Levetiracetam suppresses neuronal network dysfunction and reverses synaptic and cognitive deficits in an Alzheimer's disease model

Abstract

In light of the rising prevalence of Alzheimer's disease (AD), new strategies to prevent, halt, and reverse this condition are needed urgently. Perturbations of brain network activity are observed in AD patients and in conditions that increase the risk of developing AD, suggesting that aberrant network activity might contribute to AD-related cognitive decline. Human amyloid precursor protein (hAPP) transgenic mice simulate key aspects of AD, including pathologically elevated levels of amyloid-β peptides in brain, aberrant neural network activity, remodeling of hippocampal circuits, synaptic deficits, and behavioral abnormalities. Whether these alterations are linked in a causal chain remains unknown. To explore whether hAPP/amyloid-β-induced aberrant network activity contributes to synaptic and cognitive deficits, we treated hAPP mice with different antiepileptic drugs. Among the drugs tested, only levetiracetam (LEV) effectively reduced abnormal spike activity detected by electroencephalography. Chronic treatment with LEV also reversed hippocampal remodeling, behavioral abnormalities, synaptic dysfunction, and deficits in learning and memory in hAPP mice. Our findings support the hypothesis that aberrant network activity contributes causally to synaptic and cognitive deficits in hAPP mice. LEV might also help ameliorate related abnormalities in people who have or are at risk for AD.

Fig. 1.

LEV decreases abnormal spike activity in hAPP mice acutely and chronically. Subdural EEG activity from parietal cortices (PC) was recorded in NTG (n = 3) and hAPPJ20 (n = 16) mice before and after LEV administration. (A) Representative recordings showing typical EEG patterns in NTG and hAPP mice. hAPP mice displayed frequent epileptiform spikes (marked by magenta arrowheads). Acute LEV injection (200 mg/kg, i.p.) transiently suppressed these abnormal events in hAPP mice as illustrated by the suppression of spikes in an hAPP mouse 2 h after LEV injection. Right panel shows details of a high-voltage biphasic spike shaded in magenta on the left. (B) Time course of the LEV effect in the same hAPP mouse. Each bar represents the total number of epileptiform spikes per hour. (C) Baseline EEGs were recorded for 24 h, and drugs were injected at the end of this recording period. During the first 6 h after the injection, LEV reduced the number of epileptiform spikes in hAPP mice (n = 16) by ∼75% on average. Subsequently, the frequency of epileptiform spikes increased gradually, returning to baseline levels by 12–18 h after the injection. (D) Dose-dependent effect of LEV on spike frequency during the first 6 h after the injection (n = 5–6 hAPP mice per dose). (E) Plasma concentration of LEV in hAPP mice (n = 4) determined during and after chronic infusion of the drug (75 mg⋅kg⁻¹⋅d⁻¹) by s.c. implanted osmotic minipumps. (F) EEGs were recorded in hAPP mice (n = 10) 24 h before (baseline), during, and after continuous infusion of LEV (75 mg⋅kg⁻¹⋅d⁻¹) for 28 d. Spike frequencies were reduced to ∼50% of baseline levels throughout the LEV infusion and returned to baseline level by 12 d after the end of the treatment. **P < 0.005, ***P < 0.0005 vs. baseline or as indicated by brackets (one-way ANOVA and Bonferroni test). Values in C–F are means ± SEM.

Fig. 2.

