Retinal microglia acquire a disease-associated transcriptome in chronic mouse glaucoma, which intensifies with neuroprotective complement inhibition

Abstract

Purpose : Microglia are activated in glaucoma, but we lack understanding of their role and functional state with disease. They are also key effectors of complement signaling. Complement dysregulation is associated with early glaucoma and C3-targeted complement inhibition (AAV2.CR2-Crry) is neuroprotective (Bosco et al., 2018, Molecular Therapy), but how microglia are involved is not known. To address this, we analyzed the transcriptome of retinal microglia in DBA/2J mice during neurodegeneration and following complement inhibition.

Methods : Microglia (CX3CR1⁺/CD45⁺/CD11b⁺/CCR2^-) were FACS-sorted from 4 retinas each from female naïve DBA/2J mice at 5 mo (pre-degenerative), 10mo (during degeneration), 10mo after intravitreal injection of AAV2.CR2-Crry at 7mo, and 10mo non-glaucoma Gpnmb^WT controls. We then performed RNA sequencing and differential expression analysis using DEseq. Expression of selected genes was verified by qRT-PCR of purified microglia (n = 4/condition; ANOVA test) and by multiplex in situ hybridization chain reaction (HCR) and confocal microscopy on retinal wholemounts (n = 6/condition).

Results : Flow cytometry showed a 2-fold increase in microglial numbers in naïve 10mo retinas vs. naïve 5mo and 10mo Gpnmb^WT, and 3-fold in AAV2-CR2-Crry-treated retinas vs. 10mo naïve. Naïve 5mo DBA/2J and 10mo Gpnmb^WT retinal microglia had a largely homeostatic gene expression profile. Naïve 10mo DBA/2J retinas showed a shift to a disease-associated microglia (DAM) profile shown in brain neurodegeneration, with upregulation (>1.5 fold) of genes related to phagocytosis and lipid metabolism (Apoe, Cst7, Igf1, Ank, Itgax and Spp1), and downregulation of select homeostatic genes (Il16, Mef2a, Cst3 and Cybb1) vs. 5mo retinas. AAV2.CR2-Crry treatment resulted in further upregulation of select DAM genes (Clec7a and Axl), and further downregulation of homeostatic genes (Tmem119, P2ry12, Cx3cr1 and Cst3) relative to naïve 10mo. HCR analysis confirmed DAM gene expression to subsets of microglia in 10mo naïve mice.

Conclusions : Retinal DAM cells arise and expand during neurodegeneration in DBA/2J glaucoma, with concurrent downregulation of homeostatic gene expression. Whether DAM are detrimental or protective is unknown, but we found a more pronounced DAM gene expression signature after neuroprotective complement inhibition, suggesting a potential protective role.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.