Investigating the effect of a llama-derived transducin nanobody on mouse rod function

Abstract

Purpose : Rhodopsin mediates light detection by activating the G protein transducin, a heterotrimeric complex of G_α, G_β and G_γ subunits. Upon activation, G_α dissociates from G_βγ and initiates downstream signaling to produce the photoresponse. Proper G_α function is required for light detection, while dysregulated G_βγ signaling could lead to neurological and carcinogenic conditions. Recently, a llama-derived nanobody (Nb5) was shown to bind the G_βγ heterodimer, covering the G_α-binding site and potentially modulating G-protein signaling. Here, we investigated the function of rods in transgenic mice expressing the Nb5 nanobody.

Methods : We generated transgenic mice expressing Nb5 under the rhodopsin promoter. To facilitate detection, Nb5 was flanked by a FLAG domain. The animals were bred on a C57Bl/6 background, maintained on a 12hr light/dark cycle, and dark-adapted overnight for experiments. The Nb5 effects on rod function were studied by scotopic in-vivo electroretinography (ERG), ex-vivo transretinal recordings, and optokinetic behavior experiments. Retina histology was studied by FLAG and peanut agglutinin immunohistochemistry. Nb5 expression in the retina was quantified by western blotting.

Results : In-vivo ERG revealed a 50% reduction in maximal a-wave response amplitude in mutant mice versus controls. Dark-adapted fractional sensitivity, estimated from dim flash transretinal responses, was reduced by 30% in the mutants versus controls. Scotopic visual acuity and contrast sensitivity both were reduced by 30% in the mutants versus controls. Western blots showed robust Nb5 expression in the mutants, and immunohistochemistry confirmed Nb5 expression in rods. The presence of Nb5 down-regulated G_α subunit expression by 75% in the mutant retinas. Histological studies did not reveal any retinal degeneration in the mutants up to 5 months relative to controls. However, the immunohistochemistry revealed a non-uniform Nb5 expression in the outer nuclear layer of mutant retinas.

Conclusions : We conclude that the transducin nanobody likely remains bound to the G_βγ complex, leading to reduced rod sensitivity and visual acuity, but without inducing degeneration. Together, our results demonstrate that expression of Nb5 in rods partially suppresses their function without adverse effects on their long-term survival, advancing development of new types of G protein-coupled receptor targeting drugs.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.