Neuronal hyperactivity due to loss of inhibitory tone in APOE4 mice lacking Alzheimer's disease-like pathology

Abstract

The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer's disease (AD). However, the reason APOE4 is associated with increased AD risk remains a source of debate. Neuronal hyperactivity is an early phenotype in both AD mouse models and in human AD, which may play a direct role in the pathogenesis of the disease. Here, we have identified an APOE4-associated hyperactivity phenotype in the brains of aged APOE mice using four complimentary techniques-fMRI, in vitro electrophysiology, in vivo electrophysiology, and metabolomics-with the most prominent hyperactivity occurring in the entorhinal cortex. Further analysis revealed that this neuronal hyperactivity is driven by decreased background inhibition caused by reduced responsiveness of excitatory neurons to GABAergic inhibitory inputs. Given the observations of neuronal hyperactivity in prodromal AD, we propose that this APOE4-driven hyperactivity may be a causative factor driving increased risk of AD among APOE4 carriers.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1

APOE4 is associated with entorhinal cortex hypermetabolism in aged mice. fMRI analysis was performed on aged APOE mice (mean age = 20 months; 7 APOE3 and 8 APOE4 males), and a voxel-based analysis was performed to generate CBV maps of the whole brain of each mouse, followed by co-registration of each image, ROI selection of the hippocampal formation, and statistical comparison between genotypes by a Student’s t test, followed by multiple test correction. a Voxel-based analyses revealed that aged APOE4 mice had increased CBV in the hippocampal formation compared with APOE3 mice, primarily centered around the EC. b 3D superior view of CBV increase detected in aged APOE4 vs. APOE3 mice, as indicated using a heat scale by p value. c Right sagittal view of the same analysis. d Left sagittal view of the same analysis. For each panel, the colored regions depict the hippocampus and subiculum in green, the lateral entorhinal cortex (LEC) in blue, and the medial entorhinal cortex (MEC) in gray

Fig. 2

APOE4 is associated with increased neuronal activity in awake freely moving mice. In vivo electrophysiology was performed on aged APOE mice (mean age = 18 months; 5 APOE3 and 4 APOE4 males) using a 16-channel microdrive placed directly into the LEC. a Representative local field potentials (LFPs) in awake, freely moving (5–20 cm/s) APOE3 and APOE4 mice, recorded at a depth of 3.3 mm from the brain surface. b A Nissl-stained brain section from a mouse used in this experiment, showing a track mark made by the implanted tetrode, which was descended to a depth of 3.3 mm from the brain surface when the mouse was killed. c Power spectra of LFPs recorded at a depth of 3.1–3.3 mm from the brain surface in freely moving (5–20 cm/s) APOE3 and APOE4 mice. (Frequencies between 55–65 Hz were notch-filtered to remove electrical noise and are extrapolated in the graph using straight lines.) d Percent power distributions comparing LFPs recorded at a depth of 3.1–3.3 mm from the brain surface in freely moving (5–20 cm/s) APOE3 and APOE4 mice (mean values and SEM). A Student’s t test with Welch’s correction was used for statistical comparison between genotypes (delta, p < 0.0001; theta, p = 0.0028; beta, p = 0.0008; low gamma, p < 0.0001; high gamma, p = 0.0015). e Single unit recordings detected at a depth of 3.1–3.3 mm from the brain surface in APOE3 and APOE4 mice, separated into putative excitatory neurons and interneurons based on peak size (black lines represent mean values and SEM). An increased firing rate of excitatory neurons (p < 0.0001) was observed using a Student’s t test with Welch’s correction. (**p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 3

