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Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1α, suppress amyloid β-induced neurotoxicity - PubMed

  • ️Sat Jan 01 2011

Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1α, suppress amyloid β-induced neurotoxicity

Dayanidhi Raman et al. Toxicol Appl Pharmacol. 2011.

Abstract

Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-β (Aβ). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1α (SDF-1α), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress Aβ-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1α significantly protected neurons from Aβ-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1α. Intra-cerebroventricular (ICV) injection of Aβ led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24h following the exposure. The Aβ-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F(2)-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1α. Additionally, MIP-2 or SDF-1α was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against Aβ neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration.

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Figures

Figure 1
Figure 1. Expression profile of chemokines and their receptors in mouse neurons in vitro

CXC chemokine receptors CXCR2 and CXCR4 are present in soma and dendritic compartments in cultured neurons 21 days in vitro (DIV). MAP2 marker was used to visualize the neurons. Representative pseudo-colored confocal images of neurons were shown as cyan – CXCR2 or CXCR4 and green – MAP-2; scale bar – 20 µm.

Figure 2
Figure 2. CXC chemokines MIP-2 and CXCL12 afford neuroprotection in vitro

A. The preparation of Aβ(1–40) and Aβ(1–42) mixture was separated by 15% SDS-PAGE and analyzed by immunoblotting. B and C. Representative neurolucida traces from the confocal immunofluorescent images of MAP2-stained neurons from control, Aβ-treated and MIP-2 and CXCL12 pretreated prior to Aβ-treatment were shown depicting Aβ-induced damage and MIP2-medited and CXCL12-mediated neuroprotection. D. CXC chemokines MIP-2 and CXCL12 prevent Aβ-induced dendritic regression in vitro. The graphs show the measurements obtained for the dendritic length that were plotted against the type of treatment. The dendritic length calculated using Neuroexplorer program was expressed in microns. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. Aβ* - significant difference from the control (p<0.01); MIP-2+Aβ # - significant difference from Aβ (p<0.05); CXCL12+Aβ # -significant from Aβ (p<0.05). E. CXC chemokines MIP-2 and CXCL12 prevent Aβ–induced neuronal apoptosis in vitro. Apoptotic nuclei per 10 random high-power fields (HPF) (400X) were counted after different treatments and were plotted as % of apoptotic neurons for each respective treatment group for both CXCR2 (+/+) and CXCR2 (−/−) primary neuronal cultures. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. * - Significant difference in neuronal apoptosis was observed in Aβ-treated group; # - Significant neuroprotection was observed in individually treated MIP-2 and CXCL12 groups.

Figure 3
Figure 3. Chemokine-mediated neuroprotection is associated with activation of PI-3-kinase and ERK1/2 pathways in vitro

A and B. The specificity of the neuroprotective signaling from CXCR4 and CXCR2 was examined by treating the primary neuronal cultures with the CXCR4 antagonist AMD-3100 and CXCR2 antagonist SB225002 in terms of activation of Akt and ERK1/2 through phosphorylation. The representative blots were shown. Data from 3–4 independent experiments were quantitated using Image J program. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. A – a) pAkt - Control vs. CXCL12 - 2 min – p<0.001; Control vs. CXCL12 - 5 min – p<0.05; CXCL12 - 2 min vs. AMD-3100+CXCL12 – 2 min - p<0.05 b) pERK - Control vs. CXCL12 - 2 min – p<0.05. B – Control vs. MIP2 – p<0.001; MIP2 vs. SB225002+MIP2 – p<0.01. C. Immunoblot analysis of the chemokine-mediated neuroprotection against Aβ-induced neurotoxicity. Phosphorylation of Akt at T308 and ERK1/2 was assessed by running 50 µg of total protein from the neuronal lysates from different treatments by SDS-PAGE and Western blot analysis. In the right ERK panel, N-cadherin served as an additional loading control. Data from 3 independent experiments were quantitated using Image J program. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test - 1) Aβ vs. MIP2+ Aβ – p<0.05 2) Aβ vs. CXCL12+ Aβ – p<0.05. D. Immunoblot analysis of the stabilization of the matrix metalloproteinase ADAM17 by the CXCL12 pretreatment. 50 µg of the total protein from the neuronal lysates from different treatments was separated by SDS-PAGE and ADAM17 level and analyzed by immunoblotting. Data from 3 independent experiments were quantitated using Image J program. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test - Aβ vs. CXCL12+ Aβ – p<0.05.

