Retinal cells derived from patients with DRAM2-dependent CORD21 dystrophy exhibit key lysosomal enzyme deficiency and lysosomal content accumulation.

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.

Investigating light sensitivity in bipolar disorder (HELIOS-BD).

Many people with bipolar disorder have disrupted circadian rhythms. This means that the timing of sleep and wake activities becomes out-of-sync with the standard 24-hour cycle. Circadian rhythms are strongly influenced by light levels and previous research suggests that people with bipolar disorder might have a heightened sensitivity to light, causing more circadian rhythm disruption, increasing the potential for triggering a mood switch into mania or depression. Lithium has been in clinical use for over 70 years and is acknowledged to be the most effective long-term treatment for bipolar disorder. Lithium has many reported actions in the body but the precise mechanism of action in bipolar disorder remains an active area of research. Central to this project is recent evidence that lithium may work by stabilising circadian rhythms of mood, cognition and rest/activity. Our primary hypothesis is that people with bipolar disorder have some pathophysiological change at the level of the retina which makes them hypersensitive to the visual and non-visual effects of light, and therefore more susceptible to circadian rhythm dysfunction. We additionally hypothesise that the mood-stabilising medication lithium is effective in bipolar disorder because it reduces this hypersensitivity, making individuals less vulnerable to light-induced circadian disruption. We will recruit 180 participants into the HELIOS-BD study. Over an 18-month period, we will assess visual and non-visual responses to light, as well as retinal microstructure, in people with bipolar disorder compared to healthy controls. Further, we will assess whether individuals with bipolar disorder who are being treated with lithium have less pronounced light responses and attenuated retinal changes compared to individuals with bipolar disorder not being treated with lithium. This study represents a comprehensive investigation of visual and non-visual light responses in a large bipolar disorder population, with great translational potential for patient stratification and treatment innovation.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

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.

Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Deciphering the spatiotemporal transcriptional and chromatin accessibility of human retinal organoid development at the single-cell level.

Molecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids (ROs) provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-seq and spatial transcriptomics we present for the first-time a single cell spatiotemporal transcriptome of RO development. Our data demonstrate that ROs recapitulate key events of retinogenesis including optic vesicle/cup formation, presence of a putative ciliary margin zone, emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-seq data, we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development, and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal neurons.

Pluripotent stem cell-derived models of retinal disease: Elucidating pathogenesis, evaluating novel treatments, and estimating toxicity.

Blindness poses a growing global challenge, with approximately 26% of cases attributed to degenerative retinal diseases. While gene therapy, optogenetic tools, photosensitive switches, and retinal prostheses offer hope for vision restoration, these high-cost therapies will benefit few patients. Understanding retinal diseases is therefore key to advance effective treatments, requiring in vitro models replicating pathology and allowing quantitative assessments for drug discovery. Pluripotent stem cells (PSCs) provide a unique solution given their limitless supply and ability to differentiate into light-responsive retinal tissues encompassing all cell types. This review focuses on the history and current state of photoreceptor and retinal pigment epithelium (RPE) cell generation from PSCs. We explore the applications of this technology in disease modelling, experimental therapy testing, biomarker identification, and toxicity studies. We consider challenges in scalability, standardisation, and reproducibility, and stress the importance of incorporating vasculature and immune cells into retinal organoids. We advocate for high-throughput automation in data acquisition and analyses and underscore the value of advanced micro-physiological systems that fully capture the interactions between the neural retina, RPE, and choriocapillaris.