Circular mimics of Iron-response elements to inhibit ferroptosis

摘要：铁超载在几乎所有帕金森病患者中都存在，并可导致

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.