Seizures exacerbate excitatory: inhibitory imbalance in Alzheimer's disease and 5XFAD mice

Abstract

Approximately 22% of Alzheimer's disease (AD) patients suffer from seizures, and the co-occurrence of seizures and epileptiform activity exacerbates AD pathology and related cognitive deficits, suggesting that seizures may be a targetable component of AD progression. Given that alterations in neuronal excitatory:inhibitory (E:I) balance occur in epilepsy, we hypothesized that decreased markers of inhibition relative to those of excitation would be present in AD patients. We similarly hypothesized that in 5XFAD mice, the E:I imbalance would progress from an early stage (prodromal) to later symptomatic stages and be further exacerbated by pentylenetetrazol (PTZ) kindling. Post-mortem AD temporal cortical tissues from patients with or without seizure history were examined for changes in several markers of E:I balance, including levels of the inhibitory GABAA receptor, the sodium potassium chloride cotransporter 1 (NKCC1) and potassium chloride cotransporter 2 (KCC2) and the excitatory NMDA and AMPA type glutamate receptors. We performed patch-clamp electrophysiological recordings from CA1 neurons in hippocampal slices and examined the same markers of E:I balance in prodromal 5XFAD mice. We next examined 5XFAD mice at chronic stages, after PTZ or control protocols, and in response to chronic mTORC1 inhibitor rapamycin, administered following kindled seizures, for markers of E:I balance. We found that AD patients with comorbid seizures had worsened cognitive and functional scores and decreased GABAA receptor subunit expression, as well as increased NKCC1/KCC2 ratios, indicative of depolarizing GABA responses. Patch clamp recordings of prodromal 5XFAD CA1 neurons showed increased intrinsic excitability, along with decreased GABAergic inhibitory transmission and altered glutamatergic neurotransmission, indicating that E:I imbalance may occur in early disease stages. Furthermore, seizure induction in prodromal 5XFAD mice led to later dysregulation of NKCC1/KCC2 and a reduction in GluA2 AMPA glutamate receptor subunit expression, indicative of depolarizing GABA receptors and calcium permeable AMPA receptors. Finally, we found that chronic treatment with the mTORC1 inhibitor, rapamycin, at doses we have previously shown to attenuate seizure-induced amyloid-β pathology and cognitive deficits, could also reverse elevations of the NKCC1/KCC2 ratio in these mice. Our data demonstrate novel mechanisms of interaction between AD and epilepsy and indicate that targeting E:I balance, potentially with US Food and Drug Administration-approved mTOR inhibitors, hold therapeutic promise for AD patients with a seizure history.

Keywords: Alzheimer’s disease; GABA receptors; epilepsy; glutamate receptors; mTOR; rapamycin.

Figure 1

Seizure history is associated with worse cognitive and functional performance in patients with Alzheimer’s disease. Comparisons between Alzheimer’s disease (AD) patients without seizure history (AD−Sz) and with seizure history (AD+Sz) in final global Clinical Dementia Rating (CDR) (A), CDR-Sum of Boxes (SOB) (B) and Dementia Severity Rating Scale (DSRS) totals (C). CDR: n = 18 (AD+Sz), n = 87 (AD−Sz); DSRS: n = 14 (AD+Sz), n = 19 (AD−Sz). *P < 0.05, **P < 0.01.

Figure 2

Dysregulation of proteins involved in excitatory:inhibitory balance in Alzheimer’s disease patients with and without seizure history. (A) Representative western blot images for B–E, showing non-adjacent bands originating from the same blot of the temporal cortex from control cases (Con; n = 13) and Alzheimer’s disease patients (AD; n = 30) split into subgroups: those without (AD−Sz; n = 19) or with (AD+Sz; n = 11) known seizure history. Semi-quantitative analysis of (B) GABA_AR subunits GABA_ARα1 and GABA_ARα3, and corresponding ratio GABA_ARα1/GABA_ARα3; (C) Cl⁻ cotransporters NKCC1 and KCC2, and corresponding ratio NKCC1/KCC2; (D) AMPAR subunits GluA1 and GluA2, and corresponding ratio GluA1/GluA2; (E) NMDAR subunits GluN2A and GluN2B, and corresponding ratio GluN2B/GluN2A. *P < 0.05, **P < 0.01. (F) Pearson correlations for all AD samples, showing the relationship between GluN2A and Clinical Dementia Rating-Sum of Boxes (CDR-SOB) score and (G) the relationship between GABA_ARα1 expression and brain weight at death in grams in AD+Sz patients. Grey areas indicate 95% confidence interval for the two means.

