Immunity
ArticleApolipoprotein E4 impairs the response of neurodegenerative retinal microglia and prevents neuronal loss in glaucoma
Graphical abstract
Introduction
Glaucoma is a chronic blinding disease caused by progressive loss of retinal ganglion cells (RGCs), currently estimated to affect 80 million people worldwide (Tham et al., 2014). Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma. However, the disease often progresses despite repeated medical and surgical interventions to lower the IOP, and there are no clinically approved therapies that directly promote RGC survival (Almasieh and Levin, 2017). Despite the prevalence and morbidity of glaucoma, the underlying mechanisms that lead to RGC apoptosis in glaucomatous neurodegeneration remain poorly understood. Prior work has implicated the role of oxidative stress and mitochondrial dysfunction, neurotrophic factor deprivation, and astrocyte and microglial reactivity as key converging pathways that result in RGC death in glaucoma (Alqawlaq et al., 2019). Microglia, the resident immune cells of the central nervous system (CNS), have emerged as an important cell type critically involved in many brain neurodegenerations, including Alzheimer disease (AD) (Butovsky and Weiner, 2018; Hammond et al., 2019). In the healthy adult CNS, microglia play critical roles in maintaining homeostasis by performing constant surveillance, defense, and healthy repair (Colonna and Butovsky, 2017; Li and Barres, 2018). However, microglia can also have harmful effects on the neural tissues during disease by becoming chronically inflammatory (Butovsky and Weiner, 2018; Ising et al., 2019; Marschallinger et al., 2020).
Several prior studies have demonstrated that microglia and recruited myeloid cells play an important role in glaucoma development and progression (Soto and Howell, 2014; Williams et al., 2017; Zeng and Shi, 2018). Activated myeloid cells have been detected in the optic nerves of human glaucomatous eyes (Neufeld, 1999; Yuan and Neufeld, 2001) and early in disease course in the DBA/2J mouse model of glaucoma (Bosco et al., 2011, 2015; Howell et al., 2012). Mice with reduced levels of myeloid cell activation, either genetically (Bosco et al., 2018; Chidlow et al., 2016) or pharmacologically (Bosco et al., 2008; Cueva Vargas et al., 2016; Liu et al., 2016; Nakazawa et al., 2006; Roh et al., 2012; Wang et al., 2014), were protected from glaucomatous neurodegeneration. However, markers utilized in these studies (Iba1, CD11b, and Cx3cr1) do not differentiate between resident microglia and recruited myeloid cells. Therefore, the individual contributions of these two ontogenetically and functionally distinct cell populations to RGC degeneration in glaucoma remain unknown. Furthermore, the underlying mechanisms by which microglia contribute to RGC death remain poorly understood.
We previously identified a molecular signature of homeostatic microglia that differentiates these cells from recruited myeloid cells and other cell types in the CNS (Butovsky et al., 2014). Microglial genes P2ry12, Tmem119, and Fcrls were found to be specifically expressed in microglia (Butovsky et al., 2014). Furthermore, we and others have found that in mouse models of AD, amyotrophic lateral sclerosis (ALS), and multiple sclerosis, microglia switch from a homeostatic to microglial neurodegenerative phenotype (MGnD) (Krasemann et al., 2017), also known as disease-associated microglia (DAM) (Keren-Shaul et al., 2017). This switch is controlled by apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2) signaling (Krasemann et al., 2017). APOE is the major lipoprotein in the brain while TREM2 is a phosphatidylserine receptor expressed only by myeloid cells; both genes are well-established genetic risk factors for AD (Guerreiro et al., 2013; Jonsson et al., 2013). In particular, the APOE4 allele has been identified as the major risk factor for late-onset AD (Saunders et al., 1993; Strittmatter et al., 1993). APOE has also been linked to human glaucoma, with studies by us and others showing that APOE4 is associated with a decreased risk of glaucoma (Lam et al., 2006; Mabuchi et al., 2005; Margeta et al., 2020). This finding is consistent with the literature showing that APOE4 is also associated with a decreased risk of age-related macular degeneration (McKay et al., 2011; Xiying et al., 2017) and decreased photoreceptor degeneration in mice (Levy et al., 2015). However, why the same allele is deleterious in AD but protective in eye neurodegenerative diseases remains poorly understood.
In this study, we found that Apoe controls the microglial transition from a homeostatic to a cytotoxic neurodegenerative molecular phenotype in glaucoma, and targeting this signaling pathway ameliorates glaucomatous RGC degeneration. Similarly to Apoe−/− microglia, APOE4 microglia remained homeostatic in glaucoma, which led to decreased RGC degeneration despite elevated IOP. Our findings provide an explanation as to why APOE4 is associated with a decreased risk of glaucoma and show that the APOE signaling pathway is a promising target for neuroprotective treatments for this blinding disease.
Section snippets
Retinal microglia transition to a neurodegenerative MGnD phenotype in the microbead glaucoma model
To better understand the role of microglia in glaucoma pathogenesis, we investigated the microglial molecular signature in the microbead glaucoma model. To induce elevated IOP, magnetic microbeads were injected in the anterior chamber of wild-type (WT) mouse eyes (Chen et al., 2011; Ito et al., 2016; Sappington et al., 2010). Four experimental groups were included: (1) microbead-injected (MB) eyes, which exhibited elevated IOP and subsequent optic nerve degeneration (Figures 1A and 1B), (2)
Discussion
In this study, we investigated the role of microglia in the pathogenesis of glaucoma by identifying the microglial molecular signature in two murine glaucoma models, the microbead injection model and the DBA/2J model. We find that in both models, microglia transition from a homeostatic to a neurodegenerative transcriptional phenotype characterized by upregulation of Apoe, Lgals3, cytokines, and complement. This transcriptional signature in glaucoma overlaps with the microglial molecular
Key resources table
REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Brn-3a Antibody, POU-domain protein, clone 5A3.2 MilliporeSigma Cat#MAB1585; RRID:AB_94166 Anti Iba1, Rabbit (for Immunocytochemistry) FUJIFILM Wako Pure Chemical Corporation Cat#019-19741; RRID:AB_839504 Anti-P2ry12, Rabbit polyclonal Butovsky Lab, validated in Butovsky et al. (2014, 2015) N/A Anti-Apolipoprotein E Antibody Sigma-Aldrich Cat#AB947; RRID:AB_10770246 Purified Mouse Anti-Human Galectin-3, Clone B2C10 (RUO) BD Biosciences Cat#556904; RRID:AB_396531
Acknowledgments
This study was supported by the Cure Alzheimer's Fund (to O.B. and D.M.H.); BrightFocus Foundation 2020A016806 (O.B.); NIH/NINDS R01 NS088137 (O.B.), NIH/NIA R01 AG051812 (O.B.) and R01 AG054672 (O.B.); NIH/NEI R01 EY027921 (O.B.); and NIH/NEI K12 EY016335 (M.A.M.), NIH/NEI K08 EY030160 (M.A.M.), American Glaucoma Society Young Clinician Scientist Award (M.A.M.), Research to Prevent Blindness Career Development Award (M.A.M.), Robert M. Sinskey Foundation (M.A.M.), Ruettgers Family Charitable
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