Overexpression of monocyte chemotactic protein-1/CCL2 in beta-amyloid precursor protein transgenic mice show accelerated diffuse beta-amyloid deposition

Abstract

Microglia accumulation at the site of amyloid plaques is a strong indication that microglia play a major role in Alzheimer's disease pathogenesis. However, how microglia affect amyloid-beta peptide (Abeta) deposition remains poorly understood. To address this question, we developed a novel bigenic mouse that overexpresses both amyloid precursor protein (APP) and monocyte chemotactic protein-1 (MCP-1; CCL2 in systematic nomenclature). CCL2 expression, driven by the glial fibrillary acidic protein promoter, induced mononuclear phagocyte (MP; monocyte-derived macrophage and microglial) accumulation in the brain. When APP/CCL2 transgenic mice were compared to APP mice, a fivefold increase in Abeta deposition was present despite increased MP accumulation around hippocampal and cortical amyloid plaques. Levels of full-length APP, its C-terminal fragment, and Abeta-degrading enzymes (insulin-degrading enzyme and neprilysin) in APP/CCL2 and APP mice were indistinguishable. Sodium dodecyl sulfate-insoluble Abeta (an indicator of fibrillar Abeta) was increased in APP/CCL2 mice at 5 months of age. Apolipoprotein E, which enhances Abeta deposition, was also increased (2.2-fold) in aged APP/CCL2 as compared to APP mice. We propose that although CCL2 stimulates MP accumulation, it increases Abeta deposition by reducing Abeta clearance through increased apolipoprotein E expression. Understanding the mechanisms underlying these events could be used to modulate microglial function in Alzheimer's disease and positively affect disease outcomes.

Figure 1

CCL2 levels and Aβ deposition in APP/CCL2 mice. A: Frontal cortex of APP/CCL2, APP, CCL2, and WT mice at 14 months of age (n = 6) were dissected and subjected for protein extraction in solubilization buffer or total RNA preparation. Murine CCL2 protein and mRNA levels were determined using ELISA and real-time RT-PCR as described in the Material and Methods, respectively. *, P < 0.0001 between APP/CCL2 versus APP or CCL2 versus WT; ^#, P < 0.05 between APP/CCL2 versus CCL2. B: Ten slides of 5-μm-thick brain sections of APP or APP/CCL2 mice were immunostained with anti-Aβ antibody, developed with DAB, and counterstained with hematoxylin. The arrows indicate immunopositive Aβ deposits. C: Average percent area occupied by Aβ deposits in the hippocampus (left) and the cortex (right) as quantified by image analyses of immunostained slides (n = 6). * and **, P < 0.05 and P < 0.01 versus APP mice as determined by Student’s t-test, respectively. Original magnifications, ×40. Scale bar, 250 μm.

Figure 2

TS staining of APP/CCL2 mouse brains. A: Mice at 14 months of age were tested for compact amyloid plaques by TS staining. Images show 10-μm-thick coronal sections of the hippocampal region. B: Quantitative analysis of the number of TS-positive plaques in hippocampus and cortex. C: Quantitative analysis of the area occupied by TS-positive plaques in hippocampus and cortex. N.S. in B and C denotes no statistical significance as determined by Student’s t-test. Original magnifications, ×40.

Figure 3

Microglial accumulation in APP/CCL2 mice. A: Ten-μm-thick slices of cortical (C) and hippocampal (H) regions of mice at 14 months of age (n = 6) were immunostained with anti-IBA-1 antibody and counterstained with hematoxylin. Arrows indicate IBA-1-positive cells in the brain regions. B: Quantification of IBA-1-positive cells in brain regions. *, P < 0.05 versus other groups as determined by analysis of variance and Newman-Keuls post hoc. C: TS-positive amyloid plaques (AP) were surrounded by IBA-1-positive microglial cells in both the hippocampal and cortical regions of APP/CCL2 or APP mice. D: Quantification of IBA-1-positive cells per μm² AP. *, P < 0.05 versus APP mice (n = 6) as determined by Student’s t-test. Original magnifications: ×200 (A); ×400 (C). Scale bars, 25 μm.

