Riker, M. J. | Search bioRxiv

bioRxiv2025

Modeling MEK-inhibitor Associated Retinopathy in vitro using human induced-1 pluripotent stem cell-derived retinal pigment epithelial cells

Pharmacologic inhibitors of MEK are important anti-cancer drugs but can result in MEK inhibitor-Associated Retinopathy (MEKAR) in which vision is lost due to serous retinal detachments that form via an unknown mechanism. We hypothesized that the cause of this side effect is drug-induced dysfunction of retinal pigment epithelial (RPE) cells. To test this hypothesis, we used human induced pluripotent stem cell-derived RPE cells. We treated mature, hiPSC-derived RPE cells with selumetinib and measured impacts on RPE-specific function, structure, and gene expression. Selumetinib increases the ability of hiPSC-derived RPE to internalize bovine rod outer segments (1.9 vs 3.0, p=0.0024). It also decreases expression of aquaporin 1 during the first 10 days of treatment (2.7 vs 1.1, p=0.0015). It has no effect on the ability of hiPSC-derived RPE to maintain membrane integrity. Selumetinib alters gene expression of hiPSC-derived RPE, with significant changes in genes involved in transport of ions and small molecules regulating cell volume and lysosomal acidification. Selumetinib may lead to subretinal fluid accumulation by both increasing secretions into this space and decreasing outflow.

molecular biology↗

bioRxiv2022

Non-random distribution of mitochondrial m.3243A>G heteroplasmy in human retina and its impact on cellular phenotype

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non- random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm- derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm- derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non- random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/496449v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@10b1c41org.highwire.dtl.DTLVardef@6c2d7corg.highwire.dtl.DTLVardef@1e4d09org.highwire.dtl.DTLVardef@153a5a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