LEV treatment reverses behavioral abnormalities in hAPP mice. Behavioral testing was performed in two independent cohorts of 4- to 6-mo-old mice. Because results were similar in both cohorts, the data were pooled. (A and B) Before acute and chronic treatments, mice were prescreened in an open field for 5 min. (A) hAPPJ20 mice were more active than NTG controls (***P < 0.0001 by t test). (B) Within each genotype, mice were divided into two groups, so that baseline activity levels did not differ between groups before treatment with LEV or saline. Two-way ANOVA revealed a significant effect of the genotype (P < 0.0001) but not of group (P = 0.61) and no interaction effect (P = 0.43). ***P < 0.0005 vs. NTG (Bonferroni test). (C–F) hAPP mice and NTG controls were treated chronically with saline or LEV (75 mg⋅kg⁻¹⋅d⁻¹, s.c.; n = 21–30 mice per genotype and treatment). (C) Mice were tested in the open field 8 d (cohort 1) or 19 d (cohort 2) after the treatment started. The numbers of peripheral and central ambulatory movements and of fine movements were measured. Amb., ambulation; Mov., movements. (D–F) Mice were tested in the elevated plus maze 19 d (cohort 1) or 5 d (cohort 2) after the start of treatment. The total distance moved (D), the distance moved in the open arms (E), and the percentage of total time spent in the open arms (F) were measured. Two-way ANOVA revealed a significant interaction between genotype and treatment [C, P = 0.001 (peripheral ambulation); D, P = 0.007; E, P = 0.003; F, P = 0.012] and a significant genotype effect [C, P = 0.001 (central ambulation)]. *P < 0.05, **P < 0.005, ***P < 0.0005 vs. saline-treated NTG or as indicated by bracket (Bonferroni test). Values are means ± SEM.

Fig. 3.

LEV treatment reverses learning and memory deficits in hAPP mice. hAPPJ20 mice and NTG controls were chronically treated with saline or LEV. (A) Context-dependent learning and memory in an open field arena. In the initial phase of this test, all mice (n = 10–15 mice per genotype and treatment) showed habituation to the novel environment, reaching similar levels of activity on the fourth trial (no treatment or genotype effects and no interaction by two-way repeated-measures ANOVA). However, when reintroduced into the same arena 7 d later, saline-treated, but not LEV-treated, hAPP mice showed clear evidence of abnormal dishabituation (forgetting) compared with the control groups (P = 0.045 for interaction between genotype and treatment on trial 5 by two-way ANOVA). P < 0.005 vs. LEV-treated hAPP mice, P < 0.0005 vs. saline-treated NTG mice (Bonferroni test). Saline-treated hAPP mice were the only group that showed dishabituation in the open field 7 d after the fourth trial (P = 0.006 by paired t test, comparing total movements in trial 4 vs. trial 5). (B) Spatial learning and memory index (ratio of total movements during trials 4 and 5). Two-way ANOVA revealed a significant interaction between genotype and treatment (P = 0.0031). ***P < 0.0005 vs. saline-treated NTG (Bonferroni test). (C) Learning curves during spatial training in the Morris water maze (n = 10–15 mice per genotype and treatment). The distance each mouse swam to reach the hidden platform was recorded during 5 d. Data points represent the average performance of mice during four training trials/d. Repeated-measures ANCOVA revealed a significant interaction between genotype and day in saline-treated NTG and hAPP mice (P = 0.0063) and a significant interaction between LEV treatment and day in hAPP mice (P = 0.022) but not in NTG controls. (D) Twenty-four hours after the last training session, mice were tested in a probe trial (platform removed), and the percentage of time mice spent swimming in the target quadrant was calculated. One-tailed, one-sample t tests were performed to determine if the mean of each group was different from chance (dotted line). *P < 0.05, **P < 0.005, ***P < 0.0005 vs. 25%. Only saline-treated hAPP mice did not show a preference for the target quadrant. Two-way repeated-measures ANOVA revealed a significant interaction between genotype and treatment (P = 0.01). Bonferroni test revealed a significant difference between hAPP/saline and hAPP/LEV (P = 0.05). (E and F) Novel object recognition performed in an independent cohort of mice (n = 9–13 mice per genotype and treatment). (E) Preference index represents the time spent with the novel object (N) divided by the total time spent with both novel (N) and familiar (F) objects. One-tailed, one-sample t tests were performed to determine if the mean of each group was different from chance (dotted line). **P < 0.005, vs. 0.5. (F) Number of times mice interacted with the novel (N) versus the familiar (F) object during a 10-min test session. *P < 0.05, **P < 0.005 vs. familiar object (unpaired t test). Values are means ± SEM.

Fig. 4.

