Investigations of cAMP-dependent brain-barrier permeability in choroid plexus

项目摘要 脉络丛（ChP）包括形成关键脑屏障的细胞网络，其可以介导 某些脑部疾病和创伤的继发性损伤。Lehtinen实验室开发了一套工具， 研究离体和体内开发的ChP。该项目应用成像技术研究血液- 细胞水平上的CSF屏障通透性调节。我们的首要假设是细胞内 信使cAMP调节ChP上皮细胞之间的内皮紧密连接，从而调节血液-CSF 屏障完整性，因为它在附近的血脑屏障（BBB）。cAMP、PKA和Epac的主要效应物， 调节内皮紧密连接再分布和屏障通透性。Gi/o连接的G蛋白偶联物 受体（GPCR）是cAMP的强上游调节剂。莱赫蒂宁实验室的单核 测序数据表明，ChP上皮细胞选择性地和发育性地表达mGlur 8，其通过 Gαi在其他脑组织中的激活抑制cAMP的产生。阐明神经递质的作用机制 我们将研究神经递质激活的GPCR的细胞机制， ChP上皮细胞中的cAMP信号传导。Lehtinen实验室与共同赞助商Mark Andermann合作， 实验室，最近建立了一个协议，离体和体内成像的ChP结构和功能的基础上， 使用钙活性的荧光报告物。我将应用这些技术来揭示GPCR如何调节 使用ChP外植体中的荧光cAMP指示剂的cAMP水平（目的1）。在目标2中，我们将使用体内 制备以绘制可接近中枢配体与外周配体的受体群体。那些 位于顶膜上的细胞与CSF接触，而位于基底表面上的细胞暴露于CSF。 血通过包括低分子量荧光染料的外周递送的功能测定，我们将 评估中枢（CSF）与外周（静脉内）递送的配体如mGluR 8激动剂对 cAMP和血-CSF屏障通透性。这些研究将共同揭示机制， 神经递质，特别是谷氨酸，可能有助于健康和疾病中的血-CSF屏障完整性。 拟议的研究和培训将在波士顿儿童医院进行，这是一家世界知名的儿科医院。 医院提供了一个特殊的研究环境和无数的机会进行研究，从 长凳到床边重要的是，拟议的研究将在Maria Lehtinen博士的指导下进行， 脉络丛和脑脊液生物学领域的专家。此外，Mark Andermann博士（共同导师）是一位 活体光学成像技术的领导者。此提案的结果将导致第一作者 这些数据包括大量的出版物和大量的初步数据，可用于竞争性K99/R 00应用。该奖学金将提供 候选人有机会开始脉络丛生物学培训， 贡献，同时也帮助启动一个独立的学术生涯。

PROJECT SUMMARY The choroid plexus (ChP) comprises a network of cells that form a critical brain barrier that can mediate secondary damage in certain brain disorders and trauma. The Lehtinen lab has developed a suite of tools to study the ChP across development ex vivo and in vivo. This project applies imaging technology to study blood- CSF barrier permeability regulation at the cellular level. Our overarching hypothesis is that the intracellular messenger cAMP regulates endothelial tight junctions between ChP epithelial cells and thereby blood-CSF barrier integrity as it does in the nearby blood-brain barrier (BBB). The main effectors of cAMP, PKA and Epac, regulate endothelial tight junction redistribution and barrier permeability. Gi/o-linked G-Protein Coupled Receptors (GPCRs) are strong upstream regulators of cAMP. The Lehtinen laboratory's single nucleus sequencing data suggest that ChP epithelial cells selectively and developmentally express mGlur8, which via Gαi, activation in other brain tissues inhibits cAMP production. To elucidate mechanisms of neurotransmitter alteration of the blood-CSF barrier we will study the cellular mechanisms of neurotransmitter-activated GPCR- cAMP signaling in ChP epithelial cells. The Lehtinen lab, in collaboration with co-sponsor Mark Andermann's lab, recently established a protocol for ex vivo and in vivo imaging of ChP structure and function based on using fluorescent reporters of calcium activity. I will apply these techniques to reveal how GPCRs modulate cAMP levels using fluorescent cAMP indicators in ChP explants (Aim 1). In Aim 2 we will use an in vivo preparation to map the populations of receptors that are accessible to central vs peripheral ligands. Those located on the apical membrane are in contact with the CSF and those on the basal surface are exposed to the blood. With functional assays including peripheral delivery of low molecular weight fluorescent dyes, we will assess the effects of central (CSF) vs peripheral (intravenous) delivered ligands such as mGluR8 agonists on cAMP and blood-CSF barrier permeability. Together these studies will reveal mechanisms how neurotransmitters, specifically glutamate, may contribute to blood-CSF barrier integrity in health and disease. The research and training proposed will take place at Boston Children's Hospital, a world-renowned pediatric hospital that offers an exceptional research environment and countless opportunities to carry research from bench to bedside. Importantly, the research proposed will take place under the guidance of Dr. Maria Lehtinen, an expert in the field of choroid plexus and CSF biology. In addition, Dr. Mark Andermann (co-mentor) is a leader in in vivo optical imaging techniques. The results from this proposal will result in first-authored publications and a wealth of preliminary data for a competitive K99/R00 application. This fellowship will provide the candidate with the opportunity to begin training in choroid plexus biology and make significant scientific contributions while also helping launch an independent academic career.