The role of AGBL5 in the retina and its contribution to inherited retinal dystrophies

Abstract

Retinitis pigmentosa (RP) is the most common type of inherited retinal degeneration. It affects around 1 in 4000 individuals and account for 25% of cases of vision loss in adults. RP is a genetically heterogeneous disease and can be caused by pathogenic mutations in at least 94 different genes, including AGBL5, which encodes a dual function deglutamylase that is essential for regulating functional levels of tubulin glutamylation in cilia. Mutations in AGBL5 are associated with autosomal recessive RP, for which there are no known treatments or cure. Thus, there is a need for research to understand the disease mechanisms associated with AGBL5 loss and to identify therapeutic targets. This study hypothesised that loss of AGBL5 in human retinal cells impairs ciliogenesis, and that AGBL5 null cell models can be used to investigate opportunities for rescue therapies and obtain functional data of genetic variants. We first aimed to characterise the cellular phenotype of AGBL5 CRISPR knockout ARPE19 cells (AGBL5-/-) compared to their WT counterpart. Western blot and fluorescent immunocytochemistry experiments revealed that AGBL5-/- cells show hyper glutamylation, significantly shorter cilia and lower percentage of ciliated cells than WT cells (p<0.001). The disease phenotype can be rescued by gene augmentation, as demonstrated by transient transfection with a WT AGBL5-eGFP construct. Alternatively, we showed that CRISPR knockout (KO)and siRNA knockdown experiments to reduce the expression of the α-tubulinglutamylase TTLL5, counters for the glutamylation imbalance due to loss of AGBL5and rescued the ciliary phenotype in AGBL5-/- cells. In addition, we showed that this AGBL5-/- cell model can be used to investigate AGBL5 variants, and demonstrated that transient expression of constructs containing variants induce rescue of the disease phenotype comparable to the WT construct, and disease causing variants fail to rescue ciliogenesis. Finally, using coimmunoprecipitation of AGBL5 and mass spectrometry, we identified the exocyst component 4 EXOC4 and the deubiquitinase USP9X as potential protein interactants that could be involved in the regulation of cilia and ciliogenesis by AGBL5. This study reports a clear phenotype in AGBL5-deficient human retinal cell lines consisting of hyper glutamylation, short cilia and reduced ciliogenesis, and illustrated the value of the AGBL5-/- cell model to investigate variants. In addition, it demonstrates the potential effectiveness of AGBL5 augmentation or reduction of TTLL5 expression as therapy, and provides transcriptomic and protein interaction data that can be used by researchers in the field to continue investigating the mechanisms underlying AGBL5-associatedRP.

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