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Jonathan Mikal Skarie, Robert W Nickells; Mechanisms of Retinal Ganglion Cell Survival in Zebrafish after Axon Injury. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2555. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Optic nerve injury in mammals results in irreversible retinal ganglion cell (RGC) death and permanent vision loss. Cell death occurs via apoptosis and requires BCL2 gene family member activation. In stark contrast, the majority of RGCs in zebrafish do not die after axon injury. Instead these cells resist death, allowing axonal regeneration and restoration of visual function. The cellular mechanisms that allow zebrafish RGCs to resist cell death remain poorly understood. The purpose of this study is to investigate BCL2 family genes in zebrafish RGCs after axon injury to determine how genes essential for apoptosis in mammalian RGCs are regulated in the zebrafish.
Utility of RGC specific promoters in adult zebrafish are assessed by confocal microscopy after optic nerve injury experiments in fluorescently labeled transgenic zebrafish. Standard transcript and protein expression assays are used to assess expression levels of BCL2 genes in zebrafish retinal tissue before and after optic nerve injury. Sub-cellular localization of bax is assessed by time lapse confocal microscopy of zebrafish RGCs expressing a GFP-Bax fusion protein.
The zebrafish isl2b promoter region was identified to specifically label adult RGCs before and after optic nerve injury. Homologs of BCL2 family members important for mammalian RGC apoptosis, including anti-apoptotic BCL-X, pro-apoptotic BAX (baxa and baxb) and BH3 only domain proteins PUMA, BIM, and BAD, are present in adult zebrafish retinal tissue. RNAseq data confirms higher expression of baxa as compared to baxb in retinal tissue. The isl2b promoter is used to create a transgenic zebrafish line with GFP-Baxa fusion protein expression specifically in RGC’s. Prior work in our lab has shown that activated Bax oligomerizes at the mitochondria in RGCs and can be monitored in mouse explants. Ongoing work is being done to monitor sub-cellular localization of GFP-Baxa in zebrafish RGCs after axon injury using in vivo time-lapse confocal imaging.
Uncovering how the apoptotic machinery/BCL2 family genes are regulated in zebrafish RGCs which survive after axon injury, and comparing this to what occurs in mammals, has a strong potential for identifying novel neuro-protective therapeutic targets for glaucoma and other optic neuropathies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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