Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells

Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities.

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Pre-mRNA Processing Factors and Retinitis Pigmentosa: RNA Splicing and Beyond

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.700276 ◽

2021 ◽

Vol 9 ◽

Author(s):

Chunbo Yang ◽

Maria Georgiou ◽

Robert Atkinson ◽

Joseph Collin ◽

Jumana Al-Aama ◽

...

Keyword(s):

Retinitis Pigmentosa ◽

Rna Splicing ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Mrna Processing ◽

Retinal Cell ◽

Current Evidence ◽

Patient Specific ◽

Human Patient ◽

Processing Factors

Retinitis pigmentosa (RP) is the most common inherited retinal disease characterized by progressive degeneration of photoreceptors and/or retinal pigment epithelium that eventually results in blindness. Mutations in pre-mRNA processing factors (PRPF3, 4, 6, 8, 31, SNRNP200, and RP9) have been linked to 15–20% of autosomal dominant RP (adRP) cases. Current evidence indicates that PRPF mutations cause retinal specific global spliceosome dysregulation, leading to mis-splicing of numerous genes that are involved in a variety of retina-specific functions and/or general biological processes, including phototransduction, retinol metabolism, photoreceptor disk morphogenesis, retinal cell polarity, ciliogenesis, cytoskeleton and tight junction organization, waste disposal, inflammation, and apoptosis. Importantly, additional PRPF functions beyond RNA splicing have been documented recently, suggesting a more complex mechanism underlying PRPF-RPs driven disease pathogenesis. The current review focuses on the key RP-PRPF genes, depicting the current understanding of their roles in RNA splicing, impact of their mutations on retinal cell’s transcriptome and phenome, discussed in the context of model species including yeast, zebrafish, and mice. Importantly, information on PRPF functions beyond RNA splicing are discussed, aiming at a holistic investigation of PRPF-RP pathogenesis. Finally, work performed in human patient-specific lab models and developing gene and cell-based replacement therapies for the treatment of PRPF-RPs are thoroughly discussed to allow the reader to get a deeper understanding of the disease mechanisms, which we believe will facilitate the establishment of novel and better therapeutic strategies for PRPF-RP patients.

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Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

10.1101/232397 ◽

2017 ◽

Author(s):

Adriana Buskin ◽

Lili Zhu ◽

Valeria Chichagova ◽

Basudha Basu ◽

Sina Mozaffari-Jovin ◽

...

Keyword(s):

Alternative Splicing ◽

Retinitis Pigmentosa ◽

Gene Editing ◽

Autosomal Dominant ◽

Mrna Processing ◽

Mrna Splicing ◽

Patient Specific ◽

Rpe Cells ◽

Autosomal Dominant Retinitis Pigmentosa

SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes

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Reduction of lipid accumulation rescues Bietti’s crystalline dystrophy phenotypes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1717338115 ◽

2018 ◽

Vol 115 (15) ◽

pp. 3936-3941 ◽

Cited By ~ 11

Author(s):

Masayuki Hata ◽

Hanako O. Ikeda ◽

Sachiko Iwai ◽

Yuto Iida ◽

Norimoto Gotoh ◽

...

Keyword(s):

Free Cholesterol ◽

Cell Damage ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Patient Specific ◽

Rpe Cells ◽

Underlying Mechanisms ◽

Induced Pluripotent ◽

Bietti's Crystalline Dystrophy

Bietti’s crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.

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Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

10.1101/2021.10.11.463925 ◽

2021 ◽

Author(s):

Maria Georgiou ◽

Chunbo Yang ◽

Robert Atkinson ◽

Kuan-Ting Pan ◽

Adriana Buskin ◽

...

Keyword(s):

Waste Disposal ◽

Cell Function ◽

Core Protein ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Cell ◽

Visual Cycle ◽

Nuclear Speckles ◽

Retinal Cells ◽

Rpe Cells

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. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was 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 proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller 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 resulted in reduction of cytoplasmic aggregates and improved cell survival. 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.

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Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9

International Journal of Molecular Sciences ◽

10.3390/ijms19124127 ◽

2018 ◽

Vol 19 (12) ◽

pp. 4127 ◽

Cited By ~ 3

Author(s):

Leah Foltz ◽

Sara Howden ◽

James Thomson ◽

Dennis Clegg

Keyword(s):

Retinitis Pigmentosa ◽

Retinal Pigment Epithelial ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Splicing Factor ◽

Wild Type ◽

Rpe Cells ◽

Pigment Epithelial ◽

Apicobasal Polarity ◽

Gene And Protein Expression

Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.