LEV reverses deficits in synaptic transmission and plasticity in hAPP mice. Field recordings were made from acute hippocampal slices obtained from 4- to 5-mo-old NTG and hAPPJ20 mice treated s.c. with LEV (75 mg⋅kg⁻¹⋅d⁻¹) or saline for 20–25 d. (A and B) LTP induction in the dentate gyrus after theta-burst stimulation of the medial perforant pathway. (A) LTP curves illustrate the deficit of synaptic plasticity in saline-treated hAPP mice and the complete reversal of this deficit by LEV treatment. (B) Mean of the last 10 min of the LTP recordings. LEV treatment normalized LTP deficits in hAPP mice. Two-way ANOVA revealed a significant interaction between genotype and treatment (P = 0.045). *P < 0.05 vs. saline-treated NTG or as indicated by bracket (Bonferroni test). Number of slices per number of mice for LTP recordings: NTG/saline, 5/4; NTG/LEV, 4/3; hAPP/saline, 5/5; hAPP/LEV, 7/6. (C) LEV reversed synaptic transmission deficits in the CA1 region of hAPP mice. Linear regression analysis of input/output curves revealed that the slope of saline-treated hAPP mice (2.2 ± 0.3) was significantly lower (P < 0.0001 by F-test) than the slopes of the other groups (NTG/saline, 4.5 ± 0.4; NTG/LEV, 4.2 ± 0.3; hAPP/LEV, 4.4 ± 0.3). Number of slices per number of mice for input/output recordings: NTG/saline, 8/6; NTG/LEV, 5/3; hAPP/saline, 13/7; hAPP/LEV, 10/7. Values are mean ± SEM.

Fig. 5.

LEV reverses abnormalities in synaptic activity-related proteins in the dentate gyrus of hAPP mice. Coronal brain sections from NTG and hAPPJ20 mice treated s.c. with saline or LEV (75 mg⋅kg⁻¹⋅d⁻¹) for 28 d (n = 12–16 mice per genotype and treatment) were immunostained for calbindin, NPY, or Fos. (A–C) Photomicrographs illustrating calbindin (A), NPY (B), and Fos (C) alterations in saline-treated hAPP mice and normalization of these biomarkers in LEV-treated hAPP mice. The relative densitometric measures obtained for the sections shown in this figure were 1.06 (NTG/saline), 0.97 (NTG/LEV), 0.71 (hAPP/saline), 1.04 (hAPP/LEV) for calbindin (A) and 1.05 (NTG/saline), 0.97 (NTG/LEV), 1.23 (hAPP/saline), 1.01 (hAPP/LEV) for NPY (B). (D and E) Densitometric quantitation of calbindin in the molecular layer of the dentate gyrus (D) and of NPY in the mossy fiber pathway (E). (F) Quantification of Fos-immunoreactive cells in the granular layer of the dentate gyrus. Two-way ANOVA revealed a significant interaction between genotype and treatment: (D) P = 0.0001; (E) P = 0.029; (F) P = 0.005. *P < 0.05, **P < 0.005, ***P < 0.0005 vs. saline-treated NTG or as indicated by bracket (Bonferroni test). Values in D–F are mean ± SEM.

Fig. 6.

Prolonged LEV treatment does not alter Aβ levels in the hippocampus of hAPP mice. (A and B) Aβ1-x and Aβ1-42 levels and Aβ1–42/Aβ1-x ratios in the hippocampus of 3-mo-old hAPP mice treated s.c. for 20 d with saline or LEV (75 mg⋅kg⁻¹⋅d⁻¹; n = 8–9 mice per treatment) were determined by ELISA. LEV treatment did not alter tissue levels of Aβ1-x (P = 0.8) or Aβ1-42 (P = 0.3) or the Aβ1–42/Aβ1-x ratio (P = 0.3). (C–F) Levels of Aβ in the interstitial fluid of the hippocampus were measured by in vivo microdialysis in 5-mo-old hAPP mice treated s.c. for 14 d with saline or LEV (75 mg⋅kg⁻¹⋅d⁻¹; n = 9 mice per treatment). (C and D) Interpolated zero flow method to quantify the pool of measurable Aβx-40 and Aβx-42 in the hippocampus. (E and F) LEV treatment did not alter exchangeable levels of Aβx-40 (P = 0.3) or Aβx-42 (P = 0.5) or the Aβx-42/ Aβx-42 ratio (P = 0.5) by unpaired two-tailed t test. Values are mean ± SEM.

