Investigation and targeting of early degenerative response in retinal cells in Ischemia-Reperfusion glaucoma mouse model: paving the way for therapeutic neuroprotection strategy

Glaucoma is a group of neuropathies characterized by progressive degeneration of retinal ganglion cells (RGCs) causing vision loss. It is the leading cause of irreversible blindness with 76 million people affected worldwide. Currently, the only approved treatments aim at lowering intraocular pressure (IOP), the main modifiable risk factor. However, some patients continue losing vision even though they have a well-controlled or nonelevated IOP. Therefore, there is an unmet need to develop new neuroprotective treatments to prevent RGC loss in order to slow down disease progression and ultimately preserve vision.
The study aimed at better understanding the mechanisms involved in RGC degeneration in order to develop RGC neuroprotective strategies. To do so, we used the ischemia-reperfusion (I/R) mouse model of glaucoma.
We first characterized the I/R mouse model and showed degeneration of the RGCs with different degrees of severity: mild, moderate and severe, 7 days following the injury. Resulting visual dysfunction was observed. Performing single-cell RNA sequencing allowed us to identify initial genes and pathways transcriptionally up- or down-regulated following injury in RGCs as well as other retinal cell types.
We explored RGC surrounding cell response and evaluated the implication of cellular senescence in the retina following I/R injury. We identified at early I/R time point glial cells, rather than RGC, expressing senescence-related genes, although RGC showed hallmarks of senescence at the later time point.
We investigated further the early RGC stress response following I/R injury and identified the transcriptor factor 3 (ATF3) as a significant factor responding to I/R injury. We confirmed the relevance of this target as we observed increased ATF3 transcript expression in human glaucomatous post-mortem eyes. We decided to inhibit expression of Atf3 and relative genes that were activated in response to I/R injury to assess their effect on RGCs survival. The strategy was to use sh-miR (artificial miRNA), delivered into retinal cells using adeno-associated viral (AAV) vectors, in order to knock down target gene expression. We quantified RGC survival and observed a reduction of the degree of severity of RGC degeneration by knocking down Atf3, Ecel1 and Cdkn1a.
Our results demonstrate that targeting ATF3 and related pathways might have a neuroprotective effect on RGCs and could be considered for potential neuroprotection strategies to treat glaucoma patients with the ultimate goal of preserving patient vision.