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Updated: Mar 16, 2026
A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration
Published on: March 10, 2023
microRNA regulatory circuits in a mouse model of inherited retinal degeneration
Arpad Palfi1, Karsten Hokamp1, Stefanie M Hauck2
1Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland.
Abstract:
miRNA dysregulation is a hallmark of many neurodegenerative disorders, including those involving the retina. Up-regulation of miR-1/133 and miR-142, and down-regulation of miR-183/96/182 has been described in the RHO-P347S mouse retina, a model for a common form of inherited blindness. High-throughput LC-MS/MS was employed to analyse the protein expression of predicted targets for these miRNAs in RHO-P347S mouse retinas; 133 potential target genes were identified. Pathway over-representation analysis suggests G-protein signaling/visual transduction, and synaptic transmission for miR-1, and transmembrane transport, cell-adhesion, signal transduction and apoptosis for miR-183/96/182 as regulated functions in retina. Validation of miRNA-target mRNA interactions for miR-1, miR-96/182 and miR-96 targeting Ctbp2, Rac1 and Slc6a9, respectively, was demonstrated in vitro. In vivo interaction of miR-183/96/182 and Rac1 mRNA in retina was confirmed using miR-CATCH. Additional miRNAs (including miR-103-3p, miR-9-5p) were both predicted to target Rac1 mRNA and enriched by Rac1-miR-CATCH. Other Rac1-miR-CATCH-enriched miRNAs (including miR-125a/b-5p, miR-378a-3p) were not predicted to target Rac1. Furthermore, levels of ~25% of the retinal Rac1 interactors were determined by LC-MS/MS; expression of Rap1gds1 and Cav1 was elevated. Our data suggest significant utilisation of miRNA-based regulation in retina. Possibly more than 30 miRNAs interact with Rac1 in retina, targeting both UTRs and coding regions.
Insights
MicroRNA (miRNA) dysregulation impacts inherited blindness models. This study identified 133 miRNA targets in mouse retinas, revealing miRNA regulation of visual transduction and synaptic transmission.
Area of Science:
- Ophthalmology and Neuroscience
- Molecular Biology
- Genetics
Background:
- MicroRNA (miRNA) dysregulation is a key feature of neurodegenerative diseases, including retinal disorders.
- Specific miRNA changes (up-regulation of miR-1/133, miR-142; down-regulation of miR-183/96/182) are observed in the RHO-P347S mouse model of inherited blindness.
Purpose of the Study:
- To investigate miRNA-mediated gene regulation in the RHO-P347S mouse retina.
- To identify protein targets of dysregulated miRNAs and analyze their functional pathways.
- To validate miRNA-target interactions in vitro and in vivo.
Main Methods:
- High-throughput LC-MS/MS to analyze protein expression of predicted miRNA targets.
- Pathway over-representation analysis to identify regulated cellular functions.
- In vitro validation of miRNA-target mRNA interactions.
- miR-CATCH technique to confirm in vivo miRNA-mRNA interactions in the retina.
Main Results:
- 133 potential miRNA target genes were identified in the RHO-P347S mouse retina.
- Pathway analysis indicated regulation of G-protein signaling, visual transduction, synaptic transmission, transmembrane transport, cell adhesion, signal transduction, and apoptosis.
- In vitro and in vivo experiments confirmed interactions between specific miRNAs (miR-1, miR-96/182, miR-183/96/182) and target mRNAs (Ctbp2, Rac1, Slc6a9).
- Over 30 miRNAs were found to interact with Rac1 in the retina, affecting its UTRs and coding regions.
Conclusions:
- The study demonstrates significant miRNA-based regulation in the retina, particularly involving visual transduction and synaptic pathways.
- Rac1 is a key target interacting with numerous miRNAs in the retina.
- These findings contribute to understanding the molecular mechanisms underlying inherited retinal diseases.
