Modulation of mouse rod function by a llama-derived transducin nanobody

Abstract

Purpose : Rhodopsin initiates detection of light by activating the downstream G-protein transducin, a heterotrimeric complex of G_α, G_β and G_γ subunits. Upon activation, G_α dissociates from G_βγ complex and activates the enzyme phosphodiesterase (PDE) to produce the photoresponse. Optimum G_α function is required for light detection, while dysregulated G_βγ signaling could give rise to neuropathological conditions. Recently, a llama-derived nanobody (Nb5) was developed that binds G_βγ heterodimer, covering the Gα binding site and potentially modulating G protein signaling. Here, we investigated the function of rods in transgenic mice expressing the Nb5 nanobody.

Methods : Transgenic mice were generated by expressing Nb5 under the rhodopsin promoter on a C57Bl/6 background. Nb5 was flanked by a FLAG domain to facilitate detection. The animals were maintained on a 12hr light/dark cycle and dark-adapted overnight for experiments. The Nb5 effects on rod function were studied by optokinetic reflex behavior and ex-vivo transretinal recordings. Retina histology was studied by OCT and immunohistochemistry (IHC). Transducin and rhodopsin expression was quantified by western blotting and mass spectrophotometry.

Results : Ex-vivo transretinal recordings revealed more than 60% reduction of rod responses in mutant mice versus controls. Dark-adapted fractional sensitivity, estimated from dim flash transretinal responses, was reduced by 25% in the mutants versus controls. Scotopic visual contrast threshold was increased by three folds in the mutants versus controls. Western blots showed robust Nb5 expression in the mutants, and IHC confirmed Nb5 expression in rods. The presence of Nb5 down-regulated G_β and PDE expression by 75% and G_α expression by 95% in the mutant retina. Histological studies did not reveal any retinal degeneration in the mutants up to 5 months.

Conclusions : Our results suggest that the transducin nanobody binds to G_βγ complex, leading to reduced rod sensitivity without inducing degeneration. Together, our results demonstrate that expression of Nb5 in mouse rods partially suppresses their function without adversely affecting their long term survival. Thus, Nb5 could potentially serve as a G-protein coupled receptor targeting drug in disease conditions where reduction in the G-protein signaling would preserve cellular function.

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