Alzheimer-like amyloid and tau alterations associated with cognitive deficit in temporal lobe epilepsy

Abstract

Temporal lobe epilepsy represents a major cause of drug-resistant epilepsy. Cognitive impairment is a frequent comorbidity, but the mechanisms are not fully elucidated. We hypothesized that the cognitive impairment in drug-resistant temporal lobe epilepsy could be due to perturbations of amyloid and tau signalling pathways related to activation of stress kinases, similar to those observed in Alzheimer's disease. We examined these pathways, as well as amyloid-β and tau pathologies in the hippocampus and temporal lobe cortex of drug-resistant temporal lobe epilepsy patients who underwent temporal lobe resection (n = 19), in comparison with age- and region-matched samples from neurologically normal autopsy cases (n = 22). Post-mortem temporal cortex samples from Alzheimer's disease patients (n = 9) were used as positive controls to validate many of the neurodegeneration-related antibodies. Western blot and immunohistochemical analysis of tissue from temporal lobe epilepsy cases revealed increased phosphorylation of full-length amyloid precursor protein and its associated neurotoxic cleavage product amyloid-β*56. Pathological phosphorylation of two distinct tau species was also increased in both regions, but increases in amyloid-β1-42 peptide, the main component of amyloid plaques, were restricted to the hippocampus. Furthermore, several major stress kinases involved in the development of Alzheimer's disease pathology were significantly activated in temporal lobe epilepsy brain samples, including the c-Jun N-terminal kinase and the protein kinase R-like endoplasmic reticulum kinase. In temporal lobe epilepsy cases, hippocampal levels of phosphorylated amyloid precursor protein, its pro-amyloidogenic processing enzyme beta-site amyloid precursor protein cleaving enzyme 1, and both total and hyperphosphorylated tau expression, correlated with impaired preoperative executive function. Our study suggests that neurodegenerative and stress-related processes common to those observed in Alzheimer's disease may contribute to cognitive impairment in drug-resistant temporal lobe epilepsy. In particular, we identified several stress pathways that may represent potential novel therapeutic targets.

Keywords: beta-amyloid; cognition; stress-related kinases; tau; temporal lobe epilepsy.

Figure 1

Hypotheses for the accumulation of amyloid-β₄₂ and tau pathology linked to cognitive impairment and epileptogenesis in TLE. Following seizures, activation of surface receptors, including excitatory neurotransmitter receptors, activates the mTOR pathway leading to an increase in endoplasmic reticulum (ER) stress and oxidative stress. Chronic activation of cell stress pathways leads to neuronal death and subsequent cognitive impairment. ER stress activates PERK, which in turn phosphorylates and activates eIF2α, causing a general inhibition of protein synthesis leading to neuronal death. At the same time, phosphorylated (circled P) eIF2α de-represses the translation of BACE1 mRNA, increasing the amyloidogenic processing of APP, a process further augmented following APP phosphorylation (circled P). The amyloidogenic processing of APP (represented by large scissors) by β-secretase BACE1 results in the release of soluble APPβ (sAPPβ), while the subsequent cleavage of the remaining transmembrane APP portion (represented by small scissors) by γ-secretase generates the amyloid-β₄₂ peptide. In a similar fashion, the non-amyloidogenic processing of APP by α-secretase ADAM10 and γ-secretase results in the release of soluble APPα (sAPPα) and the p3 peptide. While also further inducing mTOR activity, ER stress, and oxidative stress, amyloid-β₄₂ peptides increase the expression of neprilysin (NEP), a major amyloid-β₄₂-degrading enzyme participating in amyloid-β₄₂ clearance. The ribosomal protein kinase p70S6K, a downstream target of activated mTOR, induces the synthesis of tau and BACE1 proteins and directly phosphorylates tau (circled P). Cellular stress also leads to activation of pro-apoptotic JNK, in addition to inhibiting PP2A activity. PP2A is a major tau phosphatase and its activation leads to decreased tau phosphorylation. JNK phosphorylates APP and tau protein (circled P), and induces BACE1 transcription (not shown).

