Activity-dependent Regulation of the Choroid Plexus-Cerebrospinal Fluid Stem Cell Niche

项目总结/摘要 构建大脑皮层需要在神经元之间进行分子信号的空间和时间协调， 祖细胞这些细胞中的每一个都沐浴在脑脊液（CSF）中，脑脊液是一种富含健康和生长的介质， 在大脑发育过程中，其成分会在几天内发生深刻变化。脉络膜 神经丛（ChP）可能对CSF成分的动态发育调节有很大贡献，但 根本的机制还不清楚。在这里，我们将使用一套现代工具来测试 胚胎ChP上皮细胞中钙浓度升高是对 外源性信号对于调节信号传导因子合成和分泌到CSF中至关重要。钙 活性调节从神经元到唾液腺上皮的多种细胞中的转录和分泌。我们 鉴定了ChP上皮细胞中表达的介导钙进入细胞的受体，如TRPM 3 受体，或驱动钙从内部储存释放，如5-羟色胺（5-羟色胺）的5 HT 2C受体（Htr 2c）。 HT，5-羟色胺）。我们的初步数据表明，5-HT调节胚胎ChP钙 活性、转录和囊泡释放，以及通过ChP中的5 HT 2C受体的完整信号传导， 对正常的大脑皮层发育至关重要。此外，母体递送用于所述抗体的选择性激动剂是有利的。 5 HT 2C受体在胚胎ChP中触发快速、稳健的立即早期基因表达。因此，我们建议 ChP钙活性由激活5 HT 2C受体（和其他受体， 作为TRPM 3）调节ChP转录和信号传导因子分泌到CSF中， 体-脑和母-胎相互作用，从而紧密协调大脑皮层发育。 为了测试这一点，我们将首先研究通过直接应用5 HT 2C和TRPM 3受体激活的作用。 在ChP外植体（Aim 1a）中钙成像期间和通过刺激 在体内两个- 贴壁胚胎的光子钙成像（Aim 1b）。然后，我们评估这种转录组学的影响， 刺激（目的1c），并测试胚胎ChP钙活性是否通过激活5 HT 2C受体和 其他受体在体外（Aim 2a）和体内（Aim 2b）触发囊泡释放和蛋白质分泌。我们还将 分析ChP受体信号传导中断对皮质发育的影响（目的3a）。CSF破坏 体积、组成和心室形成对于许多神经病症是常见的。中的扰动 母亲-胎儿的健康可能会破坏大脑发育，部分原因是神经递质和其他信号的失调 途径。我们的研究结果为阐明脑内神经元调节异常的机制提供了一个新的平台。 环境扰动的发展，从而为未来的发展提供了概念和技术路线图。 研究ChP上皮细胞的活性如何影响基因表达和蛋白质分泌， 许多神经发育疾病的后果。

PROJECT SUMMARY / ABSTRACT Building the cerebral cortex requires the spatial and temporal orchestration of molecular signals across neural progenitor cells. Each of these cells is bathed in cerebrospinal fluid (CSF), a medium rich in health- and growth- promoting factors whose composition changes profoundly across days during brain development. The choroid plexus (ChP) is likely to strongly contribute to this dynamic developmental regulation of CSF composition, but the underlying mechanisms are not understood. Here, we will use a suite of modern tools to test the hypothesis that elevations in calcium concentration in embryonic ChP epithelial cells in response to extrinsic cues is critical for regulating synthesis and secretion of signaling factors into the CSF. Calcium activity regulates transcription and secretion in diverse cells ranging from neurons to salivary gland epithelia. We identified receptors expressed in ChP epithelial cells that mediate calcium entry into the cell, such as the TRPM3 receptor, or that drive calcium release from internal stores, such as the 5HT2C receptor (Htr2c) for serotonin (5- HT, 5-hydroxytryptamine). Our preliminary data suggest that 5-HT regulates embryonic ChP calcium activity, transcription, and vesicle release, and that intact signaling via the 5HT2C receptor in ChP is essential for normal cerebral cortical development. Further, maternal delivery of a selective agonist for the 5HT2C receptor triggers rapid, robust immediate early gene expression in embryonic ChP. Thus, we propose that ChP calcium activity triggered by signals that activate the 5HT2C receptor (and other receptors such as TRPM3) regulates ChP transcription and secretion of signaling factors into the CSF and controls body-brain and maternal-fetal interactions, thereby tightly orchestrating cerebral cortical development. To test this, we will first investigate the effects of 5HT2C and TRPM3 receptor activation by direct application of ligand to the apical surface of epithelial cells during calcium imaging in ChP explants (Aim 1a) and by stimulation of the basal surface following intraperitoneal (IP) delivery of ligands to the maternal dam during in vivo two- photon calcium imaging of attached embryos (Aim 1b). We then assess the transcriptomic impact of such stimulation (Aim 1c), and test whether embryonic ChP calcium activity via activation of 5HT2C receptors and other receptors triggers vesicle release and protein secretion in vitro (Aim 2a) and in vivo (Aim 2b). We will also analyze the effects of disruption of ChP receptor signaling on cortical development (Aim 3a). Disrupted CSF volume, composition, and ventricle formation are common to many neurological conditions. Perturbations in maternal-fetal health can disrupt brain development, in part via dysregulation of serotonergic and other signaling pathways. Our findings provide a novel platform for elucidating the mechanisms underlying dysregulation of brain development by environmental perturbations, thereby providing a conceptual and technical roadmap for future studies on how activity in ChP epithelial cells influences gene expression and protein secretion, with consequences for many neurodevelopmental diseases.