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, 34 (23), 7988-98

Genetic Reduction of Mammalian Target of Rapamycin Ameliorates Alzheimer's Disease-Like Cognitive and Pathological Deficits by Restoring Hippocampal Gene Expression Signature

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Genetic Reduction of Mammalian Target of Rapamycin Ameliorates Alzheimer's Disease-Like Cognitive and Pathological Deficits by Restoring Hippocampal Gene Expression Signature

Antonella Caccamo et al. J Neurosci.

Abstract

Elevated mammalian target of rapamycin (mTOR) signaling has been found in Alzheimer's disease (AD) patients and is linked to diabetes and aging, two known risk factors for AD. However, whether hyperactive mTOR plays a role in the cognitive deficits associated with AD remains elusive. Here, we genetically reduced mTOR signaling in the brains of Tg2576 mice, a widely used animal model of AD. We found that suppression of mTOR signaling reduced amyloid-β deposits and rescued memory deficits. Mechanistically, the reduction in mTOR signaling led to an increase in autophagy induction and restored the hippocampal gene expression signature of the Tg2576 mice to wild-type levels. Our results implicate hyperactive mTOR signaling as a previous unidentified signaling pathway underlying gene-expression dysregulation and cognitive deficits in AD. Furthermore, hyperactive mTOR signaling may represent a molecular pathway by which aging contributes to the development of AD.

Keywords: Aβ; amyloid-β; autophagy; plaques; tangles; tau.

Figures

Figure 1.
Figure 1.
Schematic representation of the breeding strategy used to remove one copy of the mTOR gene from the forebrain of the Tg2576 mice. Abbreviations: “+” indicates the presence of a transgene (APP or CRE); “0” indicates the lack of such transgene; “fl” indicates the presence of a floxed allele; and “wt” indicates the presence of a wild-type allele.
Figure 2.
Figure 2.
Removing one copy of the mTOR gene decreases mTOR levels and signaling. A, Representative Western blots of proteins extracted from the hippocampi of 2-month-old mice probed with the indicated antibodies. BF, Quantification of the indicated proteins (n = 5/genotype) showed that mTOR levels and the levels of phosphorylated p70S6K and 4EBP1 were significantly decreased in the hippocampi of mice lacking one copy of the mTOR gene. G, Representative Western blots of proteins extracted from the hippocampi of 12-month-old mice probed with the indicated antibodies. H–L, Quantification of the indicated proteins (n = 5/genotype) showed that even as the mice age, mTOR levels and signaling were significantly lower in mice lacking one copy of the mTOR gene. Data were generated by normalizing the levels of the protein of interest to β-actin loading control. Results presented as means ± SEM and analyzed by one-way ANOVA with Bonferroni's correction; *p < 0.05, **p < 0.005.
Figure 3.
Figure 3.
Decreasing mTOR signaling rescues learning and memory deficits. A, Learning curve of 2-month-old mice trained in the spatial reference version of the MWM. Mice were trained to swim to a hidden platform in a tank using extramazal visual cues. All genotypes showed significant improvements over the 5 d of training. Each day represents the average of four training trials. B–D, Reference memory, tested 24 h after the last training trial was not statistically different among the four groups. E, Average swim speed during the probe trials. F, Learning curve of 12-month-old mice in the MWM. All genotypes significantly learned the task; however, at days 4 and 5 the APP mice needed significantly more time to find the hidden platform than the other three genotypes. G, H, Average distance traveled and swim speed of 12-month-old mice during day 4 of training. I, J, Average distance traveled and swim speed of 12-month-old mice during day 5 of training. K–M, Reference memory, tested 24 h after the last training trial was significantly impaired in the APP mice compared with the other three genotypes. Notably, the APP/mTOR+/− mice performed as well as the CTL mice and significantly better than the APP mice. N, Average swim speed during the probe trials was not statistically different among the four genotypes. n = 10/genotype at 2 months of age; n = 14/genotype at 12 months of age. Data are presented as means ± SEM and were analyzed by two-way ANOVA with Bonferroni's correction; *p < 0.05.
Figure 4.
Figure 4.
APP/mTOR+/− mice have less Aβ pathology than APP mice. A, B, Representative microphotographs of brain section immunostained with an Aβ42 specific antibody. C, Quantitative analysis of the Aβ immunohistochemistry showed a significant decrease in Aβ load following removal of one copy of the mTOR gene (n = 7/genotype). D, E, Soluble levels of Aβ40 and Aβ42 extracted from frozen hippocampi and measured by sandwich ELISA (n = 8/genotype). F, G, Insoluble levels of Aβ40 and Aβ42 extracted from frozen hippocampi and measured by sandwich ELISA (n = 8/genotype). Data are presented as means ± SEM and were analyzed by t test; **p = 0.002, ***p < 0.0001.
Figure 5.
Figure 5.
APP processing is not changed between APP and APP/mTOR+/− mice. A, Representative Western blots of proteins extracted from frozen hippocampi of 2- and 12-month-old mice and probed with the indicated antibodies. B–G, Quantification of the blots shows that the levels of APP, C99 and C83 were not statistically significant between APP/mTOR+/− and APP mice, at any of the age analyzed (n = 5/genotype/time point). Data are presented as means ± SEM and were analyzed by t test.
Figure 6.
Figure 6.
Increased synaptophysin levels in APP/mTOR+/− mice. A, Representative Western blots of protein extracted from 12-month-old CTL, APP and APP/mTOR+/− mice. Blots were probed with the indicated antibodies. B, C, Quantification of the blots shows that synaptophysin levels were restored to CTL levels in the APP/mTOR+/− mice. Although the same trend was apparent for PSD95, the changes were not statistically significant. Data are presented as means ± SEM and were analyzed by one-way ANOVA with Bonferroni correction; *p < 0.05.
Figure 7.
Figure 7.
Decreasing mTOR signaling increases autophagy induction. A, Representative Western blots of protein extracted from 12-month-old APP and APP/mTOR+/− mice. Blots were probed with the indicated antibodies. B–E, Quantification of the blots shows that the levels of Atg3, Atg5, Atg7 and Atg12 were significantly higher in the APP/mTOR+/− than APP mice (n = 5/genotype). F, Representative microphotographs of hippocampal sections immunostained with the indicated antibodies. G, Semiquantitative analysis showed that the number of yellow pixels (indicating a colocalization between Aβ and the lysosomal protein Lamp2A) was significantly higher in APP/mTOR± mice compared with APP mice (n = 7/genotype). Data are presented as means ± SEM and were analyzed by t test; *p < 0.01, **p < 0.001.
Figure 8.
Figure 8.
Removing one copy of the mTOR gene restores the hippocampal expression profiles of the APP mice to the CTL levels. A, The graph summarizes the expression analysis results from transcripts isolated from frozen hippocampi (n = 6/genotype), indicating the number of genes measured, how many of those were differentially expressed among the four genotypes, and how many of those were differentially expressed between APP and APP/mTOR+/− mice. B, Cluster analysis of samples from all genotypes with a fold-change ≥ 1.8-fold when comparing CTL with APP mice. Each column represents a single sample, whose genotype is color labeled above the cluster diagram. Each row represents a specific transcript, whose identity is listed to the right of the cluster diagram. C–E, Validation of microarray results of three random genes by Western blot. Each graph shows the expression levels of each transcript (relative to CTL) as found by microarray and Western blots. For the Western blots (n = 6/genotype). Data are presented as means ± SEM and were analyzed by one-way ANOVA; *p < 0.05.

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