Abstract
Purpose :
Retinal ganglion cell (RGC) death drives vision loss in glaucoma and optic nerve injury. However, RGCs are a diverse set of related neurons that demonstrate extemely different survival in animal models of degeneration and human patients. Identifying native cellular features of well-surviving RGCs can be used to develop new neuroprotective strategies.
Methods :
We use in vivo 2-photon transpupillary imaging of genetically encoded fluorescent biosensors to characterize the properties of individual RGCs before, and after neurodegeneration, We measure both Ca2+ and ATP in RGCs using FRET ratiometric biosensors Twitch2b and ATeam1.03 respectively. To express biosensors specifically in RGCs, we use VGlut2-Cre transgenic mice and intravitreal injection of AAV2 carrying a Cre-dependent biosensor expression construct, and optic nerve crush (ONC) as a degeneration model. We performed in vivo measurements of Ca2+, mitochondrial Ca2+, and ATP levels in individual RGCs at baseline and every two days after ONC for two weeks. RGC survival was tracked, and outcome related to biosensor measurements.
Results :
Well-surviving RGCs showed higher levels of cytoplasmic and mitochondrial Ca2+. Since we found high mitochondrial Ca2+ in well-surviving RGCs, we hypothesized that high mitochondrial Ca2+ levels might stimulate ATP production in these RGCs. Interestingly, we found the opposite; ATP levels are lower in well-surviving RGCs. To examine what underlies the lower ATP levels observed in well-surviving RGCs, we are screening the metabolic strategies of RGCs in vivo by observing ATP levels following inhibition of oxidative phosphorylation. Interestingly, while inhibition of Complex 4 leads to ATP reduction that is relatively uniform across all RGCs, inhibition of complex I or II leads to more specific ATP reductions in alpha RGCs. Additionally, all attempted manipulations that altered ATP levels across the RGC population failed to remove the underlying diffence between RGCs in ATP found at baseline.
Conclusions :
We successfully used in vivo imaging to construct a multifacted phenotype of well-surviving RGCs. Future experiments include gene therapy approaches to manipulate Ca2+ handling and ATP generation strategies to determine if they can protect RGCs from degeneration. Additionally we are establishing mouse glaucoma models to determine if our observations hold true in more clinically relevant models of RGC degeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.