Figure 4

Astrogliosis in APP/CCL2 mouse brains. A: APP and APP/CCL2 mice at 14 months of age (n = 3) were tested for astrogliosis by anti-GFAP staining. Images show 5-μm-thick coronal sections of the hippocampal region. B: Quantitative analysis of the number of GFAP-positive astrocytes in the hippocampus and the cortex of APP/CCL2 and APP mice. No statistical significance was observed. C: Immunoblotting analysis of GFAP at 14 months of age (n = 3 per group). *, Statistical differences (P < 0.05) between WT versus APP and CCL2 versus APP/CCL2 as determined by analysis of variance and Newman-Keuls post hoc. Original magnifications, ×40.

Figure 5

Synaptic density in APP/CCL2 mouse brains. A: Mice at 14 months of age were tested for synaptic density by anti-synaptophysin staining. Images show 5-μm-thick coronal section of hippocampal region. B: Quantitative analysis of the density of synaptophysin staining in the hippocampus and the cortex. No statistical significance was observed among the group in either brain subregion. Original magnifications, ×40.

Figure 6

APP expression and processing. A: Protein extracts from the cortex and the hippocampus of APP/CCL2 and APP mice (n = 3) were subjected to immunoblotting for APP and β-actin using anti-Aβ (6E10) and anti-β-actin monoclonal antibodies. The APP-immunoreactive band intensity was normalized by β-actin band intensity. No statistical significance was observed. B: APP α- and β-CTF protein levels were quantified by immunoblotting using anti-APP 751-770 antibody and normalized by β-actin protein levels in cortex (n = 3). No statistical significance was observed.

Figure 7

APP and Aβ in young mouse brains. A and B: APP, total Aβ40, Aβ42, and Aβ42/Aβ40 ratio in cortex quantified by specific Aβ ELISA at 5 months of age (n = 3 per group). No statistical significance was observed. C: SDS-insoluble Aβ40 ELISA in hippocampus and cortex of APP/CCL2 and APP mice at 5 months of age (n = 3). *P < 0.05 versus APP mice as determined by Student’s t-test.

Figure 8

GFAP and IBA-1 expression in young mouse brains. A: Protein extracts (30 μg/lane) from the frontal cortex of APP/CCL2 and APP mice at 5 months of age (n = 3 per group) were subjected to immunoblotting for β-actin, GFAP, and IBA-1. B: Quantification of immunoreactive bands in A. *, P < 0.05 versus APP as determined by Student’s t-test.

Figure 9

Effect of CCL2 on APP processing in vitro. Nerve growth factor-treated differentiated PC12 cells (1 × 10⁵ cells/well in poly-d-lysine-coated 24-well plate) were infected with recombinant adenovirus expressing Swedish FAD APP₆₉₅ mutant and subsequently stimulated with increasing doses of recombinant murine CCL2 for 24 hours. Media was collected and both Aβ40 and Aβ42 levels were determined by specific Aβ ELISA. Data were presented as pg/ml, ng/ml, or percent ratio of Aβ42/Aβ40.

Figure 10

Aβ-degrading enzymes in brains. A and B: Protein extracts (30 μg/lane) from the frontal cortex of four mouse groups at 14 months of age (n = 3) were subjected to immunoblotting using anti-IDE (A), anti-neprilysin (B), and anti-β-actin monoclonal antibodies (top). The band intensity of IDE and neprilysin was normalized by the β-actin signal and the average intensity ratios were presented (bottom). No statistical significance was observed.

Figure 11

ApoE levels in brains of young and aged animals. A: Protein extracts (30 μg/lane) from the frontal cortex of APP and APP/CCL2 mice at 5 and 14 months of age (n = 3 per group per age) were subjected to immunoblotting using anti-apoE (Biodesign) and anti-β-actin monoclonal antibodies. B: The band intensity of apoE was normalized by the β-actin signal and the average intensity ratios were presented (filled bar, APP/CCL2 group; open bar, APP group). *, P < 0.05 versus APP at 14 months of age as determined by Student’s t-test.