Neutralization of granulocyte macrophage colony-stimulating factor decreases amyloid beta 1-42 and suppresses microglial activity in a transgenic mouse model of Alzheimer's disease

Abstract

The purpose of our study was to investigate microglia and astrocytes that are associated with human mutant amyloid precursor protein and amyloid beta (Abeta). We investigated whether the anti-granulocyte-macrophage-colony stimulating factor (GM-CSF) antibody can suppress microglial activity and decrease Abeta production in Alzheimer's disease transgenic mice (Tg2576 line). An antibody to mouse GM-CSF was introduced by intracerebroventricular (ICV) injections into the brains of 10-month-old Tg2576 male mice. We assessed the effect of several GM-CSF-associated cytokines on microglial activities and their association with Abeta using quantitative real-time RT-PCR, immunoblotting, immunohistochemistry analyses in anti-GM-CSF antibody-injected Tg2576 mice. Using sandwich ELISA technique, we measured intraneuronal Abeta in Tg2576 mice injected with GM-CSF antibody and PBS vehicle-injected control Tg2576 mice. Using double-labeling immunofluorescence analysis of intraneuronal Abeta, Abeta deposits and pro-inflammatory cytokines, we assessed the relationship between Abeta deposits and microglial markers in the Tg2576 mice, and also in the anti-GM-CSF antibody-injected Tg2576 mice. Our real-time RT-PCR analysis showed an increase in the mRNA expression of IL6, CD11c, IL1beta, CD40 and CD11b in the cerebral cortices of the Tg2576 mice compared with their littermate non-transgenic controls. Immunohistochemistry findings of microglial markers agreed with our real-time RT-PCR results. Interestingly, we found significantly decreased levels of activated microglia and Abeta deposits in anti-GM-CSF antibody-injected Tg2576 mice compared with PBS vehicle-injected Tg2576 mice. Findings from our real-time RT-PCR and immunoblotting analysis agreed with immunohistochemistry results. Our double-labeling analyses of intraneuronal Abeta and CD40 revealed that intraneuronal Abeta is associated with neuronal expression of CD40 in Tg2576 mice. Our quantitative sandwich ELISA analysis revealed decreased levels of soluble Abeta1-42 and increased levels of Abeta1-40 in Tg2576 mice injected with the anti-GM-CSF antibody, suggesting that anti-GM-CSF antibody alone decreases soluble Abeta1-42 production and suppresses microglial activity in Tg2576 mice. These findings indicating the ability of the anti-GM-CSF antibody to reduce Abeta1-42 and microglial activity in Tg2576 mice may have therapeutic implications for Alzheimer's disease.

Figure 1.

Immunoblotting analysis of microglial proteins in Tg2576 mice injected with GM-CSF antibody and Tg2576 mice injected with PBS. (A) Twenty micrograms of protein lysate was used from each sample, and immunoblotting analysis was performed using antibodies of IL1β, IL6, CD40 and β-actin. Bottom panel represents the immunoblotting of β-actin for equal loading. (B) Densitometry values for microglial proteins IL1β, IL6 and CD40 in Tg2576 mice injected with GM-CSF antibody and Tg2576 mice injected with PBS. As shown, we found decreased protein levels for CD40 (by 48%), followed by IL1β (43%), IL6 (39%) and CD11b (11%) in Tg2576 mice injected with GM-CSF antibody compared with Tg2576 mice injected with PBS.

Figure 2.

Immunoreactivity of IL1 in different brain regions of a representative Tg2576 mouse. Accumulated immunoreactivity was found in the cerebral cortex, hippocampus and ventricular regions of the brain in Tg2576 mice. Arrows indicate the increased immunoreactivity of IL1β in the hippocampus, layers 1–3 in the cortex and ventricular regions of the midbrain. (A) Photographed at ×5 the original magnification, (B) ×10, (C and D) ×20, (E and F) ×10.

Figure 3.

Double-labeling immunofluorescence analyses of Aβ deposit and microglia marked by IL1β (A) and CD11b (B) in representative 10-month-old Tg2576 mouse. Aβ deposits are associated with microglia. (Aa) Immunoreactivity of IL1, (Ab) surrounded by Aβ deposit in the same section and (Ac) overlay of both IL1 and Aβ deposit. Arrows indicate immunoreactivity of IL1 (in white), and the arrowhead indicates Aβ deposit. (Ba) Immunoreactivity of CD11b, (Bb) surrounded by Aβ deposit and (Bc) overlay of both CD11b and Aβ deposit. Arrows indicate immunoreactivity of CD11b (in white), and the arrowhead indicate Aβ deposit.