Figure 2

Increased expression and phosphorylation of APP in human drug-resistant TLE. (A and B) Western blot quantification of (A) total APP and (B) phospho-APP [Thr668] in the hippocampus and temporal cortex from TLE, Alzheimer's disease and control patients. Data are represented as box-and-whisker plots showing the minimum value, the first quartile, the median, the third quartile, and the maximum value. Each group is compared to its respective age-matched control group using two-tailed Student t-test (normal distribution) or Mann-Whitney test (skewed distribution). *P < 0.05, ^**P < 0.01, ^****P < 0.0001. Detailed statistical data are provided in Supplementary Table 4. (C) Representative western blot images showing non-adjacent bands originating from the same blot. (D) Representative hippocampal section from a 37-year-old control subject (Ctrl 11) stained with haematoxylin and eosin (H&E) indicating the subfields imaged for staining illustrations: subiculum (SUB), Cornu Ammonis (CA1 to CA4), and dentate gyrus (DG) with its discernible granule cell layer. (E) Photomicrographs showing the hippocampal CA1 pyramidal cell layer from (E1) a 37-year-old control subject (Ctrl 11); (E2) a 29-year-old TLE patient with hippocampal sclerosis type III (TLE 12), (E3) the CA1 pyramidal cell layer, and (E4) the molecular layer of the SUB region from a 20-year-old TLE patient with hippocampal sclerosis type I (TLE 6); each immunolabelled with APP antibody (clone 22C11) or co-immunolabelled with APP (red), astrocytic marker GFAP (green), and nuclear stain DAPI (blue). Images show enhanced intra-neuronal APP labelling in the TLE hippocampus (E2) relative to control (E1), no detectable APP expression in the astrocytes (E3), and the presence of occasional extracellular APP depositions suggestive of diffuse amyloid plaques (E4). Note the similarities between the diaminobenzidine (E2) and the immunofluorescence (E3) APP signal. (F) Representative images of the temporal cortex from (F1) a 55-year-old control subject (Ctrl 15), (F2) a 29-year-old, (F3) a 24-year-old, and (F4) a 38-year-old TLE patient (TLE 12, TLE 7 and TLE 14, respectively), alongside with temporal cortex images from (F5) a 65-year-old Alzheimer's disease patient (AD 8); each labelled with APP (22C11) or co-immunolabelled with APP (red), GFAP (green), and DAPI (blue). Images demonstrate no apparent differences in neuronal APP expression between TLE (F2) and control cortex (F1), despite the presence of occasional amyloid plaque-like extracellular deposits (F3 and F4), similar to what is observed in the Alzheimer's disease case (F5). Diaminobenzidine (F3) and immunofluorescence (F4) APP labelling showing comparable signal patterns and the lack of APP co-localization with GFAP in the TLE cases (F4). Scale bars = 1000 μm in D, 100 μm in E1–3, F1–5; 10 μm in E4; insets = 10 μm.

Figure 3

Increased expression of sAPPα, amyloid-β*56 and amyloid-β₄₂ in human drug-resistant TLE. (A–D) Western blot quantification of (A) sAPPα, (B) sAPPβ, (C) amyloid-β*56, and (D) amyloid-β₄₂ (average of amyloid-β₄₂ hexamers, dimers and monomers) in the hippocampus and temporal cortex of TLE, Alzheimer’s disease (AD) and control patients. Box-and-whisker plots display the minimum value, the first quartile, the median, the third quartile, and the maximum value. Each group is compared to its respective age-matched control group using two-tailed Student t-test (normal distribution) or Mann-Whitney test (skewed distribution). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. Detailed statistical data are provided in Supplementary Table 4. (E) Representative western blot bands for the box-and-whisker plot graphs shown in A–D. Each lane shows non-adjacent bands from the same hippocampus or cortex blot. (F) Representative hippocampal section from a 38-year-old TLE patient without hippocampal sclerosis (TLE 14) stained for neurofilaments (NF) indicating the cellular layers for individual regions: stratum oriens (So), stratum pyramidale (Sp), stratum radiatum (Sr), and stratum lacunosum moleculare (Slm) for subiclulm (SUB) and CA1–4, and stratum moleculare (Sm), stratum granulare (Sg) and hilus (not shown) for dentate gyrus (DG). (G) Representative images of the hippocampal CA1 from a 55-year-old control subject (Ctrl 15) and from a 38-year-old TLE patient without hippocampal sclerosis (TLE 14) labelled with amyloid-β₄₂ antibody (clone MOAB-2), showing more frequent intracellular amyloid-β₄₂ accumulation in neuronal cell bodies and endothelial cells of blood vessels (asterisks) in the TLE case versus control. (H, middle left) Temporal lobe cortex from the same cases shown in G immunohistochemically labelled with amyloid-β₄₂ (MOAB-2), demonstrating occasional intracellular amyloid-β₄₂ expression in endothelial cells (asterisks), as well as granular extracellular amyloid-β₄₂ immunoreactivity (arrows) in TLE patient samples, but not in the control case. (H, right) Representative images of temporal lobe cortex from a 70-year-old Alzheimer’s disease patient (AD 3) labelled with the same anti-amyloid-β₄₂ monoclonal antibody as in G and H showing robust accumulation of amyloid-β₄₂ in fibrillar, compact and cored amyloid plaques. Scale bars = 200 μm in F, 100 μm in G and H; insets = 10 μm. dim. = dimers; hexam. = hexamers; mono. = monomers.

