Rationale: 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. Methods: 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. Results: 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. Conclusion: 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.

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