Role of neuronal hemoglobin in chronic stress-induced mitochondrial adaptation in hippocampal PV interneurons

项目摘要 慢性应激是一种影响大脑生理、认知和心理健康的有害状态。更改后的 长期以来，脑血管网络一直被认为是神经元功能的潜在调节器， 压力。然而，将信号从脑血管输入传递到功能通路的关键分子底物 在特定类型的神经元中，人们还没有完全了解。我们最近对慢性社会失败压力(CSDS)的研究 范式表明，a)海马区表达小白蛋白(PV)的中间神经元是CSDS的决定因素。 诱导的行为适应，b)血红蛋白(Hb)α链和β链水平在 应激敏感小鼠的海马PV神经元，以及PV中线粒体的氧化磷酸化 神经元是与应激易感性相关的最重要的功能通路。因为慢性压力 已知会导致脑血管功能障碍和脑内血流失调，我们假设PV 中间神经元是慢性应激的连续靶点，而在PV神经元中表达的Hb是一个关键的节点感觉 血管输入与PV神经元线粒体适应性的调节。这项研究的首要目标是 建立特定细胞类型的Hb缺失模型，确定Hb在慢性应激诱导的PV中的作用 神经元对慢性应激的适应和行为反应。首先，我们将使用CSDS模型，并检查 降压药卡托普利对CSDS诱导的微量出血、血脑屏障破坏、Hb诱导的影响 在PV神经元和行为结果中确定血管破裂和Hb之间的关系 PV神经元的诱导(目标1a)。海马神经元选择性缺失Hbβ链基因将 将AAV-sgRNAs载体注射到选择性表达Cas9的转基因小鼠的海马区 在PV神经元中。对照组和β链缺失小鼠将受到CSDs的影响，并对 将测量CSD(目标1b)。在行为分析之后，海马区组织将使用PV- 神经元选择性翻译核糖体亲和纯化(TRAP)/RNA-SEQ方法(目标2)。差异化的 表达的基因(Deg)将通过生物信息学的方法进行分析，以寻找改变的功能途径。 包括线粒体途径(目标2)。我们的目标是确定慢性疾病是否需要神经元Hb。 应激诱导的行为变化(目标1b)，并确定受影响的下游基因和功能通路 Hb缺失(目标2)。神经元Hb的特定细胞类型的删除工具与特定细胞类型的 本研究建立的TRAP/RNA-SEQ方法可应用于其他细胞中的Hb研究 在不同的应激或疾病模型下，不同脑区的类型。年建立的研究范式 这项研究将促进一系列新的研究，填补应激引起的脑血管缺陷之间的差距 以及特定脑细胞类型的反应。因此，拟议研究的结果可能产生广泛的影响。 在精神健康和大脑疾病领域。

Project Summary Chronic stress is a detrimental condition affecting brain physiology, cognition and mental health. Altered cerebrovascular network has long been recognized as a potential modulator of neuronal function in response to stress. However, key molecular substrates conveying signals from cerebrovascular input to functional pathways in specific types of neurons are not fully understood. Our recent study with a chronic social defeat stress (CSDS) paradigm indicates that a) hippocampal parvalbumin (PV)-expressing interneurons are a determinant of CSDS- induced behavioral adaptations, b) hemoglobin (Hb) α- and β-chain levels are drastically upregulated in hippocampal PV neurons in stress-susceptible mice, and c) mitochondrial oxidative phosphorylation in PV neurons is the most significant functional pathway associated with stress susceptibility. Because chronic stress is known to cause cerebrovascular dysfunction and dysregulated blood flow in the brain, we hypothesize that PV interneurons are a contiguous target of chronic stress, and Hb expressed in PV neurons is a critical node sensing vascular input and regulating mitochondrial adaptation in PV neurons. The overarching goal of this study is to establish a cell-type-specific deletion model of Hb and determine the role of Hb in chronic stress-induced PV neuron adaptation and behavioral responses to chronic stress. First, we will use the CSDS model, and examine the effect of an antihypertensive drug (captopril) on CSDS-induced microbleeds, BBB disruption, Hb induction in PV neurons and behavioral outcomes to determine the relationship between vascular disruption and Hb induction in PV neurons (Aim 1a). Hippocampal PV neuron-selective deletion of Hb β-chain genes will be achieved by injecting AAV-sgRNAs vector into the hippocampus of transgenic mice expressing Cas9 selectively in PV neurons. The control and β-chain-deleted mice will be subjected to CSDS, and behavioral responses to CSDS will be measured (Aim 1b). After behavioral assays, the hippocampal tissues will be analyzed using a PV- neuron-selective Translating Ribosome Affinity Purification (TRAP)/RNA-seq approach (Aim 2). Differentially expressed genes (DEGs) will be analyzed by bioinformatic approaches to find altered functional pathways including mitochondrial pathways (Aim 2). We aim to determine whether neuronal Hb is required for chronic stress-induced behavioral changes (Aim 1b) and identify downstream genes and functional pathways influenced by Hb deletion (Aim 2). Cell-type-specific deletion tools for neuronal Hb in conjunction with a cell-type-specific TRAP/RNA-seq approach established in this study can be applicable to the future studies of Hb in other cell types in different brain regions under various stress or disease models. The research paradigm established in this study will facilitate an array of new studies paving the gap between stress-induced cerebrovascular deficit and brain-cell-type-specific responses. Thus, the outcome of the proposed research could yield a broad impact on the fields of mental health and brain disorders.