Figure 4.

Double-labeling immunofluorescence analyses of Aβ deposit and microglia marked by IL6 (A) and CD11b (B) in a representative10-month-old Tg2576 mouse. Aβ deposit without microglia. (A) Microglia marked by immunoreactivity of IL6 (a), Aβ deposit without microglia in the same section (b) and overlay of both IL6 and Aβ deposit (c). (B) Immunoreactivity of microglia marked by CD11b present in the vicinity of Aβ deposit (arrows in white). Aβ deposits are present without microglial immunoreactivity of CD11b (arrows in red).

Figure 5.

Double-labeling immunofluorescence analyses of Aβ deposit and astrocytes marked by glial fibrillary acidic protein (GFAP) in a representative 10-month-old Tg2576 mouse. (A) Immunoreactivity of GFAP (in green), (B) immunoreactivity of Aβ deposits (in red) of the same section and (C) overlay of GFAP and Aβ deposits. Aβ deposits are associated with astrocytes.

Figure 6.

Double-labeling analysis of intraneuronal Aβ and CD40 in a representative 10-month-old Tg2576 mouse. (A) Immunoreactivity of CD40 (in green), (B) intraneuronal Aβ and Aβ deposits (in red) of the same section and (C) overlay of CD40 and intraneuronal Aβ. Arrows indicate localization of intraneuronal Aβ and CD40. Sections were photographed at ×40 the original magnification.

Figure 7.

Double-labeling analysis of intraneuronal NeuN and CD40 in a representative 10-month-old Tg2576 mouse. (A) Immunoreactivity of NeuN (in green) in the cerebral cortex section, (B) intraneuronal CD40 (in red) of the same section and (C) overlay of NeuN and CD40. White arrows indicate co-localized neurons with NeuN and CD40, and red arrows indicate neurons only with NeuN expression. Sections were photographed at ×40 the original magnification.

Figure 8.

Immunoreactivity of IL6 in the cerebral cortex of a representative 10-month-old anti-GM-CSF antibody-injected Tg2576 mouse and a representative PBS vehicle-injected Tg2576 mouse. (A) Increased immunoreactivity of IL6 in a PBS vehicle-injected Tg2576 mouse and (B) decreased immunoreactivity of IL6 in an anti-GM-CSF antibody-injected (25 µg) Tg2576 mouse. Sections were photographed at ×40 the original magnification.

Figure 9.

Immunoreactivity of CD11c in the cerebral cortex of a representative 10-month-old anti-GM-CSF antibody-injected Tg2576 mouse and a representative PBS vehicle-injected Tg2576 mouse. (A) Increased immunoreactivity of CD11c in a PBS vehicle-injected Tg2576 mouse and (B) decreased immunoreactivity of CD11c in an anti-GM-CSF antibody-injected (25 µg) Tg2576 mouse. Sections were photographed at ×40 the original magnification.

Figure 10.

Immunoreactivity of GFAP in a representative 10-month-old anti-GM-CSF antibody-injected Tg2576 mouse and a representative PBS vehicle-injected Tg2576 mouse. (A) Immunoreactivity of GFAP in a PBS vehicle-injected Tg2576 mouse, (B) increased immunoreactivity of GFAP in the cerebral cortex and hippocampus of an anti-GM-CSF antibody-injected Tg2576 mouse, (C) GFAP immunoreactivity of cerebral cortex from a PBS vehicle-injected Tg2576 mouse and (D) increased immunoreactivity of cerebral cortex in an anti-GM-CSF antibody-injected Tg2576 mouse. (A) and (B) were photographed at ×5 the original magnification, and (C) and (D) at ×20.

Figure 11.

Immunoreactivity of Aβ deposits in a representative 10-month-old anti-GM-CSF antibody-injected Tg2576 mouse and a representative PBS vehicle-injected Tg2576 mouse. The E610 antibody recognized both intraneuronal Aβ and Aβ deposits in mouse brain sections. (A) Aβ deposits in the cerebral cortex and hippocampus of a PBS vehicle-injected Tg2576 mouse, (B) decreased Aβ deposits in the cerebral cortex and hippocampus in an anti-GM-CSF antibody-injected mouse, (C) Aβ deposits in the hippocampus of a PBS vehicle-injected Tg2576 mouse and (D) decreased Aβ deposits in the hippocampus of an anti-GM-CSF antibody-injected mouse. (A) and (B) were photographed at ×5 the original magnification, and (C) and (D), at ×10.