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.

修改后的项目摘要/摘要部分 脑实质内血管中的内皮细胞（EC）严格调节血脑屏障（BBB）的功能以及血管系统与邻近神经元之间的通讯，以维持电路稳态。在最近的一项研究中，我们阐明 Ahnak 是抑郁行为的新型调节剂，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支架，这两者都与精神疾病的病理生理学有关。除了神经元表达之外，Ahnak 在大脑血管内皮细胞中也高度表达。然而，Ahnak 在 EC 中调节 BBB 功能的作用尚未得到研究。我们的初步数据表明，EC 特异性 Ahnak 基因敲除 (KO) 小鼠表现出抗抑郁药样行为表型。由于 EC 负责 BBB 通透性，因此我们假设 EC 中 Ahnak 缺失和 Ahnak 下游通路的改变可调节 BBB 通透性，从而影响神经元回路活动。我们的目标是阐明 Ahnak 介导的 EC 分子和功能途径。我们还旨在研究 Ahnak 在 EC 中调节 BBB 功能以及腹侧海马血管系统和神经回路之间的通讯的作用。首先，我们将使用 EC 选择性 TRAP（翻译核糖体亲和纯化）/RNA-seq 方法研究 EC 中的 Ahnak 下游基因和通路。通过研究 EC 特异性 Ahnak KO 小鼠（floxed Ahnak 小鼠与 Tek-Cre 小鼠杂交）与野生型 (WT) 对照相比的差异表达基因，我们的目标是识别下游分子和 Ahnak 介导的功能途径（具体目标 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研究 EC 特异性 Ahnak KO 对腹侧海马 BBB 通透性的影响（具体目标 2）。最后，我们将使用 WT 和 EC-in-vDG (EC[vDG]) 特异性 Ahnak KO 小鼠的高密度硅探针记录，研究 EC 中 Ahnak 缺失对腹侧齿状回 (vDG) 神经元活动的影响。 EC[vDG] 特异性 Ahnak 缺失将通过 CRISPR/Cas9 方法实现（具体目标 3）。拟议研究的结果将确定调节腹侧海马回路稳态的新分子因素和机制。