Immunity
Volume 55, Issue 9, 13 September 2022, Pages 1627-1644.e7
Journal home page for Immunity

Article
Apolipoprotein E4 impairs the response of neurodegenerative retinal microglia and prevents neuronal loss in glaucoma

https://doi.org/10.1016/j.immuni.2022.07.014Get rights and content

Highlights

  • Microglia switch from a homeostatic to a neurodegenerative phenotype in glaucoma

  • Apoe and APOE3 induce Galectin-3, a key effector of neurodegenerative microglia

  • Apoe−/− and APOE4 retinal microglia have a decreased response to neurodegeneration

  • APOE4 allele protects against retinal ganglion cell (RGC) loss in glaucoma

Summary

The apolipoprotein E4 (APOE4) allele is associated with an increased risk of Alzheimer disease and a decreased risk of glaucoma, but the underlying mechanisms remain poorly understood. Here, we found that in two mouse glaucoma models, microglia transitioned to a neurodegenerative phenotype characterized by upregulation of Apoe and Lgals3 (Galectin-3), which were also upregulated in human glaucomatous retinas. Mice with targeted deletion of Apoe in microglia or carrying the human APOE4 allele were protected from retinal ganglion cell (RGC) loss, despite elevated intraocular pressure (IOP). Similarly to Apoe−/− retinal microglia, APOE4-expressing microglia did not upregulate neurodegeneration-associated genes, including Lgals3, following IOP elevation. Genetic and pharmacologic targeting of Galectin-3 ameliorated RGC degeneration, and Galectin-3 expression was attenuated in human APOE4 glaucoma samples. These results demonstrate that impaired activation of APOE4 microglia is protective in glaucoma and that the APOE-Galectin-3 signaling can be targeted to treat this blinding disease.

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 RESOURCESOURCEIDENTIFIER
Antibodies
Anti-Brn-3a Antibody, POU-domain protein, clone 5A3.2MilliporeSigmaCat#MAB1585; RRID:AB_94166
Anti Iba1, Rabbit (for Immunocytochemistry)FUJIFILM Wako Pure Chemical CorporationCat#019-19741; RRID:AB_839504
Anti-P2ry12, Rabbit polyclonalButovsky Lab, validated in Butovsky et al. (2014, 2015)N/A
Anti-Apolipoprotein E AntibodySigma-AldrichCat#AB947; RRID:AB_10770246
Purified Mouse Anti-Human Galectin-3, Clone B2C10 (RUO)BD BiosciencesCat#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

References (101)

  • Y. Liu et al.

    Galectin-3 regulates microglial activation and promotes inflammation through TLR4/MyD88/NF-kB in experimental autoimmune uveitis

    Clin. Immunol.

    (2022)
  • S. Nagata et al.

    Sensing and clearance of apoptotic cells

    Curr. Opin. Immunol.

    (2021)
  • K. Omodaka et al.

    Neuroprotective effect against axonal damage-induced retinal ganglion cell death in apolipoprotein E-deficient mice through the suppression of kainate receptor signaling

    Brain Res

    (2014)
  • C.N. Parkhurst et al.

    Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor

    Cell

    (2013)
  • Y. Qin et al.

    A milieu molecule for TGF-beta required for microglia function in the nervous system

    Cell

    (2018)
  • Y.C. Tham et al.

    Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis

    Ophthalmology

    (2014)
  • S.G. Utz et al.

    Early fate defines microglia and non-parenchymal brain macrophage development

    Cell

    (2020)
  • Y. Wang et al.

    TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model

    Cell

    (2015)
  • P.A. Williams et al.

    Neuroinflammation in glaucoma: a new opportunity

    Exp. Eye Res.

    (2017)
  • M. Xiying et al.

    Association of apolipoprotein E polymorphisms with age-related macular degeneration subtypes: an updated systematic review and meta-analysis

    Arch. Med. Res.

    (2017)
  • C.A. Abreu et al.

    Absence of galectin-3 promotes neuroprotection in retinal ganglion cells after optic nerve injury

    Histol. Histopathol.

    (2017)
  • F. Albalawi et al.

    The P2X7 receptor primes IL-1beta and the NLRP3 inflammasome in astrocytes exposed to mechanical strain

    Front. Cell. Neurosci.

    (2017)
  • M. Almasieh et al.

    Neuroprotection in glaucoma: animal models and clinical trials

    Annu. Rev. Vis. Sci.

    (2017)
  • M.G. Anderson et al.

