TBCD is required for the photoreceptor and vasculature development in the retina

Abstract

Purpose : TBCD is one of tubulin cofactors that is required for the correct folding of β-tubulin folding and further assembly of tubulin dimers. Mutations of β-tubulin may impair the formation of tubulin cofactor complex and lead to the abnormal assembly of tubulin dimers and microtubules . Patients carrying these mutations present neurodegenerative encephalopath. Some of these mutations cause visual problems, including deep set eyes, absent visual tracking, and optokinetic nystagmus. The purpose of this study was to explore the function of TBCD in the retina.

Methods : Mice with floxed Tbcd was generate by CRISPR/CAS9 technology. Tbcd^flox/flox mice were crossed to with Six3-Cre to establish retina specific Tbcd knockout (^retTbcd-/- ). The localization of proteins was examined by immunostaining of retinal cryosections of P15 ^retTbcd-/-.. Retinal function was tested by electroretinography (ERG).Apoptosis was assessed by the TUNEL assay. Knockdown of TBCD in ARPE-19 was performed by using the shRNA technology. Cell viability and proliferation were examined by the CCK8 assay. Cilium in ARPE-19 was induced by serum starvation, and the length and number of cilia were determined.

Results : ^RetTbcd-/- mice developed microphthalmia and exhibited early retinal degeneration and retinal dysfunction. Immunofluorescence showed a reduced length of connecting cilium and shorted outer segments. Trafficking of phototransduction proteins, including GRK1, PDE6B, rhodopsin, and s-opsin, from inner segments to outer segments was normal, but with reduced expression levels. In addition, abnormal vascular development and vascular leakage were observed in ^retTbcd-/-- mice, accompanied by inflammation and activation of gliocytes. In vitro knockdown of TBCD in ARPE-19 cells inhibited ciliogenesis and suppressed cilium growth. Moreover, attenuated TBCD expression resulted in decreased cell viability and proliferation. It is interesting to find that decreasing expression of TBCD may down-regulate tight junction in ARPE-19, which probably explained the vascular leakage in retina.

Conclusions : TBCD-mediated tubulin synthesis is required for in vitro ciliogenesis, cell proliferation and tight junction. The absence of TBCD in the retina impairs the development of photoreceptor connecting cilia and outer segments, leading to retinal dysfunction, retinal degeneration, and abnormal retinal vasculatures.

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