Abstract
Purpose :
Failure of retinal ganglion cell (RGC) axon regeneration prevents recovery of vision in optic neuropathy conditions like glaucoma. Due to the poor regenerative capacity of the adult mammalian central nervous system, finding ways to stimulate successful axonal regeneration in humans remains challenging. Unlike patients and mammalian model organisms, adult teleost fish can fully regenerate RGC axons enabling functional recovery from optic nerve injury. We are using the zebrafish model to probe the mechanisms of successful axon regeneration with the hopes of translating these findings to improve regeneration in mammals and eventually patients.
Methods :
Laser Capture Microdissection RNA-seq (LCM-seq) was used to determine differentially expressed genes in the retinal ganglion cell layer of adult zebrafish 3 days post optic nerve crush versus uninjured control. Bioinformatic analysis was used to identify pathways and gene sets that are enriched in differentially expressed genes. To test the importance of srebf2 specifically, fatostatin drug treatment and morpholino antisense were used to antagonize its function. Axon regeneration into optic nerve at 3 days and optic tectum at 7 days post injury was analyzed by measuring GFP intensity using gap43:GFP transgenic zebrafish. The dorsal light response test was used to measure visual recovery at multiple times post injury. To assay RGC survival, whole mount retina with Hoechst stain was used to counting cell densities after each treatment.
Results :
Retinal ganglion cell layer LCM-seq identified genes in the mevalonate/cholesterol synthesis pathway that are upregulated during axon regeneration. The master transcriptional regulator of this pathway, srebf2, was also highly upregulated. In comparison to untreated or vehicle treated control groups, srebf2 inhibited by either fatostatin or morpholino decreased axon regeneration into the optic tectum at both 3 days and 7 days post injury and delayed recovery of the dorsal light response over the course of normal optic nerve regeneration without causing any significant loss of RGCs.
Conclusions :
These data indicate that srebf2 plays an important role in regulating efficient axon regeneration in the zebrafish visual system and suggests that the mevalonate/cholesterol synthesis pathway and its downstream products may have a role in this effect.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.