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

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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.