Ferroptosis modulation enhances astrocyte-to-neuron conversion induced by a novel chemical cocktail in the hemorrhagic brain

Intracerebral hemorrhage (ICH) leads to significant neuronal loss and glial scar formation, but the regenerative capacity of the adult brain remains limited. Although small-molecule-induced astrocyte-to-neuron (AtN) conversion has shown promise in vitro, in vivo applications—particularly under pathological conditions—are still scarce. We aimed to develop and validate a small-molecule cocktail for inducing astrocyte-to-neuron reprogramming in vivo following ICH. We identified a seven-compound cocktail (DFGKLRV) capable of converting astrocytes into neurons under both physiological and ICH conditions. Using immunostaining, RT‒qPCR, electrophysiology, RNA sequencing, neural circuit tracing, and behavioral assessment, we assessed the identity and functionality of induced neurons. In vivo reprogramming was achieved via continuous intracerebral infusion of the cocktail using osmotic pumps. Lineage tracing with aldehyde dehydrogenase 1 family member L1 ( Aldh1l1 ) -Cre ERT2 / Rosa-CAG-tdTomato mice confirmed the astrocytic origin of the reprogrammed neurons. Additionally, we monitored ferroptosis dynamics during reprogramming and evaluated the effect of ferroptosis inhibition on conversion efficiency. DFGKLRV successfully reprogrammed astrocytes into functional, electrophysiologically active neurons. This reprogramming was effective both in vitro and in vivo, including in the hemorrhagic brain environment. Pharmacological inhibition of ferroptosis significantly improved reprogramming efficiency. Mechanistically, ferroptosis inhibition promoted astrocyte-to-neuron conversion at least in part through suppression of the TGF-β/SMAD3/SOX9 axis, whereas exogenous TGF-β1 treatment or Sox9 overexpression reversed this pro-reprogramming effect. Our findings demonstrate that the DFGKLRV cocktail enables efficient in vivo astrocyte-to-neuron reprogramming following ICH. Moreover, ferroptosis represents a key regulatory mechanism and potential therapeutic target for enhancing chemical reprogramming strategies.