Regulation of brain cholesterol homeostasis following neonatal hypoxia-ischemia

项目概要/摘要 我们的长期目标是确定负责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）的模型，并寻找 新的和特异性的诊断或治疗HIE/NE的目标。 在这个建议中，我们将研究新生儿HI如何影响胆固醇稳态，这对大脑至关重要。 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，其在发育中起重要作用。 我们的初步数据表明，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稳态。 它的新陈代谢我们假设HI干扰胆固醇的生物合成，上调CYP 46 A1，脑- 负责胆固醇清除的特异性羟化酶，有助于HI脑损伤; 24 S-HC是CYP 46 A1的酶促产物，可用作新生儿HI的血液生物标志物 脑损伤我们将提供一个全面的研究，在胆固醇合成的变化， 新生儿HI（Aim 1）从转录水平到关键酶的蛋白质水平;我们将量化 胆固醇中间体沿着其生物合成途径使用HPLC-MS和GC-MS。 将通过以下方法测定体内和体外（氧葡萄糖剥夺）CYP 46 A1对HI脑损伤（目的2）： 用慢病毒介导的诱导型CRISPR/cas9敲除方法操纵CYP 46 A1表达 在体外和体内与特异性CYP 46 A1抑制剂联用。最后，我们将评估血浆24 S水平的价值- HC作为新生儿HI脑损伤的生物标志物（目的3）。在纵向研究中，我们将确定 HI后6周，血液24 S-HC与运动和认知障碍（行为测试）之间存在差异， 术后6天和6周通过MRI（T2 W和DTI）和组织学染色评价白色损伤 行为和核磁共振检查 该提案旨在深入了解脑胆固醇合成的分子调控， 代谢，以更好地了解脂质紊乱，蛋白质-脂质相互作用及其在 新生儿HI。这些重要的问题在围产期脑损伤中很大程度上未被探索。这些临床前 数据可以帮助确定新的和早发性循环生物标志物， 改善HIE患儿脑损伤的药理学靶点。我们的研究有相当大的翻译 对严重脑损伤的儿童有益。

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.