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Qing Wang, Irene Harutyunyan, S. Thomas Carmichael; Activity-dependent molecular programs for optic nerve regeneration. Invest. Ophthalmol. Vis. Sci. 2019;60(9):646. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In the mammalian visual system, retinal ganglion cells (RGCs) are unable to regenerate their axonal projections to targets in the brain following traumatic or ischemic damage or in degenerative diseases such as glaucoma. RGC axons can be coaxed into regrowing following optic nerve axotomy by manipulating both extrinsic and intrinsic molecular pathways; however, despite these efforts, RGC axons are still inefficient at growing past the optic chiasm and innervating their visual targets. More recently, increasing neuronal activity through visual stimulation or chemogenetic activation has been shown to further promote axonal regrowth after optic nerve crush. Genetic programs that modulate this form of regeneration remains to be identified. In other CNS injury models, such as stroke, increasing neuronal activity through forced limb overuse has been similarly shown to promote the formation of new circuits associated with improved motor recovery. Our lab has generated an RNA-Seq dataset of genes differentially regulated in new neuronal connections formed in the limb overuse stroke model. We investigate whether these activity-dependent genes can similarly promote reformation of retinofugal pathways following injury.
We used an AAV-mediated approach for in vivo overexpression and knockout of candidate genes in RGCs. AAV2 carrying an expression cassette for a GFP reporter with or without a gene candidate of interest under the control of the human synapsin (hSyn) promoter was injected intravitreally into unilateral adult mouse eyes. Similarly, AAV2 carrying an SaCas9 and guide RNAs of gene candidates (or scrambled control) were used for CRISPR knockout models. The optic nerve of the injected eye was then crushed 2 weeks after expression injection of the viruses. GFP signal, CTB anterograde tracing, and GAP-43 staining was used to quantify regenerating axons in optic nerve sections. Whole mount retinas were stained with TUJ1 or RBPMS for analysis of RGC survival.
We demonstrate efficient and selective expression of our candidate genes in RGCs as well as successful knockdown of candidate genes through the CRISPR. Our preliminary results show improved regeneration of crushed RGCs axons through overexpression or knockout of a subset of our candidate genes.
Our results show novel molecular mechanisms downstream of activity that can improve regeneration in glaucoma and neurodegenerative diseases of the CNS.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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