Investigating the role of apolipoprotein E in brain ferroptosis sensitivity

与衰老相关的神经疾病影响着全球数百万人，这一数字预计还会增加 随着全球平均年龄的持续增长，这一数字急剧上升。虽然需要开发新的神经保护措施 治疗学从来没有达到过更高的水平，我们目前的努力只依赖于对分子的部分了解， 驱动老化大脑变化的细胞和电路机制。其中一个变化是氧化作用增强。 应激部分由大脑的高代谢需求引起，并因大脑中氧化催化过渡金属的丰富和过氧化敏感的多不饱和脂肪酸(PUFA)的加入而加剧 物种进入磷脂膜。过氧化脂质的积聚会导致膜损伤和 铁下垂，一种铁依赖的、非凋亡的细胞死亡模式。我们的初步数据表明神经元， 星形胶质细胞和小胶质细胞对铁性下垂易感，而少突胶质细胞则有抵抗力。然而， 调节这些细胞类型对铁性下垂的不同易感性的机制尚不清楚。 铁性下垂和神经退行性变之间的一个可能的联系是载脂蛋白E(ApoE)，它是主要的 中枢神经系统(CNS)的脂质和胆固醇转运蛋白。载脂蛋白E基因变异体调节 发生神经退行性疾病的可能性，每个apoE4等位基因大约2倍于 与更常见的apoE3等位基因相比，晚发性阿尔茨海默病的风险增加。相比之下， APOE2变异体在认知完好的老年人中丰富。虽然apoE4的许多特征与 其他异构体，包括蛋白质稳定性降低和脂质运输改变，没有一个机制被考虑 导致载脂蛋白E4携带者神经变性风险增加。最近的研究表明，载脂蛋白E在 神经元增强期过氧化敏感脂肪酸从神经元向星形胶质细胞的转移 活性，表明脂质运输能力不足可能导致这些潜在有毒物质的积累 神经元中的物种。有趣的是，我们的初步数据表明，表达apoE3的星形胶质细胞可能是 与apoE基因敲除的星形胶质细胞相比，可以预防某些形式的铁性下垂，但这种易感性 可以通过外源性应用血浆高密度脂蛋白(HDL)来挽救。 我们建议使用中枢神经系统细胞的原代培养来验证我们的初步发现。因为细胞之间的相互作用 脑中的类型对于建立脑脂代谢模型至关重要，我们将使用器官型切片培养来 研究载脂蛋白E亚型对铁性下垂敏感性的影响。描述异构体依赖程度的特征 载脂蛋白E-脂质相互作用的差异影响中枢神经系统铁下垂的敏感性，我们将载脂蛋白E与纯化的血浆复合 高密度脂蛋白，并测试这些颗粒如何影响铁下垂敏感性。最后，我们将鉴定与之有关的脂类。 在我们的初步数据中，通过构建包含以下内容的载脂蛋白纳米粒来抵抗铁下垂 个体高密度脂蛋白脂质种类及其在细胞铁性下垂敏感性检测中的作用。

Aging-related neurological diseases impact millions people worldwide, and this number is expected to increase drastically as the global average age continues to increase. While the need to develop new neuroprotective therapeutics has never been higher, our current efforts rely on only a partial understanding of the molecular, cellular, and circuit mechanism that drive changes in the aging brain. One such change is increased oxidative stress arising partially from the brain’s high metabolic needs and exacerbated by brain enrichment of oxidation-catalyzing transition metals and incorporation of peroxidation-sensitive polyunsaturated fatty acid (PUFA) species into phospholipid membranes. Accumulation of peroxidized lipids results in membrane damage and ferroptosis, an iron-dependent, non-apoptotic mode of cell death. Our preliminary data suggest that neurons, astrocytes, and microglia are susceptible to ferroptosis, whereas oligodendrocytes are resistant. However, the mechanisms regulating differential susceptibility to ferroptosis in these cell types remain unknown. One possible connection between ferroptosis and neurodegeneration is apolipoprotein E (apoE), the primary lipid and cholesterol transport protein of the central nervous system (CNS). APOE gene variants modulate the probability of developing neurodegenerative disease, with each apoE4 allele conferring an approximately 2-fold increase in risk for late-onset Alzheimer’s disease compared the more common apoE3 allele. In contrast, the apoE2 variant is enriched in cognitively-intact elders. While many characteristics differentiate apoE4 from the other isoforms, including decreased protein stability and altered lipid transport, no one mechanism is considered responsible for increased risk of neurodegeneration in apoE4 carriers. Recent work shows that apoE mediates transfer of peroxidation-sensitive fatty acids from neurons to astrocytes during periods of enhanced neuronal activity, suggesting that deficient capacity for lipid transport could lead to accumulation of these potentially toxic species in neurons. Interestingly, our preliminary data suggest that astrocytes expressing apoE3 may be protected against some forms of ferroptosis compared to apoE knockout astrocytes, but that this susceptibility can be rescued by exogenous application of plasma high density lipoproteins (HDL). We propose to validate our initial findings using primary culture of CNS cells. Because interactions among cell types in the brain are critical for modeling of brain lipid metabolism, we will use organotypic slice culture to investigate the impact of apoE isoform on ferroptosis sensitivity. To characterize how isoform-dependent differences in apoE-lipid interactions impact CNS ferroptosis sensitivity, we will complex apoE to purified plasma HDL and test how these particles impact ferroptosis sensitivity. Lastly, we will identify the lipid species conferring resistance to ferroptosis in our preliminary data by constructing apoE lipoprotein nanoparticles containing individual HDL lipid species and testing their function in cellular assays of ferroptosis sensitivity.