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. 2009 Oct;175(4):1586-97.
doi: 10.2353/ajpath.2009.081113. Epub 2009 Sep 3.

Role of the Macrophage Inflammatory protein-1alpha/CC Chemokine Receptor 5 Signaling Pathway in the Neuroinflammatory Response and Cognitive Deficits Induced by Beta-Amyloid Peptide

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Free PMC article

Role of the Macrophage Inflammatory protein-1alpha/CC Chemokine Receptor 5 Signaling Pathway in the Neuroinflammatory Response and Cognitive Deficits Induced by Beta-Amyloid Peptide

Giselle Fazzioni Passos et al. Am J Pathol. .
Free PMC article

Abstract

The hallmarks of Alzheimer's disease include the deposition of beta-amyloid (Abeta), neuroinflammation, and cognitive deficits. The accumulation of activated glial cells in cognitive-related areas is critical for these alterations, although little is known about the mechanisms driving this event. Herein we used macrophage inflammatory protein-1alpha (MIP-1alpha(-/-))- or CC-chemokine receptor 5 (CCR5(-/-))-deficient mice to address the role played by chemokines in molecular and behavioral alterations induced by Abeta(1-40). Abeta(1-40) induced a time-dependent increase of MIP-1alpha mRNA followed by accumulation of activated glial cells in the hippocampus of wild-type mice. MIP-1alpha(-/-) and CCR5(-/-) mice displayed reduced astrocytosis and microgliosis in the hippocampus after Abeta(1-40) administration that was associated with decreased expression of cyclooxygenase-2 and inducible nitric oxide synthase, as well as reduced activation of nuclear factor-kappaB, activator protein-1 and cyclic AMP response element-binding protein. Furthermore, MIP-1alpha(-/-) and CCR5(-/-) macrophages showed impaired chemotaxis in vitro, although cytokine production in response to Abeta(1-40) was unaffected. Notably, the cognitive deficits and synaptic dysfunction induced by Abeta(1-40) were also attenuated in MIP-1alpha(-/-) and CCR5(-/-) mice. Collectively, these results indicate that the MIP-1alpha/CCR5 signaling pathway is critical for the accumulation of activated glial cells in the hippocampus and, therefore, for the inflammation and cognitive failure induced by Abeta(1-40). Our data suggest MIP-1alpha and CCR5 as potential therapeutic targets for Alzheimer's disease treatment.

