ABSTRACT Failure of central nervous system projection neurons to spontaneously regenerate long-distance axons underlies irreversibility of white matter pathologies. A barrier to axonal regenerative research is that the axons regenerating in response to experimental treatments stall growth before reaching post-synaptic targets. Here, we test the hypothesis that the interaction of regenerating axons with live oligodendrocytes, which were absent during developmental axon growth, contributes to stalling axonal growth. To test this hypothesis, first, we used single cell RNA-seq (scRNA-seq) and immunohistology to investigate whether post-injury born oligodendrocytes incorporate into the glial scar after optic nerve injury. Then, we administered demyelination-inducing cuprizone and stimulated axon regeneration by Pten knockdown (KD) after optic nerve crush. We found that post-injury born oligodendrocyte lineage cells incorporate into the glial scar, where they are susceptible to the demyelination diet, which reduced their presence in the glial scar. We further found that the demyelination diet enhanced Pten KD-stimulated axon regeneration and that localized cuprizone injection promoted axon regeneration. We also present a resource for comparing the gene expression of scRNA-seq-profiled normal and injured optic nerve oligodendrocyte lineage cells.

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

ABSTRACT Central nervous system projection neurons fail to spontaneously regenerate injured axons. Targeting developmentally regulated genes in order to reactivate embryonic intrinsic axon growth capacity or targeting pro-growth tumor suppressor genes such as Pten promotes long-distance axon regeneration in only a small subset of injured retinal ganglion cells (RGCs), despite many RGCs regenerating short-distance axons. A recent study identified αRGCs as the primary type that regenerates short-distance axons in response to Pten inhibition, but the rare types which regenerate long-distance axons, and cellular features that enable such response, remained unknown. Here, we used a new method for capturing specifically the rare long-distance axon-regenerating RGCs, and also compared their transcriptomes with embryonic RGCs, in order to answer these questions. We found the existence of adult non-α intrinsically photosensitive M1 RGC subtypes that retained features of embryonic cell state, and showed that these subtypes partially dedifferentiated towards an embryonic state and regenerated long-distance axons in response to Pten inhibition. We also identified Pten inhibition-upregulated mitochondria-associated genes, Dynlt1a and Lars2, which promote axon regeneration on their own, and thus present novel therapeutic targets.

Pten inhibition dedifferentiates long-distance axon-regenerating intrinsically photosensitive retinal ganglion cells and upregulates mitochondria-associated Dynlt1a and Lars2

The failure of mature central nervous system (CNS) projection neurons to regenerate axons over long distances drastically limits the recovery of functions lost after various CNS injuries and diseases. A major barrier in axon regeneration research is that, in most neurons, the axonal regenerative response to experimental treatments stalls before the axons reach their post-synaptic targets. Here, we tested the hypothesis that premature de novo myelination of the injured axons that are experimentally stimulated to regenerate stalls their growth, even after the glial scar is bypassed. To test this hypothesis, we used single cell RNA-seq (scRNA-seq) and immunohistological analysis to investigate whether post-injury born oligodendrocytes integrate into the glial scar. We also used a multiple sclerosis model of demyelination concurrently with the stimulation of axon regeneration by Pten knockdown (KD) in projection neurons after traumatic optic nerve injury. We found that post-injury born oligodendrocytes integrate into the glial scar, where they are susceptible to the demyelination treatment, which prevented premature myelination, and thereby enhanced Pten KD-stimulated axon regeneration. We also present a website for comparing the gene expression of scRNA-seq-profiled optic nerve oligodendrocytes under physiological and pathophysiological conditions. SIGNIFICANCE STATEMENT Myelin debris from degenerating axons along with reactive astrocytes in the glial scar inhibit CNS axon regeneration. However, even with the recently developed experimental approaches which activate axons to regenerate passed the glial scar, almost all axons still stall growth before reaching their post-synaptic targets. Here, we show that post-injury born oligodendrocytes integrate into the glial scar, and that other than myelin debris, live oligodendrocytes prematurely myelinating the regenerating axons inhibit growth, even if the axons have already regenerated passed the glial scar.

https://www.biorxiv.org/content/biorxiv/early/2021/10/20/2021.10.15.464557.full.pdf

Post-injury born oligodendrocytes integrate into the glial scar and inhibit growth of regenerating axons by premature myelination

Transcriptomic profiling of retinal cells reveals a subpopulation of microglia/macrophages expressing Rbpms marker of retinal ganglion cells (RGCs) that confound identification of RGCs

Experimental gene expression of developmentally downregulated Crmp1, Crmp4, and Crmp5 promotes axon regeneration and retinal ganglion cell survival after optic nerve injury

Jian Xing

Papers

Developmentally upregulated transcriptional elongation factor a like 3 suppresses axon regeneration after optic nerve injury.

Post-injury born oligodendrocytes integrate into the glial scar and inhibit growth of regenerating axons by premature myelination