Chakraborty, S., Sarma, J., Roy, S.S., Mitra, S., Bagchi, S., Das, S., Saha, S., Mahapatra, S., Bhattacharjee, S., Maulik, M., & Acharya, M. (2024). Functional investigation suggests CNTNAP5 involvement in glaucomatous neurodegeneration obtained from a GWAS in primary angle closure glaucoma. PLoS Genetics, 20(12): e1011502.
Abstract
Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.
Author summary
Glaucoma is the most common irreversible blindness worldwide where neurodegeneration manifests in the retina and optic nerve head. Primary angle closure glaucoma (PACG) is one of the major subsets of glaucoma, prevalent mostly in east and south-east Asia. In the current study we report a putative candidate CNTNAP5, a neurexin family gene, obtained from our previously published GWAS on PACG, involved in glaucomatous neurodegeneration. We have used antisense oligo mediated knockdown of the human CNTNAP5 ortholog cntnap5a and cntnap5b in the zebrafish. We have observed disrupted retinal cytoarchitecture with higher expression of acetylated tubulin and increased chromatolysis and cell death in zebrafish larvae injected with antisense oligos against cntnap5a and 5b at 4 days post-fertilization when compared with the mismatch control oligo injected zebrafish larvae, pointing towards neurodegeneration. Moreover, the double knockdown zebrafish larvae showed restricted movement in the light phase only in a simple set-up with software and camera to record and analyse the movement of zebrafish larvae. This phenomenon further validates the loss of vision in zebrafish larvae lacking both cntnap5 orthologs. Altogether, this study, for the first time, provides direct evidence of CNTNAP5 involvement in neuronal loss in the retina and glaucoma.
