The role of mTORC1 activation in seizure-induced exacerbation of Alzheimer's disease

Abstract

The risk of seizures is 10-fold higher in patients with Alzheimer's disease than the general population, yet the mechanisms underlying this susceptibility and the effects of these seizures are poorly understood. To elucidate the proposed bidirectional relationship between Alzheimer's disease and seizures, we studied human brain samples (n = 34) from patients with Alzheimer's disease and found that those with a history of seizures (n = 14) had increased amyloid-β and tau pathology, with upregulation of the mechanistic target of rapamycin (mTOR) pathway, compared with patients without a known history of seizures (n = 20). To establish whether seizures accelerate the progression of Alzheimer's disease, we induced chronic hyperexcitability in the five times familial Alzheimer's disease mouse model by kindling with the chemoconvulsant pentylenetetrazol and observed that the mouse model exhibited more severe seizures than the wild-type. Furthermore, kindled seizures exacerbated later cognitive impairment, Alzheimer's disease neuropathology and mTOR complex 1 activation. Finally, we demonstrated that the administration of the mTOR inhibitor rapamycin following kindled seizures rescued enhanced remote and long-term memory deficits associated with earlier kindling and prevented seizure-induced increases in Alzheimer's disease neuropathology. These data demonstrated an important link between chronic hyperexcitability and progressive Alzheimer's disease pathology and suggest a mechanism whereby rapamycin may serve as an adjunct therapy to attenuate progression of the disease.

Keywords: Alzheimer’s disease; cognition; mTOR; neuropathology; seizures.

Figure 1

Seizures are associated with increased brain atrophy, neuropathology and mTORC1 activity in patients with Alzheimer’s disease. Study cohorts included control cases (Con, circles, n = 7–13), and patients with Alzheimer’s disease (AD, squares, n = 18–34), split into subgroups: those without (AD−Sz, n = 9–20) or with (AD+Sz, n = 9–14) known seizure history. In these patients, we evaluated: (A) brain weight at the time of death in grams; (B) ventricular enlargement pathology graded by severity (0 = none, 1 = mild, 2 = moderate, 3 = severe); (C) soluble human amyloid-β₄₂ (Aβ₄₂) in pg/ml in the temporal cortex; quantification of amyloid plaque deposition in the (D) grey matter (GM) of the temporal cortex and (E) subcortical white matter (WM) from staining performed in I; (F) pTau [Thr212/Ser214] AT100/Tau in the temporal cortex; and (G) phosphorylated-S6 [Ser235/Ser236]/S6 in the temporal cortex. (H) Representative western blot images of the temporal cortex for F and G showing non-adjacent bands originating from the same blot. (I) Representative images of the temporal cortex from a control subject, an AD−Sz patient and an AD+Sz patient immunolabelled with amyloid-β, showing increased amyloid plaques in cases of Alzheimer's disease, with increased subcortical plaque deposition in the AD+Sz patient. Scale bar = 500 μm. (J) Representative images of the temporal cortex from a control subject, an AD−Sz patient and an AD+Sz patient co-immunolabelled with pTau AT100 (red) and pS6 (green), showing increased pTau AT100 accumulation in neurofibrillary tangles in cases of Alzheimer's disease co-localizing with the mTORC1 activity marker pS6. (K) Quantification of the percentage of cells with pTau inclusions from staining performed in J. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. ^#Alzheimer’s disease effect, P < 0.0001; ^$Sex effect, P < 0.0001; ^†Seizure effect, P < 0.001. Box and whisker plots display minimum, maximum and all quartiles. Scatter plots display all data-points, with mean ± standard error of the mean (SEM). Closed symbols represent males and open symbols represent females.

