Nfe2l3 promotes neuroprotection and long-distance axon regeneration after injury in vivo [Third party: scRNA-seq]

Nuclear factor erythroid 2 like (Nfe2l) gene family members 1-3 mediate cellular response to oxidative stress, including in the central nervous system (CNS). However, neuronal functions of Nfe2l3 are unknown. Here, we comparatively evaluated expression of Nfe2l1, Nfe2l2, and Nfe2l3 in singe cell RNA-seq (scRNA-seq)-profiled cortical and retinal ganglion cell (RGC) CNS projection neurons, investigated whether Nfe2l3 regulates neuroprotection and axon regeneration after CNS injury in vivo, and characterized a gene network associated with Nfe2l3 in neurons. We showed that, Nfe2l3 expression transiently peaks in developing immature cortical and RGC projection neurons, but is nearly abolished in adult neurons and is not upregulated after injury. Furthermore, within the retina, Nfe2l3 is enriched in RGCs, whereas Nfe2l1 and Nfe2l2 are expressed robustly in other retinal cell types as well, and are also upregulated after injury. We also found that, expressing Nfe2l3 in injured RGCs through localized intralocular viral vector delivery promotes neuroprotection and long-distance axon regeneration after optic nerve injury in vivo. Moreover, Nfe2l3 treatment provided a similar extent of neuroprotection and axon regeneration as viral vector-targeting of Pten and Klf9, which are prominent regulators of neuroprotection and long-distance axon regeneration. Finally, we bioinformatically characterized a gene network associated with Nfe2l3 in neurons, which revealed the association of Nfe2l3 with established mechanisms of neuroprotection and axon regeneration. Thus, Nfe2l3 is a novel neuroprotection and axon regeneration-promoting factor with a therapeutic potential for treating CNS injury and disease. Normalized matrices and cell metadata (e.g., type assignment) was obtained from the GEO accession numbers as follows: for cortical embryonic, postnatal, and adult projection neurons from GSE143949, GSE123335, and GSE116470; for RGC embryonic, postnatal, adult projection neurons, and injured adult RGCs from GSE185671 and GSE137400; and for all adult normal and injured retinal cell types from GSE199317. Seurat v4.3.0 was used to merge and normalize the datasets mapped to the mm10 genome. Datasets were batch adjusted and integrated using Harmony. Cell type identity of cells that passed scRNA-seq QCs was confirmed based on established cell type markers. Data processing details: Cell-counts matrices were obtained from the GEO submissions from the previously published data. Matrices were loaded as Seurat objects in R. Seurat objects were merged and expression was normalized. A cell counts matrix for the merged dataset was then generating by extracting counts matrix from Seurat object. The condition each cell belongs to was appended to the cell ID in the columns of the matrix genome assembly: mm10 RDS files: Containing cortical or RGC embryonic, postnatal, adult projection neurons, injured adult RGCs, and all adult normal and injured retinal cell types, with cells' normalized expression values for each gene.