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Differentially Expressed MicroRNAs in the Development of Early Diabetic Retinopathy

Journal of Diabetes Research ◽

10.1155/2017/4727942 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 25

Author(s):

Qiaoyun Gong ◽

Jia’nan Xie ◽

Yang Liu ◽

Ying Li ◽

Guanfang Su

Keyword(s):

Diabetic Retinopathy ◽

High Glucose ◽

Retinal Pigment ◽

Treatment Strategies ◽

Diabetic Rats ◽

Retinal Cell ◽

Rpe Cells ◽

Hoechst Staining

The pathological mechanisms of diabetic retinopathy (DR), a leading cause of blindness in adults with diabetes mellitus, remain incompletely understood. Because microRNAs (miRNAs) represent effective DR therapeutic targets, we identified aberrantly expressed miRNAs associated with cellular dysfunction in early DR and detected their potential targets. We exposed human retinal endothelial cells (HRECs) and a cell line of retinal pigment epithelial (RPE) cells to high glucose (25 mmol/L, 1–7 days) to mimic DR progression and used streptozotocin-injected rats (4–8 weeks) for an in vivo diabetes model. HREC/RPE viability decreased after 24 h incubation and diminished further over 6 days, and Hoechst staining revealed hyperglycemia-induced HREC/RPE apoptosis. Although miR-124/-125b expression decreased with DR progression in vitro and in vivo, miR-135b/-199a levels decreased in retinal cells under hyperglycemia exposure, but increased in diabetic retinas. Moreover, miR-145/-146a expression decreased gradually in high-glucose-treated HRECs, but increased in hyperglycemia-exposed RPE cells and in diabetic rats. Our findings suggested that aberrant miRNA expression could be involved in hyperglycemia-induced retinal-cell dysfunction, and the identified miRNAs might vary in different retinal layers, with expression changes associated with DR development. Therefore, miRNA modulation and the targeting of miRNA effects on transcription factors could represent novel and effective DR-treatment strategies.

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Transcriptome Analyses of lncRNAs in A2E-Stressed Retinal Epithelial Cells Unveil Advanced Links between Metabolic Impairments Related to Oxidative Stress and Retinitis Pigmentosa

Antioxidants ◽

10.3390/antiox9040318 ◽

2020 ◽

Vol 9 (4) ◽

pp. 318 ◽

Cited By ~ 14

Author(s):

Luigi Donato ◽

Concetta Scimone ◽

Simona Alibrandi ◽

Carmela Rinaldi ◽

Antonina Sidoti ◽

...

Keyword(s):

Oxidative Stress ◽

Retinitis Pigmentosa ◽

Cellular Response ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Differentially Expressed ◽

Retinal Cell ◽

Relevant Role ◽

Human Disorders ◽

Epithelium Cells

Long non-coding RNAs (lncRNAs) are untranslated transcripts which regulate many biological processes. Changes in lncRNA expression pattern are well-known related to various human disorders, such as ocular diseases. Among them, retinitis pigmentosa, one of the most heterogeneous inherited disorder, is strictly related to oxidative stress. However, little is known about regulative aspects able to link oxidative stress to etiopathogenesis of retinitis. Thus, we realized a total RNA-Seq experiment, analyzing human retinal pigment epithelium cells treated by the oxidant agent N-retinylidene-N-retinylethanolamine (A2E), considering three independent experimental groups (untreated control cells, cells treated for 3 h and cells treated for 6 h). Differentially expressed lncRNAs were filtered out, explored with specific tools and databases, and finally subjected to pathway analysis. We detected 3,3’-overlapping ncRNAs, 107 antisense, 24 sense-intronic, four sense-overlapping and 227 lincRNAs very differentially expressed throughout all considered time points. Analyzed lncRNAs could be involved in several biochemical pathways related to compromised response to oxidative stress, carbohydrate and lipid metabolism impairment, melanin biosynthetic process alteration, deficiency in cellular response to amino acid starvation, unbalanced regulation of cofactor metabolic process, all leading to retinal cell death. The explored lncRNAs could play a relevant role in retinitis pigmentosa etiopathogenesis, and seem to be the ideal candidate for novel molecular markers and therapeutic strategies.

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Combinatorial library of biodegradable polyesters enables delivery of plasmid DNA to polarized human RPE monolayers for retinal gene therapy

10.1101/264390 ◽

2018 ◽

Author(s):

Bibhudatta Mishra ◽

David R. Wilson ◽

Srinivas R. Sripathi ◽

Mark P. Suprenant ◽

Yuan Rui ◽

...

Keyword(s):

Gene Delivery ◽

Plasmid Dna ◽

High Throughput Screening ◽

High Efficiency ◽

Polymeric Nanoparticles ◽

Retinal Pigment ◽

Weight Ratio ◽

Retinal Cell ◽

Viral Gene ◽

Rpe Cells

Abstract:Efficient gene delivery into hard-to-transfect cells is still a challenge despite significant progress in the development of various gene delivery tools. Non-viral and synthetic polymeric nanoparticles offer an array of advantages for gene delivery over the viral vectors and high in demand as they are safe to use, easy to synthesize and highly cell-type specific. Here we demonstrate the use of a high-throughput screening (HTS) platform to screen for biodegradable polymeric nanoparticles (NPs) that can transfect human retinal pigment epithelial (RPE) cells with high efficiency and low toxicity. These NPs can deliver plasmid DNA (pDNA) to RPE monolayers more efficiently compared to the commercially available transfection reagents without interfering the global gene expression profile of RPE cells. In this work, we have established an HTS platform and identified synthetic polymers that can be used for high efficacy non-viral gene delivery to human RPE monolayers, enabling gene loss- and gain-of-function studies of cell signaling and developmental pathways. This platform can be used to identify the optimum polymer, weight-to-weight ratio of polymer to DNA, and the dose of NP for various retinal cell types.

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