Departments of Ophthalmology and Physiology & Biophysics

University of California, Irvine

Irvine, California

BASIC RESEARCH PROJECT

Understanding how the G90D and G90V rhodopsin mutations cause blindness

Research Interests

Vision begins in photoreceptor cells with the activation by light of the visual pigment, rhodopsin. The exquisite sensitivity of our rod photoreceptors that enables us to see in dim to moderately bright light requires that rhodopsin is extremely stable and does not activate the rods in the absence of light. Recently, two mutations in rhodopsin have been identified that appear to compromise its stability: Glycine 90 to Aspartate (G90D) reported to cause congenital stationary night blindness (CSNB), and Glycine 90 to Valine (G90V) found in patients with retinitis pigmentosa (RP). Structural and biochemical in vitro studies have suggested that the G90D/G90V mutations cause lower stability of both chromophore-bound and chromophore-free rhodopsin. However, previous studies with transgenic animal models have yielded conflicting results about the form of rhodopsin responsible for its abnormally high activity. Thus, despite decades of research, the molecular mechanism by which these mutations cause abnormal photoreceptor function and degeneration remains a subject of debate. and, as a result, effective treatments for people carrying these mutations are not available.

The purpose of the project is to identify the molecular mechanism by which these mutations cause abnormal photoreceptor function and degeneration as a first step in developing effective treatments for people carrying these mutations. Ultimately, Dr. Kefalov’s goal is to develop effective treatments targeted specifically towards patients carrying each of these two mutations and test their efficacy in human clinical studies.

Plans for 2024

Dr. Kefalov has now completed the initial physiological and morphological
characterization of G90D and G90V mice as proposed and have obtained additional
molecular data. He plans to extend these findings to investigate further the mechanism by which these mutations lead to blindness and possible retinal degeneration.

Specific Aims: To investigate the mechanisms by which the G90 mutations affect rod function and health by performing single-cell suction recordings from G90D (Aim 1) and G90V (Aim 2) heterozygous and homozygous mouse rods. The rigorous analysis of the frequency, amplitude, and kinetics of quantal events will allow Dr. Kefalov to determine the level of G90 mutation-driven spontaneous activity in G90D and G90V rods.

Progress in 2023

We continued to make steady progress in our studies on G90D/G90V rhodopsins by performing extensive in vivo and ex vivo electroretinography (ERG) recordings. First, we investigated the kinetics of rod dark adaptation of WT, G90D and G90V homozygous mice using in vivo ERG recordings. We found that following exposure to bright light estimated to bleach >90% of rhodopsin, the amplitude of rod a-wave recovered to 50% of its prebleached level in 63 min for WT rods; 22 min for G90V rods; and 10 min for G90D rods (shown). Thus, both mutations greatly accelerate the regeneration of rhodopsin in vivo, while also producing phenotypes distinct from each other. Second, we examined the effect of exogenous 11-cis-retinal chromophore on the physiology of G90 mutant rods. We found that this treatment promoted increases sensitivity in G90V rods but not in G90D rods (not shown). Thus, only the G90V mutant desensitizes mouse rods due to the presence of free opsin in the dark.

The purpose of the project is to identify the molecular mechanism by which these mutations cause abnormal photoreceptor function and degeneration as a first step in developing effective treatments for people carrying these mutations.

Dr. Kefalov’s laboratory has now completed the quantification of the physiological and morphological characterization of G90D and G90V mice as proposed and have obtained additional molecular data. In 2023, they plan to extend these findings and begin addressing the mechanism by which these mutations lead to blindness and possible retinal degeneration.

Specific Aims:

Aim 1. To investigate the disease mechanism of the G90D/G90V rhodopsin mutations by evaluating whether exogenous chromophore is able to restore normal rod function.

Aim 2. To determine the effect of the G90D and G90V mutations on chromophore binding to rhodopsin by measuring rod dark adaptation.

Progress in 2022

Throughout 2022, Dr. Kefalov expanded his studies on G90D/G90V rhodopsins over the past year by performing microspectrophotometry and biochemistry experiments that were not part of the proposed research plan but provided significant insight into their properties. First, in collaboration with Dr. Clint Makino from Boston University, he measured absorption spectra of rod outer segments from patches of freshly dissected mouse retinas to capture the time course of production and decay of metarhodopsin III (Meta III) after photobleaching (shown). Dr. Kefalov observed normal production and decay of Meta III in wild type (WT) rods. In striking contrast, photobleached mutant rhodopsins decay was monophasic. Second, in collaboration with Dr. Pere Garriga from Universitat Politecnica de Catalunya, Spain, Dr. Kefalov’s team found that the chemical stability of both mutants is dramatically reduced compared to WT. Curiously, we also found normal WT-like thermal stability of G90D but 50-fold lower thermal stability of G90V rhodopsin (not shown).

Progress in 2021

Dr. Kefalov relocated his lab from Washington University in St. Louis to the University of California, Irvine in June 2021. Nevertheless, he made significant progress in analyzing the structure and function of the photoreceptors in G90D and G90V mutant mice. Using electroretinography, his team found that both G90D and G90V rhodopsin mutations cause suppressed scotopic light responses in 4 months-old mice (shown). The rod-driven responses from younger 2 months-old mutant animals were similarly reduced (not shown), suggesting that this functional deficit is not caused by progressive retinal degeneration but rather by abnormal mutant opsin stability. Indeed, retina morphology in 4 months-old mutant animals appeared normal (not shown). These findings demonstrate the feasibility of our approach and, encouragingly, are consistent with the human phenotype associated with the G90D and G90V mutations.