Kim Hyehyun, Le Bryan, Goshi Noah, Zhu Kan, Grodzki Ana Cristina, Lein Pamela J, Zhao Min, Seker Erkin
bioRxiv. 2024 Mar 17:2024.03.15.584736. doi: 10.1101/2024.03.15.584736.
The etiology and progression of sporadic Alzheimer's Disease (AD) have been studied for decades. One proposed mechanism is that amyloid-beta (Aβ) proteins induce neuroinflammation, synapse loss, and neuronal cell death. Microglia play an especially important role in Aβ clearance, and alterations in microglial function due to aging or disease may result in Aβ accumulation and deleterious effects on neuronal function. However, studying these complex factors , where numerous confounding processes exist, is challenging, and until recently, models have not allowed sustained culture of microglia, astrocytes and neurons in the same culture. Here, we employ a tri-culture model of rat primary neurons, astrocytes, and microglia and compare it to co-culture (neurons and astrocytes) and mono-culture enriched for microglia to study microglial function (i.e., motility and Aβ clearance) and proteomic response to exogenous Aβ.
We established cortical co-culture (neurons and astrocytes), tri-culture (neurons, astrocytes, and microglia), and mono-culture (microglia) from perinatal rat pups. On days (DIV) 7 - 14, the cultures were exposed to fluorescently-labeled Aβ (FITC-Aβ) particles for varying durations. Images were analyzed to determine the number of FITC-Aβ particles after specific lengths of exposure. A group of cells were stained for βIII-tubulin, GFAP, and Iba1 for morphological analysis via quantitative fluorescence microscopy. Cytokine profiles from conditioned media were obtained. Live-cell imaging with images acquired every 5 minutes for 4 hours was employed to extract microglia motility parameters (e.g., Euclidean distance, migration speed, directionality ratio).
FITC-Aβ particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aβ particles, as confirmed via epifluorescence and confocal microscopy. Adding FITC-Aβ significantly increased the size of microglia, but had no significant effect on neuronal surface coverage or astrocyte size. Analysis of the cytokine profile upon FITC-Aβ addition revealed a significant increase in proinflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6) in tri-culture, but not co-culture. In addition, Aβ addition altered microglia motility marked by swarming-like motion with decreased Euclidean distance yet unaltered speed. These results highlight the importance of cell-cell communication in microglia function (e.g., motility and Aβ clearance) and the utility of the tri-culture model to further investigate microglia dysfunction in AD.
散发性阿尔茨海默病（AD）的病因和进展已研究数十年。一种提出的机制是β淀粉样蛋白（Aβ）诱导神经炎症、突触丧失和神经元细胞死亡。小胶质细胞在Aβ清除中起特别重要的作用，衰老或疾病导致的小胶质细胞功能改变可能导致Aβ积累并对神经元功能产生有害影响。然而，研究这些存在众多混杂过程的复杂因素具有挑战性，直到最近，模型还不允许在同一培养物中持续培养小胶质细胞、星形胶质细胞和神经元。在此，我们采用大鼠原代神经元、星形胶质细胞和小胶质细胞的三培养模型，并将其与共培养（神经元和星形胶质细胞）以及富含小胶质细胞的单培养进行比较，以研究小胶质细胞功能（即运动性和Aβ清除）以及对外源性Aβ的蛋白质组学反应。
我们从围产期大鼠幼崽建立了皮质共培养（神经元和星形胶质细胞）、三培养（神经元、星形胶质细胞和小胶质细胞）和单培养（小胶质细胞）。在第7 - 14天（体外培养天数，DIV），将培养物暴露于荧光标记的Aβ（FITC - Aβ）颗粒不同时间。分析图像以确定特定暴露时间后FITC - Aβ颗粒的数量。一组细胞用βIII - 微管蛋白、胶质纤维酸性蛋白（GFAP）和离子钙结合衔接分子1（Iba1）染色，通过定量荧光显微镜进行形态分析。获取条件培养基中的细胞因子谱。采用每5分钟采集一次图像、共采集4小时的活细胞成像来提取小胶质细胞运动参数（如欧几里得距离、迁移速度、方向性比率）。
与共培养相比，FITC - Aβ颗粒在三培养中清除更有效。这归因于小胶质细胞吞噬FITC - Aβ颗粒，经落射荧光和共聚焦显微镜证实。添加FITC - Aβ显著增加了小胶质细胞的大小，但对神经元表面覆盖率或星形胶质细胞大小无显著影响。添加FITC - Aβ后细胞因子谱分析显示，三培养中促炎细胞因子（肿瘤坏死因子 - α、白细胞介素 - 1α、白细胞介素 - 1β、白细胞介素 - 6）显著增加，而共培养中未增加。此外，添加Aβ改变了小胶质细胞的运动性，其特征为类似集群的运动，欧几里得距离减小但速度未改变。这些结果突出了细胞间通讯在小胶质细胞功能（如运动性和Aβ清除）中的重要性以及三培养模型在进一步研究AD中小胶质细胞功能障碍方面的实用性。
