Abstract
Purpose :
Microtubules, polymers of αβ-tubulin heterodimers, are essential for cell division, intracellular protein trafficking, ciliogenesis, and ciliary function. Recent studies showed a link between autosomal dominant missense mutations in β4B-tubulin isotype (TUBB4B) and Leber congenital amaurosis (LCA) and severe hearing loss. However, the mechanism underlying the diseases caused by TUBB4B mutations and the role of β4B-tubulin in photoreceptor cells remains to be elucidated and is the focus of this study.
Methods :
CRISPR-Cas9 generated Tubb4b knockout (KO) and Tubb4b R391H or R391C knockin (KI) murine models were utilized to understand the importance of β4B-tubulin in photoreceptors and the mechanisms behind LCA. Protein and mRNA levels were measured by quantitative immunoblotting and reverse transcription PCR. Electroretinography was performed to measure the response of photoreceptors to light stimuli. Immunofluorescence analysis was used to assess the morphology of the retina. All experiments were performed with wild-type littermates as controls. A two-tailed Student’s t-test was used for statistical analysis.
Results :
Photoreceptor function and the morphology of the retina were not affected in mice lacking β4B-tubulin, suggesting that other β-tubulin isotypes may compensate for the loss of this particular tubulin isotype in the retina. Indeed, β6-tubulin transcripts and protein levels were upregulated in the Tubb4b-/- retina. Intriguingly, Tubb4b KI mice did not recapitulate patient phenotype as photoreceptor function was normal in these animals. Analysis of published single-cell RNA sequencing of the human and murine retina revealed a species-specific difference in β4B-tubulin expression in photoreceptor cells. Indeed, β4B-tubulin was localized to photoreceptors only in non-human primate retina, while in the mouse retina, it was expressed throughout the retina and localized to photoreceptors and downstream neurons.
Conclusions :
This study shows that the β4B-tubulin is not essential for photoreceptor development and maintenance in mice. We found that β6-tubulin transcript and protein levels were upregulated in the absence of β4B-isotype, suggesting functional compensation by β6-tubulin. We demonstrated that the β4B-tubulin is differentially expressed in the mouse and non-human retina. Altogether, our findings indicate that the Tubb4b mice model may not be suitable for investigating mechanisms behind LCA linked to TUBB4B mutations.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.