Voltage Imaging of Astrocyte-Neuron Interactions

摘要。 星形胶质细胞控制神经传递，有助于形成强大的血脑屏障（BBB），并在代谢上支持 神经元活动在阿尔茨海默氏病（AD）中，星形胶质细胞在A β斑块附近变得反应性，有助于 神经炎症，并有助于突触和电路异常。星形胶质细胞在 通过从细胞外空间去除谷氨酸来控制突触兴奋在AD中特别有趣。 星形胶质细胞表达高水平的兴奋性氨基酸转运蛋白（EAAT），包括GLT 1和GLAST， 它们的外周过程结合并去除细胞外谷氨酸以限制突触兴奋。EAAT，作为 以及其他星形胶质细胞蛋白，已知在AD进展的后期降低。异常谷氨酸信号传导 被认为有助于AD的进展，如谷氨酸受体美金刚的临床使用所证明的 拮抗剂作为AD治疗，强烈暗示EAAT参与AD的进展。最后，可溶性A β抑制 EAAT在神经元活动增强时发挥作用。有趣的是，我们最近发现， 导致星形胶质细胞去极化，从而驱动EAAT功能的电压依赖性抑制， 神经元激活在亲本R 01中，我们使用星形胶质细胞表达的遗传编码电压指示剂 （GEVI）光学定量Vm，并显示神经元活动使外周星形胶质细胞过程去极化 （PAPs）具有突触特异性。在本补充申请中，我们提供了在正常老化期间 星形胶质细胞偶发性地失去GLT 1、GLAST和Kir4.1的表达，并呈现称为 非典型星形胶质细胞（AtAs）。重要的是，AtA不是反应性星形细胞增多症的一种形式。初步数据提示 在AD的APPNL-G-F小鼠模型中，AtA更丰富且出现更早，并且在老年人中可见 个脑袋在这里，我们将测试他们的假设，因为缺乏Kir4.1和GLT-1的AtAs在大脑中丰富， 在APPNL-G-F小鼠的海马中，星形胶质细胞经历增加的活性诱导的去极化，导致 谷氨酸摄取的过度抑制。这造成了一种情况，在这种情况下，增加的神经元活动可以 在AD中驱动协同的电压依赖性和A β介导的EAAT抑制。我们选择使用APPNL-G-F 模型由于其发病早，与AtAs的正常发育重叠，没有APP过表达。 使用APPNL-G-F小鼠，我们将确定：（SA 1）“APPNL-G-F小鼠中的海马星形胶质细胞是否显示增加的 VM对神经元活动的反应？”和（SA 2）“谷氨酸摄取的活性依赖性抑制是否增强， APPNL-G-F小鼠的海马体“.活动诱导的PAP去极化增加和A β升高都可能导致 改变APPNL-G-F小鼠中活性诱导的EAAT抑制。如果是这样，这将定位星形胶质细胞-神经元相互作用， K+和谷氨酸稳态是早期AD进展的关键因素， 和K+摄取以减少AD相关的病理。完成后，我们将知道新发现的变化 在PAP中，Vm被夸大并导致APPNL-G-F小鼠中异常的谷氨酸兴奋。我们会准备好 利用这些发现来增强K+和谷氨酸稳态，以防止AD中的病理性兴奋。

ABSTRACT. Astrocytes control neurotransmission, contribute to a robust blood-brain-barrier (BBB), and metabolically support neuronal activity. In Alzheimer’s Disease (AD), astrocytes become reactive near A plaques, contribute to neuroinflammation, and contribute to synaptic and circuit abnormalities. The role that astrocytes play in controlling synaptic excitation by removing glutamate from the extracellular space is particularly intriguing in AD. Astrocytes express high levels of excitatory amino acid transporters (EAATs), including GLT1 and GLAST, in their peripheral processes that bind and remove extracellular glutamate to limit synaptic excitation. EAATs, as well as other astrocyte proteins, are known to be decreased late in AD progression. Aberrant glutamate signaling is thought to contribute to AD progression, as evidenced by the clinical use of memantine, a glutamate receptor antagonist, as an AD therapy, strongly implicating EAATs in the progression of AD. Finally, soluble A inhibits EAAT function during heightened neuronal activity. Interestingly, we recently showed that neuronal activity causes astrocyte depolarization, which drives voltage-dependent inhibition of EAAT function to enhance neuronal activation. In the parent R01, we use astrocyte-expressed genetically encoded voltage indicators (GEVIs) to optically quantify Vm and showed that neuronal activity depolarizes peripheral astrocyte processes (PAPs) with synapse-specificity. In this supplement request, we provide preliminary data that during normal aging astrocytes sporadically lose the expression of GLT1, GLAST, and Kir4.1 and take on a phenotype known as atypical astrocytes (AtAs). Importantly, AtAs are not a form of reactive astrocytosis. Preliminary data suggests AtAs are more abundant and occur earlier in the APPNL-G-F mouse model of AD and are seen in the aged human brain. Here we will test they hypothesis that because AtAs, which lack Kir4.1 and GLT-1, are abundant in the hippocampus of APPNL-G-F mice, astrocytes experience increased activity-induced depolarization, leading to exaggerated inhibition of glutamate uptake. This creates a situation in which increased neuronal activity could drive synergistic voltage-dependent and A-mediated EAAT inhibition in AD. We chose to use the APPNL-G-F model due to its early onset, overlapping with the normal development of AtAs, without APP overexpression. Using the APPNL-G-F mice, we will determine: (SA1) “Do hippocampal astrocytes in APPNL-G-F mice show increased Vm responses to neuronal activity?” and (SA2) “Is activity-dependent inhibition of glutamate uptake enhanced in the hippocampus of APPNL-G-F mice?“. Both increased activity-induced PAP depolarization and elevated A could alter activity-induced EAAT inhibition in APPNL-G-F mice. If so, this would position astrocyte-neuron interactions, K+ and glutamate homeostasis as key players in early AD progression and would motivate enhancing glutamate and K+ uptake to reduce AD-related pathology. When complete, we will know whether newly discovered changes in PAP Vm are exaggerated and contribute to aberrant glutamate excitation in APPNL-G-F mice. We will be poised to leverage these findings to enhance K+ and glutamate homeostasis to prevent pathological excitation in AD.