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.

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 2023

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.