Post-injury born oligodendrocytes incorporate into the glial scar and contribute to the inhibition of axon regeneration

Pathologies of the central nervous system (CNS) white matter often result in permanent functional deficits because mature mammalian projection neurons fail to regenerate long-distance axons after injury. A major barrier to axonal regenerative research is that the CNS axons that regenerate in response to experimental treatments stall growth before reaching their post-synaptic targets. Here, we test the hypothesis that interaction of regenerating axons with live oligodendrocytes, which were absent during developmental axon growth, stalls axonal growth. To test this hypothesis, first, we used single cell RNA-seq (scRNA-seq) and immunohistological analysis to investigate whether post-injury born oligodendrocytes incorporate into the glial scar after optic nerve injury. Then, we administered demyelination-inducing cuprizone (used in studies of multiple sclerosis), concurrently with the stimulation of axon regeneration by Pten knockdown (KD) in projection neurons after optic nerve injury. We found that post-injury born oligodendrocyte lineage cells incorporate into the glial scar, where they are susceptible to the demyelination diet treatment, which reduced their presence in the glial scar. We further found that the demyelination diet enhanced Pten KD-stimulated axon regeneration, and localized injection of cuprizone promoted axon regeneration. We also present a website for comparing the gene expression of scRNA-seq-profiled optic nerve oligodendrocyte lineage cells under physiological and pathophysiological conditions. Annotation of oligodendrocyte lineage cell subtypes, and normal or injured condition, are indicated in the last two rows of the cell matrix CSV file. Cells were purified from uninjured (n = 5) and injured (n = 5) optic nerves of both sexes 10-week old mice. ONC injury was performed at 8 weeks. 6,000 cells from each sample were loaded in parallel for capture onto the Chromium System using the v2 single cell reagent kit (10X Genomics). Following capture and lysis, cDNA was synthesized and amplified (12 cycles) as per manufacturer's protocol (10X Genomics).