Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient‐induced pluripotent stem cell‐derived retinal pigment epithelium cells

Abstract

Introduction Mutations in pre‐mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri‐snRNP complex, cause autosomal‐dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri‐snRNP proteins result in retina‐specific disorders, and so far, the underlying mechanism of splicing factors‐related RP is poorly understood. Methods We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31‐adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. Results To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle‐related pathways to be significantly affected. Strikingly, the patient‐derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin‐conjugated proteins including key visual cycle and other RP‐linked tri‐snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild‐type levels led to tri‐snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. Conclusions Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31‐initiated mis‐splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31‐adRP patients.

Progressive accumulation of cytoplasmic aggregates in PRPF31 retinal pigment epithelium cells interferes with cell survival

“…To gain a better understanding, our group evaluated the activity of autophagy and the proteasome degradation system using multiple assays. Strikingly, our findings indicated that these waste disposal mechanisms are impaired, and hence cytoplasmic aggregates accumulate over time 1 . Similar observations were reported in other neurodegenerative diseases, where cytoplasmic accumulation of misfolded proteins have been associated with reduced or blocked activity of the waste disposal mechanisms 16,17 .…”

“…AAV, aggregate formation, gene therapy, iPSC-RPE, PRPF31, retinal organoids, retinitis pigmentosa, RPE A recent publication by our group in Clinical and Translational Medicine titled 'Activation of autophagy reverses progressive and deleterious protein aggregation in in pre-mRNA (Messenger RNA) processing factors 31 (PRPF31) patient iPSC-derived retinal pigment epithelium (RPE) cells' provides new insights into why mutations in PRPFs cause retinitis pigmentosa (RP). 1 RP is an inherited retinal disease that affects more than 1 million individuals globally. 2 Approximately 15% of autosomal-dominant RP (adRP) cases are linked to genetic mutations in PRPFs.…”

“…To further identify the impact of cytoplasmic aggregates on cell survival, we used immunoblotting and IF analysis to assess the expression and localisation of the apoptosis regulator Caspase‐3 in control and patient RPE cells. The predominant expression of Caspase‐3 in the nucleus of patient RPE cells and disruption of tight junctions in areas with cytoplasmic aggregates suggests that progressive accumulation of cytoplasmic aggregates leads to cell death 1 …”

Aberrant Retinal Pigment Epithelial Cells Derived from Induced Pluripotent Stem Cells of a Retinitis Pigmentosa Patient with the PRPF6 Mutation

“…Additionally, a decreased degree of polarity, phagocytic ability and barrier function was observed in the RP-iRPE group compared to the NC-iRPE group. Such an aberrant phenotype was also observed in iPSC-RPE cells with PRPF31 mutation [ 38 , 39 ]. Their results also revealed that splicing defects appeared to be correlated with the ultrastructural, cellular and functional deficiencies that are characteristic of RPE in the RP disease state [ 38 ].…”

Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders

“…They observed that HDR-based CRISPR-correction resulted in the rescue of molecular and cellular phenotypes. The same iPSC lines were further characterized by Georgiou and colleagues, who showed localization of mutant PRPF31 in cytoplasmic aggregates in RP11 iPSC-RPE cells, in contrast to control cells where PRPF31 was localized in nuclear speckles required for splicing activity [ 103 ]. The authors also demonstrated that the dysregulation of splicing promoted protein misfolding and contributed to aggregate formation.…”