Chemogenetic-induced retinal ganglion cell excitation reveals IP3R-dependent dysregulation of calcium dynamics linked to neuronal metabolic stress in glaucoma

Abstract

Purpose : The Gq-coupled DREADD (designer receptors solely activated by designer drugs) hM3Dq increases neuronal activity as a result of elevated cytosolic calcium (Ca²⁺) levels. Inositol 1,4,5-trisphosphate receptor (IP3R)-driven Ca²⁺ release from the endoplasmic reticulum (ER) or mitochondria is the primary mechanism for hM3Dq-induced neuronal excitation. Here, we tested the hypothesis that IP3R-dependent retinal ganglion cell (RGC) Ca²⁺ dynamics and subsequent ATP production are affected in the early stages of ocular hypertension (OHT).

Methods : An adeno-associated virus vector type 2 encoding hM3Dq (AAV.hM3Dq) was delivered intravitreally to target RGCs. Live two-photon laser scanning microscopy (TPLSM) trans-scleral imaging was used to record the following hM3Dq-evoked responses: i) single-RGC Ca²⁺ dynamics in transgenic mice carrying the Ca²⁺ sensor GCaMP6f, and ii) RGC-specific ATP changes using an AAV vector encoding the ATP sensor Ateam. OHT was induced by intracameral injection of magnetic microbeads, and Ca²⁺ and ATP signals were recorded two weeks later, prior to RGC loss. Longitudinal TPLSM imaging was performed before and after clozapine N-oxide administration (CNO, 5mg/kg, i.p.). Ca²⁺ and ATP signals were extracted and spike frequency, inter-spike interval, mean amplitude, amplitude variability, and ATP signals were computed.

Results : In vivo longitudinal Ca²⁺ and ATP TPLSM imaging revealed disrupted Ca²⁺ dynamics and compromised energy balance following CNO administration in glaucomatous RGCs (N=4-8 mice/group, n>700 RGCs, p<0.01). RGCs subjected to OHT displayed a marked increase in spontaneous spike frequency (sham: 0.20 ± 0.04 Hz, OHT: 0.30 ± 0.06 Hz, N=4-8 mice/group, n=98-102 cells/group, p<0.01). Neuronal hyperexcitability coincided with a substantial decrease in ATeam FRET signals, indicative of reduced ATP levels in RGCs (N=5 mice/group, n=213-287 cells/group, p<0.01). Analysis of molecular pathways by single-cell (sc)-RNAseq, qRT-PCR, flow cytometry, and immunohistochemistry revealed RGC-specific downregulation of IP3R1 gene and protein expression.

Conclusions : Our findings reveal early RGC hyperexcitability and energy deficits during OHT-induced stress, and suggest that IP3R1 dysregulation has a detrimental effect on the ability of these neurons to maintain Ca²⁺ and energy homeostasis.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.