Imbalanced Unfolded Protein Response Signaling Contributes to 1-Deoxysphingolipid Retinal Toxicity
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE213948 | GEO | 2023/05/30
REPOSITORIES: GEO
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2020-02-01 | E-MTAB-6490 | biostudies-arrayexpress
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Project description:Mutations in RP2 lead to a severe form of X-linked retinitis pigmentosa (XLRP). RP2 functions as a GTPase activating protein (GAP) for the small GTPase ARL3, which is essential for cilia function and for photoreceptor development and maintenance. The mechanisms of RP2 associated retinal degeneration in humans are poorly understood, and genetically engineered animal models of RP2 XLRP present with differing retinal phenotypes and slow degeneration suggesting potential species differences. Here, we developed CRISPR gene edited isogenic RP2 knock-out (RP2 KO) induced pluripotent stem cells (iPSC) and RP2 patient derived iPSC to produce 3D retinal organoids as a human retinal disease model. The isogenic RP2 KO retinal organoids and two unrelated RP2 patient iPSC lines produced retinal organoids with a defined photoreceptor cell layer (ONL) containing rod and cone photoreceptors. Strikingly the RP2 KO and RP2 patient derived organoids showed a thinning of the ONL by 180 days (D180) of culture, which was associated with a spike in cell death in the ONL at D150 of differentiation. RNA sequencing confirmed induction of cell death pathways in the RP2 null organoids at this stage. Photoreceptor cell death and ONL thinning was attributed to cells undergoing terminal rod cell differentiation characterized by reduced RHO expression and fewer rhodopsin immunoreactive photoreceptors. Gene augmentation with a human RP2 transgene in an AAV2/5 vector efficiently transduced RP2 KO organoids and led to high levels of RP2 expression in both rods and cones. Importantly, the viral transduction significantly increased ONL thickness and restored rhodopsin expression, suggesting rescue of the RP2 degeneration phenotype. These data show that 3D retinal organoids can be used to model molecular defects associated with inherited retinal disease, photoreceptor cell death and also to test potential therapies targeted to prevent photoreceptor degeneration.
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Project description:Background. Recurrence or persistence of retinoblastoma (RB) is common following chemoreduction and often be managed with intra-arterial/ intravitreal chemotherapy. However, no/minimal response to local chemotherapy has been documented, with enucleation needed for tumor control. Otherwise, the globe is preserved with effective tumor control but at the cost of vision due to retinal toxicity. The aim of this study was to discover novel drug with improved antitumor activity and retinal safety profiles for RB via drug reprofiling in patient-derived tumor organoids. Methods. Five tumor organoid lines were fully characterized using CGH/SNP array and whole exome sequencing and used as a model to test 133 FDA-approved compounds using two-step screening processes. RNA sequencing was conducted to generate drug signature in conjunction with cell cycle and apoptotic assays. An ability of candidate drug to restrict proliferative tumor cone were examined together with retinal toxicity testing in human ESC-derived retinal organoids, in comparison with clinically used drugs by immunostaining. Results. Sunitinib was identified as high cytotoxic agent for the classical RB1-deficient and novel MYCN-amplified RBs. Gene signature indicated that anti-tumoral activity of sunitinib was associated with MAPK activity. Additionally, sunitinib was more effective to suppress proliferative tumor cones and had less retinal toxicity compared with melphalan and topotecan. Conclusions. Sunitinib may be a “universal” drug for all subtypes of RB. With high efficacy and low retinal toxicity, sunitinib is potential agent used in local chemotherapy for treating RB.
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Project description:Drug toxicity screening on retina is essential for the development of safe therapies for a large number of diseases, whilst preserving visual acuity and function. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to human retina and the ease of generation in large-scale formats, offering almost unlimited excess of tissue. Two hPSC cell lines were differrentiated to retinal organoids which comprised all key retinal cell types in multiple nuclear and synaptic layers, enabling the maintenance of retinal ganglion and bipolar cells and moreover allowed the development of subtypes as revealed by the single cell RNA-Seq analysis. Ketorolac, Digoxin, Thioridazine, Sildenafil, Ethanol and Methanol were used to screen drug effects on retinal organoids. Exposure of the hPSC-derived retinal organoids to Diogxin, Thioridazine and Sildenafil exposure resulted in photoreceptor cell death, while Digoxin and Thioridazine additionally affected all other cell types, including Müller glia cells. Ethanol and Methanol caused an upregulation in retinal ganglion cell related geneexpression. All drug treatments activated astrocytes, indicated by dendrites sprouting into neuroepithelium and upregulation of astrocyte related genes. The ability to resond to light was presereved in organoids although the number of active retinal ganglion cells decreased after drug expsoure. These data indicate comparable drug effects in organoids to those reported in in vitro models and/or in humans, thus providing first robust experimental evidence of their suitability for toxicological studies.
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