Chronic rapamycin restores brain vascular integrity and function through NO synthase activation and improves memory in symptomatic mice modeling Alzheimer's disease

Abstract

Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, which extends lifespan and delays aging, halts the progression of AD-like disease in transgenic human (h)APP mice modeling AD when administered before disease onset. Here we demonstrate that chronic reduction of TOR activity by rapamycin treatment started after disease onset restored cerebral blood flow (CBF) and brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and improved cognitive function in symptomatic hAPP (AD) mice. Like acetylcholine (ACh), a potent vasodilator, acute rapamycin treatment induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO release in brain endothelium. Administration of the NOS inhibitor L-NG-Nitroarginine methyl ester reversed vasodilation as well as the protective effects of rapamycin on CBF and vasculature integrity, indicating that rapamycin preserves vascular density and CBF in AD mouse brains through NOS activation. Taken together, our data suggest that chronic reduction of TOR activity by rapamycin blocked the progression of AD-like cognitive and histopathological deficits by preserving brain vascular integrity and function. Drugs that inhibit the TOR pathway may have promise as a therapy for AD and possibly for vascular dementias.

Figures

Figure 1

Improved memory and restored cerebral blood flow (CBF) in AD mice treated with rapamycin after the onset of disease. (A) Spatial learning. While learning in AD mice was impaired, , (*P<0.001 and P<0.01, Bonferroni's post hoc test applied to a significant effect of genotype and treatment, F(3,188)=6.04, P=0.0014, repeated measures (RM) two-way ANOVA), performance of rapamycin-fed AD mice was indistinguishable from WT littermates. No significant interaction was observed between day number and genotype (P=0.96), thus genotype and treatment had the same effect at all times during training. Overall, learning was effective in all groups (F(4,188)=3.36, P=0.01, RM two-way ANOVA). (B) Spatial memory is restored by rapamycin treatment. While memory in control-fed AD mice was impaired, , (P values as indicated, Tukey's test applied to a significant effect of genotype and treatment (P<0.0001), one-way ANOVA), memory in rapamycin-fed AD mice was indistinguishable from WT littermate groups and was significantly improved compared with control-fed AD mice (P=0.03). (C–G) Rapamycin restores CBF in AD mice. (C) CBF maps and regional CBF maps (E) of representative control- and rapamycin-treated WT and AD mice obtained by MRI. (D) Decreases in CBF in AD mice are abrogated by rapamycin treatment (P as indicated, Bonferroni's test on a significant effect of genotype and treatment on CBF, F(1,16)=14.54, P=0.0015, two-way ANOVA). (F and G) Decreased hippocampal (F) but not the thalamic (G) CBF in AD mice is restored by rapamycin treatment (P as indicated, Bonferroni's test on a significant effect of treatment on CBF, F(1,16)=13.62, P=0.0020, two-way ANOVA). Data are means±s.e.m. Panels (A–B), n=10–17 per group. Panels (C–G), n=6 per group.

Figure 2

Increased vascular density without changes in glucose metabolism in rapamycin-treated brains of AD mice. (A) Cerebral metabolic rate of glucose (CMRGlc) maps of representative control- and rapamycin-treated WT and AD mice obtained by positron emission tomography. (B) Cerebral metabolic rate of glucose as standardized uptake values (SUV) for the region of interest were not different among experimental groups (F(1,20)=0.77, P=0.39 for the effect of genotype and F(1,20)=3.63, P=0.071 for the effect of treatment, two-way analysis of variance). (C) Magnetic resonance angiography images of brains of rapamycin-treated WT and AD mice. Representative regions showing loss of vasculature in control-treated AD mice and its restoration in rapamycin-treated animals are denoted by arrows. (D) Decreased cerebral vessel density in control-treated AD mice is abrogated by rapamycin treatment (P as indicated, Bonferroni's post hoc test applied to a significant effect of treatment on vascular density, F(1,16)=24.47, P=0.0001, two-way ANOVA). Data are means±s.e.m. n=6 per group.

