Reformulating Pro-Oxidant Microglia in Neurodegeneration

Abstract

In neurodegenerative diseases, microglia-mediated neuroinflammation and oxidative stress are central events. Recent genome-wide transcriptomic analyses of microglial cells under different disease conditions have uncovered a new subpopulation named disease-associated microglia (DAM). These studies have challenged the classical view of the microglia polarization state's proinflammatory M1 (classical activation) and immunosuppressive M2 (alternative activation). Molecular signatures of DAM and proinflammatory microglia (highly pro-oxidant) have shown clear differences, yet a partial overlapping gene profile is evident between both phenotypes. The switch activation of homeostatic microglia into reactive microglia relies on the selective activation of key surface receptors involved in the maintenance of brain homeostasis (a.k.a. pattern recognition receptors, PRRs). Two relevant PRRs are toll-like receptors (TLRs) and triggering receptors expressed on myeloid cells-2 (TREM2), whose selective activation is believed to generate either a proinflammatory or a DAM phenotype, respectively. However, the recent identification of endogenous disease-related ligands, which bind to and activate both TLRs and TREM2, anticipates the existence of rather complex microglia responses. Examples of potential endogenous dual ligands include amyloid β, galectin-3, and apolipoprotein E. These pleiotropic ligands induce a microglia polarization that is more complicated than initially expected, suggesting the possibility that different microglia subtypes may coexist. This review highlights the main microglia polarization states under disease conditions and their leading role orchestrating oxidative stress.

Keywords: RNS; ROS; disease-associated microglia (DAM); inflammation; microglia; neurodegeneration; oxidative stress.

Figure 1

Potential cross-interactions between different disease-associated microglia polarization subtypes. It is well established that microglia sense the disease environment through different pattern recognition receptors (PRRs). Two illustrative examples are toll-like receptors (TLRs) and triggering receptors expressed on myeloid cells-2 (TREM2). Specific ligands of PRRs are different danger-associated molecular patterns (DAMPs), including aggregated proteins (amyloid β, Aβ, and α-synuclein, αS); high-mobility group box protein 1 (HMGB1); nucleic acids (NA); and ATP. From these, Aβ and HMGB1 are believed to activate TLRs, thus driving microglia to a M1-proinflammatory phenotype, which is highly pro-oxidant. More recently, the term neurodegeneration-associated molecular patterns (NAMPs) has been introduced to highlight endogenous disease-associated ligands of TREM2. Examples of NAMPS include phosphatdyil serine (PS), present in apoptotic cells and glycolipids sphingomyelin and sulfatide derived from damaged myelin; Aβ; several lipoproteins like apolipoprotein E (APOE); and negatively charged phospholipids like phosphatidylinositol (PI) and phosphatidylcholine (PS). TREM2 signaling is suggested to drive the disease-associated microglia (DAM) phenotype, leading to downregulation of microglia homeostatic genes (not shown) and strong upregulation of DAM genes, including Apoe, Lgals3 (galectin-3; GAL3), Clec7a, etc., and thus, driving microglia to an anti-inflammatory (DAM-1) phenotype. From these, APOE and GAL3 can be released by reactive microglia and govern microglia immune responses. The possibility exists that GAL3 and APOE, along with other endogenous ligands like Aβ, drive TLR-associated signaling (solid blue arrows) further in either pro-oxidative DAM phenotypes (DAM-2; dashed blue arrow) or classic M1 proinflammatory microglias (red arrows). In addition, different classical microglia proinflammatory mediators like tumor necrosis factor (TNF)-α, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) may affect the DAM phenotype, which may thus evolve into a pro-oxidative DAM phenotype.

Figure 2

Pro-oxidant microglia under disease conditions. TLR signaling drives NF-κB activation and transcription of proinflammatory and pro-oxidant molecules including iNOS, COX2, NADPH oxidase (NOX2), TNF-α, and pro-IL-1β. Assembly of NOX2 and ulterior activation constitutes an important source of superoxide anion and subsequent formation of radical oxygen (ROS) and nitrogen species. The figure illustrates how extracellular superoxide dismutates to H₂O₂ through the extracellular activity of superoxide dismutase (SOD) 3 and formation of hydroxyl radicals through the Fenton and Haber–Weiss reactions. Alternatively, superoxide anion may react with nitric oxide (NO) to form the highly toxic reactive peroxynitrites. The figure also illustrates the important role of COX2 in generating ROS. Thus, phospholipase A2 (PLA2) supplies arachidonic acid (AA) to COX2 for prostanoid biosynthesis (PGE2) along with ROS. NF-κB activation also leads to NLRP3 upregulation (the main inflammasome component). Upon appropriate stimulation (not shown; examples include K⁺ efflux or cathepsin release from damaged lysosomes), NLRP3 assembles a multiprotein platform resulting in caspase-1/caspase-8 activation and subsequent cleavage of pro-IL-1β into an active mature form (IL-1β). The figure also illustrates how different multivalent ligands, including Aβ, galectin-3 (GAL3), and APOE, may drive both TLR- and TREM2-signaling pathways. The switch from homeostatic to disease-associated microglia (DAM) is believed to be TREM2-dependent and it is accompanied by strong upregulation of different genes including GAL3 and APOE. These proteins can be released to the extracellular space, which together with Aβ and other DAMPS, may bind to and activate TLRs and trigger the microglia pro-oxidant response.