Abstract
Purpose :
Microglia, the immune cells of central nervous system and the retina, respond to changes in the environment by altering their molecular and transcriptomic profiles, resulting in distinct functional states. However, little is known about how these transitions between states are regulated during regeneration of rod photoreceptors. Our lab has established a transgenic P23H rhodopsin zebrafish model of Retinitis Pigmentosa with degeneration and ongoing regeneration of rod photoreceptors. We hypothesize that microglia undergo transcriptomic and phenotypic changes that might promote rod regeneration.
Methods :
Analysis of whole retina single-cell transcriptomes from three P23H and WT zebrafish was performed using Seurat. Intravitreal injection of siRNAs was used to knock down lncRNA LOC110439915. To visualize microglia, retinal sections from adult P23H and WT fish were stained with antibody 7.4.C4 and lcp1 antibodies; photoreceptors were stained with retp1 antibody.
Results :
Immunostaining revealed that microglia in P23H retinas showed more activated, ameboid morphology compared to the WT and accumulated in the ONL and subretinal space. Furthermore, microglia’s number in the INL was reduced. scRNA seq analysis identified three distinct microglia clusters in both WT and P23H with unique gene signatures. The relative proportion of the three clusters was altered. Expression of several lncRNAs, si:ch211-214p16.2, LOC103909107, LOC103910136, LOC103909099 and LOC110439915 was microglia cluster-specific and was upregulated by 3.5 to 17.6 fold in P23H microglia compared to WT. LOC110439915 is highly expressed in all three microglia clusters in P23H, with predicted localization to the nucleus. Knock-down of LOC110439915 in P23H retina led to an increase in microglia/macrophages number and disruption of cone outer segments. The cell bodies of the rods appeared to be smaller compared to P23H controls.
Conclusions :
P23H microglia undergo transcriptomic and phenotypic changes as well as changes in their localization during retinal degeneration and regeneration. The altered expression of lncRNAs in P23H microglia suggests that they might regulate these changes. We observed a more advanced retinal degenerative phenotype following the knock-down of LOC110439915, suggesting that LOC110439915 might suppress processes that cause degeneration, creating an environment more conducive to rod regeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.