Abstract
Purpose :
Failure of retinal ganglion cell (RGC) axon regeneration prevents recovery of vision in optic neuropathy conditions like glaucoma. Finding ways to stimulate successful axonal regeneration in humans remains challenging. Adult teleost fish, like zebrafish, can fully regenerate RGC axons enabling functional recovery making it an optimal model to probe the mechanisms of successful axon regeneration. Here we identify the transcription factor Srebf2 as necessary for efficient optic nerve (ON) regeneration and explore the mechanisms downstream of its action.
Methods :
Laser Capture Microdissection RNA-seq (LCM-seq) was used to determine differentially expressed genes in the RGCs of adult zebrafish 3 days post optic nerve crush injury. RNAscope was used to measure time-course srebf2 expression post-injury. Srebf2 loss of function was accomplished with fatostatin drug treatment or morpholino antisense. Transgenic hsp70l:nSrebf2.2AmCherry zebrafish were used to overexpress the bHLH domain of Srebf2 for rescue experiments. Axon regeneration was analyzed by measuring GFP intensity using gap43:GFP transgenic zebrafish. The dorsal light response test was used to measure the visual recovery time course. Whole mount retinas with Hoechst stain were used to assay RGC densities after each treatment. Finally, LCM-seq for RGCs was used to detect changes in gene expression in response to the treatments mentioned above.
Results :
RGC LCM-seq identified genes in the mevalonate/cholesterol synthesis pathway that are upregulated during axon regeneration including the master transcriptional regulator of this pathway, srebf2. srebf2 expression is elevated throughout the whole regeneration phase. Blocking Srebf2 activity decreased axon regeneration and delayed visual recovery without causing any significant loss of RGCs. Re-expression of nSrebf2 rescued delayed axon regeneration and visual recovery by inhibitor or MO. LCM-seq results for each experimental treatment group will be reported.
Conclusions :
These data indicate that Srebf2 plays an important role in regulating efficient axon regeneration in the zebrafish visual system and suggest that the mevalonate/cholesterol synthesis pathway and its downstream products may have a role in this effect.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.