Extracellular signal-regulated kinase regulates microglial immune responses in Alzheimer's disease

Abstract

The importance of mitogen-activated protein kinase (MAPK) pathway signaling in regulating microglia-mediated neuroinflammation in Alzheimer's disease (AD) remains unclear. We examined the role of MAPK signaling in microglia using a preclinical model of AD pathology and quantitative proteomics studies of postmortem human brains. In multiplex immunoassay analyses of MAPK phosphoproteins in acutely isolated microglia and brain tissue from 5xFAD mice, we found phosphorylated extracellular signal-regulated kinase (ERK) was the most strongly upregulated phosphoprotein within the MAPK pathway in acutely isolated microglia, but not whole-brain tissue from 5xFAD mice. The importance of ERK signaling in primary microglia cultures was next investigated using transcriptomic profiling and functional assays of amyloid-β and neuronal phagocytosis, which confirmed that ERK is a critical regulator of IFNγ-mediated pro-inflammatory activation of microglia, although it was also partly important for constitutive microglial functions. Phospho-ERK was an upstream regulator of disease-associated microglial gene expression (Trem2, Tyrobp), as well as several human AD risk genes (Bin1, Cd33, Trem2, Cnn2), indicative of the importance of microglial ERK signaling in AD pathology. Quantitative proteomic analyses of postmortem human brain showed that ERK1 and ERK2 were the only MAPK proteins with increased protein expression and positive associations with neuropathological grade. In a human brain phosphoproteomic study, we found evidence for increased flux through the ERK signaling pathway in AD. Overall, our analyses strongly suggest that ERK phosphorylation, particularly in microglia in mouse models, is a regulator of pro-inflammatory immune responses in AD pathogenesis.

Keywords: Alzheimer's disease; ERK; RRID:AB_331646; RRID:AB_354872; RRID:AB_394489; RRID:AB_396772; RRID:CVCL_0470; RRID:IMSR_JAX:000664; RRID:MMRRC_034840-JAX; RRID:SCR_002798; RRID:SCR_002865; RRID:SCR_003420; RRID:SCR_017386; microglia; neuroinflammation; proteomics.

FIGURE 1

Increased activation of ERK1/2 and p38 mitogen-activated protein kinase (MAPK) signaling in microglia in the 5xFAD mouse Alzheimer’s disease model. (a) Schematic summarizing key MAPK signaling pathways including ERK1/2, p38, and c-Jun N-terminal kinase (JNK) signaling cascades, the three families of MAPKs, all of which result in the activation of transcription factors which regulate gene regulation. (b) Experimental outline for Luminex studies of acutely isolated CD11b⁺ CNS myeloid cells and frontal cortex brain samples isolated from age-matched WT and 5xFAD mice (n = 7–8 mice/group). CD11b⁺ cells were isolated by Percoll density centrifugation followed by CD11b enrichment using MACS columns. *p < 0.05, ^**p < 0.01, ^***p < 0.005. (c) Summary of phospho-protein signaling data for phosphoERK1/2 (p-ERK1/2), p-p38, and p-JNK signaling cascades in CD11b⁺ CNS myeloid cells from WT and 5xFAD mice. Mean and SD of the expression of phospho (p)-proteins, as represented by the intensity of fluorescence, are represented. *p < 0.05, ^**p < 0.01, ^***p < 0.005

FIGURE 2

Confirmation of increased ERK1/2 activation in 5xFAD microglia. (a,b) Bar plot showing the relative abundance of 17 mitogen-activated protein kinase (MAPK) proteins as compared to non-MAPK proteins in microglia from WT, 5xFAD, and lipopolysaccharide (LPS)-treated WT mice (a) and BV2 microglia (b) using published proteomic data sets. (c) Differentially expressed MAPK proteins identified in PBS-treated WT, 5xFAD, and LPS-treated WT mice. Error bars represent SD. Proteins differentially expressed across groups (ANOVA p < 0.05) and meeting statistical significance (post hoc Tukey’s HSD p < 0.05) as compared to the WT-PBS group are indicated. *p < 0.05, ^**p < 0.01, ^***p < 0.005. (d) Intracellular flow cytometric analysis of acutely isolated CNS myeloid cells for p-ERK1/2 labeling. Gating strategy used is shown (top). (e) Quantitative analyses of flow cytometric studies of p-ERK1/2 and total ERK1/2 expression in WT and 5xFAD CD11b⁺ CD45int microglia. *p < 0.05, ^**p < 0.01, ^***p < 0.005

FIGURE 3

Transcriptomic profiling of microglia reveals distinct clusters of genes regulated by ERK1/2. (a) Heat map showing K-means clustering analysis of 465 genes with differential expression across four treatment groups, as identified by one-way ANOVA, using NanoString gene expression data. Cluster 1: inhibited by ERK; Cluster 2: activated by ERK and inhibited by IFNγ; Cluster 3: activated by ERK and IFNγ; Cluster 4: inhibited by ERK. (b) Gene ontology (GO) analysis depicting enrichment of GO terms in genes belonging to each cluster.

