Understanding the mechanisms of ferroptosis in direct neuronal reprogramming – towards improving neuronal replacement in vivo

在哺乳动物的大多数大脑区域，由于脑损伤或神经退行性疾病而失去的神经元是无法替代的。我们已经开创了从局部神经胶质细胞直接进行神经元重编程的新方法，并确定铁性下垂是这一过程中的一个主要障碍。在这里，我们的目标是进一步剖析当小鼠和人源性星形胶质细胞在体外转化为神经元时引发铁下垂的途径。我们将进一步探索与未折叠蛋白反应的可能联系，并分析在我们最近发现的星形胶质细胞和神经元线粒体蛋白质组中神经元特有的抗氧化蛋白的作用。基于体外最有希望的结果，我们的目标是在体内通过过表达FSP1、Gpx4、删除Acsl4或使用CRISPRa激活神经元特异性线粒体蛋白的表达来特异性地抑制小鼠大脑皮层刺伤后反应性星形胶质细胞的转化。这些基因操作将与药物治疗的效果进行比较，例如使用第三代利普生抑素或罗格列酮等抑制剂，两者都能通过血脑屏障。在最终目标中，我们将建立一个体内移植方案，将人IPSC来源的星形胶质细胞移植到新生小鼠体内，并在成年阶段诱导它们的神经元转化。这将为人类星形胶质细胞转化为神经元提供体内实验，以探索铁性下垂在这一过程中的作用。这一开创性的方法研究铁性下垂在神经元直接重编程中的作用，将极大地受益于关于铁性下垂的SPP网络，并为探索人类细胞的铁性下垂机制提供一个独特的平台。

Neurons lost upon brain injury or in neurodegenerative disease cannot be replaced in most brain regions of mammals. We have pioneered the novel approach of direct neuronal reprogramming from local glial cells and identified ferroptosis as a major hurdle in this process. Here we aim to further dissect the pathways involved in eliciting ferroptosis when astrocytes of murine and human origin are converted into neurons in vitro. We will further explore a possible link to the unfolded protein response and analyze the role of neuron-specific antioxidant proteins identified in our recent mitochondrial proteome of astrocytes and neurons. Based on in vitro most promising results, we aim to inhibit these pathways specifically when converting reactive astrocytes after stab wound injury in the murine cerebral cortex in vivo, e.g. by overexpressing FSP1, Gpx4, deleting Acsl4 or using CRISPRa to activate the expression of neuron-specific mitochondrial proteins. These genetic manipulations will be compared to the effects of pharmacological treatments, e.g. by using inhibitors like of 3rd generation liproxstatin or Rosiglitazone, both passing the blood-brain barrier. In the final aim we will establish an in vivo protocol of transplanting human iPSC-derived astrocytes into neonatal mice and induce their neuronal conversion at adult stages. This will provide an in vivo assay for human astrocyte conversion into neurons to explore the role of ferroptosis in this process.This pioneering approach of examining the role of ferroptosis in direct neuronal reprogramming will greatly profit from the SPP network on ferroptosis and provide a unique platform for exploring ferroptosis mechanisms in human cells.