Promoting axon regeneration by enhancing the non-coding function of the injury-responsive coding gene Gpr151

Abstract

Gene expression profiling in response to nerve injury has been mainly focused on protein functions of coding genes to understand mechanisms of axon regeneration and to identify targets of potential therapeutics for nerve repair. However, the protein functions of several highly injury-induced genes including Gpr151 for regulating the regenerative ability remain unclear. Here we present an alternative approach focused on non-coding functions of the coding genes, which led to the identification of the Gpr151 RNA function as a molecular sponge via its interaction with RNA-binding proteins such as CSDE1. Gpr151 promotes axon regeneration by the function of its 5’- untranslated region (5’UTR) and expression of an engineered form of the 5’UTR improves regenerative capacity in vitro and in vivo in both sciatic nerve and optic nerve injury models. Our data suggest that searching injury-induced coding genes potentially functioning by their non-coding regions is required for the RNA-based gene therapy for improving axon regeneration.

Construction of a searchable database for gene expression changes in spinal cord injury experiments

“…The top 25 up-regulated genes are shown in Supplemental Table S3, while the top 25 down-regulated genes are shown in Supplemental Table S4. Included in the up-regulated genes in the DRG for the mouse and rat meta-analysis are GADD45A, which is involved in cell cycle and neuronal cell death [64] and may act in a neuroprotective fashion [65][66][67]; GPR151 which is involved in neuropathic pain, and may promote axon regeneration [68,69]; FLRT3 which promotes neurite outgrowth [70][71][72], CRLF1 which forms complexes with neurotrophic factors to promote survival of neuronal cells [73], and the neuropeptide GAL [74,75]. At the top of the down-regulated genes are two leucine zipper proteins (FOSB and FOS), two zinc fingers (EGR1 and ZFP36), and CYR61.…”

FKBPL and FKBP8 regulate DLK degradation and neuronal responses to axon injury

“…Considering the DLK functions regulating axon regeneration and degeneration in dorsal root ganglion neurons, we reviewed the abundance of FKBPs' mRNA in mouse L4,5 DRG tissues and mouse sciatic nerves from our previously pulished datasets (Shin et al, 2019;Shin et al, 2018a;Lee et al, 2021). To consider the neuronal expression of the FKBPs, the microarray dataset from cultured mouse embryonic DRG neurons was presented as the relative sizes of the circles (Figure 3A).…”

“…When HEK293T cells were incubated with bafilomycin A1, FKBPL-and FKBP8-induced DLK degradation was inhibited indicating that FKBPL-and FKBP8-induced DLK degradation was regulated by lysosomal protein degradation functions. Moreover, the basal DLK protein level without co-expressing FKBP8 or FKBPL was increased by bafilomycin A1 treatement, suggesting that the baseline DLK turnover is analysis of relative expression levels in mouse DRG (Shin et al, 2019;Lee et al, 2021), sciatic nerve 192 tissue (Shin et al, 2018b), and cultured embryonic DRG neurons (Cho et al, 2013) The kinase domain was a major target of DLK ubiquitination and sumoylation DLK protein degradation is regulated by PHR1 E3 ligase (Collins et al, 2006;Nakata et al, 2005). As FKBPL-or FKBP8-induced DLK protein reduction required lysosomal degradation function, we tested if FKBP8-dependent DLK protein reduction was regulated by the ubiquitin-dependent protein degradation pathway.…”

“…To knock down DLK in vitro, shRNA targeting sequences identified by the BROAD Institute (TRCN0000322150) were synthesized (Bionics) and ligated into a pLKO.1 lentiviral vector with the restriction sites AgeI/EcoRI. Lentivirus was produced using Lenti-XTM Packaging Single Shot (Qiagen, 631276), concentrated using a Lenti-XTM Concentrator (Qiagen, 631232), and quantified using a Lenti-XTM GoStixTM Plus kit (Qiagen, 631280) as previously described (Jeon et al, 2021). The efficiency of the knockdown process was confirmed using RT-qPCR.…”