Interdependence of neural network dysfunction and microglial alterations in Alzheimer's disease-related models

Abstract

Nonconvulsive epileptiform activity and microglial alterations have been detected in people with Alzheimer's disease (AD) and related mouse models. However, the relationship between these abnormalities remains to be elucidated. We suppressed epileptiform activity by treatment with the antiepileptic drug levetiracetam or by genetic ablation of tau and found that these interventions reversed or prevented aberrant microglial gene expression in brain tissues of aged human amyloid precursor protein transgenic mice, which simulate several key aspects of AD. The most robustly modulated genes included multiple factors previously implicated in AD pathogenesis, including TREM2, the hypofunction of which increases disease risk. Genetic reduction of TREM2 exacerbated epileptiform activity after mice were injected with kainate. We conclude that AD-related epileptiform activity markedly changes the molecular profile of microglia, inducing both maladaptive and adaptive alterations in their activities. Increased expression of TREM2 seems to support microglial activities that counteract this type of network dysfunction.

Keywords: Clinical neuroscience; Molecular neuroscience; Neuroscience.

Graphical abstract

Figure 1

LEV treatment reduces epileptiform activity, behavioral abnormalities and alterations in inflammation-related gene expression in aged, plaque-laden hAPP-J20 mice (A) Coronal brain sections from 30-month-old mice of the indicated genotypes were labeled with an antibody against human Aβ (3D6). Scale bar: 500 μm. (B–Q) Intracranial EEGs were recorded on two occasions from 24–30-month-old male hAPP-J20 mice and WT controls while they were treated with levetiracetam (LEV) or placebo. After the second EEG recording, their hippocampal mRNA was analyzed with a NanoString Neuroinflammation panel and their cortical mRNA was analyzed by qRT-PCR. Behavioral testing was carried out in a replicate cohort of mice that were also treated with LEV or placebo but were not implanted with EEG electrodes. (B) Schematic of experiment. mo, months relative to beginning of LEV treatment. (C and D) Frequency of epileptiform spikes after 2 (C) or 4 (D) months of LEV (+; 2 g/kg of chow) or placebo (−) treatment. (E) Locomotor activity in the open field quantified as total movements per 10 min. (F–H) Learning and memory assessment in the active place avoidance (APA) paradigm. (F) Number of entries into the aversive zone during habituation (Habit., shock inactivated), training days 1–4 (shock activated), and a probe trial (shock inactivated). (G) Rank summary score of aversive zone entries during training. (H) Number of aversive zone entries during probe trial. (I and J) Volcano plots of transcripts whose levels differed in placebo-treated mice that did or did not express hAPP (I) and of transcripts whose levels differed in LEV- vs. placebo-treated hAPP-J20 mice (J). Transcripts with a p value < 0.01 are indicated in blue. Transcripts in orange and identified by name denote the ten genes with the largest log₂ fold change and a p value < 0.01. (K) Venn diagram indicating the number of transcripts whose levels were significantly changed by one or both of the indicated experimental manipulations. (L) Log-fold change in expression for transcripts in (K). Transcripts whose levels were changed by both manipulations are highlighted in green or magenta. Transcripts in green and identified by name were selected for RT-qPCR analysis of cortical tissues from the same mice (N–Q). (M) STRING network analysis of proteins encoded by transcripts identified by green dots in (L). The darker the green, the greater the change LEV treatment caused in hAPP-J20 mice. Line thickness indicates the confidence score of the associations between nodes. Three additional proteins identified by STRING as potentially related to this network are shown in white boxes. Five proteins listed at the bottom left had no known associations with any of the other gene products. (N–Q) Cortical levels of the indicated transcripts were determined by qRT-PCR. Mean levels in placebo-treated mice without hAPP expression were defined as 1.0. n = 5–7 mice per group for (C, D, I, J, L, and N–Q) and 6–8 mice per group for (E–H). p values in (I and J) were obtained from a linear regression model followed by Benjamini–Yekutieli adjusted t-tests. Two-way ANOVA of the data in (C–H) revealed significant effects of genotype (p < 0.05) and treatment (p < 0.05), and a trend toward an interaction between them for (C, D; p = 0.08) but not (E–H; p > 0.3). Two-way ANOVA of the data in (N–Q) revealed significant effects of genotype (N–Q; p < 0.001) and treatment (N; p < 0.05 and P, Q; p < 0.01), and a significant interaction between these variables for some transcripts (N; p < 0.05; and P; p < 0.01) and a trend toward an interaction for another (O; p = 0.06). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by two-way ANOVA and Holm-Sidak test in (C–H, N–Q). Dots represent individual mice (C–H and N–Q) or transcripts (I, J, and L) and bars means ± s.e.m.

