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

脉络丛 cAMP 依赖性脑屏障通透性的研究

• 批准号: 10753098
• 负责人: Dario Xavier Figueroa Velez
• 金额: $ 7.41万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10753098
• 关键词: Adult; Agonist; Apical; Biochemical; Biological Assay; Biology; Blood; Blood - brain barrier anatomy; Blood Circulation; Blood-Borne Pathogens; Boston; Brain; Brain Diseases; Brain Hypoxia-Ischemia; Calcium; Cell Nucleus; Cells; Central Nervous System; Cerebral Ventricles; Cerebrospinal Fluid; Choroid Plexus Epithelium; Collaborations; Coupled; Cyclic AMP; Cyclic AMP Receptors; Cyclic AMP-Dependent Protein Kinases; Data; Development; Dextrans; Disease; Electron Microscopy; Endothelial Cells; Endothelium; Environment; Epilepsy; Epithelial Cells; Exposure to; Fellowship; Fluorescent Dyes; G-Protein-Coupled Receptors; Glutamates; Goals; Health; Image; Imaging Techniques; Imaging technology; In Vitro; Individual; Intravenous; Investigation; Laboratories; Ligands; Link; Maps; Measures; Mediating; Mental Depression; Mental disorders; Mentors; Methods; Molecular; Molecular Weight; Mus; Neuroglia; Neurologic; Neurotransmitters; Pediatric Hospitals; Peripheral; Peripheral Nervous System; Permeability; Play; Population; Predisposition; Preparation; Process; Production; Property; Protocols documentation; Publications; Regulation; Reporter; Research; Resolution; Role; Schizophrenia; Signal Transduction; Source; Stains; Structure of choroid plexus; Surface; Techniques; Testing; Therapeutic Intervention; Tight Junctions; Tissues; Training; Traumatic Brain Injury; Vascular Endothelial Cell; Vascularization; Visualization; apical membrane; awake; bench to bedside; blood cerebrospinal fluid barrier; brain tissue; career; cell type; experimental study; imaging modality; improved; in vivo; in vivo imaging; in vivo optical imaging; in vivo two-photon imaging; intravenous injection; lateral ventricle; metabotropic glutamate receptor 8; nervous system disorder; neuroprotection; novel strategies; receptor; selective expression; sensor; tool; two-photon

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.

项目概要 脉络丛 (ChP) 包含一个细胞网络，形成一个关键的脑屏障，可以介导 Lehtinen 实验室开发了一套工具来治疗某些脑部疾病和创伤的继发性损伤。 研究离体和体内发育过程中的 ChP 该项目应用成像技术来研究血液。 我们的首要假设是细胞内的脑脊液屏障通透性调节。 信使 cAMP 调节 ChP 上皮细胞之间的内皮紧密连接，从而调节血脑脊液 屏障完整性，如附近的血脑屏障 (BBB) cAMP、PKA 和 Epac 的主要效应器， 调节内皮紧密连接重新分布和屏障通透性。 受体 (GPCR) 是 Lehtinen 实验室单核的强大上游调节因子。 测序数据表明 ChP 上皮细胞选择性地、发育性地表达 mGlur8，其通过 Gαi，其他脑组织中的激活抑制 cAMP 产生，以阐明神经递质的机制。 改变血脑脊液屏障，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 实验室与共同发起人 Mark Andermann 的合作。 实验室最近建立了基于 ChP 结构和功能的离体和体内成像协议 我将应用这些技术来揭示 GPCR 如何调节钙活性。 使用 ChP 外植体中的荧光 cAMP 指示剂测定 cAMP 水平（目标 1），在目标 2 中，我们将使用体内荧光 cAMP 指示剂。 准备绘制中枢配体与外周配体可接近的受体群体图。 位于顶膜上的部分与脑脊液接触，而位于基底表面的部分则暴露于脑脊液中。 通过功能测定，包括低分子量荧光染料的外周递送，我们将 评估中枢 (CSF) 与外周（静脉内）递送配体（例如 mGluR8 激动剂）对 这些研究将共同​​揭示 cAMP 和血液-脑脊液屏障通透性的机制。 神经递质，特别是谷氨酸，可能有助于健康和疾病中血脑脊液屏障的完整性。 拟议的研究和培训将在世界著名的儿科医院波士顿儿童医院进行 医院提供卓越的研究环境和无数开展研究的机会 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，Mark Andermann 博士（共同导师）是一位脉络丛和脑脊液生物学领域的专家。 该提案的结果将产生第一作者。 该奖学金将为竞争性 K99/R00 申请提供出版物和大量初步数据。 候选人有机会开始脉络丛生物学培训并做出重大科学研究 做出贡献，同时还帮助开展独立的学术生涯。