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

项目摘要 慢性压力是影响大脑生理、认知和心理健康的有害条件。改变 脑血管网络长期以来被认为是神经元功能的潜在调节剂， 应力然而，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还没有完全理解。我们最近的研究与慢性社会失败压力（CSDS） 范例表明a）海马小清蛋白（PV）表达的中间神经元是CSDS的决定因素， 诱导的行为适应，B）血红蛋白（Hb）α-和β-链水平在 应激敏感小鼠中海马PV神经元，和c）PV中线粒体氧化磷酸化 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PV可引起脑血管功能障碍和脑血流失调，我们假设PV 中间神经元是慢性应激的连续靶点，PV神经元中表达的Hb是关键节点感知 血管输入和调节PV神经元的线粒体适应。本研究的总体目标是 建立细胞类型特异性Hb缺失模型，并确定Hb在慢性应激诱导PV中的作用 神经元适应和行为反应的慢性压力。首先，我们将使用CSDS模型，并检查 抗高血压药物（卡托普利）对CSD诱导的微出血、BBB破坏、Hb诱导的影响 PV神经元和行为结果，以确定血管破裂和Hb之间的关系 PV神经元中的诱导（Aim 1a）。海马PV神经元选择性缺失Hb β链基因将是 通过将AAV-sgRNA载体注射到选择性表达Cas9的转基因小鼠的海马中来实现 在PV神经元中。对照和β-链缺失小鼠将经受CSDS，并且行为反应为 将测量CSDS（目标1b）。在行为测定后，将使用PV-1000分析海马组织。 神经元选择性翻译核糖体亲和纯化（TRAP）/RNA-seq方法（Aim 2）。差异 表达基因（DEG）将通过生物信息学方法进行分析，以发现改变的功能途径 包括线粒体途径（Aim 2）。我们的目的是确定是否神经元血红蛋白是需要慢性 应激诱导的行为变化（目标1b），并确定下游基因和功能途径的影响 通过Hb缺失（目的2）。用于神经元Hb的细胞类型特异性缺失工具结合细胞类型特异性 本研究建立的TRAP/RNA-seq方法可应用于其他细胞血红蛋白的研究 在不同的压力或疾病模型下，不同的大脑区域的类型。建立的研究范式 这项研究将促进一系列新的研究，填补应激诱导的脑血管功能障碍之间的差距， 和脑细胞类型特异性反应。因此，拟议研究的结果可能产生广泛的影响 精神健康和脑部疾病领域的研究

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.