Augmenting fibronectin levels in injured adult CNS promotes axon regeneration in vivo [scRNA-seq]

In an attempt to repair injured central nervous system (CNS) nerves/tracts, immune cells are recruited into the injury site, but endogenous response in adult mammals is insufficient for promoting regeneration of severed axons. Here, we found that a portion of retinal ganglion cell (RGC) CNS projection neurons that survive after optic nerve crush (ONC) injury are enriched for and upregulate fibronectin (Fn)-interacting integrins Itga5 and ItgaV, and that Fn promotes long-term survival and long-distance axon regeneration of a portion of axotomized adult RGCs in culture. We then show that, Fn is developmentally downregulated in the axonal tracts of optic nerve and spinal cord, but injury-activated macrophages/microglia upregulate Fn while axon regeneration-promoting zymosan augments their recruitment (and thereby increases Fn levels) in the injured optic nerve. Finally, we found that Fn’s RGD motif, established to interact with Itga5 and ItgaV, promotes long-term survival and long-distance axon regeneration of adult RGCs after ONC in vivo, with some axons reaching the optic chiasm when co-treated with Rpl7a gene therapy. Thus, experimentally augmenting Fn levels in the injured CNS is a promising approach for therapeutic neuroprotection and axon regeneration of at least a portion of neurons. We generated 10x Genomics 3’-droplet based scRNA-seq dataset of macrophage/microglia from adult (10 weeks old) uninjured and injured (2 weeks after ONC) optic nerves from mice of both sexes using methods we previously described (Rheaume et al 2018; Xing et al, 2023). ONC injury was performed at 8 weeks. Cells from were loaded 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). After demultiplexing and aligning to the mouse genome and transcriptome (mm10) using the CellRanger pipeline and merging/integration in Rstuduio, 778 macrophage/microglia passed quality control (including filters of a minimum of 200 genes and a maximum of 6500 genes). Batches were merged, normalized and scaled using Seurat. Each bam file is comprised of a single paired end fastq files. After each is aligned to the mm10 genome using Cell Ranger Count, batches were merged, normalized and scaled using Seurat with default parameters. Seurat's RunUmap function was performed with parameters "dims = 1:4" and all other parameters as default.