APOE4 is associated with increased expression of small molecules related to energy metabolism. Targeted metabolite profiling was performed on small-molecule metabolites extracted from the EC and PVC of aged APOE mice (14–15 months old; 8 APOE3/3, 9 APOE3/4, and 7 APOE4/4 males). a Scatter dot plots depicting metabolite abundance in the EC for five energy-related metabolites found to be upregulated (Mann–Whitney; malate, p = 0.037; citrate/isocitrate, p = 0.028; ATP, p = 0.049; fructose-6-phosphate, p = 0.011; carnitine, p = 0.021) in APOE4 EC (black lines represent mean values and SEM). b Schematic of the energy metabolism-related metabolites found to be dysregulation in APOE4 EC. (*p ≤ 0.05)

Fig. 4

APOE4 is associated with abnormal excitability and synaptic plasticity in the hippocampal formation. In vitro electrophysiology was performed on horizontal hippocampal brain slices from aged APOE mice (mean age = 20 months; 5 APOE3 and 4 APOE4 males). a Representative traces of spontaneous extracellular field potentials (sEFPs) recorded in the subiculum (Sub), the dentate gyrus (DG), superficial layers (II/III) of the EC (sEC), and deep layers (V/VI) of the EC (dEC) in APOE3 and APOE4 mice. b Event durations and frequencies comparing APOE3 and APOE4 mice in the different regions recorded (mean values and SEM). Event durations in the Sub, DG, and sEC were found to be increased in aged APOE4 mice (Mann–Whitney; p < 0.001). c Representative sEFPs in APOE4 sEC before and after bath application (25 min) of AP-V (30 μM) and NBQX (10 μM). d A representative horizontal hippocampal brain slice used in this study, with asterisks denoting the location where each electrode was placed and the insert depicting the location of the horizontal slice in the mouse brain. e Cortico-cortical LTP in superficial EC induced by high-frequency stimulation (HFS, 3 trains of 100 pulses at 100 Hz, 10 s interval). Stimulation electrode was placed in lateral EC layer II, and the recording electrode was placed in lateral EC layers III/II. Stimulus intensity was selected to be 50–60% of the maximum amplitude of the fEPSP observed in the input–output curve, and a baseline for 15 mins was obtained before HFS. The slope of the initial component of the fEPSP (10–90%) was normalized to baseline and compared between APOE3 and APOE4 mice (repeated measures ANOVA; F(1,6) = 15.001, p = 0.008). f Mean value of potentiation between 40 and 60 min after LTP induction, comparing APOE3 and APOE4 (t(6) = 3.878, p = 0.008). (**p < 0.01)

Fig. 5

APOE4 is associated with decreased background synaptic inhibition in layer II entorhinal cortex pyramidal neurons. Miniature inhibitory post-synaptic currents (mIPSCs) were recorded using the patch-clamp whole-cell modality on pyramidal cells (n = 7 cells from APOE3 and n = 8 cells from APOE4) in the layer II EC of aged APOE mice (mean age = 20 months). a Representative traces of mIPSCs of layer II EC pyramidal neurons of APOE3 and APOE4 mice from recordings in voltage-clamp modality (holding at −65 mV) in the presence of APV (10 µM), NBQX (16 µM), and TTX (1 µM). b Representative average mIPSCs from a 3 min recording of an APOE3 and APOE4 mouse. c Plotted are mean + SEM values of peak amplitude of mIPSCs in the different genotypes as indicated (APOE3 26.09 ± 0.73 pA; APOE4 21.6 ± 0.48 pA; p < 0.001). d Cumulative probability distribution of peak amplitude in APOE3 and APOE4 mice, showing lower values for APOE4 in the entire distribution. e Shown are mean + SEM values of instantaneous frequencies in the different genotypes (APOE3 4.92 ± 0.3 Hz; APOE4 5.36 ± 0.31 Hz; t(748) = −1.035, p = 0.301). f Cumulative probability distribution of instantaneous frequencies in APOE3 and APOE4 mice, showing no significant differences. g Plotted are mean + SEM values of decay time constant in both groups (APOE3 9.56 ± 1.61 ms; APOE4 9.19 ± 0.53; p = 0.819). (***p < 0.001)