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Oxygen-dependent alternative mRNA splicing and a cone-specific motor protein revealed by single-cell RNA sequencing in hypoxic retinas

ABSTRACT: Restricted oxygen supply from the choroid may lead to hypoxic conditions in the outer retina of the ageing eye and contribute not only to physiological aging but also to nonhereditary degenerative retinal diseases. To understand the hypoxic response of specific retinal cell types, we performed droplet-based single-cell RNA sequencing (scRNA-Seq) of retinas isolated from mice exposed to hypoxia. Except for ganglion cells, we identified all retinal cell types and a small RPE cluster among the 9410 normoxic and 9033 hypoxic cells in the analysis. Expression of hypoxic marker genes including Bnip3, Higd1a, and Egln1 was significantly upregulated in hypoxic clusters, confirming a general transcriptional response to hypoxia. Interestingly, RNA-binding proteins Rbm3 and Cirbp were differentially expressed across clusters and demonstrated isoform switching in hypoxia. The resulting short variants of these gene transcripts are connected to epitranscriptomic regulation, a notion supported by the differential expression of writers, readers and erasers of m 6 A RNA methylations in the hypoxic retina. By focusing on the hypoxic response in photoreceptors, we identified and confirmed a kinesin motor protein (Kif4) that was specifically and strongly induced in hypoxic cones. Retinal cells adapted to hypoxic conditions by adjusting their gene expression profile. Epitranscriptomic regulation of m 6 A RNA methylations may support the adaptational process. The cone-specific upregulation of Kif4, a motor protein with multiple functions, indicated a cone-specific response to hypoxia that may include cargo-delivery to specific intracellular locations, enrichment of transmembrane receptors or channels and/or modulation of chromatin structure.

ORGANISM(S): Mus musculus

PROVIDER: GSE242982 | GEO | 2024/12/03

REPOSITORIES: GEO

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Project description:Reduced oxygenation of photoreceptors and the RPE in the ageing eye may be a risk factor for the development of both neovascular and dry AMD. Chronic activation of the molecular response to hypoxia in RPE or photoreceptors leads to retinal degeneration that depends on hypoxia inducible transcription factors. For approaching the identification of accessible markers characteristic for photoreceptors with an activated hypoxic response, we used a proteomics approach to determine the protein composition of the vitreous humor in mice. To discriminate between rod and cone-specific effects we used genetically modified mice that had the hypoxic response activated specifically in rods of a rod-dominant retina (rodΔVhl) or a genetically engineered all-cone retina (coneΔVhl). For comparison, we used wild-type mice exposed for 6 hours to acute hypoxia. We identified 1,357 unique proteins in the vitreous of mice after acute hypoxia, 1,624 in rodΔVhl and 1,895 in coneΔVhl mice. Of these, 1,043 proteins were common to all three types of mice. Of the identified proteins, 257 were significantly regulated by a factor of 1.5 or more in hypoxic mice, 258 in rodΔVhl and 356 in coneΔVhl mice in at least one of the three analyzed time points. Only 51 of the significantly regulated proteins were common to the vitreous of rodΔVhl and coneΔVhl mice, suggesting different consequences of the activated hypoxic response for rods and cones. Guanylate binding protein 2 (GBP2) was found at increased levels in the vitreous of both rodΔVhl and coneΔVhl mice at all time points tested. This was also reflected by increased gene expression in the retina. Although retinal expression of the AMD-associated gene alpha-2 macroglobulin (A2M) appeared increased in both types of mice, the protein was only found elevated in the vitreous of rodΔVhl mice. Other proteins found increased included Serpina3n, synaptosome associated protein 25 (SNAP25) and others. The distinct protein compositions present at early and late time points, suggest a well-regulated process in our models. We hypothesize that some of the proteins identified at early time points may potentially be used as markers for the chronic hypoxic response of photoreceptors.

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Oxygen-dependent alternative mRNA splicing and a cone-specific motor protein revealed by single-cell RNA sequencing in hypoxic retinas

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