Figure 3

Excitation:inhibition dysfunction in CA1 pyramidal neurons from prodromal 5XFAD mice. (A) Representative miniature inhibitory postsynaptic current (mIPSC) traces from wild-type (WT) and 5XFAD CA1 neurons. (B and C) Quantifications of mIPSC amplitudes. (D and E) Quantifications of mIPSC decay times. (F and G) Quantifications of inter-mIPSC intervals. n = 20 cells from three WT mice, 20 cells from three 5XFAD mice. ****P < 0.0001. (H) Representative miniature excitatory postsynaptic current (mEPSC) traces from WT and 5XFAD CA1 neurons. (I and J) Quantifications of mEPSC amplitudes. (K and L) Quantifications of inter-mEPSC intervals. n = 19 cells from three WT mice, 21 cells from three 5XFAD mice, ****P < 0.0001.

Figure 4

Excitatory:inhibitory (E:I) markers in prodromal 5XFAD mice. (A) Representative western blot images for B–I, showing non-adjacent bands originating from the same blot. Analysis of bands from western blots for E:I markers from the hippocampus (B–E) and cortex (F–I) of 5XFAD and wild-type (WT) mice. (B and F) GABA_AR subunits GABA_ARα1 and GABA_ARα3 and corresponding GABA_ARα1/GABA_ARα3 ratio; (C and G) Cl⁻ cotransporters NKCC1 and KCC2 and corresponding NKCC1/KCC2 ratio; (D and H) AMPAR subunits GluA1 and GluA2 and corresponding GluA1/GluA2 ratio; (E and I) NMDAR subunits GluN2A, GluN2B and corresponding GluN2B/GluN2A ratio. Error bars represent the standard error of the mean. n = 8–15 per group. *P < 0.05.

Figure 5

Excitatory:inhibitory imbalance in the hippocampus of 5XFAD mice following induced seizures. (A–E) Quantification of (A) GABA_AR subunits GABA_ARα1 and GABA_ARα3 and corresponding ratio GABA_ARα1/GABA_ARα3; (B) Cl⁻cotransporters NKCC1 and KCC2 and corresponding ratio NKCC1/KCC2; (C) AMPAR subunits GluA1 and GluA2 and corresponding ratio GluA1/GluA2; and (D) NMDAR subunits GluN2A and GluN2B and corresponding ratio GluN2B/GluN2A. (E) Representative western blot images for A–D showing non-adjacent bands originating from the same blot. (F) Pearson correlation of pentylenetetrazol (PTZ)-kindled 5XFAD mice showing the relationship between NKCC1/KCC2 and Aβ normalized to 5XFAD-vehicle (Veh), (G) pTau AT100 (Thr212, Ser14)/Tau normalized to wild-type (WT)-Veh (H) and percentage of spontaneous alternations in the Y-maze. Group comparisons for Aβ ELISA, pTau western blots and the Y-maze have been published previously (H). Grey areas indicate 95% confidence interval for the two means. n = 12–19 WT-vehicle, 12–17 WT-PTZ, 12 5XFAD-vehicle and 17 5XFAD-PTZ. *P < 0.05, **P < 0.01. Females and males are designated by square and triangle data-points, respectively, where sex effects were found.

Figure 6

Differential effects of mTORC1 inhibition on seizure-induced excitation:inhibition imbalance in 5XFAD mice. (A–D) Quantification of (A) GABA_ARα1 and GABA_ARα3 and corresponding GABA_ARα1/GABA_ARα3 ratio; (B) Cl⁻ cotransporters NKCC1 and KCC2 and corresponding NKCC1/KCC2 ratio; (C) AMPAR subunits GluA1 and GluA2 and corresponding ratio GluA1/GluA2; and (D) NMDAR subunits GluN2A and GluN2B and corresponding GluN2B/GluN2A ratio. (E) Representative western blot images for A–D showing non-adjacent bands originating from the same blot. n = 12–13 for each group. *P < 0.05, **P < 0.01.