Figures

Figure 1
Figure 1
Effect of Aβ1–40 on the expression of MIP-1α and CCR5 and in glial cell activation in mouse hippocampus. Wild-type C57BL/6 mice were left untreated (naive mice, N) or were treated i.c.v. with Aβ1–40 or Aβ40–1 (400 pmol/mouse), and brains were harvested at the time points indicated. Total RNA was isolated from hippocampuses for evaluation of MIP-1α and CCR5 mRNA expression, and β-actin mRNA was assessed as an internal control for the amount of RNA in each sample. Representative RT-PCR analysis showing MIP-1α (A) and CCR5 mRNA (B) expression. Densitometric analysis is expressed as the MIP-1α/β-actin (C) and the CCR5/β-actin ratio (D). E: Representative images of GFAP and CD68 immunostaining in the CA1 subregion of the hippocampus. Original magnification, ×100. Graphic representation of the number of GFAP- (F) and CD68-positive cells (G) determined in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampus. The values represent the mean ± SEM (N = 3 to 5 mice/group). *P < 0.05 and **P < 0.01 compared with the naive group.
Figure 2
Figure 2
Involvement of MIP-1α and CCR5 in Aβ1–40-induced glial cell activation in mouse hippocampus. Immunohistochemical analysis for GFAP and CD68 was performed 6 hours or 8 days after Aβ1–40 (400 pmol/mouse) or PBS i.c.v. injection, respectively. Mice lacking MIP-1α or CCR5 showed a reduced number of GFAP- (A and C) and CD68-positive cells (B and D) in the hippocampus. Representative images of GFAP (A) and CD68 immunostaining (B) in the CA1 subregion of the hippocampus. Original magnification, ×100. Graphic representation of the number of GFAP- (C) and CD68-positive cells (D) determined in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampus. The values represent the mean ± SEM (N = 5 mice/group). **P < 0.01 compared with the PBS-treated wild-type mouse group. ##P < 0.01 compared with the Aβ1–40-treated wild-type mouse group.
Figure 3
Figure 3
MIP-1α and CCR5 modulate Aβ1–40-induced iNOS and COX-2 up-regulation. A:1–40 induced time-dependent iNOS and COX-2 protein expression in the hippocampus. Western blot analysis for iNOS, COX-2, and β-actin (loading control) was performed in the hippocampuses of wild-type C57BL/6 mice 1 and 8 days after Aβ1–40 (400 pmol/mouse) i.c.v. injection. Some mice were left untreated (naive mice, N) or were treated with the reverse Aβ40–1 peptide (400 pmol/mouse i.c.v.). B: MIP-1α and CCR5 genetic deletion resulted in a reduction of iNOS up-regulation induced by Aβ1–40, when evaluated 1 day after treatment. C: Graph showing quantification of iNOS protein normalized by β-actin protein (loading control). For COX-2 protein, immunohistochemical analysis was performed 1 day after Aβ1–40 (400 pmol/mouse) or PBS i.c.v. injection. D: Representative images of COX-2 immunostaining, demonstrating diminished levels of this enzyme in the CA1 hippocampal subregion of MIP-1α−/− and CCR5−/− mice. Original magnification, ×40 E: Graphic representation of the average immunostaining for COX-2 evaluated in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampus. The values represent the mean ± SEM (N = 6 to 7 mice/group). *P < 0.05 and **P < 0.01 compared with the PBS-treated wild-type mouse group. #P < 0.05 and ##P < 0.01 compared with the Aβ1–40-treated wild-type mouse group.
Figure 4
Figure 4
Role of MIP-1α and CCR5 in intracellular pathways activated in response to Aβ1–40. Immunohistochemical analysis for p-CREB, p-p65 NF-κB, and p-c-Jun/AP-1 protein was performed 6 hours after Aβ1–40 (400 pmol/mouse), Aβ40–1 (400 pmol/mouse), or PBS i.c.v. injection. A: Graphic representation of the average immunostaining for p-CREB, demonstrating reduced activation in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampus of MIP-1α−/− and CCR5−/− mice. Similar results were observed when the number of positive cells for p-p65 NF-κB (B) and p-c-Jun AP-1 (C) per section was determined. The values represent the mean ± SEM (N = 5 mice/group). **P < 0.01 compared with the PBS-treated wild-type mouse group. #P < 0.05 and ##P < 0.01 compared with the Aβ1–40-treated wild-type mouse group. D: Representative images of nuclear p-CREB, p-p65 NF-κB, and p-c-Jun AP-1 immunostaining in the CA1 hippocampal subregion of wild-type, MIP-1α−/−, and CCR5−/− mice. Original magnification, ×40.
Figure 5
Figure 5
MIP-1α and CCR5 are required for cellular migration but not for activation induced by Aβ1–40 in vitro. Peritoneal macrophages were isolated from wild-type (WT), MIP-1α−/−, or CCR5−/− mice, cultured on 24-well plates, and incubated with 30 μmol/L Aβ1–40 or Aβ40–1 for 24 hours, and the supernatant was collected and assayed for chemotactic activity. Chemotaxis of wild-type, MIP-1α−/−, and CCR5−/− macrophages in response to the supernatants was assayed using the Neuro Probe transwell assay. Chemotactic index represents the number of cells that migrated in response to supernatant from stimulated macrophages/number of cells that migrated in response to supernatant from unstimulated macrophages. A: MIP-1α−/− macrophages produce reduced chemotactic activity for wild-type, MIP-1α−/−, and CCR5−/− cells when stimulated with Aβ1–40 compared with wild-type and CCR5−/− macrophages. Macrophages isolated from CCR5−/− mice show a diminished chemotactic index in response to supernatant of Aβ1–40-stimulated wild-type, MIP-1α−/−, or CCR5−/− cells. No significant chemotactic activity for macrophages was induced in response to the Aβ40–1. B: Macrophage chemotaxis toward C5a (10 nmol/L) was not affected by genetic deletion of MIP-1α or CCR5. IL-1β (C) or TNF-α (D) released into the supernatant of macrophages of wild-type, MIP-1α−/−, and CCR5−/− mice stimulated for 24 hours with 30 μmol/L Aβ1–40 or Aβ40–1, LPS (100 ng/ml), or C5a (10 nmol/L) was measured by enzyme-linked immunosorbent assay. E: Cell viability assessed after stimulation with Aβ1–40, Aβ40–1, LPS, or C5a. “Control” refers to unstimulated cells. Values represent the mean ± SEM (N = 3/group). Chemotaxis assays were performed in triplicate, and ELISA experiments in duplicate. **P < 0.01 compared with the control group. #P < 0.05 compared with the Aβ1–40-treated wild-type group. N.D., not determined.
Figure 6
Figure 6
Contribution of MIP-1α and CCR5 to cognitive deficits induced by Aβ1–40 in mice. The spatial reference memory version of the Morris water maze test was used as a measure of cognition. Training trials were performed on day 7 after a single i.c.v. administration of Aβ1–40 (400 pmol/mouse) or vehicle (PBS). Data are presented as means ± SEM latency (seconds) for escape to a hidden platform (n = 8−10 mice/group). The probe test session was performed 24 hours after training trials. Data are presented as means ± SEM of the frequency of time spent in the correct quadrant. MIP-1α−/− (A and B) and CCR5−/− (C and D) mice were significantly more resistant than wild-type (WT) C57Bl/6 mice to the deleterious effect of Aβ1–40 in spatial learning (A and C) and spatial retrieval (B and D). *P < 0.05 compared with the PBS-treated wild-type mouse group; #P < 0.05 compared with the Aβ1–40-treated wild-type mouse group.
Figure 7
Figure 7
Role of MIP-1α and CCR5 in Aβ1–40-induced synaptic disruption. A: Representative images of synaptophysin immunostaining in the CA1 subregion of the hippocampus evaluated 8 days after PBS, Aβ1–40, or Aβ40–1 i.c.v. administration (400 pmol/mouse). Original magnification, ×40. B: Graphic representation of the average optical density (O.D.) of the immunostaining for synaptophysin evaluated in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampus, demonstrating that MIP-1α−/− and CCR5−/− mice were significantly more resistant than wild-type mice to Aβ1–40-induced synaptic disruption. The values represent the mean ± SEM (N = 5 mice/group). **P < 0.01 compared with the PBS-treated wild-type mouse group. ##P < 0.01 compared with the Aβ1–40-treated wild-type mouse group.

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