Figure 3

Reduced cerebral amyloid angiopathy (CAA) and Aß plaques in rapamycin-treated AD mice. (A–F). Reduced Aß plaques in rapamycin-treated AD mice. (A and B) Representative images of hippocampi of control- (A) and rapamycin-treated (B) AD mice incubated with an Aß-specific antibody. (C and D) secondary antibodies only. (D) 4',6-diamidino-2-phenylindole fluorescence of the field in C. (E and F) Quantitative analyses of Aß immunoreactivity (P as indicated). (G) Decreased numbers of Thioflavin S-positive amyloid deposits in rapamycin-treated AD mice. (H–I) Improved brain tissue integrity in rapamycin-treated AD mice. (H) Representative images of heat maps displaying apparent diffusion coefficients for water in brains of control- and rapamycin-treated mouse AD or WT brains. (I) Quantitative analyses of whole-brain average diffusivity values. (J–L) Reduced CAA in rapamycin-treated AD mouse brains. Representative maximum intensity projections of stacks of confocal images of control- (J) and rapamycin- (K) treated AD mouse brain sections reacted with Aß-specific antibodies and with tomato lectin to illuminate brain vasculature. (L) Quantitative analyses of colocalization of Aß immunoreactivity and tomato lectin labeling of brain vasculature indicate reduced Aß deposition on vessels in rapamycin-treated AD mice (P as indicated). (M and N) Reduced microhemorrhages in rapamycin-treated AD mouse brains. (M) Representative hemosiderin deposit. (N) Quantitative analyses of numbers of hemosiderin deposits (P as indicated). Significance of differences between group means was determined using two-tailed unpaired Student's t-test, or one-way ANOVA followed by Tukey's multiple comparisons test or two-way ANOVA followed by Bonferroni's test. Data are means±s.e.m. n=6 to 8 per experimental group.

Figure 4

Rapamycin-induced nitric oxide (NO)-dependent vasodilation in brain. (A) Rapamycin-induced cortical vasodilation. In vivo imaging of cortical vasculature illuminated by FITC-Dextran (green). Arrows indicate areas of maximal vasodilatory effect 10 minutes after rapamycin administration (tabbed white lines). (B) Quantitative analyses of changes in diameter for cortical vessels of different sizes (P as indicated, Bonferroni's test applied to a significant effect of treatment, F(1,20)=154.12, P<0.0001, two-way ANOVA). (C) Quantitative analyses of changes in diameter for cortical vessels of different sizes 10 minutes after treatment with acetylcholine (ACh, P as indicated, Bonferroni's test applied to a significant effect of treatment, F(1,15)=2900.20, P<0.0001, two-way ANOVA). (D) Rapamycin-induced vasodilation is preceded by NO release. Arrowheads indicate regions of local NO release by DAF-FM fluorescence (green) followed by dilation of rhodamine-dextran labeled vasculature (red) in vivo. (E) Rapamycin-induced vasodilation requires NO synthase activity. L-NG-Nitroarginine methyl ester (L-NAME) administration abolished rapamycin-induced NO release (DAF-FM fluorescence) and dilation of cortical vasculature. (F) Acetylcholine-induced vasodilation is preceded by NO release. Uniform NO release (DAF-FM fluorescence, green) preceded vasodilation induced by ACh. (G and H) Ser1176 phosphorylation of endothelial nitric oxide synthase (eNOS) in brain vasculature. (G) eNOS and phospho-eNOS (p-eNOS, Ser1176) immunoreactivity in lysates of vasculature purified from brains at the indicated times after injection of rapamycin or ACh. (H) The relative ratio of p-eNOS(Ser1176) to total eNOS was quantified (P as indicated, Tukey's test applied to a significant effect of treatment, P<0.0052, one-way ANOVA). n=3 to 4 per group. (I) NO synthase activity is required for rapamycin-induced preservation of cerebral blood flow (CBF). Four weeks of intermittent L-NAME administration (once every other day) abolished rapamycin-mediated preservation of CBF in AD mice (P as indicated, Tukey's test applied to a significant effect of treatment, P<0.0001, one-way ANOVA). n=6 per group. (J and K) Chronic rapamycin treatment does not affect eNOS levels in AD and WT mouse brains. (J) Endothelial nitric oxide synthase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoreactivity in lysates of brains from control- and rapamycin-treated mouse brains. (K) Quantitative analyses of eNOS immunoreactivity normalized to GAPDH levels. n=4 per group. Data are means±s.e.m.

Figure 5

Proposed pathway for target-of-rapamycin (TOR) inhibition of nitric oxide synthase (NOS)-dependent regulation of cerebral blood flow (CBF) in AD mouse brains.