Circular mimics of Iron-response elements to inhibit ferroptosis

综述：几乎所有帕金森病患者都会发现铁超载，并可能导致 铁下垂，一种铁依赖的细胞死亡形式。铁在帕金森氏病病理中的重要性 铁络合剂在帕金森氏病的培养和动物模型中的有益作用支持了这一点。 然而，铁络合剂的药代动力学较差，血脑屏障通透性较差，而且它们 激活细胞的代偿性反应，包括激活细胞内的铁摄取。因此，细胞的 对螯合剂的补偿反应最终可能抵消任何有益的除铁效果 螯合剂。因此，开发可能更有效的替代除铁疗法是很重要的 而不是传统的螯合剂。在这项提案中，我们提出了一种全新的方法来移除 来自神经元的铁。我们不是使用螯合剂，而是激活细胞的内源性铁去除和 戒毒计划。细胞中的铁稳态依赖于两种铁感应蛋白IRP1和IRP2 (铁反应蛋白1和2)。IRP1和IRP2是RNA结合蛋白，可与含有 IRE(铁反应元件)发夹序列。抑制Irp1和Irp2会导致细胞激活 降低细胞内铁水平的途径。我们正在创造一种新型的RNA疗法，在这种疗法中我们使用 IRE RNA发夹作为“诱饵”阻止IRP1和IRP2与其靶标mRNAs结合。虽然小RNA 在细胞中不稳定，Chimerna已经开发出一种新的技术，允许小RNA快速 在体外或在细胞中表达时形成环状的。我们在HEK293细胞中的研究表明，环状IRES 诱导一个强大的除铁程序，并提供对铁下垂的抵抗力。在这一点上，主要问题是 环形IRES是否能阻断帕金森病模型中的铁性下垂。为了测试这一想法， 该建议的具体目的是：(1)优化环状IRE RNA和环状IRE-RNA的转染。 中脑神经元铁耗竭的表达载体。在这个目标中，我们将优化两个 环状IRE RNA的不同递送方式：(A)直接转染环状IRE RNA；(B)质粒- 基于循环IRES的表达式。我们将测试总铁还原效率，转铁蛋白受体， 培养神经元中铁蛋白和铁蛋白的水平。总的来说，这些实验将优化两个不同的 在神经元中实现环状ire RNA的方法。(2)比较去铁胺和环状IRE作为 培养的帕金森氏病模型神经元中神经退行性变的抑制剂。在这里，我们将使用 两种帕金森病模型：MPTP毒性和α-突触核蛋白毒性。我们将比较环形IRES 去铁胺和铁络合剂，以确定环状IRES是否为或可能更有效， 而不是标准的基于螯合剂的方法。如果这些方法是成功的，这将表明 Irp1/2是一个治疗靶点，而环状RNA代表了有别于小分子新模式 治疗帕金森氏症的分子螯合剂。

SUMMARY: Iron overload is found in nearly all patients with Parkinson's disease and can lead to ferroptosis, an iron-dependent form of cell death. The importance of iron in Parkinson's disease pathology is supported by the beneficial effects of iron chelators in culture and animal models of Parkinson's disease. However, iron chelators have poor pharmacokinetics, poor blood-brain barrier permeability, and they activate a compensatory cellular response that involves activating iron uptake into cells. Thus, the cell's compensatory response to chelators could eventually counteract any beneficial iron-removal effect of the chelator. Therefore, it is important to develop alternative iron removal therapies that may be more effective than conventional chelators. In this proposal, we are proposing a completely novel approach for removing iron from neurons. Rather than using a chelator, we are activating the cell's endogenous iron removal and detoxification programs. Iron homeostasis in the cell relies on two iron-sensing proteins, IRP1 and IRP2 (iron-response protein 1 and 2). IRP1 and 2 are RNA-binding proteins that bind to mRNAs that contain an IRE (iron-response element) hairpin sequence. Inhibiting IRP1 and 2 would cause the cell to activate pathways that reduce intracellular iron levels. We are creating a new type of RNA therapy in which we use the IRE RNA hairpin as a “decoy” to block IRP1 and 2 from binding its target mRNAs. Although small RNAs are unstable in cells, Chimerna has developed a novel technology that allows small RNAs to be rapidly circularized, either in vitro, or when expressed in cells. Our studies in HEK293 cells show that circular IREs induce a robust iron removal program and confer resistance to ferroptosis. At this point, the major question is whether circular IREs can block ferroptosis in models of Parkinson's disease. In order to test this idea, the specific aims of this proposal are: (1) To optimize transfection of circular IRE RNA and circular IRE- expressing plasmids for iron depletion in mesencephalic neurons. In this aim, we will optimize two distinct delivery modes for circular IRE RNAs: (A) direct transfection of circular IRE RNAs; and (B) plasmid- based expression of circular IREs. We will test the efficiency of total iron reduction, transferrin receptor, ferritin and ferroportin levels in cultured neurons. Overall, these experiments will optimize two different approaches for achieving circular IRE RNA in neurons. (2) To compare deferoxamine and circular IRE as inhibitors of neurodegeneration in a cultured Parkinson's disease model neurons. Here, we will use two Parkinson's disease models: MPTP toxicity and alpha-synuclein toxicity. We will compare circular IREs to desferoxamine, and iron chelator, to determine whether circular IREs are as, or potentially more effective, than standard chelator-based approaches. If these approaches are successful, it would suggest that IRP1/2 is a therapeutic target, and that circular RNAs represented new modality distinct from small molecule chelators for Parkinson's disease.