Abstract
Purpose :
Non-coding regulatory variants, at the microRNA miR9-2 gene locus, have the strongest genome-wide association with macular thickness in the UK Biobank and have been associated with Age-Related Macular Degeneration, Macular Telangiectasia type 2, and increased retinal vascular caliber. In the mature retina, miR9-2 is expressed in Müller glia. We tested the hypothesis that disruption of miR9-2 gene expression contributes to dysregulation of Müller glial cells and retinal-disease phenotypes.
Methods :
miR9-2 constitutive (KO) and conditional (cKO) knockout mouse retinas were harvested at 3-5 weeks of age. The cKO strain expresses tamoxifen-inducible cre under control of the Glast promoter allowing for deletion of miR9-2 specifically in Müller glia cells. cKO mice were harvested one-week after tamoxifen induction. We compared wildtype (WT), KO, and cKO retinas, using immunofluorescence, electron microscopy, and single cell sequencing to assess morphological and transcriptional changes in mature Müller glia.
Results :
Loss of miR9-2 in KO mice led to dysregulation of retinal organization and cell class proportions. By immunofluorescence and single cell sequencing, the most disrupted cells were Müller glia-like, and these cells also appeared to express markers of bipolar cell fate. Further, in cKO animals, Müller glia had abnormal morphology in their endfeet, apical processes, and cell bodies compared to wildtype cells. Additionally, in glia of KO animals, gene regulatory networks were significantly altered compared to wildtype, suggesting that miR9-2 may be important for control of the intrinsic regulatory programs of these cells.
Conclusions :
The disease-associated locus, miR9-2 appears to play an important role in the development and maintenance of Müller cell identity. While alterations to Müller glial fate likely arise developmentally in KO animals, in cKO mice, loss of miR9-2 after development still leads to disruption of Müller glia cells. As individual microRNAs are known to act on a variety of potential gene targets, it is likely these changes arise due to derepression of miR9-2 target transcripts. These results highlight the role of miR9-2 in retinal development and homeostasis and demonstrate how dysregulation of this microRNA may contribute to the disrupted cell types and pathways in MacTel and AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.