Figure 4

Differential expression of APP processing enzymes ADAM10 and BACE1 in human drug-resistant TLE. (A and B) Western blot quantification of (A) ADAM10 and (B) BACE1 in the hippocampus and temporal cortex of TLE, Alzheimer’s disease (AD) and control patients. (C) Representative western blot images of non-adjacent bands from the same hippocampus or cortex blot. Box-and-whisker plots display the minimum value, the first quartile, the median, the third quartile, and the maximum value. Each group is compared to its respective age-matched control group using two-tailed Student t-test (normal distribution) or Mann-Whitney test (skewed distribution). ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001. Detailed statistical data are provided in the Supplementary Table 4. (D) Representative images of the hippocampal CA3 pyramidal cell layer from a 55-year-old control subject (Ctrl 15) and a 38-year-old TLE patient with no hippocampal sclerosis (TLE 14) immunohistochemically labelled with ADAM10 (top row) showing comparable expression patterns in pyramidal neuron cell bodies of control and TLE cases. Photomicrographs of the hippocampal CA3 pyramidal cell layer from the same control subject (Ctrl 15) and of a 55-year-old TLE patient with hippocampal sclerosis type II (TLE 18) immunohistochemically labelled with BACE1 (bottom row) demonstrating increased BACE1 labelling in pyramidal cell bodies and processes in the TLE case relative to control, and occasional BACE1 accumulation in granular-like structures surrounding the nucleus. (E) Temporal lobe cortex from a 72-year-old Alzheimer’s disease patient immunolabelled with ADAM10 (top) and BACE1 (bottom) showing faint ADAM10 labelling in neuronal cell bodies and accumulation of BACE1 mostly in fibrillar amyloid plaques. Scale bars = 100 μm in D and E; insets = 10 μm. Insets show higher magnification images of the same areas. Sl = stratum lucidum; Sp = stratum pyramidale.

Figure 5

Increased expression of total tau protein in human drug-resistant TLE. (A–C) Western blot quantification of (A) total tau (antibody clone Tau 5), (B) tau 3-repeat (Tau 3R), and (C) tau 4-repeat (Tau 4R) isoforms in the hippocampus and temporal cortex of TLE, Alzheimer’s disease (AD) and control patients. Box-and-whisker plots display the minimum value, the first quartile, the median, the third quartile, and the maximum value. Each group is compared to its respective age-matched control group using two-tailed Student t-test (normal distribution) or Mann-Whitney test (skewed distribution). *P < 0.05, ^**P < 0.01. Detailed statistical data are provided in Supplementary Table 4. (D) Representative images of the hippocampal CA1 pyramidal cell layer (top row) from a 55-year-old control subject (Ctrl 15) and a 20-year-old TLE patient with hippocampal sclerosis type I (TLE 6) and of the dentate gyrus (bottom row) from a 37-year-old control subject (Ctrl 11) and a 49-year-old TLE patient with hippocampal sclerosis type II (TLE 17), each immunohistochemically labelled with Tau 5 antibody. Images show stronger labelling of neuronal cell bodies and processes in TLE cases compared to controls within both hippocampal regions. (E) Temporal lobe cortex from a 65-year-old Alzheimer’s disease patient (AD 2) showing typical tau accumulation in neurofibrillary tangles and neuropil threads surrounding amyloid plaques. Scale bars = 100 μm in D and E; insets = 10 μm. Insets show higher magnification images of the same areas. (F) Representative western blot images for the box-and-whisker plot graphs shown in A–C representing non-adjacent bands from the same hippocampus or cortex blot. Images show up to four bands, corresponding to tau isoforms 0N3R (55 kDa), 0N4R or 1N3R (64 kDa), 1N4R or 2N3R (69 kDa), and 2N4R (74 kDa). Sg = stratum granulare; Sm = stratum moleculare; Sp = stratum pyramidale.

