Single-cell transcriptome analysis of regenerating RGCs reveals potent glaucoma neural repair genes

Axon regeneration holds great promise for neural repair of CNS axonopathies, including glaucoma. Pten deletion in retinal ganglion cells (RGCs) promotes potent optic nerve regeneration, but only a small population of Pten-null RGCs are actually regenerating RGCs (regRGCs); most surviving RGCs (surRGCs) remain non-regenerative. Here, we developed a strategy to specifically label and purify regRGCs and surRGCs, respectively, from the same Pten-deletion mice after optic nerve crush, in which they differ only in their regeneration capability. Smart-Seq2 single-cell transcriptome analysis revealed novel regeneration-associated genes that significantly promote axon regeneration. The most potent of these, Anxa2, acts synergistically with its ligand tPA in Pten-deletion-induced axon regeneration. Anxa2, its downstream effector ILK, and Mpp1 dramatically protect RGC somata and axons and preserve visual function in a clinically relevant model of glaucoma, demonstrating the exciting potential of this innovative strategy to identify novel effective neural repair candidates. •Powerful single-cell regRGCs-seq to profile Pten KO-regeneration-associated genes•In vivo demonstration of multiple pro-axon regeneration genes•Striking neuroprotection in glaucoma by pro-regeneration genes Anxa2 and Mpp1•The tPA-Anxa2-ILK pathway in axon regeneration and neuroprotection Combined retrograde regenerating axon tracing and Smart-Seq2 single-cell sequencing approaches revealed multiple pro-regeneration genes downstream of Pten deletion. Two of these genes, Anxa2 and Mpp1, also presented striking neuroprotection and visual function preservation in a mouse glaucoma model. Additionally, we established the role of tPA-Anxa2-ILK axis in neural repair.