Applying human in vitro models to understand the link between trauma and tau pathology

创伤性脑损伤是阿尔茨海默病最重要的环境危险因素， 阿尔茨海默病相关痴呆(AD/ADRD)。TBI事件可能发生在出现之前数年 AD/ADRD，所以有时间进行治疗。不幸的是，没有合适的治疗方法，因为 TBI与AD/ADRD之间的联系知之甚少。Tau蛋白的变化在 AD/ADRD。钙超载会导致tau的变化。钙超载也是脑外伤的一个后果。这 提案假设脑损伤导致钙超载，从而导致tau变化，这一序列有助于 解释为什么颅脑损伤会增加AD/ADRD的风险。PIEZO1是一种钙通道，在细胞变形时开放。 因此，它可能会被创伤打开。这项提议将在大脑皮层星形胶质细胞、大脑皮层星形胶质细胞 神经元和内皮细胞。这些细胞将从人类诱导的多能干细胞中产生。这个 第一个目标将测量每种细胞类型对创伤的脆弱性。细胞也会以同样的方式变形 它们发生在脑损伤事件期间，将测量由此导致的细胞死亡和细胞损伤。钙超载和 炎症信号也将被量化。在患者的大脑中，tau的变化积累在血管周围。 但目前尚不清楚这种模式是否反映了血液或血管内皮细胞的毒性影响 血管。因此，内皮细胞对邻近星形胶质细胞和神经元的影响将是 通过在培养物中混合细胞或在细胞培养物之间转移细胞培养液的实验来测量。 在Piezo1从细胞中去除后，将重复进行创伤敏感结果的实验 以确定创伤反应是否需要Piezo1。此外，同样的结果将在 化学激活Piezo1而不会造成创伤的实验。第二个目标将使用脑有机化合物。 这些脑细胞簇大约是圆形的，大约1毫米宽。它们包含 皮质神经元和星形胶质细胞，在某些情况下，内皮细胞将被添加到它们中。这些有机化合物 可以复制钙超载依赖的tau变化，这种变化被认为是导致脑损伤后疾病的原因 AD/ADRD。它们将以相同的方式机械变形，就像它们在TBI事件中变形一样。 由此产生的细胞死亡和细胞损伤将被量化。分泌已知的指示脑损伤的蛋白质 还将测量自发电活动的变化。此外，总tau蛋白和 创伤后将对磷酸化的tau蛋白进行定量。显微成像将确定tau是否发生变化 当内皮细胞存在时，它们会聚集在内皮细胞周围。和以前一样，对 在Piezo1从细胞中消除后，创伤将重复发生。然后，它们将在以下情况下重复 PIEZO1已经被化学激活，而不会造成创伤。结合起来，这些实验将重现 从机械性创伤进展到tau改变，驱动AD/ADRD并揭示Piezo1在其中的作用 进程。这些结果将是朝着治疗这些患者迈出的重要一步，以中断这种进展。

Traumatic brain injury (TBI) is the most important environmental risk factor for Alzheimer’s disease and Alzheimer’s disease related dementias (AD/ADRD). The TBI event may occur years before the emergence of AD/ADRD so there is time to apply treatments. Unfortunately, no appropriate treatments exist because the connection between TBI and AD/ADRD is poorly understood. Changes in tau proteins play an important role in AD/ADRD. Calcium overload drives changes in tau. Calcium overload is also a consequence of TBI. This proposal hypothesizes that TBI causes calcium overload that leads to tau changes and this sequence helps explain why TBI increases the risk of AD/ADRD. Piezo1 is a calcium channel that opens when cells deform. Therefore, it could be opened by trauma. This proposal will test this hypothesis in cortical astrocytes, cortical neurons, and endothelial cells. These cells will be generated from human induced pluripotent stem cells. The first Aim will measure how vulnerable each cell type is to trauma. The cells will be deformed in the same way they are during a TBI event and resulting cell death and cell damage will be measured. Calcium overload and inflammatory signaling will also be quantified. In patient brains, tau changes accumulate around blood vessels after TBI but it is not clear if this pattern reflects the toxic influence of blood or the endothelial cells that line blood vessels. Therefore, the influence of endothelial cells on neighboring astrocytes and neurons will be measured with experiments that either mix cells in culture or transfer cell culture media between cell cultures. Experiments with trauma-sensitive outcomes will be repeated after Piezo1 has been eliminated from the cells to determine if Piezo1 is required for a trauma response. In addition, the same outcomes will be measured in experiments that activate Piezo1 chemically without trauma. The second Aim will employ brain organoids. These are clusters of brain cells that are approximately round and about 1 millimeter wide. They contain cortical neurons and astrocytes and, in some cases, endothelial cells will be added to them. These organoids can reproduce the calcium overload-dependent tau changes that are hypothesized to drive disease in post-TBI AD/ADRD. They will be mechanically deformed in the same way they would be deformed during a TBI event. Resulting cell death and cell damage will be quantified. Secretion of proteins known to indicate brain damage and changes in spontaneous electrical activity will also be measured. In addition, total tau protein and phosphorylated tau protein will be quantified after trauma. Microscopic imaging will determine if tau changes accumulate around endothelial cells when they are present. As before, experiments that show sensitivity to trauma will be repeated after Piezo1 has been eliminated from the cells. Then, they will be repeated when Piezo1 has been activated chemically without trauma. In combination, these experiments will reproduce the progression from mechanical trauma to tau changes that drive AD/ADRD and reveal the role of Piezo1 in that process. These results will be an important step toward treating these patients to interrupt this progression.