C, D and F). TG = transgenic, WT = wild-type. n = 8-10. p-values: * = p < 0.05, ** = p < 0.01.

Figure 6

Everolimus decreases soluble mutant huntingtin levels in muscle, but not brain. Relative quantification of soluble mutant huntingtin by time-resolved FRET. There is no effect of everolimus on soluble mutant huntingtin levels in striatum and cortex (A). Everolimus significantly decreases mean soluble mutant huntingtin in quadriceps muscle (B). X-axes = dose (μmoles/kg). ** = p < 0.01, n = 12