Calcium clearance deficits linked to endoplasmic reticulum stress are signature features of early retinal ganglion cell damage in glaucoma

Abstract

Purpose : The mechanisms underlying retinal ganglion cell (RGC) vulnerability in glaucoma are poorly understood. We tested the hypothesis that retinal ganglion cell (RGC) calcium (Ca²⁺) dynamics are affected in the early stages of ocular hypertension (OHT) and probed the mechanisms underlying this response.

Methods : Two-photon laser scanning microscopy (TPLSM) was used to record light-evoked single-RGC Ca²⁺ dynamics in transgenic mice carrying the Ca²⁺ sensor GCaMP6f. OHT was induced by intracameral injection of magnetic microbeads, and Ca²⁺ signals were recorded two weeks later, prior to RGC loss. TPLSM imaging was performed in living anesthetized mice by longitudinal trans-scleral imaging as well as ex vivo in retinal explants. Ca²⁺ signals were extracted, and the parameters computed included rise time (Ca²⁺ influx), decay time (Ca²⁺ clearance), and amplitude. A set of approaches was used to examine RGC-specific pathways, including single-cell-RNAseq, qRTPCR, FACS, flow cytometry, and immunohistochemistry.

Results : Live trans-scleral and ex vivo imaging showed consistent defects in Ca²⁺ clearance across all RGC types (N=5-8 mice/group, n>1,200 RGCs, p<0.01). For example, ON-RGCs subjected to OHT displayed an increase in Ca²⁺ decay time relative to sham controls (sham: 1.1 ± 0.08 sec, OHT: 2.8 ± 0.5 sec, N=7-8 mice/group, n=62 cells/group, p<0.01). Analysis of molecular pathways revealed an RGC-specific reduction in gene and protein expression of the endoplasmic reticulum (ER) Ca²⁺ ATPase 2 (SERCA2), which is responsible for pumping Ca²⁺ from the cytoplasm to the ER. Loss of SERCA2 was accompanied by upregulation of the ER stress markers pPERK, pEIF-2a, ATF4, and CHOP. TPLSM recordings in naïve mice treated with a pharmacological inhibitor of SERCA2 showed Ca²⁺ clearance impairment in RGCs, recapitulating the effect of OHT. In contrast, glaucomatous eyes treated with a SERCA2-specific activator restored the ability of RGCs to effectively reduce cytoplasmic Ca²⁺ to physiological levels.

Conclusions : Our study reveals defective Ca²⁺ clearance as a signature feature of early RGC damage, a trait conserved across RGC subtypes, and suggests that loss of SERCA2 has a profoundly detrimental effect on the ability of these neurons to regulate cytoplasmic Ca²⁺.

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