Fig. 7.

Behavioral abnormalities and hippocampal remodeling in hAPP mice 35 d after the end of LEV treatment. Four- to six-month-old NTG and hAPPJ20 mice were treated s.c. with saline or LEV (75 mg⋅kg⁻¹⋅d⁻¹) for 28 d (n = 7–9 mice per genotype and treatment). (A and B) Thirty-five days after the end of the treatment, mice were retested in the open field (A) and the elevated plus maze (B). Two-way ANOVA revealed a significant effect of genotype (A, P = 0.0009; B, P = 0.0054) but not treatment (A, P = 0.88; B, P = 0.58) and no interaction between genotype and treatment (A, P = 0.48; B, P = 0.56). (C and D) Coronal brain sections from these mice were immunostained for calbindin and Fos. Calbindin levels in the molecular layer of the dentate gyrus were quantified by densitometry (C), and the average number of Fos-immunoreactive cells in the granular layer of the dentate gyrus per section was counted (D). Two-way ANOVA revealed a significant effect of genotype (C and D, P < 0.0001) but not treatment (C, P = 0.22; D, P = 0.73) and no interaction between genotype and treatment (C, P = 0.7; D, P = 0.45). *P < 0.05, **P < 0.005, ***P < 0.0005 vs. saline-treated NTG (Bonferroni test). Values are means ± SEM.

Fig. 8.

Loss of antiepileptic efficacy at high doses is associated with a loss of beneficial LEV effects on behavioral and molecular abnormalities in hAPP mice. hAPPJ20 mice were chronically treated with saline or LEV. Two doses of LEV were compared: 75 mg⋅kg⁻¹⋅d⁻¹ delivered s.c. via implanted osmotic minipumps (low) versus 150 mg⋅kg⁻¹⋅d⁻¹ delivered s.c. via implanted osmotic minipumps plus LEV added to the drinking water at 1.8 mg/mL (high). (A) EEG activity from parietal cortices was recorded in hAPP mice (n = 4) before and during high-dose LEV administration. Loss of efficacy in reducing abnormal spike activity was first observed at 14 d of treatment. *P < 0.05, **P < 0.005 vs. baseline (one-way ANOVA and Bonferroni test). (B) Plasma concentration of LEV in hAPP mice (n = 4) determined after 21 d of chronic infusion of the drug at low versus high doses. **P < 0.005 (unpaired t test). (C–F) In an independent cohort of mice, NTG and hAPPJ20 mice were treated with saline or LEV at low or high doses (n = 10–15 mice per genotype and treatment). The gray dotted lines in graphs represent the mean of measurements obtained in NTG controls. (C and D) Low-dose but not high-dose LEV reversed the hyperactivity of hAPPJ20 mice in the open field (C) and their disinhibition-like behavior in the elevated plus maze (D). (E and F) Low-dose but not high-dose LEV reversed calbindin depletion in the molecular layer of the dentate gyrus (E) and ectopic expression of NPY in the mossy fibers (F) of hAPP mice. *P < 0.05, ***P < 0.0005 vs. hAPP treated with saline (one-way ANOVA followed by Bonferroni test). Values are means ± SEM.

Fig. P1.

Beneficial effects of LEV treatment in hAPP mice. hAPPJ20 mice, which are genetically modified to express human amyloid precursor protein (hAPP) and amyloid-β (Aβ) peptides in the brain, recapitulate key aspects of AD. They have abnormal brain network activity (e.g., spikes on EEG recordings shown in first row), which leads to remodeling of neuronal circuits in the hippocampus, a major memory center of the brain. This remodeling is reflected, in part, by reduction in the neuronal activity-regulated protein calbindin (brown immunostaining in the second row). hAPPJ20 mice also have prominent impairments in synaptic function and spatial memory. The downward deflection in the electrical traces in the third row reflects synaptic transmission strength, and the swim paths shown in the fourth row reflect the distance mice traveled in a water maze before they found the location of a hidden platform. In the current study we show that LEV treatment eliminates or ameliorates all these abnormalities in hAPPJ20 mice.