Homeostatic mitochondrial and cytoplasmic Ca²⁺ levels are associated with retinal ganglion cell survival

Abstract

Purpose : Retinal Ganglion Cells (RGCs) are the sole projection neurons from the retina to the brain. Only a small portion of RGCs survive after optic nerve crush (ONC), a strong CNS neurodegeneration model. We recently discovered that cytoplasmic Ca²⁺ set-points are differential across RGCs within the mouse retina, and that high Ca²⁺ levels correlate with survival after ONC. Since multiple intracellular stores contribute to cyto-Ca²⁺, we tested the hypothesis that mitochondrial Ca²⁺ levels contribute to survival.

Methods : Ca²⁺ levels in mouse RGCs were visualized in-vivo at single-cell resolution with two-photon microscopy. The ratiometric Ca²⁺ sensor, Twitch-2b, was expressed in RGCs using an intersectional approach consisting of Vglut2-Cre transgenic mice and a Cre-dependent AAV2 expression vector. A mitochondrial localization signal peptide from Cox8 was used to localize Twitch-2b to the mitochondria. Baseline Ca²⁺ was measured in RGC somas and then ONC was performed to assess survival. Cells were tracked longitudinally for up to two weeks after injury. To assess the contribution of Ca²⁺ related to neuronal activity, acute Ca²⁺ responses to tetrodotoxin (TTX) were evaluated and then ONC was performed to determine survival.

Results : Mito- Ca²⁺ levels varied across individual RGCs at baseline similar to cyto- Ca²⁺. Baseline Ca²⁺ levels corresponded with survival in both stores, where Ca²⁺ levels of surviving cells had significantly higher homeostatic Ca²⁺ levels than those that died (Cyto: p=.00056, Mito: p=.00047). After TTX injections, cyto- Ca²⁺ decreased by 20.4 ± 1.7%, whereas mito- Ca²⁺ levels decreased by only 10.4% ± 1.3%. Most cells recovered cyto- Ca²⁺ within 20 minutes after TTX injection (74% of cells), and recovered cells survived ONC injury almost 2x better than those that did not (59 ± 6.1% and 35 ± 13% survival).

Conclusions : Our findings confirm our hypothesis that homeostatic Ca²⁺ set-points in mitochondria positively correlate with survival after axon injury. Ca²⁺ responses to TTX show that while physiological mito-Ca²⁺ and cyto-Ca²⁺ are both influenced by neuronal activity, mito- Ca²⁺ is impacted less-so and thus may act as a buffer for cyto-Ca²⁺ fluxes. Taken together, our data suggest that baseline mitochondrial Ca²⁺, which is coupled to cytoplasmic Ca²⁺, predicts survival in RGCs where higher Ca²⁺cells have increased resilience to axon injury.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.