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Asha Kumari, Raul Ayala, Juan Carlos Zenteno, Sasik Roman, Radha Ayyagari, Shyamanga Borooah; Single cell RNA sequencing of retinal immune cells identifies activation of resident retinal microglia associated with subretinal pseudodrusen-like deposits. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2748.
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Mutations in the retinal pigment epithelium expressed gene, membrane frizzled related protein (MFRP), are associated with early-onset retinal degeneration associated with microphthalmia, foveoschisis and optic disc drusen in humans. Mouse models demonstrate widespread fundus autofluorescent (AF) spots. The hypothesis for this set of studies was that the AF spots were associated with cell mediated immune response.
Ophthalmic examination of 4 siblings with homozygous c.498_499 mutations in MFRP gene was performed using wide-field color, ophthalmic scanning laser microscopy (SLO) and SD-OCT. For murine studies, mice homozygous for the same c.498_499 mutation in Mfrp (MfrpKI/KI) were bred on a C57BL/6J background. Age-matched wild type (WT) controls were used. Mice underwent fundus imaging using SLO and SD-OCT (n=3). Single cell RNA sequencing (ScRNAseq) of CD45+Lo/CD11b+Hi FACS sorted retinal cells was performed on the 10x genomics chromium platform (n=5). To validate scRNAseq, we performed immunostaining of retinal cryosections (n=3) and retinal wholemounts (n=3) for microglial markers and qRT-PCR.
Regularly interspersed pseudodrusen-like spots were seen in the fundus of two early stage patients. MfrpKI/KI mice had significantly increased subretinal AF spots confirmed by wholemounts. FACS analysis showed increased numbers of CD45+Lo/CD11b+Hi cells in MfrpKI/KI mice retina (p<0.05). ScRNAseq identified that the major immune cell subpopulations were of resident microglial rather than bone marrow origin and had increased expression of activation and phagocytic markers. The findings were validated by immunostaining with Iba-1 and CD68 antibodies which demonstrated activated subretinal and retinal microglia in MfrpKI/KI mice.
Subretinal pseudodrusen deposits have been described in a number of diseases including age-related macular degeneration. However, study of these deposits has been limited by the lack of models. In the present study, using the MfrpKI/KI model, we found evidence of an association between sub-retinal deposits and activated resident retinal microglia. Prolonged activation of these cells could increase retinal degeneration in this model. MfrpKI/KI mice may provide a good model to better understand pathology associated with subretinal deposit formation.
This is a 2020 ARVO Annual Meeting abstract.
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