    GpnmbR150X allele must be present in bone marrow derived cells to mediate DBA/2J glaucoma

    BMC Genet

    (2008)
  • M.G. Anderson et al.

    Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice

    Nat. Genet.

    (2002)
  • H. Asai et al.

    Depletion of microglia and inhibition of exosome synthesis halt tau propagation

    Nat. Neurosci.

    (2015)
  • A. Bosco et al.

    Reduced retina microglial activation and improved optic nerve integrity with minocycline treatment in the DBA/2J mouse model of glaucoma

    Invest. Ophthalmol. Vis. Sci.

    (2008)
  • A. Bosco et al.

    Neurodegeneration severity can be predicted from early microglia alterations monitored in vivo in a mouse model of chronic glaucoma

    Dis. Model. Mech.

    (2015)
  • A. Bosco et al.

    Early microglia activation in a mouse model of chronic glaucoma

    J. Comp. Neurol.

    (2011)
  • A. Boza-Serrano et al.

    Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer's disease

    Acta Neuropathol

    (2019)
  • B.V. Bui et al.

    Ganglion cell contributions to the rat full-field electroretinogram

    J. Physiol.

    (2004)
  • O. Butovsky et al.

    Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice

    Ann. Neurol.

    (2015)
  • O. Butovsky et al.

    Identification of a unique TGF-beta-dependent molecular and functional signature in microglia

    Nat. Neurosci.

    (2014)
  • O. Butovsky et al.

    Microglial signatures and their role in health and disease

    Nat. Rev. Neurosci.

    (2018)
  • N.B. Caberoy et al.

    Galectin-3 is a new MerTK-specific eat-me signal

    J. Cell. Physiol.

    (2012)
  • K.E. Campagno et al.

    Rapid morphologic changes to microglial cells and upregulation of mixed microglial activation state markers induced by P2X7 receptor stimulation and increased intraocular pressure

    J. Neuroinflammation

    (2021)
  • H. Chen et al.

    Optic neuropathy due to microbead-induced elevated intraocular pressure in the mouse

    Invest. Ophthalmol. Vis. Sci.

    (2011)
  • W.S. Chen et al.

    Galectin-3 inhibition by a small-molecule inhibitor reduces both pathological corneal neovascularization and fibrosis

    Invest. Ophthalmol. Vis. Sci.

    (2017)
  • G. Chidlow et al.

    Evidence supporting an association Between expression of major histocompatibility complex II by microglia and optic nerve degeneration During experimental glaucoma

    J. Glaucoma

    (2016)
  • W.S. Chung et al.

    Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways

    Nature

    (2013)
  • M. Colonna et al.

    Microglia function in the central nervous system during health and neurodegeneration

    Annu. Rev. Immunol.

    (2017)
  • B. Fortune et al.

    Selective ganglion cell functional loss in rats with experimental glaucoma

    Invest. Ophthalmol. Vis. Sci.

    (2004)
  • M. Gratuze et al.

    Activated microglia mitigate Abeta-associated tau seeding and spreading

    J. Exp. Med.

    (2021)
  • R. Guerreiro et al.

    TREM2 variants in Alzheimer's disease

    N. Engl. J. Med.

    (2013)
  • J.M. Harder et al.

    Early immune responses are independent of RGC dysfunction in glaucoma with complement component C3 being protective

    Proc. Natl. Acad. Sci. USA

    (2017)
  • G.R. Howell et al.

    Absence of glaucoma in DBA/2J mice homozygous for wild-type versions of Gpnmb and Tyrp1

    BMC Genet

    (2007)
  • G.R. Howell et al.

    Deficiency of complement component 5 ameliorates glaucoma in DBA/2J mice

    J. Neuroinflammation

    (2013)
  • G.R. Howell et al.

    Radiation treatment inhibits monocyte entry into the optic nerve head and prevents neuronal damage in a mouse model of glaucoma

    J. Clin. Invest.

    (2012)
  • Y. Huang et al.

    Microglia use TAM receptors to detect and engulf amyloid beta plaques

    Nat. Immunol.

    (2021)
  • T. Inoue et al.

    Elevated levels of multiple biomarkers of Alzheimer's disease in the aqueous humor of eyes with open-angle glaucoma

    Invest. Ophthalmol. Vis. Sci.

    (2013)
  • Cited by (33)

    • Role of APOE in glaucoma

      2024, Biochemical and Biophysical Research Communications
    View all citing articles on Scopus
    13

    Lead contact

    View full text