Regulation of brain cholesterol homeostasis following neonatal hypoxia-ischemia

项目摘要/摘要 我们的长期目标是确定导致大脑损伤和修复的复杂机制 新生儿缺氧缺血(HI)后作为新生儿脑病(NE)模型的研究 针对HIE/NE的新的特异性诊断或治疗靶点。 在这个方案中，我们将研究新生儿缺氧缺血性脑病如何影响对大脑至关重要的胆固醇稳态。 由于其在膜完整性、髓鞘形成、突触发生和神经传递中的重要性而发育。 我们的初步数据显示，HI通过抑制胆固醇的合成和加速胆固醇的合成来扰乱胆固醇的动态平衡 它的新陈代谢。我们假设HI干扰胆固醇的生物合成；CYP46A1的上调，大脑- 负责清除胆固醇的特定羟基酶，导致缺氧脑损伤；血浆中 CYP46A1的酶产物24S-HC可作为新生儿缺氧缺血性脑病的血液标志物 脑部受损。我们将对以下胆固醇合成的变化进行全面的研究 新生儿缺氧缺血性脑病(目标1)从转录水平到关键酶的蛋白质水平；我们将量化 用高效液相色谱-质谱仪和气相色谱-质谱法研究胆固醇生物合成途径中的中间产物。的贡献 CYP46A1在体内和体外对HI脑损伤(氧糖剥夺)(AIM 2)的作用将通过以下方法确定 慢病毒介导的可诱导CRISPR/Cas9基因敲除方法调控细胞色素P46A1的表达 体外和体内应用特异性的细胞色素P46A1抑制剂。最后，我们将评估24小时血浆水平的价值- HC作为新生儿缺氧缺血性脑损伤的生物标志物(目标3)。在纵向研究中，我们将确定两者之间的相关性 在HI后6周有运动和认知障碍(行为测试)的血液24S-HC之间，灰色和 术后6天和6周行MRI(T2W和DTI)和组织学染色评价脑白质损伤 行为和核磁共振检查。 这项提议旨在深入了解大脑胆固醇合成的分子调控和 代谢有助于更好地了解血脂紊乱、蛋白质-脂质相互作用及其在糖尿病中的意义 新生儿缺氧缺血性脑病。这些重要的问题在围产期脑损伤中很大程度上是未知的。这些临床前研究 数据可以帮助识别新的和早期发病的循环生物标记物，以及潜在的基于脂类的新标记物 改善HIE婴儿脑损伤的药理靶点。我们的研究有相当大的翻译性 有益于严重脑损伤的儿童。

Project summary/Abstract Our long-term objectives are to define the complex mechanisms responsible for brain damage and repair following neonatal hypoxia-ischemia (HI), as a model for neonatal encephalopathy (NE), and to search for novel and specific diagnostic or therapeutic targets for HIE/NE. In this proposal, we will investigate how neonatal HI impacts cholesterol homeostasis, which is crucial for brain development due to its importance in membrane integrity, myelination, synaptogenesis and neurotransmission. Our preliminary data shows that HI disturbs cholesterol homeostasis by inhibiting its synthesis and accelerating its metabolism. We hypothesize that HI disturbs cholesterol biosynthesis; upregulation of CYP46A1, the brain- specific hydroxylase responsible for cholesterol removal, contributes to HI brain injury; and the plasma level of 24S-HC, the enzymatic product of CYP46A1, can be used as a blood biomarker for evaluation of neonatal HI brain damage. We will provide a comprehensive study of the changes in cholesterol synthesis following neonatal HI (Aim 1) from the transcriptional level to the protein levels of the key enzymes; we will quantify the cholesterol intermediates along its biosynthetic pathway using HPLC-MS & GC-MS. The contribution of CYP46A1 to HI brain injury in vivo and in vitro (oxygen glucose deprivation) (Aim 2) will be determined by manipulation of the CYP46A1 expression with lentiviral-mediated inducible CRISPR/cas9 knockout approach in vitro and with specific CYP46A1 inhibitor in vivo. Finally, we will evaluate the value of plasma levels of 24S- HC as a biomarker for neonatal HI brain injury (Aim 3). In a longitudinal study, we will determine the correlation between blood 24S-HC with motor and cognitive impairments (behavioral tests) 6 weeks post-HI, with gray and white matter injury evaluated by both MRI (T2W and DTI) and histological staining at 6 days and 6 weeks after behavioral and MRI exams. This proposal is designed to gain deep insights into the molecular regulation of brain cholesterol synthesis and metabolism for a better understanding of lipid disorders, protein-lipid interactions and their implication in neonatal HI. These important questions are largely unexplored in perinatal brain damage. These preclinical data could help identify new and early-onset circulating biomarkers and potentially novel lipid-based pharmacologic targets to ameliorate brain injury in HIE babies. Our studies have considerable translational benefit to severely brain-damaged children.