Luchena Celia, Zuazo-Ibarra Jone, Valero Jorge, Matute Carlos, Alberdi Elena, Capetillo-Zarate Estibaliz
Department of Neuroscience, University of the Basque Country (UPV/EHU), Leioa, Spain.
Achucarro Basque Center for Neuroscience, Leioa, Spain.
Front Aging Neurosci. 2022 Apr 14;14:844534. doi: 10.3389/fnagi.2022.844534. eCollection 2022.
Glial cells are essential to understand Alzheimer's disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as co-cultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward setting with neurons and glial cells to study AD. We generated and optimized a 2D triple co-culture model with murine astrocytes, neurons and microglia, based on sequential seeding of each cell type. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric Aβ (oAβ) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory marker Arginase I, and reduced pro-inflammatory iNOS and IL-1β, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Anti-inflammatory marker TGF-β1 was also increased in the triple co-culture. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oAβ in the triple co-culture reduced synaptic markers and increased CD11b in microglia, which are hallmarks of AD. Consequently, we developed a straightforward and reproducible triple co-cultured model, where cells resemble physiological conditions better than in individual primary cultures: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology. Moreover, we are able to recapitulate Aβ-induced synaptic loss and CD11b increase. This model emerges as a powerful tool to study neurodegeneration and neuroinflammation in the context of AD and other neurodegenerative diseases.
鉴于神经胶质细胞在神经炎症和神经退行性变中的作用，对于理解阿尔茨海默病（AD）的进展至关重要。需要可靠且易于操作的模型来研究神经元与神经胶质细胞之间通讯背后的机制。目前可用的模型，如共培养模型，需要复杂的方法，而且可能并非所有实验室都能负担得起。考虑到这一点，我们旨在建立一个包含神经元和神经胶质细胞的简单体系来研究AD。我们基于每种细胞类型的顺序接种，生成并优化了一种包含小鼠星形胶质细胞、神经元和小胶质细胞的二维三重共培养模型。使用免疫荧光、蛋白质印迹和酶联免疫吸附测定技术来表征该模型中寡聚Aβ（oAβ）的作用。我们发现，在三重共培养中，与单独的小胶质细胞相比，小胶质细胞增加了抗炎标志物精氨酸酶I的表达，并降低了促炎标志物诱导型一氧化氮合酶（iNOS）和白细胞介素-1β（IL-1β）的表达。星形胶质细胞降低了促炎A1标志物AMIGO2和C3的表达，并呈现出类似于生理状态的分支形态。抗炎标志物转化生长因子-β1（TGF-β1）在三重共培养中也有所增加。最后，与单独的原代培养相比，神经元增加了突触后标志物的表达，并长出了更多更长的分支。在三重共培养中添加oAβ会降低突触标志物，并增加小胶质细胞中CD11b的表达，这些都是AD的特征。因此，我们开发了一种简单且可重复的三重共培养模型，其中细胞比单独的原代培养更接近生理状态：小胶质细胞炎症反应较轻，星形胶质细胞反应性较低，神经元呈现出更成熟的形态。此外，我们能够重现Aβ诱导的突触损失和CD11b增加。该模型成为研究AD和其他神经退行性疾病背景下神经退行性变和神经炎症的有力工具。
