Biology subjects

Keuthan, C. J.

Publications and source records attributed to Keuthan, C. J..

4 recordsLinked to original sources

bioRxiv2025

Dynamic changes in mRNA isoform usage during human retinal development

BackgroundAlternative mRNA splicing is a key mechanism for generating isoform diversity in eukaryotic cells. However, the extent of the splicing changes that occur during complex regulatory processes like neurodevelopment are still incompletely characterized. ResultsWe performed nanopore-based long-read RNA sequencing on differentiating human stem cell-derived retinal organoids to identify temporal patterns of isoform usage across developmental stages. We found that retinal organoids undergo dynamic shifts in isoform usage throughout differentiation, which were not necessarily accompanied with changes in overall gene expression, as was observed for many genes involved in the regulation of mRNA splicing itself. Further analysis of human stem cell-derived retinal ganglion cells uncovered neuron-specific splicing signatures. Additionally, allele-specific gene expression analysis revealed extensive allelic imbalance in induced pluripotent stem cell-derived organoid cultures. ConclusionsBy combining direct long-read RNA sequencing with human stem cell retinal models we were able to develop a comprehensive database of isoform-level changes in differentiating human retinal cells during development. These results uncovered dynamic shifts in transcript usage during retinal differentiation, adding to our knowledge base of post-transcriptional RNA processing in the developing central nervous system and human in vitro culture systems.

molecular biology↗

bioRxiv2023

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Experimental Glaucoma and Optic Nerve Crush

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and in human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and on separately micro-dissected unmyelinated and myelinated optic nerve regions from naive C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naive unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor {beta} pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

molecular biology↗

bioRxiv2020

AMP-activated-kinase (AMPK) is essential sensor and metabolic regulator of retinal neurons and their integrated metabolism with RPE.

The retina has one of the highest energy demands in the human body, and proper regulation of metabolism among the cell types of the retina is required for functional vision. Recent studies have reported that retinal metabolism is disrupted during retinal degeneration and aging, while therapies designed to enhance metabolism can slow or prevent degeneration. Here, we show that multiple metabolic pathways, including those regulated by a key ATP sensor and regulator of metabolism, AMP-activated-kinase (AMPK), were dysregulated in a model of inherited retinal degeneration. In order to assess the direct role of AMPK in regulating retinal metabolism, we used mice with retina-specific knockout of AMPK activity. Conditional loss of AMPK resulted in impaired visual function at an early age, with slow photoreceptor loss observed in older mice. Moreover, we found that loss of AMPK resulted in decreased metabolic flux from glucose, decreased mitochondrial DNA copy number, decreased mitochondrial-related gene expression, and alterations in mitochondrial morphology in the photoreceptors, all of which preceded degeneration. Surprisingly, metabolic changes from the loss of AMPK in retinal neurons also resulted in secondary degeneration of retinal pigment epithelial (RPE) cells. Together, these data show that AMPK signaling is important for maintaining metabolic homeostasis of the retina and support the hypothesis that photoreceptors and RPE have a shared metabolic ecosystem that is controlled, in part, by AMPK.

neuroscience↗

bioRxiv2018

A drug-tunable gene therapy for broad-spectrum protection against retinal degeneration

Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of light-sensitive photoreceptors that impairs the vision of 9.1 million people in the US. An attractive treatment option is to use gene therapy to deliver broad-spectrum neuroprotective factors. However, this approach has had limited clinical translation because of the inability to control transgene expression. To address this problem, we generated an adeno-associated virus vector named RPF2 that was engineered to express domains of leukemia inhibitory factor fused to the destabilization domain of bacterial dihydrofolate reductase. Fusion proteins containing the destabilization domain are degraded in mammalian cells but can be stabilized with the binding of the drug trimethoprim. Our data show that expression levels of RPF2 are tightly regulated by the dose of trimethoprim, and can be reversed by trimethoprim withdrawal. We further show that stabilized RPF2 can protect photoreceptors and prevent blindness in treated mice.

bioengineering↗