Abstract
Purpose :
Most cases of dominant optic atrophy (DOA) are due to OPA1 gene mutations that affect mitochondrial dynamics and function. Despite its wide expression, OPA1 deficiency primarily affects retinal ganglion cells (RGCs). Since human RGCs are difficult to procure, the impact of OPA1 mutations on RGC physiology has not been adequately characterized. This investigation sought to identify possible DOA caused RGC dysfunctions by comparing the electrophysiological properties of pluripotent stem cell (PSC)-derived neurons harboring OPA1 mutations and their corresponding isogenic controls.
Methods :
OPA1 mutant PSC lines were created by reprogramming DOA patient blood cells or by CRISPR-Cas editing of the OPA1 gene in a control embryonic stem cell line. An isogenic control was established by correcting the OPA1 mutation carried by DOA patients. Current- and voltage-clamp recordings were made in monolayer culture from PSC-derived retinal organoids and Neurog2 directly-induced neurons using whole-cell patch clamp to assess membrane potential and specific ion channel activity.
Results :
Properties of the voltage-activated Nav and Kv channel currents responsible for spiking activity appeared to be the same in the mutant and control RGCs when recorded under voltage-clamp. Under current-clamp, electrophysiological differences between control and OPA1 mutant RGCs were immediately noted with mutant RGCs being significantly more hyperpolarized at resting potential (p<0.0001). The mutant RGCs fire more spikes in response to injected current, consistent with them having less Nav channel inactivation due to their increased hyperpolarization at rest, but cannot sustain the activity. When the resting potential of all RGC types was forced to -60 mV, injected current-evoked spiking was indistinguishable in the mutant and control RGCs. Glibenclamide, which inhibits K-ATP channels, depolarized OPA1 mutant neurons.
Conclusions :
Together with the knowledge that OPA1 mutant retinal organoids have reduced levels of ATP compared to isogenic controls, our results support a conclusion that the control RGCs and Neurog2-induced neurons have fewer active K-ATP channels, contributing to their more depolarized resting potentials. Resting potentials are more negative in OPA1 mutant RGCs, consistent with greater K-ATP channel activation due to reduced ATP levels, increasing RGC spiking.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.