Cheng Qian (Johns Hopkins School of Medicine)| Ying Xin (Johns Hopkins School of Medicine)| Cheng Qi (Johns Hopkins School of Medicine)| Bryan Dong (Johns Hopkins University)| Feng-Quan Zhou (Johns Hopkins School of Medicine)
Weak inherent axonal regrowth in adult CNS hampers circuit reconnection and neuronal function recovery after various types of injuries or axonopathies. Emerging evidence showed that developmentally downregulated transcriptomic and epigenomic regulators, including Klf6/7, Sox11, Jun, Myc, Uhrf1 and Lin28, functioned to promote CNS axon regeneration sufficiently. We thus hypothesize that manipulating developmentally regulated transcriptional factors (TFs) in retinal ganglion cells (RGCs) may act to promote optic nerve regeneration. An advanced integrative bioinformatics analysis approach was established to explore a mouse retinal embryonic and postnatal developmental deep-sequencing dataset (GSE87064) including RNA-seq, ChIP-seqs of histone markers and ATAC-seq. The RNA-seq was used to sort out differential TFs across development. By characterizing both the chromatin accessibility transition and active enhancer regions, a motif-based footprinting analysis (notches in the ATAC peaks) was utilized to predict TF-promoter as well as TF-enhancer-gene regulations. We then used a scoring algorithm to identify a group potential candidate TFs that might be manipulated to enhance optic nerve regeneration. Among all the candidate TFs, Ascl1 was predicted with prioritized score in interacting with the regeneration-related transcriptome via either promoter or enhancer regions. Beside Ascl1, some other novel TFs as well as some known regeneration promoting TFs, such as Sox11, Myc, Fos and Id2, also received high scores.
We then used optic nerve injury and regeneration as a model system to perform functional screening of these candidate TFs. In vivo optic nerve crush injury model in adult CF-1 mice (6 weeks, N=10) showed that Ascl1 overexpression (AAV2-Ef1a-mAscl1) significantly promoted optic nerve regeneration after injury compared with the control group (AAV2-CMV-GFP, N=4), at each measured length point (100mm P <0.0001; 250mm P <0.0001; 500mmP=0.002; 750mm P=0.002). Functional validation experiments of other candidate TFs are currently ongoing, and we expect to identify additional novel TFs that can be manipulated to enhance optic nerve regeneration.
Our results provided evidence that developmentally regulated TFs (either up or down regulated) are promising candidates for enhancing optic nerve regeneration via modifying the transcriptome of mature RGCs. Additional multiomics sequencing experiments using purified RGCs, in which functional TFs (i.e. Ascl1) were manipulated, will allow us to elucidate the optimal chromatin and transcriptomics landscape supporting optic nerve regeneration.
Support or Funding Information
NIH (R01NS085176, R01GM111514, R01EY027347, R01EY030883, R01EY031779), the Craig H. Neilsen Foundation (259450), and the BrightFocus Foundation (G2017037)
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