MicroRNA-mediated neuronal detargeting alters astrocyte cell fate conversion trajectories in vivo.

Neuronal reprogramming using adeno-associated viruses with a GFAP mini-promoter offers a promising strategy for astrocyte-to-neuron conversion; however, specificity remains a challenge due to off-target transgene expression in endogenous neurons. To address this issue, here we incorporated microRNA-124 target sequences (124T) into a transcriptional cassette containing the GFAP mini-promoter and Ascl1(SA6), a potent reprogramming transcription factor. Lineage tracing via Slc1a3-CreERT and Aldh1l1-CreERT2, used to pre-label astrocytes prior to conversion, confirmed the glial derivation of reprogrammed neuron-like cells, even with 124T. Single-cell transcriptomics identified a transitional cluster emerging from a proliferative astrocyte population with low GSK3 signaling, which branched towards hybrid neuronal and oligodendrocyte fates. Pseudotime trajectory analysis revealed that Ascl1(SA6) drives rapid neuronal transitions, whereas 124T delays conversion and introduces lineage bifurcation. Ascl1(SA6) favors a GABAergic interneuron-like identity, while Ascl1(SA6) -124T biases fate transitions towards an oligodendrocyte-like fate, and to a lesser extent, glutamatergic neuronal-like cells. SeeSawPred linked these distinct trajectories to transcription factor shifts, including Foxo1 in neuron-like cell fates and Stat1 in oligodendrocyte lineages. 124 T thus effectively detargets endogenous neurons, refining target cell specificity, while further guiding reprogramming outcomes. This approach establishes a foundation for precision reprogramming platforms aimed at restoring specific neural cell types.