Figure 2

Hyperexcitability phenotype and seizure-induced effect on cognition in 5XFAD mice. (A) Experimental design in wild-type (WT) and 5XFAD mice involving PTZ kindling starting from 3 months, cognitive assessment from 6.5–7 months and euthanasia for biochemical analyses of the hippocampus at 7 months. The schematic provides additional background on the 5XFAD mice: amyloid-β₄₂ accumulation starting from 1.5 months of age, epileptiform spikes and cognitive impairment starting from 4 months and neuronal and synaptic loss starting from 9 months. (B) Maximal Racine score reached per day of PTZ injection. (C) Maximal Racine score reached per minute during the 1-h post-PTZ injection video recording (average of eight PTZ injections). Inset represents evaluation of the area under the corresponding generated curves. (D) Latency to seizure in minutes. (E) Associative long-term (tested at 24 h post-training) and remote memory (tested at 14 days post-training) assessed with contextual fear conditioning test, measuring % of time freezing adjusted by individual baseline freezing on training day. (F) Spatial working memory evaluated by % spontaneous alternation in the Y-maze. (G) Mice self-care assessed by nest building. Box and whisker plots display minimum, maximum and all quartiles. n = 19 WT-vehicle, 17 WT-PTZ, 12 5XFAD-vehicle and 17 5XFAD-PTZ. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ^#Genotype effect, P < 0.01; ^†Kindling effect, P < 0.05; ^††Kindling effect, P < 0.0001.

Figure 3

PTZ kindling worsens amyloid pathology in 5XFAD mice. (A) ELISA assessment of soluble human amyloid-β₄₂ (Aβ₄₂) in the hippocampus of 5XFAD mice. (B) Pearson correlation between seizure severity and human amyloid-β₄₂ concentration in pg/ml in the hippocampus of 5XFAD-PTZ mice. Grey area indicates 95% confidence interval. (C) ELISA assessment of soluble human amyloid-β₄₂ in the cortex of 5XFAD mice. (D) Representative images of the hippocampus and cortex with insets of the dentate gyrus (DG) immunohistochemically (IHC) co-labelled with human amyloid-β₄₂ (red) and nuclear DAPI stain (blue). Images show no expression in the wild-type mice, amyloid plaque accumulation in 5XFAD mice and increased amyloid deposition in the 5XFAD-PTZ group compared to 5XFAD-vehicle. Scale bars = 500 μm for entire images; and 20 μm for insets. (E) Corresponding immunohistochemical quantification of amyloid load (% area amyloid-β₄₂-positive) in the hippocampus (left) and cortical layers IV–VI (right) of 5XFAD mice. (F) ELISA assessment of hippocampal soluble mouse amyloid-β₄₂ levels. Western blot quantification of (G) total APP and (H) phosphorylated APP [Thr668] in wild-type and 5XFAD hippocampus. (I) Representative western blot images for G and H showing non-adjacent bands originating from the same blot. Box and whisker plots display minimum, maximum and all quartiles. Scatter plots display all data-points, with mean ± SEM. Closed symbols represent males and open symbols represent females. n = 12–19 WT-vehicle, 12–17 WT-PTZ, 12 5XFAD-vehicle and 17 5XFAD-PTZ. *P < 0.05, ****P < 0.0001. ^$Sex effect, P < 0.0001; ^†Kindling effect, P < 0.0001 for hippocampus, P < 0.05 for cortex; ^#Genotype effect, P < 0.001; ^‡Genotype × Kindling interaction, P < 0.05.

Figure 4

PTZ kindling worsens neuronal death and upregulates mTORC1 activity in 5XFAD mice. (A) Representative images of the hippocampus and cortex with insets of the dentate gyrus (DG) stained with Fluoro-Jade B. Images show no neuronal death in the wild-type mice, moderate neuronal death in 5XFAD mice and severe neuronal death in the 5XFAD-PTZ group compared with 5XFAD-vehicle. Scale bars = 500 μm for whole images; and 20 μm for insets. (B) Corresponding quantification of hippocampal (left) and cortical (right) Fluoro-Jade B staining, expressed as the number of positive cells per 0.25 mm². (C) Pearson correlation between seizure severity and the number of Fluoro-Jade B-positive cells in 5XFAD-PTZ hippocampus. (D) Immunohistochemical quantification of the number of neurons (NeuN-positive cells) in wild-type and 5XFAD hippocampus (left) and cortical layers IV–VI (right). (E) Representative images of the hippocampus and cortex with insets of the DG from wild-type and 5XFAD mice, co-labelled with NeuN (red), pS6 [Ser240/Ser244] (green) and nuclear DAPI stain (blue), showing increased pS6 staining in neuronal cell bodies in 5XFAD mice compared with the wild-type, as well as an increase in PTZ groups compared with the vehicle-treated mice in both genotypes. Scale bars = 500 μm for entire images; and 20 μm for insets. (F) Corresponding quantitative analysis of phosphorylated S6, expressed as a % of phosphorylated S6-positive cells relative to the number of NeuN-positive neurons per analysed area of the hippocampus (left) and cortical layers IV–VI (right). (G) Pearson correlation between seizure severity and the percentage of pS6-positive cells in the hippocampus of the 5XFAD-PTZ group. Scatter plots display all data-points, with mean ± SEM. Closed symbols represent males and open symbols represent females. Grey area in Pearson correlations indicates 95% confidence interval. n = 12–19 WT-vehicle, 12–17 WT-PTZ, 12 5XFAD-vehicle and 17 5XFAD-PTZ. *P < 0.05, ****P < 0.0001. ^$Sex effect, P < 0.05; ^†Kindling effect, P < 0.05; ^#Genotype effect, P < 0.001.

