Assessment of tau phosphorylation and β-amyloid pathology in human drug-resistant epilepsy

Abstract

Objective: Epilepsy can be comorbid with cognitive impairments. Recent evidence suggests the possibility that cognitive decline in epilepsy may be associated with mechanisms typical of Alzheimer's disease (AD). Neuropathological hallmarks of AD have been found in brain biopsies surgically resected from patients with drug-resistant epilepsies. These include hyperphosphorylation of the tau protein (p-tau) that aggregates into neuropil threads (NT) or neurofibrillary tangles (NFT), as well as the presence of β-amyloid (Aβ) deposits. While recent studies agree on these AD neuropathological findings in epilepsy, some contrast in their correlation to cognitive decline. Thus, to further address this question we determined the abundance of p-tau and Aβ proteins along with their association with cognitive function in 12 cases of refractory epilepsy.

Methods: Cortical biopsies surgically extracted from the temporal lobes of patients with refractory epilepsy were processed for immunohistology and enzyme-linked immunoassays to assess distribution and levels, respectively, of p-tau (Antibodies: Ser202/Thr205; Thr205; Thr181) and Aβ proteins. In parallel, we measured the activation of mechanistic target of rapamycin (mTOR) via p-S6 (Antibodies: Ser240/244; Ser235/236). Pearson correlation coefficient analysis determined associations between these proteins and neurophysiological scores for full-scale intelligence quotient (FSIQ).

Results: We found a robust presence of p-tau (Ser202/Thr205)-related NT and NFT pathology, as well as Aβ deposits, and p-S6 (Ser240/244; Ser235/236) in the epilepsy biopsies. We found no significant correlations between p-tau (Thr205; Thr181), Aβ, or mTOR markers with FSIQ scores, although some correlation coefficients were modest to strong.

Significance: These findings strongly support the existence of hyperphosphorylated tau protein and Aβ deposits in patients with human refractory epilepsy. However, their relation to cognitive decline is still unclear and requires further investigation.

Keywords: Alzheimer's disease; mTOR; refractory epilepsy; tau protein; β-amyloid.

FIGURE 1

NeuN immunostaining shows altered neuronal densities and abnormal neurons in cortical samples from human epilepsy cases. Representative images immunostained with antibodies against NeuN, a neuronal marker, are shown for cortical tissues surgically resected from different patients (P) with FCD with drug‐resistant seizures (A‐Ei). NeuN immunoreactivity is shown in brown and nuclear Nissl staining is shown in blue. NeuN signal (Ai) shows normal appearing cortical layering along with areas of dispersed NeuN staining (Aii). NeuN staining within the cortex revealed organized cortical layering with normal appearing neurons compared (B‐Bi) to enlarged dysmorphic neurons (C‐Ci) observed within the same patient sample (Patient 2; P2). Representative cortical tissue samples from two additional different patients show NeuN‐positive neuronal staining with characteristics of radial microcolumnar disorganization with abnormal cell size in P8 (D‐Di) and P12 (E‐Ei). Representative higher magnification images containing cortical layers II & III are shown in panels (Bi, Ci, Di, Ei). Scale bars: A, 1 mm; Ai and Aii, 500 μm; (B‐E), 250 μm, and (Bi‐Ei), 25 μm; white matter; wm; gray matter, gm. Pearson correlation coefficient (r) analyses between FSIQ scores and the age at surgery (F) and the epilepsy duration (G) are shown.

FIGURE 2

Phosphorylated tau (p‐tau) is abundant in cortical samples from human refractory epilepsy. Representative images immunostained with antibodies against AT8, a marker for p‐tau (Ser202/Thr205), are shown for cortical tissues surgically resected from different patients (P) with drug‐resistant seizures (A‐Di). P‐tau staining is shown in brown while nuclear Nissl staining is shown in blue. P‐tau signal is observed in the form of neurofibrillary tangles localized in pyramidal neuron‐like cells (black arrowheads) and neuropil threads (white arrowheads) within the brain parenchyma (Ai, Bi, Ci, Di). Scale bars: A, B, C, and D, 250 μm; Ai, Bi, Ci, and Di, 25 μm. Protein levels for p‐tau (Thr205) (E) and p‐tau (Thr181) (F) in each brain biopsy are shown as relative signal intensity (rsi). Pearson correlation coefficient (r) analysis between both p‐tau phosphorylated sites is shown in (G).

FIGURE 3

Correlation analysis of p‐tau abundance with age, epilepsy duration, and FSIQ scores. Pearson correlation coefficient (r) analysis of p‐tau (Thr205) and p‐tau (Thr181) to age at surgery (A, D), epilepsy duration (B, E), and FSIQ (C, F) are shown.

FIGURE 4

β‐amyloid protein is evident in cortical samples from human refractory epilepsy. Representative images immunostained with antibodies against Aβ are shown for cortical tissues surgically resected from different patients (P) drug‐resistant seizures (A‐Di). Aβ immunoreactivity is shown in brown and nuclear Nissl staining is shown in blue. Variable patterns of Aβ signal are evident in different patient biopsies (P) (A‐Di). Most tissues showed small circular structures positive for Aβ signal (black arrowheads) (A, C, D). One sample displayed a robust accumulation of Aβ in the form of plaques throughout the cortex of the entire sample (B). Representative higher magnification images are shown in panels Ai, Bi, Ci, Di. Scale bars: A, B, C, and D, 250 μm; Ai, Bi, Ci, and Di, 25 μm. Protein levels for Aβ are shown as ng/ml (E). Pearson correlation coefficient (r) analysis between Aβ and the age at surgery (F), the epilepsy duration (G), and FSIQ scores (H) are shown.

FIGURE 5

Phosphorylated ribosomal S6 (P‐S6) is evident in cortical samples from human refractory epilepsy. Representative images immunostained with antibodies against p‐S6 (Ser240/244), a marker for mTOR activity, are shown for cortical tissues surgically resected from different patients (P) with drug‐resistant seizures (A‐Di). P‐S6 (Ser240/244) immunoreactivity is shown in brown and nuclear Nissl staining is shown in blue. Variable intensities of p‐S6 signal are evident across the different patient samples (A‐Di). Black arrows point to neurons with robust p‐S6 (Ser240/244) staining while the white arrows point to cells with comparable weaker signal (Ai‐Di). Representative higher magnification images of cortical layer III are shown in panels Ai‐Ci. Scale bars: A, B, C, D, 250 μm; Ai, Bi, Ci, and Di, 25 μm. Representative immunoblots are shown for p‐S6 (Ser240/244), p‐S6 (Ser 235/235), S6, and Actin (loading control) (E). Quantification of the relative signal intensity (rsi) for p‐S6/S6 is shown in (F). Pearson correlation coefficient (r) analysis between both p‐S6 phosphorylated sites is shown in (G).

FIGURE 6

Correlation analysis of p‐S6 abundance with age, epilepsy duration, and FSIQ scores. Pearson correlation coefficient (r) analysis of p‐S6 (Ser240/244) and p‐S6 (Ser 235/235) to the age at surgery (A, D), the epilepsy duration (B, E), and FSIQ (C, F) are shown.