SINGLE-CELL ANALYSIS REVEALS TRANSCRIPTIONAL PROGRAMS GOVERNING IN VIVO GLIA-TO-NEURON REPROGRAMMING IN THE ADULT MOUSE EPILEPTIC HIPPOCAMPUS

Direct lineage reprogramming holds promise as a new cell replacement therapy in several disease contexts. We recently demonstrated that reactive glial cells, proliferating in the hippocampus of a mouse model of drug-resistant epilepsy, can be efficiently converted into GABAergic induced neurons (iNs) upon retrovirally-mediated expression of the transcription factors (TFs) Ascl1 and Dlx2. Those newly generated iNs fully integrated into diseased networks ultimately reducing seizures (Lentini et al, 2021). How TFs impose a new neurogenic program onto glial cells remains largely uncharacterized. Using single-nuclei RNA Seq, we obtained the transcriptomes of glial cells undergoing reprogramming in the epileptic hippocampus at different stages of neuronal conversion (4,7,14,30 days after retrovirus injection). We reconstructed two separate reprogramming trajectories originating from astrocytes and oligodendrocyte precursor cells (OPCs) towards distinct subtypes of GABAergic iNs. Combined with genetic fate-mapping analysis, these data demonstrate genuine in vivo reprogramming of glial cells into interneurons. Conversely, microglia remained resistant to reprogramming. GABAergic iNs of both glial origin could be distinguished by expression of distinct interneuron subtype markers, which was associated with induction of distinct transcriptional programs. Intriguingly, we identified a subset of OPCs that remained refractory to reprogramming, characterized by a pro-inflammatory phenotype, suggesting that the glial cell reactivity status influences conversion. Those results uncover that the identity and state of source cell types play a crucial role in reprogramming efficiency and iN subtype specification. Further elucidating glia-to-iN reprogramming limitations will help define strategies to manipulate this process to generate disease-relevant iNs and broaden its scope of application.