Lineage tracing and live-cell imaging reveal that NeuroD1 does not reprogram microglia into neurons

<jats:p>Induction of glia-to-neuron conversion is a promising regenerative strategy for treating brain injuries and neurodegenerative diseases. Previous studies have suggested that NeuroD1 can induce microglia-to-neuron cross-lineage conversion. However, it remains highly controversial. To conclusively determine whether NeuroD1 can convert microglia into neurons, we used genetic fate mapping to track the cell fate of microglia ectopically expressing NeuroD1. Furthermore, we performed two-photon imaging to trace the fate of the NeuroD1-expressing microglia. Our findings revealed that cells ectopically expressing NeuroD1 are bona fide microglia, not neurons, regardless of injury preconditioning. Additionally, NeuroD1 overexpression in microglia did not promote brain injury recovery, and the microglial identity remained intact. These results provide solid evidence that NeuroD1 cannot convert microglia into neurons. Our study underscores the necessity of lineage-tracing and cell fate mapping strategies to verify glia-to-neuron conversion, highlighting the importance of rigorous validation in regenerative research.</jats:p>