Impairments of GABAergic transmission in hippocampus mediate increased susceptibility of epilepsy in the early stage of Alzheimer's disease

Abstract

Background: Patients with Alzheimer's disease (AD) are often co-morbid with unprovoked seizures, making clinical diagnosis and management difficult. Although it has an important role in both AD and epilepsy, abnormal γ-aminobutyric acid (GABA)ergic transmission is recognized only as a compensative change for glutamatergic damage. Neuregulin 1 (NRG1)-ErbB4 signaling can promote GABA release and suppress epileptogenesis, but its effects on cognition in AD are still controversial.

Methods: Four-month-old APPswe/PS1dE9 mice (APP mice) were used as animal models in the early stage of AD in this study. Acute/chronic chemical-kindling epilepsy models were established with pentylenetetrazol. Electroencephalogram and Racine scores were performed to assess seizures. Behavioral tests were used to assess cognition and emotion. Electrophysiology, western blot and immunofluorescence were performed to detect the alterations in synapses, GABAergic system components and NRG1-ErbB4 signaling. Furthermore, NRG1 was administrated intracerebroventricularly into APP mice and then its antiepileptic and cognitive effects were evaluated.

Results: APP mice had increased susceptibility to epilepsy and resulting hippocampal synaptic damage and cognitive impairment. Electrophysiological analysis revealed decreased GABAergic transmission in the hippocampus. This abnormal GABAergic transmission involved a reduction in the number of parvalbumin interneurons (PV⁺ Ins) and decreased levels of GABA synthesis and transport. We also found impaired NRG1-ErbB4 signaling which mediated by PV⁺ Ins loss. And NRG1 administration could effectively reduce seizures and improve cognition in four-month-old APP mice.

Conclusion: Our results indicated that abnormal GABAergic transmission mediated hippocampal hyperexcitability, further excitation/inhibition imbalance, and promoted epileptogenesis in the early stage of AD. Appropriate NRG1 administration could down-regulate seizure susceptibility and rescue cognitive function. Our study provided a potential direction for intervening in the co-morbidity of AD and epilepsy.

Keywords: Alzheimer's disease; Cognition; Epilepsy; ErbB4; GABAergic transmission; NRG1; Parvalbumin interneurons.

Fig. 1

Higher susceptibility to epilepsy of APP/PS1 mice. A Experimental design of PTZ acute kindling model. B-D The latency of seizure phenotypes (B), time to reach Racine V (C) and required dose of PTZ (D) in 4-month-old WT and APP group. N = 6. E Maximal Racine score reached every 5 min within 90 min. N = 6. F Experimental design of PTZ chronic kindling model. G Maximal Racine score reached per day. N = 20. H Survival curve for PTZ chronic kindling model. N = 20. Data were shown as means ± SEM. Mann Whitney test for (B). Unpaired t test for (C, D). Two-way ANOVA for (E and G). Log-rank test for (H). *p < 0.05, ** p < 0.01, ***p < 0.001

Fig. 2

EEG characteristics in PTZ acute kindling model of APP/PS1 and WT mice. A and B Representative epileptiform activities, including interictal spike (A) and polyspikes (B). C-E The number (C), normalized power (D), and total duration (E) of polyspikes. N = 6. F and G The number (F) and normalized power (G) of interictal spikes. N = 6. H and I The typical spectrum analysis of polyspikes (H) and interictal spike (I) in WT (blue) and APP (red) mice. Data were shown as means ± SEM. Unpaired t test for (C-G). *p < 0.05, ** p < 0.01, ***p < 0.001

Fig. 3

Seizures impair behavioral performances of APP/PS1 mice. A-C The mean speed (A), time in the corner zone (B) and time in the center zone (C) in the Open Field tests. D The percentage of time to explore new objects in the NOR tests. E The percentage of spontaneous alternation in the Y maze tests. F and G The percentage of entries into the open arm (F) and time in the open arm (G) in the EPM tests. H In the MWM tests, the escape latency in the acquisition trial. I-M In the MWM tests, the number of platform crosses (I), latency to platform (J), the mean swimming speed (K), time in the target quadrant (L) and latency to target quadrant (M) during the probe test. N Representative track images of mice in the probe test. N = 11, 10, 10, 10. Data were shown as means ± SEM. One-way ANOVA for (C-G, I-M). Kruskal-Wallis test for (A and B). Two-way ANOVA for (H). *p < 0.05, ** p < 0.01, ***p < 0.001

