Interrogation and Modulation of the Epigenome after Prenatal Hypoxic Brain Injury

项目总结 新生儿缺氧缺血性脑病(HIE)，一种常见的缺氧和缺氧所致的脑损伤 出生前的营养物质会导致长期的神经缺陷，即使在没有明显 围产期细胞死亡。围产期损伤领域的一个主要空白是对如何造成围产期损伤的机制理解有限 短暂的低氧会导致持续的细胞缺陷。一种令人信服的机制将产前缺氧与 持续性缺陷是产前缺氧永久性地改变了表观基因组，因为表观基因组整合 发展与环境相适应。 为了研究缺氧缺血性脑病的发病机制，我建立了一种新的短暂性脑缺血发作小鼠模型。 妊娠晚期表现为轻度HIE的产前缺氧性损伤。产前缺氧导致持续性行为 以及成年小鼠的结构性缺陷，尽管胎儿大脑中的细胞死亡没有显著增加。我的初选 数据显示出生后28天(P28)、周龄皮质丘脑神经元内树突棘密度降低 缺氧后。胎儿期缺氧与表观基因组相关基因上调有关 胎儿单核RNA和转座酶可及染色质测序(SnRNA/ATAC-seq) 暴露后一小时内的大脑皮层。组蛋白变异体H3F3B是最上调的基因之一，是一种 保护大脑免受更严重的脑损伤的候选人。然而，表观遗传学的轨迹 甚至在低氧暴露后，皮质丘脑神经元似乎立即发生了移位。因此，我的中央 假设H3F3B上调是保护神经元免受严重损伤的必要和充分条件 出生前缺氧和不同的表观遗传调节因素会导致持久的神经元损伤。 为了验证我的假设，我提出了三个目标。在目标1中，我将使用条件基因敲除小鼠来测试H3F3B 缺氧后耗竭会立即增加细胞死亡，并加剧脊髓密度的持续不足。 P28小鼠和成年小鼠的行为。在目标2中，我将使用宫内电穿孔来测试是否过度表达 H3F3B可改善P28小鼠因缺氧造成的脊柱密度缺陷和成年小鼠的行为。最后，在目标3中， 我建议使用p28小鼠大脑皮质中的snRNA/atac-seq来确定产前缺氧是否具有永久性 对皮质丘脑神经元表观基因组的影响。 我的长期目标是成为一名儿童神经科医生，利用我在分子生物学方面的研究专长。 和临床技能在围产期脑损伤中开发出针对性的治疗方法，以应对这种经常具有破坏性的 受伤。我的导师埃里克·马什博士和米哈尔·埃洛维茨博士都是致力于使用 旨在改善神经系统疾病儿童预后的转化性研究。在他们的指导下 在K08提案中，我将在生物信息学方面获得更多技能，在翻译动物研究方面获得更深层次的专业知识， 和实验室管理技能，成为神经疾病表观遗传学的专家，并 准备成为一名独立的R01资助的内科科学家。

PROJECT SUMMARY Neonatal hypoxic ischemic encephalopathy (HIE), a common brain injury from loss of oxygen and nutrients immediately prior to birth, results in long-term neurologic deficits even in the absence of significant perinatal cell death. A major gap in the perinatal injury field is the limited mechanistic understanding of how transient hypoxia results in persistent cellular deficits. A compelling mechanism linking prenatal hypoxia to persistent deficits is that prenatal hypoxia permanently alters the epigenome because the epigenome integrates development with response to the environment. To study the mechanisms underlying the pathology of HIE, I developed a novel mouse model of transient late gestation prenatal hypoxic injury that phenocopies mild HIE. Prenatal hypoxia leads to persistent behavioral and structural deficits in adult mice despite no significant increase in cell death in the fetal brain. My preliminary data showed decrease in dendritic spine density in corticothalamic neurons at postnatal day 28 (P28), weeks after hypoxia. Prenatal hypoxia was associated with upregulation of genes associated with the epigenome in single nucleus RNA and assay for transposase-accessible chromatin sequencing (snRNA/ATAC-seq) in the fetal neocortex within one hour of exposure. One of the most upregulated genes, the histone variant H3f3b, is a candidate for protecting the brain from more severe brain injury. However, the epigenetic trajectories of corticothalamic neurons seemed to be shifted even immediately after hypoxic exposure. Therefore, my central hypothesis is that H3f3b upregulation is necessary and sufficient to protect neurons from severe injury after prenatal hypoxia and distinct epigenetic regulators contribute to lasting neuronal injury. To test my hypothesis, I propose three aims. In Aim 1, I will use conditional knockout mice to test if H3f3b depletion increases cell death immediately after hypoxia and worsens the persistent deficits in spine density in P28 mice and behaviors in adult mice. In Aim 2, I will use in utero electroporation to test if overexpression of H3f3b improves deficits from hypoxia in spine density in P28 mice and behaviors in adult mice. Lastly, in Aim 3, I propose to use snRNA/ATAC-seq in the cortex of P28 mice to determine if prenatal hypoxia has a permanent effect on the epigenome of corticothalamic neurons. My long-term goal is to be a child neurologist who leverages my research expertise in molecular biology and clinical skills in perinatal brain injury both to develop targeted therapies towards this frequently devastating injury. My mentors, Drs. Eric Marsh and Michal Elovitz, are excellent physician scientists devoted to using translational research to improving outcomes in children with neurological disorders. Under their guidance for this K08 proposal, I will gain additional skills in bioinformatics, a deeper expertise in translational animal studies, and lab management skills necessary to become an expert in the epigenetics of neurological disorders and be prepared for a career as an independent R01-funded physician scientist.