Figure 5

Inhibition of mTORC1 rescued long term and remote memory deficits in 5XFAD mice following PTZ kindling. (A) Experimental design in wild-type (WT) and 5XFAD mice, involving PTZ kindling starting from 3 months, rapamycin treatment starting from 3.5 months, cognitive assessment from 6.5–7 months and euthanasia for biochemical analyses at 7 months. (B) Associative long-term (test at 24 h post-training) and (C) remote memory (test at 14 days post-training) assessed with contextual fear conditioning test measuring % of time freezing relative to freezing pre-stimuli on training day. (D) Spatial working memory evaluated by % spontaneous alternation in the Y-maze. (E) Self-care assessed by nest building. Box and whisker plots display minimum, maximum and all quartiles. Scatter plots display all data-points, with mean ± SEM. Closed symbols represent males and open symbols represent females. n = 12–13 for each group. *P < 0.05, **P < 0.01, ***P < 0.001. ^!Treatment × Kindling interaction, P < 0.01; ^&Genotype × Sex interaction, P < 0.01; ^XGenotype × Kindling × Treatment interaction, P < 0.01; ^†Kindling effect, P < 0.05; ^#Genotype effect, P < 0.01.

Figure 6

Inhibition of mTORC1 protects against the effect of seizures on amyloid pathology and neuronal death in 5XFAD mice. (A) Representative western blot images for B showing non-adjacent bands originating from the same blot. (B) Western blot quantification of phosphorylated S6_S240/total S6. (C) ELISA assessment of hippocampal soluble human amyloid-β₄₂ in pg/ml. (D) Pearson correlation between seizure severity and hippocampal amyloid-β₄₂ concentration. (E) Representative images of the hippocampus and cortex with insets of the dentate gyrus (DG) immunohistochemically (IHC) co-labelled with human amyloid-β₄₂ (red) and DAPI stain (blue). (F) Corresponding quantification of hippocampal amyloid load. (G) Quantification of Fluoro-Jade B-positive cells per 0.25 mm² in the 5XFAD hippocampus (left) and the CA1/2 region (right). (H) Representative hippocampal section labelled with nuclear DAPI stain showing various regions of interest. (I) Representative images of the hippocampus and cortex with insets of the dentate gyrus stained with Fluoro-Jade B. Scale bars = 500 μm for entire images; and 20 μm for insets. Box and whisker plots display minimum, maximum and all quartiles. Scatter plots display all data-points, with mean ± SEM. Closed symbols represent males and open symbols represent females. n = 12–13 for each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ^$Sex effect, P < 0.05; ^&Genotype × Sex interaction, P < 0.05; ^XGenotype × Kindling × Treatment interaction, P < 0.05; ^†Kindling effect, P < 0.01; ^RTreatment effect, P < 0.05.

Figure 7

Proposed mechanism of the bidirectional relationship of seizures and Alzheimer’s disease with contributions of mTOR. A diagram that summarizes and synthesizes the findings from our work in this paper and previous work in models of Alzheimer’s disease and epilepsy. In this paper, we demonstrated that epilepsy/seizures can result in increased mTOR activity, amyloid-β (Aβ) and tau pathology and neuronal death. Previous work in epilepsy models has demonstrated that mTOR is involved in epileptogenesis, and our work here shows that mTOR is also increased by seizures. The mTOR pathway has been implicated in amyloid-β deposition via autophagy, and in this paper was shown to be co-localized at sites of pTau inclusions. Finally, amyloid pathology, tau pathology, mTOR and neuronal death all contribute to cognitive impairment in patients with Alzheimer’s disease and seizures.