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

Reviewer #1 (Public review):

Summary:

This study revisits an important and controversial question in brain repair: whether NeuroD1 can convert brain immune cells into nerve cells in vivo. Using a virus-free genetic system, in vivo imaging, injury experiments, and single-cell profiling, the authors provide convincing evidence that NeuroD1-expressing cells do not become nerve cells under the tested conditions. Instead, these cells largely retain their original immune-cell identity, and some appear to undergo cellular stress or loss.

Strengths:

The main strength of the work is that it tests this question with a cleaner genetic strategy, avoiding some of the concerns associated with viral delivery and unintended cell labeling. Although the overall conclusion is consistent with the authors' previous work, the current study adds useful independent evidence, particularly through the virus-free fate-mapping system and live imaging in the brain.

Weaknesses:

There are some limitations. In the injury experiment, the labeled cells may include both resident brain immune cells and blood-derived immune cells recruited after injury, so the authors should be cautious when referring to all labeled cells as microglia. The level of NeuroD1 expression achieved by the genetic system is also not fully defined, which matters because the effects of such a cell-fate regulator may depend on expression level. Finally, the tested time window may not fully address very delayed or incomplete neuronal differentiation.

Overall, this is a useful and careful study that supports the conclusion that NeuroD1 does not drive brain immune cells to become nerve cells in the tested settings. It should be valuable for researchers studying brain repair, cell fate conversion, and genetic fate mapping, and it provides a clear caution against overinterpreting reprogramming results based only on viral labeling.

Reviewer #2 (Public review):

Summary:

In vivo glia-to-neuron conversion emerges as a potential regeneration-based therapeutic strategy for neural injuries and diseases. However, controversies exist in this exciting field, largely arising from the non-stringent methods employed for analyzing in vivo neuronal conversions. The study by Li et al. directly addressed this controversy regarding Neurod1-mediated microglia-to-neuron conversion. They took advantage of two transgenic mouse lines to specifically express Neurod1 in the microglia of adult mouse brains. Results from immunohistochemistry, in vivo live-cell imaging, and scRNA-seq convincingly demonstrate that microglia cannot be converted in vivo to neurons by ectopic Neurod1 expression under both normal and injury conditions. Instead, it induces microglia death, consistent with their earlier findings. These solid results, though negative, are critical additions to the field and further support that stringent lineage tracing methods are essential for studying in vivo cell reprogramming. Overall, the studies are rigorously designed and executed. Only minor issues need to be dealt with.