Figure 2

Transcripts modulated by hAPP expression and LEV treatment colocalize with IBA1-positive cells. (A–C) Coronal brain sections from 26–30-month-old placebo-treated hAPP-J20 mice (Figure 1) were stained with ThioS (green), an IBA1 antibody (blue), and probes (magenta) against C1qa (A), Trem2 (B), or Tyrobp (C) mRNA. Representative images are shown at different levels of magnification. Scale bars: 100 μm.

Figure 3

LEV does not alter amyloid plaque loads or plaque-associated microgliosis in hAPP-J20 mice (A–I) Coronal brain sections from 26–30-month-old hAPP-J20 mice that had been treated with LEV or placebo (Figure 1) were stained with ThioS (magenta) to label plaques and with TO-PRO3 to label nuclei (cyan) (A–E) or with ThioS (magenta) and an IBA1 antibody to label microglia/macrophages (blue) (F–I). (A) Representative images of the hippocampus. Scale bars: 500 μm, 50 μm (inset). (B) Number of plaques per mm² of hippocampal area. (C) Fluorescence intensity of ThioS-positive plaques. Mean levels of fluorescence intensity in placebo-treated hAPP-J20 mice were defined as 1.0. (D) Area covered by ThioS positive plaques. (E) Plaque circularity. A perfect circle would equal 1.0. (F) Representative images of microglia/macrophages. Scale bar: 50 μm. (G) Number of IBA1-positive cell bodies within 25 μm of a ThioS-positive plaque. (H) Fraction of IBA1-positive pixels that were also positive for ThioS. (I) Soma size (area) of IBA1-postive cells that were located > 25 μm (distal, Dist.) or < 25 μm (adjacent, Adj.) of a ThioS-positive plaque. n = 7 mice per group (3 sections were analyzed per mouse) for (B–E and G–I). For (G–I), three plaques were analyzed in each section. For (I), three microglia distal to a plaque and three microglia adjacent to a plaque were analyzed. Unpaired, two-tailed Student’s t-test of the data in (B–E and G–H) revealed no significant differences between groups. Two-way ANOVA of the data in (I) revealed an effect of distance to plaques (p < 0.001), but no significant effect of LEV treatment (p = 0.41) and no interaction between these variables (p = 0.52). ∗p < 0.05, ∗∗p < 0.01 vs. leftmost bar or as indicated by brackets by two-way ANOVA and Holm-Sidak test. Dots in (B–E and G–I) represent individual mice. Bars are means ± s.e.m.

Figure 4

Effects of LEV treatment on microglial gene expression changes in young hAPP-J20 mice (A–L) Male hAPP-J20 mice and WT controls were treated for 4 or 12 weeks with LEV (+; 2 g/kg of chow) or placebo (−) starting at 6 months of age. At the end of treatments, hippocampal and cortical mRNA was analyzed by qRT-PCR. (A–F) Hippocampal (A–C) and cortical (D–F) levels of the indicated transcripts after 4 weeks of LEV or placebo treatment. (G–L) Hippocampal (G–I) and cortical (J–L) levels of the indicated transcripts after 12 weeks of LEV or placebo treatment. Mean levels of the respective transcripts in placebo-treated mice without hAPP expression were defined as 1.0. n = 4–5 mice per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. leftmost bar or as indicated by brackets (two-way ANOVA and Holm-Sidak test). Two-way ANOVA revealed a significant effect of genotype on all transcripts but one (F, p = 0.18; E,K,L, p < 0.05; A,D, p < 0.01; C, p < 0.001; B,G,H,I,J, p < 0.0001), a significant effect of LEV treatment on some transcripts (B,E,H,I, p < 0.05; G, p < 0.01) but not others (A,C,D,F,J–L, p > 0.05), and a significant interaction between these variables for some transcripts (I, p < 0.05; D, p < 0.01) but not others (A–C,E–H,J–L, p > 0.05). Dots represent individual mice and bars means ± s.e.m.

