CCL2 Overexpression in the Brain Promotes Glial Activation and Accelerates Tau Pathology in a Mouse Model of Tauopathy

Abstract

Innate immune activation is a major contributor to Alzheimer's Disease (AD) pathophysiology, although the mechanisms involved are poorly understood. Chemokine C-C motif ligand (CCL) 2 is produced by neurons and glial cells and is upregulated in the AD brain. Transgene expression of CCL2 in mouse models of amyloidosis produces microglia-induced amyloid β oligomerization, a strong indication of the role of these activation pathways in the amyloidogenic processes of AD. We have previously shown that CCL2 polarizes microglia in wild type mice. However, how CCL2 signaling contributes to tau pathogenesis remains unknown. To address this question, CCL2 was delivered via recombinant adeno-associated virus serotype 9 into both cortex and hippocampus of a mouse model with tau pathology (rTg4510). We report that CCL2 overexpression aggravated tau pathology in rTg4510 as shown by the increase in Gallyas stained neurofibrillary tangles as well as phosphorylated tau-positive inclusions. In addition, biochemical analysis showed a reduction in the levels of detergent-soluble tau species followed by increase in the insoluble fraction, indicating a shift toward larger tau aggregates. Indeed, increased levels of high molecular weight species of phosphorylated tau were found in the mice injected with CCL2. We also report that worsening of tau pathology following CCL2 overexpression was accompanied by a distinct inflammatory response. We report an increase in leukocyte common antigen (CD45) and Cluster of differentiation 68 (CD68) expression in the brain of rTg4510 mice without altering the expression levels of a cell-surface protein Transmembrane Protein 119 (Tmem119) and ionized calcium-binding adaptor molecule 1 (Iba-1) in resident microglia. Furthermore, the analysis of cytokines in brain extract showed a significant increase in interleukin (IL)-6 and CCL3, while CCL5 levels were decreased in CCL2 mice. No changes were observed in IL-1α, IL-1β, TNF-α. IL-4, Vascular endothelial growth factor-VEGF, IL-13 and CCL11. Taken together our data report for the first time that overexpression of CCL2 promotes the increase of pathogenic tau species and is associated with glial neuroinflammatory changes that are deleterious. We propose that these events may contribute to the pathogenesis of Alzheimer's disease and other tauopathies.

Keywords: Alzheimer's disease; Aβ; gene therapy; monocyte chemoattractant protein-1 (MCP-1); neuroinflammation; tau.

Figure 1

Intracranial injections of AAV9-RFP or AAV9-CCL2 in cortex and hippocampus in rTg4510 mice. Five-mo-old rTg4510 mice underwent intracranial bilateral injection of rAAV9-CCL2 (n = 6) or red fluorescent protein (RFP, n = 6) in both the hippocampus and the anterior cortex. Two months after the intracranial injections, brain tissue was collected. Micrographs represent the immunohistochemistry staining for CCL2 in RFP injected (A–C) and CCL2- injected animals (D–F). Higher magnification shows cell localization of CCL2 (insets). (G) Percent positive area of distribution in anterior cortex (ACX, boxed area), hippocampus (DG and CA3, boxed area) and entorhinal cortex (EC) of injected animals (n = 6, **p < 0.01 and ***p < 0.001). (H) CCL2 levels were measured in the hippocampus of injected mice by multiplex assay and the results were normalized to the amount of protein (ng/mg of protein). Student's t-test were performed between RFP and CCL2 groups for each dependent variable and each brain region separately. Scale bar represents 1,000 μm in (A,D), 200 μm in (B,C,E,F) and 20 μm in insets.

Figure 2

CCL2 overexpression does not affect microglia abundance in rTg4510. Micrograph representation of hippocampal area (A,B) and cortical area (C,D) stained for Iba-1 in RFP injected animals (A,C) and CCL2 injected animals (B,D). Quantification of positive area stained is presented for anterior cortex (ACX), hippocampus (HPC) entorhinal cortex (EC) and meninges (MNG) for Iba-1 (mean ± S.E.M, n = 6) (E). Student's t-test were prformed between RFP and CCL2 groups for each dependent variable and each brain region separately (n = 6). The scale bar represents 50 μm in panels and 20 μm in insets. Meningeal area is outlined in the cortical images (C,D, m).

Figure 3

CCL2 overexpression induces microglia activation in rTg4510 brain. Micrograph representation of cortical area stained for CD45 and CD68 in RFP injected animals (A,C) and CCL2 injected animals (B,D). Quantification of positive area stained is presented for anterior cortex (ACX), hippocampus (HPC) and entorhinal cortex (EC) for CD45 (E) and CD68 (F) (mean ± S.E.M, n = 6). Student's t-test were performed between RFP and CCL2 groups for each dependent variable and each brain region separately (n = 6, *p < 0.05, **p < 0.01). The scale bar represents 200 and 20 μm in panels and insets, respectively.

