Rapamycin increases survival in ALS mice lacking mature lymphocytes

Abstract

Background: Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. Disease pathophysiology is complex and not yet fully understood, but is proposed to include the accumulation of misfolded proteins, as aggregates are present in spinal cords from ALS patients and in ALS model organisms. Increasing autophagy is hypothesized to be protective in ALS as it removes these aggregates. Rapamycin is frequently used to increase autophagy, but is also a potent immune suppressor. To properly assess the role of rapamycin-induced autophagy, the immune suppressive role of rapamycin should be negated.

Findings: Autophagy is increased in the spinal cord of ALS mice. Dietary supplementation of rapamycin increases autophagy, but does not increase the survival of mutant SOD1 mice. To measure the effect of rapamycin in ALS independent of immunosuppression, we tested the effect of rapamycin in ALS mice deficient of mature lymphocytes. Our results show that rapamycin moderately increases the survival of these ALS mice deficient of mature lymphocytes.

Conclusions: Rapamycin could suppress protective immune responses while enhancing protective autophagy reactions during the ALS disease process. While these opposing effects can cancel each other out, the use of immunodeficient mice allows segregation of effects. Our results indicate that maximal therapeutic benefit may be achieved through the use of compounds that enhance autophagy without causing immune suppression.

Figures

Figure 1

Autophagy is increased in ALS mouse spinal cord. Western blot analysis of age-matched spinal cords of non-transgenic (non-tg, n = 4) and end stage SOD1G93A mice (n = 4) (A). Quantification of Western blot signal of LC3-II for non-transgenic and end stage SOD1G93A mice (B), mTOR (C) and p-mTOR (D). **p < 0.01.

Figure 2

Rapamycin delivered in chow increases autophagy in the spinal cord of RAG1−/− mice. Western blot analysis of LC3-II, mTOR and p-mTOR in the spinal cords of RAG1−/− mice fed vehicle or rapamycin containing chow for 3 months (A). Quantification of the levels of LC3-II in spinal cords of mice fed chow containing rapamycin for 3 months (n = 4) or vehicle chow (n = 4) (B). Quantification of mTOR (C), p-mTOR (D), ATG5 (E) and beclin-1 (F). *p < 0.05, **p < 0.01.

Figure 3

Rapamycin does not affect survival of SOD1G93A mice, but increases survival of SOD1G93A mice lacking mature lymphocytes. SOD1G93A mice fed vehicle (n = 9) or rapamycin chow (n = 7) relative weight (A), onset-free survival (B) and disease duration (C). RAG1−/− SOD1G93A mice fed vehicle (n = 7) or rapamycin chow (n = 12) relative weight (E), onset-free survival (F) and disease duration (G). Survival analysis of SOD1G93A mice that were fed vehicle (152.6 ± 1.8 days, n = 17) or rapamycin chow (153.1 ± 2.5 days, n = 17) (D). Survival analysis of RAG−/− SOD1G93A mice fed with vehicle (148.6 ± 2.0 days, n = 23) or rapamycin chow (155.1 ± 1.8 days, n = 19, p = 0.04) (H). Western blot analysis of the levels of LC3-II (I), mTOR (J), phosphorylated mTOR (p-mTOR) (K), p62 (L) and NeuN (M) for end stage RAG1−/− SOD1G93A mice fed vehicle (n = 4) or rapamycin-containing chow (n = 4 and n = 5 for the analysis of p62 and NeuN). *p < 0.05, **p < 0.01.