FIGURE 4

ERK1/2 positively regulates homeostatic, anti-inflammatory disease-associated microglia (DAM), and pro-inflammatory DAM gene expression in microglia. (a) t-SNE plots showing clusters of genes based on NanoString expression data. Color codes indicate K-means clusters identified in Figure 3. Clusters 2, 3, and 4 are positively regulated by ERK while Cluster 1 is negatively regulated by ERK. (b) Distribution of homeostatic, pro-inflammatory DAM, anti-inflammatory DAM, M1-like, and M2-like microglial genes in each cluster. (c) Expression of synthetic eigengenes of each microglial state (homeostatic, pro-inflammatory DAM, and anti-inflammatory DAM) across experimental conditions. (d) Plot showing the magnitude of change in overall gene expression level in each cluster (averaging across normalized gene expression within each cluster) between ERK inhibitor treated and nontreated conditions in the presence of IFNγ. N = 3 replicates per group (Error bars represent SD). *p < 0.05, ^**p < 0.01, ^***p < 0.005 based on Tukey’s HSD post hoc pairwise comparisons

FIGURE 5

Roles of extracellular signal-regulated kinase (ERK) activity in regulating microglial phagocytosis of fibrillar Aβ42 and of neuronal phagocytosis. (a) Flow cytometry experimental data showing the uptake of fluorescent Aβ42 fibrils by live CD45+ microglia that had been treated with an ERK1/2 inhibitor and/or IFNγ. (b) Dotplots showing the reduction of microglial phagocytosis of Aβ42 by ERK inhibition under unstimulated and stimulated conditions in-vitro. N = 3 replicates per group. Error bars represent SD. *p < 0.05, ^**p < 0.01, ^***p < 0.005. (c) Experimental protocol for microglia-N2a coculture study: GFP-positive N2a cells were differentiated for 4 days using retinoic acid. Meanwhile, primary microglia were isolated, and after 24 hr, were incubated with the ERK inhibitor with or without IFNγ. After incubation for 24 hr, the microglia were seeded into the differentiated N2a cultures overnight and then harvested for antibody staining and flow cytometry. This figure was created using modified images from Servier Medical Art. (d) Bar plot demonstrating the viability of N2a cells when cocultured with microglia. N = 3 replicates per group. Error bars represent SD. *p < 0.05, ^**p < 0.01, ^***p < 0.005. (e) Bar plots showing a reduction of microglialphagocytosis of differentiated N2a cells in the ERK inhibited group. GFP⁺ N2A cells were differentiated and then cocultured with primary microglia that were pretreated with an ERK inhibitor with or without IFNγ. N = 3 replicates per group. Error bars represent SD. *p < 0.05, ^**p < 0.01, ^***p < 0.005

FIGURE 6

Multi-marker analysis of GenoMic annotation (MAGMA) and TMT proteomics reveals role for extracellular signal-regulated kinase (ERK) activation in human Alzheimer’s disease (AD). (a) t-SNE plot showing late-onset AD risk genes identified in human GWAS analyses that are present in mouse microglia from the NanoString data set (*p < 0.05). (b) Differential expression analysis of human frontal cortex proteomes showing that MAPK1 (ERK2) and MAPK3 (ERK1), both shown in red, are expressed more highly in AD brains than in control brains while the opposite trend is observed in other notable MAPKs, shown in blue. (c) Box plots (median, min to max range, and individual values) showing that MAPK3 expression levels are increased in AD and AD/Parkinson’s disease (PD) brains as compared to PD or control brains (n = 10 per group). (d) Correlation between AD Braak stage and MAPK3 expression. Spearman’s R and p value are indicated. (e) Box plots (median, min to max range, and individual values) showing that MAPK1 expression levels are increased in brains with AD pathology (n = 10 per group). (f) Correlation between Braak stage and MAPK1 expression. Spearman’s R and p value are indicated

FIGURE 7

Phosphoproteomic analysis of human postmortem brain reveals extracellular signal-regulated kinase (ERK) activation in Alzheimer’s disease (AD). (a) Heat map showing k-means clustering of 57 differentially expressed phosphopeptides that mapped to a mitogen-activated protein kinase (MAPK) reference gene list, across frontal cortex samples from six non-AD control, six asymptomatic AD, and six symptomatic AD cases. (b) Box and whisker plot quantifying the trajectory of change in expression for each cluster across stages of AD progression. (c) Diagram showing the number of peptides (in parenthesis) and their respective gene symbols (outside of parenthesis) are involved in ERK, c-Jun N-terminal kinase (JNK), and p38 signaling. (d) Visual representation and k-means clustering of key phosphosites within the differentially expressed ERK-related peptides in the IMAC data set, mapping to all levels of the ERK cascade, that are relevant in AD

FIGURE 8

Phosphoproteomics reveals progressively upregulated flux through MAPK pathway in asymptomatic Alzheimer’s disease (AD) and cognitively impaired AD cases. Colored circles represent fold-change of each detected phosphoprotein protein compared to control cases (six non-AD control, six asymptomatic AD, and six symptomatic AD cases)