RESUMEN
Biallelic mutations in DRAM2 lead to an autosomal recessive cone-rod dystrophy known as CORD21, which typically presents between the third and sixth decades of life. Although DRAM2 localizes to the lysosomes of photoreceptor and retinal pigment epithelium (RPE) cells, its specific role in retinal degeneration has not been fully elucidated. In this study, we generated and characterized retinal organoids (ROs) and RPE cells from induced pluripotent stem cells (iPSCs) derived from two CORD21 patients. Our investigation revealed that CORD21-ROs and RPE cells exhibit abnormalities in lipid metabolism, defects in autophagic flux, accumulation of aberrant lysosomal content, and reduced lysosomal enzyme activity. We identified potential interactions of DRAM2 with vesicular trafficking proteins, suggesting its involvement in this cellular process. These findings collectively suggest that DRAM2 plays a crucial role in maintaining the integrity of photoreceptors and RPE cells by regulating lysosomal function, autophagy, and potentially vesicular trafficking.
Asunto(s)
Autofagia , Células Madre Pluripotentes Inducidas , Lisosomas , Proteínas de la Membrana , Epitelio Pigmentado de la Retina , Humanos , Lisosomas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Autofagia/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Organoides/metabolismo , Organoides/patología , Distrofias de Conos y Bastones/genética , Distrofias de Conos y Bastones/metabolismo , Distrofias de Conos y Bastones/patología , Retina/metabolismo , Retina/patología , Metabolismo de los Lípidos , MutaciónRESUMEN
The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.