Figure 6

Increased phosphorylation of tau in human drug-resistant TLE. (A and B) Western blot quantification of (A) phospho-tau [Ser202/Thr205] (antibody clone AT8) and (B) phospho-tau [Thr231] (antibody clone AT180) in the hippocampus and temporal cortex of TLE, Alzheimer’s disease (AD) and control patients. Box-and-whisker plots display the minimum value, the first quartile, the median, the third quartile, and the maximum value. Each group is compared to its respective age-matched control group using two-tailed Student t-test (normal distribution) or Mann-Whitney test (skewed distribution). ^***P < 0.001, ^****P < 0.0001. Detailed statistical data are provided in Supplementary Table 4. (C) Representative western blot images of individual proteins depicting non-adjacent bands originating from the same blot. (D) Photomicrographs of hippocampal CA1 pyramidal cell layer (top row) and of granular layer of the dentate gyrus (bottom row) from a 37-year-old control subject (Ctrl 11) and a 49-year-old TLE patient with hippocampal sclerosis type II (TLE 17) immunohistochemically labelled with p-tau AT180. Images show greater accumulation of p-tau AT180 in neuronal cell bodies and processes within the hippocampal CA1 pyramidal cell layer and DG granular layer in the TLE case compared to control. (E, top) Hippocampal CA1 pyramidal cell layer of a 49-year-old TLE patient with hippocampal sclerosis type II (TLE 17), co-immunolabelled with p-tau AT180 (green), astrocytic marker GFAP (red), and nuclear stain DAPI (blue) showing no colocalization of p-tau AT180 with GFAP. (E, bottom) Representative section of the dentate gyrus (DG) region from a 12-year-old TLE patient with hippocampal sclerosis type II (TLE 3), co-immunolabelled with p-tau AT180 (green), MAP2 (red) and nuclear stain DAPI (blue) demonstrating co-localization of p-tau AT180 with neuronal cell bodies and processes. A similar p-tau AT180 expression pattern is evident with both diaminobenzidine (D) and immunofluorescence (E) labelling. (F) Representative temporal cortex sections from a 20-year-old control subject (Ctrl 6) and a 49-year-old TLE patient (TLE 17) immunohistochemically labelled with p-tau AT180 show more robust labelling of neuronal cell bodies and processes in the TLE case compared to the control subject. (G) Temporal lobe cortex images from a 65-year-old Alzheimer’s disease patient (AD 2) showing accumulation of p-tau AT180 in neurofibrillary tangles and neuropil threads surrounding amyloid plaques. Scale bars = 100 μm in D–G; insets = 10 μm. Insets show higher magnification images of the same areas. Sg = stratum granulare; Sm = stratum moleculare; So = stratum oriens; Sp = stratum pyramidale; Sr = stratum radiatum.

Figure 7

Dysregulation of JNK, p70S6K, GSK-3β, p25/p35 and PERK/eIF2α signalling in human drug-resistant TLE. (A–F) Western blot quantification of (A) phospho-JNK [Thr183/Tyr185]/JNK, (B) phospho-p70S6K [Thr389]/p70S6K, (C) phospho-GSK-3β [Tyr216]/GSK-3β, (D) p25/p35, (E) phospho-PERK [Ser713]/PERK, and (F) phospho-eIF2α [Ser51]/eIF2α ratios in the hippocampus and temporal cortex of TLE, Alzheimer’s disease (AD) and control (Ctrl) patients. Box-and-whisker plots display the minimum value, the first quartile, the median, the third quartile, and the maximum value. Each group is compared to its respective age-matched control group using two-tailed Student t test (normal distribution) or Mann-Whitney test (skewed distribution). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. Detailed statistical data are provided in the Supplementary Table 4. (G) Representative western blot images of individual proteins depicting non-adjacent bands originating from the same blot.

Figure 8

Correlations between Alzheimer’s disease-like pathology markers, age at surgery and cognitive scores from pre-operative assessment in human drug-resistant TLE. (A) Heat map showing the Pearson correlation matrix between the biological markers analysed by western blot in the hippocampus (top) and cortex (bottom) from TLE cases adjusted for gender, age at surgery and disease duration (n = 11–18). (B and C) Pearson correlations in TLE hippocampus show positive relationships between age at surgery and unadjusted hippocampal (B) amyloid-β*56 (n = 12) and (C) tau 4-repeat isoforms (Tau 4R, n = 11) expression. (D-F) Pearson correlations in adult TLE hippocampus showing negative relationships between executive function assessed by the processing speed efficiency test, expressed as z-scores, and unadjusted hippocampal (D) pAPP (n = 8), (E) BACE1 (n = 8), and (F) total tau (Tau 5, n = 8) protein expression. (G) Pearson correlation in adult TLE hippocampus showing negative relationships between executive function assessed by the digit span backward test, expressed as z-scores, and unadjusted hippocampal p-tau AT180 (n = 6) expression. Grey area indicates 95% confidence interval for the two means for each graph.