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

创伤性脑损伤（TBI）是阿尔茨海默病最重要的环境风险因素， 阿尔茨海默病相关性痴呆（AD/ADRD）。TBI事件可能发生在 AD/ADRD，以便有时间进行治疗。不幸的是，没有合适的治疗方法，因为 TBI和AD/ADRD之间的联系知之甚少。tau蛋白的变化在 AD/ADRD。钙超载驱动tau蛋白的变化。钙超载也是TBI的结果。这 一项提案假设TBI导致钙超载，导致tau蛋白改变，而这一序列有助于 解释为什么TBI会增加AD/ADRD的风险。Piezo 1是一种钙通道，当细胞变形时会打开。 因此，可能是外伤造成的。这项提议将在皮质星形胶质细胞，皮质 神经元和内皮细胞。这些细胞将由人诱导多能干细胞产生。的 第一个目标是测量每种细胞类型对创伤的脆弱程度。细胞也会以同样的方式变形 它们处于TBI事件期间，并且将测量所导致的细胞死亡和细胞损伤。钙超载和 炎症信号也将被定量。在患者的大脑中，tau蛋白的变化在血管周围积累 但尚不清楚这种模式是否反映了血液或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重要的。 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基的实验来测量。 在从细胞中消除Piezo 1后，将重复具有创伤敏感结果的实验 以确定创伤反应是否需要Piezo 1。此外，同样的结果将在 化学激活Piezo 1的实验没有创伤。第二个目标将使用脑类器官。 这些是脑细胞簇，近似圆形，约1毫米宽。它们含有 将向其中加入皮质神经元和星形胶质细胞，并且在某些情况下加入内皮细胞。这些类器官 可以重现钙超载依赖性tau蛋白的变化，这种变化被认为是TBI后疾病的驱动因素。 AD/ADRD。它们将以与TBI事件期间相同的方式发生机械变形。 将量化所导致的细胞死亡和细胞损伤。蛋白质的分泌表明脑损伤 并且还将测量自发电活动的变化。此外，总tau蛋白和 在创伤后定量磷酸化的tau蛋白。显微成像将确定tau蛋白是否改变 当它们存在时在内皮细胞周围积聚。和以前一样，实验表明， 在Piezo 1从细胞中消除后，创伤将重复。然后，它们将被重复， Piezo 1已经被化学激活，没有创伤。结合起来，这些实验将重现 从机械性创伤到tau蛋白的变化，驱动AD/ADRD，并揭示了Piezo 1在其中的作用， 过程这些结果将是治疗这些患者以中断这种进展的重要一步。

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.