Fig. 4

Seizures induce synaptic loss and worsen neuronal death in the hippocampus of APP/PS1 mice. A The I-O curve of hippocampal CA1. N = 3–4 mice per group, n = 7–9 slices per mouse. B and C LTP induced by high-frequency stimulation was evaluated in hippocampal CA1. N = 3–4 mice per group, n = 7–9 slices per mouse. D-F Western blot and quantitative analysis for synapse-associated proteins in the hippocampus, including PSD-95 (E) and Syn-1 (F). N = 6. G Representative images of neuronal apoptosis in the hippocampal CA1 and CA3 regions. Scale bar, 200 μm. Data were shown as means ± SEM. Two-way ANOVA for (A, C, E and F). *p < 0.05, ***p < 0.001

Fig. 5

Impaired GABAergic inputs in the hippocampus of APP/PS1 mice. A Representative AP traces of evoked spikes. B Average AP firing frequency of CA1 pyramidal neurons in response to 0- to 200-pA depolarizing current steps. N = 3 mice per group, n = 6–7 neurons per mouse. C Evoked threshold of CA1 pyramidal neurons of APP and WT mice. N = 3 mice per group, n = 6–7 neurons per mouse. D Rest membrane potential of CA1 pyramidal neurons of APP and WT mice. N = 3 mice per group, n = 6–7 neurons per mouse. E Representative traces of mIPSC recordings in hippocampal CA1 region. F, G Mean mIPSC amplitude (F) and frequency (G) in CA1 pyramidal neurons. N = 3 mice per group, n = 3–5 neurons per mouse. H Representative fluorescence images showing the PV⁺ Ins (green) and SOM⁺ Ins (red) in the hippocampal CA1 region of APP and WT mice. Scale bar, 50 μm. I Comparable number of PV⁺ Ins in the hippocampal CA1 region of APP and WT mice. N = 3 mice per group, n = 6 (average of 12 slices) per mouse. J Comparable number of SOM⁺ Ins in the hippocampal CA1 region of APP and WT mice. N = 3 mice per group, n = 6 (average of 12 slices) per mouse. K-P Western blot and quantitative analysis for vGAT (L), GAD65/67 (M), NRG1 (O) and ErbB4 (P). N = 3. Q, R Quantitative analysis for number of ErbB4⁺ cells per unit area (Q), and mean fluorescent intensity of ErbB4 particularly in PV⁺ Ins (R). N = 3 mice per group, n = 3 (average of 9 slices, 72–81 cells) per mouse. S Representative fluorescence images showing the PV⁺ Ins (green) and ErbB4 (red) in the hippocampal CA1 region of APP and WT mice. Scale bar, 50 μm. Data were shown as means ± SEM. Two-way ANOVA for (B). Mann Whitney test for (C). Unpaired t test for (D, F, G, I, J, L, M, O-R). *p < 0.05, ** p < 0.01, ***p < 0.001

Fig. 6

Administration of NRG1 down-regulates seizure susceptibility and rescues cognitive function of APP/PS1 mice. A Schematic diagram of intracerebroventricular injection. B-D The latency of seizure phenotypes (B), time to reach Racine V (C) and required dose of PTZ (D) in APP + PBS and APP + NRG1 acute kindling models. N = 3, 4. E Maximal Racine score reached every 5 min within 75 min. N = 3, 4. F Maximal Racine score reached per day. N = 13, 10. G Survival curve for PTZ chronic kindling model. N = 13, 10. H-J The mean speed (H), time in the corner zone (I) and time in the center zone (J) were recorded in the Open Field tests. N = 6, 7. K The percentage of time to explore new objects was recorded in the NOR tests. N = 6, 7. L The percentage of spontaneous alternation was recorded in the Y maze tests. N = 6, 7. M-Q In the MWM tests, the number of platform crosses (M), latency to platform (N), the mean swimming speed (O), time in the target quadrant (P) and latency to target quadrant (Q) were recorded during the probe test. R In the MWM tests, the escape latency was recorded in the acquisition trial. S Representative track images of mice in the probe test. N = 6, 7. T-V Western blot and quantitative analysis for synapse-associated proteins in the hippocampus, including PSD-95 (U) and Syn-1 (V). N = 5. Data were shown as means ± SEM. Two-way ANOVA for (E, F, R). Mann Whitney test for (D, P). Unpaired t test for (B, C, H-O, Q, U, V). Log-rank test for (G). *p < 0.05, ** p < 0.01, ***p < 0.001