Figure 5

Tau reduction prevents network dysfunction and microglial gene changes in aged hAPP-J20 mice (A–J) EEG recordings were obtained from male and female Mapt^+/+ and Mapt^–/– mice that did or did not express hAPP at 26–30 months of age. Hippocampal mRNA was analyzed with a NanoString Neuroinflammation panel. (A) Frequency of epileptiform spikes in male (black) and female (white) mice of the indicated genotypes. (B and C) Volcano plots of transcripts whose levels differed between Mapt^+/+ mice that did or did not express hAPP (B) and of transcripts whose levels differed between hAPP-J20 mice of the Mapt^–/– vs. Mapt^+/+ background (C). Transcripts with a p value < 0.01 are indicated in blue. Transcripts in orange and identified by name denote the ten genes with the largest log₂ fold change and a p value < 0.01. For (C), Mapt mRNA data was excluded before plotting to avoid disproportional scale expansion. (D) Venn diagram indicating the number of transcripts whose levels were significantly changed by one or both of the indicated genetic modifications. (E) Log-fold change in expression for transcripts in (D). Transcripts whose levels were changed by both manipulations are highlighted in green or magenta. Transcripts in green and identified by name were selected for RT-qPCR analysis of cortical tissues from the same mice (G–J). (F) STRING network analysis of proteins encoded by transcripts identified by green dots in (E). The darker the green, the greater the change tau ablation caused in hAPP-J20 mice. Line thickness indicates the confidence score of the associations between nodes. Three additional proteins identified by STRING as potentially related to this network are shown in white boxes. Three proteins listed at the bottom right had no known associations with any of the other gene products. (G–J) Cortical levels of the indicated transcripts were determined by RT-qPCR in the same mice whose hippocampi were analyzed with the NanoString Neuroinflammation panel (A–F). Mean levels of the respective transcripts in Mapt^+/+ mice without hAPP expression were defined as 1.0. (K–M) LEV treatment and tau reduction prevented or reversed an overlapping set of inflammation-related gene expression changes in hAPP-J20 mice. (K) Venn diagram indicating the number of transcripts whose hippocampal levels in 26–30-month-old hAPP-J20 mice were significantly changed by LEV treatment, tau ablation, or both (Figures 1 and 5A–5F). (L) Log-fold change in expression for transcripts in (K). Dots represent individual transcripts. Transcripts whose levels were changed by both manipulations are highlighted in green. (M) STRING network analysis of proteins encoded by transcripts identified by green dots in (L). Line thickness indicates the confidence score of the associations between nodes. Three additional proteins identified by STRING as potentially related to this network are shown in white boxes. Two proteins listed at the bottom left had no known associations with any of the other gene products. n = 10–14 (A–F), 4–8 (G–J), or 5–14 (K–M) mice per group. p values in (B and C) were obtained from a linear regression model adjusted for sex as a covariate followed by Benjamini–Yekutieli t-tests. Two-way ANOVA of the data in (A) revealed a significant effect of hAPP-J20 genotype (p < 0.01), a significant effect of Mapt genotype (p < 0.01), and a significant interaction between them (p < 0.05). Two-way ANOVA of the data in (G–J) revealed a significant effect of hAPP-J20 genotype (I,J, p < 0.05; G,H, p < 0.01), a significant effect of Mapt genotype for some transcripts (G,I, p < 0.05), a trend in this direction for others (H = 0.07, J = 0.06), and a significant interaction between these variables for some transcripts (G,I, p < 0.05) but not others (H = 0.57, J = 0.07). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. leftmost bar or as indicated by brackets by two-way ANOVA and Holm-Sidak test. Dots represent individual mice (A and G–J) transcripts (B, C, and E). Bars are means ± s.e.m.

Figure 6

Microglial/macrophage gene expression changes correlate with the frequency of epileptiform spikes in hAPP-J20 mice (A) List of genes whose expression levels correlated positively (white) or negatively (black) with the frequency of epileptiform spikes (p < 0.05 calculated from Pearson correlation coefficients) in at least two of the following groups of mice: placebo-treated male hAPP-J20 mice (n = 7) (Figure 1) or untreated male (n = 8) or female (n = 6) hAPP-J20 mice (Figures 5A–5J). (B–D) Hippocampal Tyrobp mRNA levels correlated positively with the frequency of epileptiform spikes in placebo-treated male hAPP-J20 mice (B) and in untreated male (C) or female (D) hAPP-J20 mice. Pearson correlation coefficients (r) and p values are shown in the graphs. Dots represent individual mice.

Figure 7

Genetic reduction of Trem2 exacerbates epileptiform activity after low-dose kainate challenge (A–I) EEG recordings were obtained from 6-month-old male Trem2^+/− mice and WT (Trem2^+/+) controls before and after a single IP injection of kainate (10 mg/kg body weight). (A) Schematic of experiment. (B) Sample EEG traces during the first hour after kainate injection. (C) Spike frequency 24 h before and after kainate injection. (D) Number of spikes per min during the first hour after kainate injection. (E) Peak spike frequency after kainate injection. (F) Time to reach peak spike frequency after kainate injection. (G) Change in spike frequency over 35 days after kainate injection. (H) Cumulative spikes estimated by calculating the area under the curve in (G). (I) Kaplan–Meier curves indicating the percentage of mice with abnormally increased epileptiform activity relative to their own baseline at different days after the kainate injection. p < 0.05 by Mantel-Cox logrank test. n = 15 mice per group at the start of the experiment. Tick marks indicate mice euthanized for reasons unrelated to the experiment. Two-way ANOVA of the data in (C) revealed a significant effect of genotype (p < 0.001) and treatment (p < 0.001) and a significant interaction between them (p < 0.001). Dots in (C, E, F, and H) represent individual mice. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs similarly treated WT mice or as indicated by the bracket, based on two-way ANOVA and Holm-Sidak test (C) or unpaired, two-tailed Student’s t test (E, F, and H). Values in (C–H) are means ± s.e.m.