Figure 4

Tmem119 is stably expressed and co-localizes with Iba-1⁺ microglia in rTg4510 mice. Imaging of fluorescence labeled microglia with (A,E) Tmem119 (green) and (B,F) Iba-1 (purple) in the hippocampus of RFP- and CCL2-injected rTg4510 mice. Nuclei is stained with DAPI (C,G). Merged images of Tmem119 and Iba1 immunoreactivity in brain sections from treated mice (D,H). N = 6, 8 section per animal. Scale bar, 100 μm (I,J,L,M). High magnification (60x) co-localization images utilizing z-stack image intensity of highly ramified Tmem119⁺ cells and Iba-1⁺ microglia. Scale bar, 10 μm. (K,N) The intensity correlation analysis represented by the scatter plots of the fluorescence intensities of Tmem119 (Alexa Fluor 488) and Iba-1 (Alexa Fluor 647) of confocal z-stacks. The degree of co-localization is estimated by Mander's overlap coefficient.

Figure 5

CCL2 induces astrocytic ramification in rTg4510 mice. (A) Images of fluorescence immunohistochemical stain of astrocytes (GFAP, green) in the cortices of rTg4510 mice following CCL2 and (B) RFP overexpression. Individual astrocytes were subjected to Sholl analysis (box inset). (C,D) Maximum intensity projection of each cell tiled micrographs extracting 2D images of astrocytes. Sholl-based metrics of arborization using a 50 μm radii area from the soma of each astrocyte measured the number of intersections. (E) Quantification of positive area stained for GFAP is presented for anterior cortex (ACX). (F) Sholl analysis retrieved curve-fitting and regression analysis of astrocytes within the region of interest, demonstrated induced astrocytic intersection in CCL2 overexpressing mice compared to the RFP mice. (G) Scattered plot of the number of astrocytic intersections in each group, Student t-test, Mann-Whitney unpaired parametric test, ****p < 0.0001.

Figure 6

Cytokine levels following expression of CCL2 in rTg4510 mice. The concentrations of Interleukin 1 alpha (IL-1α), Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6), Interleukin 10 (IL-10), Interleukin 13 (IL-13), C-C motif ligand 11 (CCL11), Keratinocyte chemoattractant (KC), C-C motif ligand 3 (CCL3), C-C motif ligand 5 (CCL5), Vascular endothelial growth factor (VEGF) and Tumor necrosis factor alpha (TNF-α) were measured using the mouse cytokine/chemokine panel (MILLIPLEX MAP kit; Millipore, Billerica, MA, USA) in CCL2 injected mice and RFP controls. (mean ± S.E.M, n = 6). Statistical analysis was performed using multiple t-test analysis (*p < 0.05) with alpha = 0.05. and without assuming a consistent SD (df = N−2) followed by post-hoc test.

Figure 7

CCL2 overexpression induces tau accumulation in rTg4510 mice. Images of immunostaining in the cortical area for H150 tau (A,B), p-tau AT8 (C,D) and pSer396 (E,F) as well as Gallyas staining (aggregated tau G,H) in mice injected with either rAAV9-RFP or rAAV9-CCL2. Quantification of positive area stained is shown in anterior cortex (ACX), hippocampus (HPC), entorhinal cortex (EC) for total tau H150 (I), p-tau AT8 (J), pSer396 (K) and Gallyas (L). Scale bar represents 100 and 20 μm. Statistical analysis by Student t-test (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 8

CCL2 overexpression worsens tau pathology in rTg4510 mice. Western blots analyses of total tau (H150), and p-tau (AT180, PHF1, pSer396, and pSer199/202) in the soluble hippocampal brain fraction (A) and insoluble fraction (B). Band pixel densitometry values normalized to GAPDH and control mice for soluble fraction (C), and to controls for insoluble fraction (D) (n = 6, *p < 0.05, **p < 0.01, Student t-test). Overall, CCL2 overexpression resulted in increased high molecular weight tau in soluble fractions together with increased intensity of phosphorylated epitopes in insoluble fractions.

Figure 9

Amyloidosis following CCL2 overexpression in APP/PS1 mice. Micrograph representation of cortical area stained for amyloid beta (Aβ, A,B) and Congo red (C,D) in GFP injected animals (A,C) and CCL2 injected animals (B,D). Quantification of positive area stained is presented for anterior cortex amyloid beta (E) and Congo red (F) (Avg ± S.E.M, n = 10). Statistical analysis was performed using Student's t-test (n = 6, *p < 0.05) followed by Fisher's PLSD multiple comparison test. Scale bar represents 200 μm and insets represent 20 μm.