Investigating the role of apolipoprotein E in brain ferroptosis sensitivity

与衰老有关的神经系统疾病影响着全球数百万人，预计这一数字还会增加 随着全球平均年龄的不断增长，虽然需要开发新的神经保护 治疗方法从未如此之高，我们目前的努力仅依赖于对分子的部分理解， 细胞和电路机制，驱动老化大脑的变化。其中一个变化是氧化性增加 部分由大脑的高代谢需求引起的压力，并因大脑富集氧化催化过渡金属和掺入过氧化敏感性多不饱和脂肪酸（PUFA）而加剧 进入磷脂膜。过氧化脂质的积累导致膜损伤， 铁凋亡，一种铁依赖性的非凋亡性细胞死亡模式。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凋亡敏感，而少突胶质细胞具有抗性。但 在这些细胞类型中调节对铁凋亡的不同易感性的机制仍然未知。 铁凋亡和神经退行性变之间的一种可能的联系是载脂蛋白E（apoE），其主要的神经退行性变是铁凋亡。 中枢神经系统（CNS）的脂质和胆固醇转运蛋白。APOE基因变异体调节 发生神经退行性疾病的可能性，每个apoE 4等位基因赋予大约2倍的 与更常见的apoE 3等位基因相比，晚发性阿尔茨海默病的风险增加。而反观 apoE 2变异体在认知功能完整的老年人中富集。虽然apoE 4与apoE 4有许多不同的特征， 其他亚型，包括蛋白质稳定性降低和脂质转运改变，没有考虑一种机制 导致apoE 4携带者神经变性风险增加。最近的研究表明，apoE介导 在增强的神经元刺激期间，过氧化敏感脂肪酸从神经元转移到星形胶质细胞 活性，表明脂质转运能力不足可能导致这些潜在毒性物质的积累， 神经元中的物种。有趣的是，我们的初步数据表明，表达apoE 3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CNS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 脑中的器官型对于脑脂质代谢的建模至关重要，我们将使用器官型切片培养， 研究apoE亚型对铁缺乏症敏感性的影响。为了表征异构体依赖性 apoE-脂质相互作用的差异影响CNS铁凋亡敏感性，我们将apoE与纯化的血浆复合， HDL和测试这些颗粒如何影响铁凋亡敏感性。最后，我们将确定赋予 在我们的初步数据中，通过构建含有载脂蛋白E的脂蛋白纳米颗粒， 单个HDL脂质种类并在铁凋亡敏感性的细胞测定中测试它们的功能。

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.