Single-cell profiling of brain pericyte heterogeneity following ischemic stroke unveils distinct pericyte subtype-targeted neural reprogramming potential and its underlying mechanisms

Brain pericytes can acquire multipotency to produce multi-lineage cells following injury. However, pericytes are a heterogenous population and it remains unknown whether there are different potencies from different subsets of pericytes in response to injury. We used an ischemic stroke model combined with pericyte lineage tracing animal models to investigate brain pericyte heterogeneity under both naïve and brain injury conditions via single-cell RNA-sequencing and immunohistochemistry analysis. In addition, we developed an NG2 pericyte neural reprogramming culture model from both murine and humans to unveil the role of energy sensor, AMP-dependent kinase (AMPK), activity in modulating the reprogramming/differentiation process to convert pericytes to functional neurons by targeting a Ser 436 phosphorylation on CREB-binding protein (CBP), a histone acetyltransferase. We showed that two distinct pericyte subpopulations, marked by NG2 and Tbx18 , had different potency following brain injury. NG2 pericytes expressed dominant neural reprogramming potential to produce newborn neurons, while Tbx18 pericytes displayed dominant multipotency to produce endothelial cells, fibroblasts, and microglia following ischemic stroke. In addition, we discovered that AMPK modulators facilitated pericyte-to-neuron conversion by modulating Ser436 phosphorylation status of CBP, to coordinate an acetylation shift between Sox2 and histone H2B, and to regulate Sox2 nuclear-cytoplasmic trafficking during the reprogramming/differentiation process. Finally, we showed that sequential treatment of compound C (CpdC) and metformin, AMPK inhibitor and activator respectively, robustly facilitated the conversion of human pericytes into functional neurons. We revealed that two distinct subtypes of pericytes possess different reprogramming potencies in response to physical and ischemic injuries. We also developed a genomic integration-free methodology to reprogram human pericytes into functional neurons by targeting NG2 pericytes.