Rapamycin rescues vascular, metabolic and learning deficits in apolipoprotein E4 transgenic mice with pre-symptomatic Alzheimer's disease

Abstract

Apolipoprotein E ɛ4 allele is a common susceptibility gene for late-onset Alzheimer's disease. Brain vascular and metabolic deficits can occur in cognitively normal apolipoprotein E ɛ4 carriers decades before the onset of Alzheimer's disease. The goal of this study was to determine whether early intervention using rapamycin could restore neurovascular and neurometabolic functions, and thus impede pathological progression of Alzheimer's disease-like symptoms in pre-symptomatic Apolipoprotein E ɛ4 transgenic mice. Using in vivo, multimodal neuroimaging, we found that apolipoprotein E ɛ4 mice treated with rapamycin had restored cerebral blood flow, blood-brain barrier integrity and glucose metabolism, compared to age- and gender-matched wild-type controls. The preserved vasculature and metabolism were associated with amelioration of incipient learning deficits. We also found that rapamycin restored the levels of the proinflammatory cyclophilin A in vasculature, which may contribute to the preservation of cerebrovascular function in the apolipoprotein E ɛ4 transgenics. Our results show that rapamycin improves functional outcomes in this mouse model and may have potential as an effective intervention to block progression of vascular, metabolic and early cognitive deficits in human Apolipoprotein E ɛ4 carriers. As rapamycin is FDA-approved and neuroimaging is readily used in humans, the results of the present study may provide the basis for future Alzheimer's disease intervention studies in human subjects.

Keywords: APOE4; Alzheimer’s disease; Brain imaging; blood–brain barrier; cerebral blood flow; cerebral glucose metabolism; cognition; inflammation; rapamycin.

Figure 1.

Experimental design and timeline. APOE4 transgenic mice can have vascular defects as early as two weeks (0.5 months of age); metabolic/synaptic dysfunctions at four months of age; memory decline at 12 months of age.^, We obtained female mice at one month of age to test the hypothesis that restoring vascular functions can further impede the decline of metabolic and cognitive functions. After baseline cerebral blood flow (CBF) was measured, rapamycin diet was continuously supplied for six months. CBF was measured longitudinally using MRI after 1, 3, and 6 months of feeding. At the end-point of the study (i.e. mice at 7 months of age), blood–brain barrier (BBB) integrity was evaluated using MRI and cerebral metabolic rate of glucose (CMR_Glc) was measured by PET (N = 6). A separate group of mice (N = 15) underwent behavioral assessment with Morris water maze (MWM). After MWM, mice were sacrificed and brain tissues were used for mechanistic pathway analyses using Western blot.

Figure 2.

Rapamycin restoring brain vascular functions of APOE4 mice. (a) Representative baseline (pre-treated) CBF images of a WT-control and APOE4-control mice. The color code indicates the level of CBF in a linear scale; (b) comparison of CBF between pre- and one-month-post-treatment of a APOE4 mouse; (c) the time course of the global CBF changes among the three groups; (d) cortical CBF (in ml/g/min) of the mice at seven months of age; (e) hippocampal CBF (in ml/g/min) of the mice at seven months of age; (f) temporal lobe CBF (in ml/g/min) of the mice at seven months of age; (g) BBB leakage (indicated by the arrows) of the mice at seven months of age. Data are presented as mean ± standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001;^# no difference between WT-control and APOE4-Rapa. n.s.: non-significant; APOE4: apolipoprotein E4.

Figure 3.

Rapamycin restoring brain metabolic functions of APOE4 mice. (a) CMR_Glc maps of mice at seven months of age; the color code indicates the level of CMR_Glc in a linear scale. Quantitative CMR_Glc in the (b) cortex; (c) hippocampus; and, (d) temporal lobe of the three groups of mice. Data are presented as mean ± standard error of the mean. **P < 0.01; ***P < 0.001; n.s.: non-significant; APOE4: apolipoprotein E4.

Figure 4.

Rapamycin ameliorates incipient learning phenotypes of APOE4 mice. (a) Time in seconds to reach a hidden platform. Time F (3, 117) = 37.51, P < 0.0001; Treatment F (2, 39) = 0.496, P = 0.613; Interaction F (6, 117) = 5.919, P < 0.0001; (b) total distance swam during trial. Distance F (3, 117) = 30.06, P < 0.0001; treatment F (2, 39) = 0.079, P = 0.024; interaction F (6, 117) = 5.704, P < 0.0001; (c) number of times mice crossed over the platform location in the probe trial. n.s., P = 0.091; (d) average swim speeds during training. Speed F (3, 117) = 0.416, P = 0.742; treatment F (2, 39) = 1.59, P = 0.217; interaction F (6, 117) = 4.986, P < 0.0001; (e) percent of trial spent floating. Time floating F (3, 117) = 0.821, P = 0.485; treatment F (2, 39) = 2.601, P = 0.087; interaction F (6, 117) = 1.19, P = 0.316; (f) percent of trial spent in thigmotaxis. Time in thigmotaxis F (3, 117) = 39.25, P < 0.0001; treatment F (2, 39) = 5.92, P = 0.006; interaction F (6, 117) = 2.212, P = 0.047. Data are presented mean ± standard error of the mean of 4 trials/animal/day. All asterisks (*) indicate a significant difference between WT-control vs. APOE4-control, all pound signs (^#) indicate a significant difference between APOE4-control vs. APOE4-Rapa, and all money signs ($) indicate a significant difference between WT-control vs. APOE4-Rapa. Behavioral data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. APOE4: apolipoprotein E4.

Figure 5.

Rapamycin restoring CypA levels in brain vasculature of APOE4 mice. (a,b) Immunoblots of cortical CypA lysates and the corresponding quantitative analyses; (c,d) immunoblots of microvascular CypA lysates and the corresponding quantitative analyses; (e,f) immunoblots of cortical NF-κB lysates and the corresponding quantitative analyses; (g,h) immunoblots of microvascular NF-κB lysates and the corresponding quantitative analyses. Data are presented as mean ± standard error of the mean. *P < 0.05. CypA: cyclophilin A; NF-κB: nuclear factor-κb.