Figure 4
Figure 4. Expression profile of chemokines and their receptors in mouse neurons in vivo

The brain sections were examined for the expression of chemokines MIP2 andCXCL12 and the chemokine receptors CXCR2 and CXCR4 in the hippocampal neurons in vivo. The left panel depicts the immunolocalization in the hippocampus of CXCR2 (+/+) mice and the right panel shows CXCR2 (−/−) mice. The hippocampal sections in which positively stained neurons were observed were indicated by red arrows.

Figure 5
Figure 5. Golgi-Cox staining of neurons for the assessment of quantitative neuroprotection by the chemokines in vivo

A. Photomicrographs of mouse hippocampus (2.5X) with pyramidal neurons (10X) and neuronal tracing from CA1 hippocampal area of brain 24 h after saline (control) injection. Tracing and counting were done using a Neurolucida system at 100 x under oil immersion (MicroBrightField, VT). Colors indicate the degree of dendritic branching (yellow=1°, red=2°, purple=3°, green=4°, turquoise=5°). B. Chemokine MIP-2 and CXCL12 pretreatment preserved the dendritic spines from Aβ-induced neurotoxicity in vivo. The panel depicts high power photomicrographs (100 X) of Golgi-impregnated dendritic segments with spines from hippocampal neurons that were treated with either i) saline, ii) Aβ or iii) combined MIP-2 and CXCL12 before Aβ exposure.

Figure 6
Figure 6. Morphometric evaluation of chemokine-mediated neuroprotection in vivo

A & B. Preservation of dendritic length of hippocampal CA1 neurons upon MIP-2 and CXCL12 pretreatment in CXCR2 (+/+) and CXCR2 (−/−) mice. The dendritic length (microns) obtained was plotted against the different treatment groups. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. * - Significant difference observed in Aβ-treated group when compared to the control; # - Significant neuroprotection observed in individually treated MIP-2 and CXCL12 groups compared to the Aβ–treated group. C & D. Preservation of dendritic spine density by MIP-2 and CXCL12 pretreatment. The dendritic spine density of CA1 hippocampal neurons was plotted against respective treatment group. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. * - Significant difference observed in Aβ-treated group when compared to the control; # - Significant neuroprotection observed in individually treated MIP-2 and CXCL12 groups compared to the Aβ-treated group.

Figure 7
Figure 7. Increased expression of CXCR1 mRNA in the adult CXCR2 (−/−) brain

A. The level of CXCR1 and CXCR2 mRNA from CXCR2 (+/+) and CXCR2 (−/−) brain was analyzed by qRT-PCR of the total mRNA obtained by the TRIzol method. The level was normalized to the β-tubulin mRNA and expressed as fold change. B. The semi-quantitative RT-PCR analysis of the level of CXCR1 mRNA from CXCR2 (+/+) and CXCR2 (−/−) brain was performed. The lung sample served as the positive control and the actin served as the loading control.

Figure 8
Figure 8. MIP2 or CXCL12 pretreatment prevented the Aβ-induced oxidative damage in vivo

The oxidative damage by Aβ and the prevention of such oxidative damage by the chemokine pretreatment was assessed by the quantitative measurement of F2-isoprostanes in the cerebrum in the adult CXCR2 (+/+) brain. The level of F2-isoprostanes was expressed as ng / g of brain tissue and was plotted against different treatments. Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test – Aβ vs. control – p<0.001; Aβ vs. Aβ+MIP2 – p<0.01; Aβ vs. Aβ+CXCL12 – p<0.001.

Figure 9
Figure 9. Differential distribution of p21-activated kinase (PAK) in CA1 hippocampal neurons in vivo

A: Light photomicrographs of hippocampal CA1 neurons from control brain sections (20 X) depicting the total PAK distribution; Aβ–treated neurons showed intense or darkly stained pattern for PAK1 in the cytoplasm; MIP-2 (1 µg) or CXCL12 (1 µg) pretreatment depicts a diffuse and smooth distribution of PAK. B: Sections from the brain that were treated with Aβ only (40 X and 100 X of two different sections) - Aβ affected neurons displayed mislocalized, intense and particulate distribution of PAK and also nuclear condensation was observed indicated by dark blue nuclei. C: Boxed inset area in ‘B’ was magnified and shown. Note that the Aβ affected neurons pointed out by closed arrows have dark and granular PAK distribution; some have condensed dark blue nuclei. The normal adjacent neurons are indicated by open arrows with clear nuclei shown to contrast the Aβ affected CA1 hippocampal neurons in the field.

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