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

修改后的项目摘要/摘要部分 脑实质内血管中的内皮细胞(ECs)密切调节血脑屏障(BBB)的功能以及血管系统与邻近神经元之间的通讯，以维持回路的动态平衡。在最近的一项研究中，我们阐明了AHNAK是一种新的抑郁行为调节因子，是S100A10(P11)/Anxa2蛋白复合体和L型电压门控钙通道的内源性神经支架，两者都参与了精神障碍的病理生理学。除了神经元的表达外，AHNAK还在大脑血管内皮细胞中高表达。然而，AHNAK在内皮细胞中对血脑屏障功能的调节作用尚未被研究。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的行为表型。由于内皮细胞负责血脑屏障通透性，我们假设内皮细胞中AHNAK缺失和AHNAK下游通路的改变调节血脑屏障通透性，从而影响神经元的电路活动。我们的目标是阐明AHNAK在内皮细胞中的分子和功能途径。我们还旨在研究AHNAK在调节血脑屏障功能以及腹侧海马区血管系统和神经回路之间的通讯中的作用。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ECs中AHNAK下游基因和途径。通过研究EC特异性AHNAK KO小鼠(与TEK-CRE小鼠杂交的FLOXED AHNAK小鼠)与野生型(WT)对照小鼠的差异表达基因，我们的目的是识别下游分子和AHNAK介导的功能通路(特定目标1)。其次，我们将利用核磁共振成像(MRI)扫描、免疫组织化学、生化分析和透射电子显微镜(SP2)研究EC特异性AHNAK KO对腹侧海马区血脑屏障通透性的影响。最后，我们将使用高密度硅探针记录WT和EC-in-VDG(EC[VDG])特异的AHNAK KO小鼠，研究ECs中AHNAK缺失对腹侧齿状回(VDG)神经元活动的影响。将通过CRISPR/CAS9办法(具体目标3)实现欧共体[VDG]特定的AHNAK删除。拟议的研究结果将确定新的分子因素和机制，调节腹侧海马回路的动态平衡。

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.