Oregon National Primate Research Center, Beaverton, 97006, USA.
Hum Mol Genet. 2009 Oct 15;18(20):3876-93. doi: 10.1093/hmg/ddp331. Epub 2009 Jul 19.
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.
我们的研究目的是研究与人突变淀粉样前体蛋白和淀粉样 β（Abeta）相关的小胶质细胞和星形胶质细胞。我们研究了抗粒细胞-巨噬细胞集落刺激因子（GM-CSF）抗体是否可以抑制阿尔茨海默病转基因小鼠（Tg2576 品系）中的小胶质细胞活性并减少 Abeta 的产生。通过脑室内（ICV）注射将针对小鼠 GM-CSF 的抗体引入 10 月龄 Tg2576 雄性小鼠的大脑中。我们使用定量实时 RT-PCR、免疫印迹、免疫组织化学分析来评估几种与 GM-CSF 相关的细胞因子对小胶质细胞活性的影响及其与 Abeta 的关联，在接受抗 GM-CSF 抗体注射的 Tg2576 小鼠中。使用夹心 ELISA 技术，我们测量了 GM-CSF 抗体注射的 Tg2576 小鼠和 PBS 载体注射的对照 Tg2576 小鼠中的神经元内 Abeta。通过神经元内 Abeta、Abeta 沉积和促炎细胞因子的双重标记免疫荧光分析，我们评估了 Tg2576 小鼠中 Abeta 沉积与小胶质细胞标志物之间的关系，以及抗 GM-CSF 抗体注射的 Tg2576 小鼠中的关系。我们的实时 RT-PCR 分析显示，与同窝非转基因对照相比，Tg2576 小鼠大脑皮质中 IL6、CD11c、IL1beta、CD40 和 CD11b 的 mRNA 表达增加。小胶质细胞标志物的免疫组织化学发现与我们的实时 RT-PCR 结果一致。有趣的是，与 PBS 载体注射的 Tg2576 小鼠相比，抗 GM-CSF 抗体注射的 Tg2576 小鼠中激活的小胶质细胞和 Abeta 沉积水平显着降低。我们的实时 RT-PCR 和免疫印迹分析结果与免疫组织化学结果一致。我们对神经元内 Abeta 和 CD40 的双重标记分析表明，神经元内 Abeta 与 Tg2576 小鼠中神经元表达的 CD40 相关。我们的定量夹心 ELISA 分析显示，抗 GM-CSF 抗体注射的 Tg2576 小鼠中可溶性 Abeta1-42 水平降低，Abeta1-40 水平升高，表明抗 GM-CSF 抗体单独降低可溶性 Abeta1-42 的产生并抑制 Tg2576 小鼠中的小胶质细胞活性。这些发现表明抗 GM-CSF 抗体能够降低 Tg2576 小鼠中的 Abeta1-42 和小胶质细胞活性，这可能对阿尔茨海默病具有治疗意义。
