Single cell RNA sequencing confirms retinal microglia activation associated with early onset retinal degeneration

• Published in: Scientific reports 2022-09, Vol.12 (1), p.15273-15273, Article 15273
• Main Authors: Kumari, Asha, Ayala-Ramirez, Raul, Zenteno, Juan Carlos, Huffman, Kristyn, Sasik, Roman, Ayyagari, Radha, Borooah, Shyamanga
• Format: Article
• Language: English
• Subjects: 692/699/3161/3165; 692/699/3161/3175; Age; Animals; Apolipoprotein E; Apolipoproteins E - genetics; Cell activation; Fovea Centralis - metabolism; Frizzled protein; Humanities and Social Sciences; Humans; Immunofluorescence; Macrophages; Membrane proteins; Membrane Proteins - metabolism; Mice; Microglia; Microglia - metabolism; Microphthalmia; multidisciplinary; Mutation; Retina; Retinal degeneration; Retinal Degeneration - genetics; Retinal Degeneration - metabolism; Retinitis; Retinitis pigmentosa; Science; Science (multidisciplinary); Sequence Analysis, RNA; Transcriptomics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-022-19351-w

Mutations in the Membrane-type frizzled related protein ( Mfrp) gene results in an early-onset retinal degeneration associated with retinitis pigmentosa, microphthalmia, optic disc drusen and foveal schisis. In the current study, a previously characterized mouse model of human retinal degeneration carrying homozygous c.498_499insC mutations in Mfrp ( Mfrp KI/KI ) was used. Patients carrying this mutation have retinal degeneration at an early age. The model demonstrates subretinal deposits and develops early-onset photoreceptor degeneration. We observed large subretinal deposits in Mfrp KI/KI mice which were strongly CD68 positive and co-localized with autofluorescent spots. Single cell RNA sequencing of Mfrp KI/KI mice retinal microglia showed a significantly higher number of pan-macrophage marker Iba-1 and F4/80 positive cells with increased expression of activation marker ( CD68) and lowered microglial homeostatic markers ( TMEM119, P2ry13, P2ry13, Siglech ) compared with wild type mice confirming microglial activation as observed in retinal immunostaining showing microglia activation in subretinal region. Trajectory analysis identified a small cluster of microglial cells with activation transcriptomic signatures that could represent a subretinal microglia population in Mfrp KI/KI mice expressing higher levels of APOE. We validated these findings using immunofluorescence staining of retinal cryosections and found a significantly higher number of subretinal Iba-1/ApoE positive microglia in Mfrp KI/KI mice with some subretinal microglia also expressing lowered levels of microglial homeostatic marker TMEM119 , confirming microglial origin. In summary, we confirm that Mfrp KI/KI mice carrying the c.498_499insC mutation had a significantly higher population of activated microglia in their retina with distinct subsets of subretinal microglia. Further, studies are required to confirm whether the association of increased subretinal microglia in MfrpKI/KI mice are causal in degeneration.