Studies of Ahnak pathways in endothelial cells and blood-brain barrier regulation

内皮细胞 Ahnak 通路和血脑屏障调节的研究

• 批准号: 10669565
• 负责人: Yong Kim
• 金额: $ 27.48万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-25 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10669565
• 关键词: ANXA2 gene; Affect; Affinity Chromatography; Antidepressive Agents; Astrocytes; Biochemical; Biological Assay; Blood; Blood - brain barrier anatomy; Blood Cells; Blood Vessels; Brain region; CRISPR/Cas technology; Calcium Channel; Candidate Disease Gene; Cells; Central Nervous System; Cerebrovascular system; Communication; Contrast Media; Data; Dyes; Endothelial Cells; Enterobacteria phage P1 Cre recombinase; Exclusion; Functional disorder; Gadolinium; Genes; Hippocampus; Homeostasis; Horseradish Peroxidase; Immunohistochemistry; Knock-in; Knock-out; Knockout Mice; LoxP-flanked allele; MRI Scans; Measures; Mediating; Mental disorders; Messenger RNA; Methods; Molecular; Mood Disorders; Moods; Mus; Neurons; Ontology; Outcome; Outcome Study; Pathway interactions; Pattern; Penetration; Pericytes; Psychiatry; Regulation; Ribosomes; Role; Silicon; Silicones; Tight Junctions; Translating; Transmission Electron Microscopy; Vascular System; Visualization; Wild Type Mouse; behavioral phenotyping; blood-brain barrier function; blood-brain barrier permeabilization; brain parenchyma; brain tissue; density; dentate gyrus; depressive behavior; differential expression; granule cell; insight; intravenous administration; intravenous injection; meter; neural circuit; neuronal circuitry; novel; novel strategies; prevent; protein complex; public health relevance; transcriptome sequencing; voltage

Modified Project Summary/Abstract Section Endothelial cells (ECs) in blood vessels within brain parenchyma tightly regulate the function of the blood-brain barrier (BBB) and the communication between the vascular system and neighboring neurons to maintain circuitry homeostasis. In a recent study, we elucidated that Ahnak is a novel regulator of depressive behavior and is an endogenous neuronal scaffolder of the S100A10 (p11)/Anxa2 protein complex and L-type voltage-gated calcium channels, both of which have been implicated in the pathophysiology of psychiatric disorders. In addition to neuronal expression, Ahnak is highly expressed in ECs in blood vessels in the brain. However, Ahnak’s role in the ECs in the regulation of BBB function has not yet been investigated. Our preliminary data indicate that EC-specific Ahnak knockout (KO) mice display an antidepressant-like behavioral phenotype. Because ECs are responsible for BBB permeability, we hypothesize that Ahnak deletion and alterations of Ahnak downstream pathways in ECs modulate BBB permeability and thereby affect neuronal circuit activities. We aim to elucidate Ahnak-mediated molecular and functional pathways in the ECs. We also aim to investigate the roles of Ahnak in ECs in the regulation of BBB function and the communication between the vascular system and neural circuits in the ventral hippocampus. First, we will investigate Ahnak downstream genes and pathways in ECs using an EC-selective TRAP (Translating Ribosome Affinity Purification)/RNA-seq approach. By investigating differentially expressed genes in EC-specific Ahnak KO mice (floxed Ahnak mice crossed with Tek-Cre mice) compared to wild type (WT) controls, we aim to identify downstream molecules and Ahnak-mediated functional pathways (Specific Aim 1). Second, we will investigate the effect of EC-specific Ahnak KO on the BBB permeability in the ventral hippocampus using magnetic resonance imaging (MRI) scans, immunohistochemistry, biochemical assays and transmission electron microscopy (Specific Aim 2). Lastly, we will investigate the effect of Ahnak deletion in ECs on neuronal activity in the ventral dentate gyrus (vDG) using high-density silicon probes recordings of WT and EC-in-vDG (EC[vDG])-specific Ahnak KO mice. EC[vDG]-specific Ahnak deletion will be achieved by a CRISPR/Cas9 approach (Specific Aim 3). The outcome of the proposed studies will identify novel molecular factors and mechanisms regulating homeostasis of the ventral hippocampal circuitry.

修改的项目摘要/摘要部分 脑实质内血管中的内皮细胞（ECS）紧密调节血脑屏障（BBB）的功能以及血管系统与邻近神经元之间的通信以维持电路稳态。在最近的一项研究中，我们阐明了AHNAK是抑郁行为的新调节剂，并且是S100A10（P11）/AnxA2蛋白复合物和L型电压门控钙通道的内源性神经元脚手骨架，除了神经元表达外，AHNAK在血液中的ECS中都表现出了高度的表达。但是，尚未研究AHNAK在ECS中的ECS调节中的作用。我们的初步数据表明EC特异性的AHNAK敲除（KO）小鼠显示出抗抑郁药样的行为表型。由于EC是负责BBB渗透性的原因，因此我们假设AHNAK缺失和ECS下游途径的变化调节了BBB渗透性，从而影响神经元电路活动。我们旨在阐明EC中的AHNAK介导的分子和功能途径。我们还旨在研究AHNAK在ECS中的作用在BBB功能调节中的作用，以及腹侧海马中血管系统与神经元回路之间的通信。首先，我们将使用EC选择性陷阱（翻译核糖体亲和力纯化）/RNA-SEQ方法研究ECS的AHNAK下游基因和途径。通过研究与野生型（WT）对照相比，通过研究EC特异性AHNAK KO小鼠（与Tek-Cre小鼠的Floxed Ahnak小鼠）中不同表达的基因，我们旨在鉴定下游分子和AHNAK介导的功能途径（特定AIM 1）。其次，我们将使用磁共振成像（MRI）扫描，免疫组织化学，生化测定和透射电子显微镜（特定的AIM 2），研究EC特异性AHNAK KO对腹侧海马中BBB渗透性的影响（特定AIM 2）。最后，我们将使用WT和EC-IN-IN-VDG（EC [VDG]）的高密度硅问题记录 - 特定的AHNAK KO小鼠，研究AHNAK缺失对ECS对ECS神经元活性的影响。 EC [VDG] - 特定的AHNAK删除将通过CRISPR/CAS9方法（特定目标3）实现。拟议研究的结果将确定调节腹侧海马电路体内稳态的新分子因子和机制。