Reformulating Pro-Oxidant Microglia in Neurodegeneration

Journal of Clinical Medicine Review Reformulating Pro-Oxidant Microglia in Neurodegeneration Juan García-Revilla 1,2,†, Isabel M. Alonso-Bellido 1,2,†, Miguel A. Burguillos 1,2 , Antonio J. Herrera 1,2, Ana M. Espinosa-Oliva 1,2, Rocío Ruiz 1,2 , Luis Cruz-Hernández 1,2, Irene García-Domínguez 1,2, María A. Roca-Ceballos 1,2, Marti Santiago 1,2, JoséA. Rodríguez-Gómez 2,3 , Manuel Sarmiento Soto 1,2, Rocío M. de Pablos 1,2 and JoséL. Venero 1,2,* 1 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain; [email protected] (J.G.-R.); [email protected] (I.M.A.-B.); mabur[email protected] (M.A.B.); [email protected] (A.J.H.); [email protected] (A.M.E.-O.); rr[email protected] (R.R.); [email protected] (L.C.-H.); ir[email protected] (I.G.D.); [email protected] (M.A.R.-C.); [email protected] (M.S.); [email protected] (M.S.S.); [email protected] (R.M.d.P.) 2 Institute of Biomedicine of Seville (IBIS)-Hospital Universitario Virgen del Roc í o/CSIC/University of Seville, 41012 Seville, Spain; [email protected] 3Departament of Medical Physiology and Biophysics, Faculty of Medicine, University of Seville, 41009 Sevilla, Spain *Correspondence: jlvener[email protected] †Shared first authorship. Received: 17 September 2019; Accepted: 12 October 2019; Published: 17 October 2019   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: microglia; oxidative stress; neurodegeneration; inflammation; disease-associated microglia (DAM); ROS; RNS J. Clin. Med. 2019,8, 1719; doi:10.3390/jcm8101719 www.mdpi.com/journal/jcm J. Clin. Med. 2019,8, 1719 2 of 32 1. Microglia: Origin and Roles in Health and Disease; Triggering Receptors Expressed on Myeloid Cells-2 (TREM2) Significance A century ago, del Rio-Hortega described, for the first time, the existence of cells that he considered “the third element of nerve centers” [ 1 ] and later termed ‘microglia’ [ 2 ]. These are immune cells, macrophages residing in the central nervous system (CNS). Microglial cells derive from progenitors that originate in the yolk sac [ 3 , 4 ] and later proliferate and migrate to the CNS [ 5 ], continuously monitoring the brain parenchyma to maintain the correct homeostasis of the nervous tissue [6]. Microglial cells, reactive oxygen species (ROS), and pathological aggregated proteins have been long suspected to belong to a detrimental loop intrinsically involved in neurodegeneration. However, the strongest evidence about the role of microglia in neurodegeneration has been obtained by different genome-wide association studies (GWAS). These studies have identified different risk genes that are either enriched or uniquely expressed in immune cells, including microglia [ 7 – 9 ]. Representative examples are triggering receptor expressed on myeloid cells-2 (Trem2), Cd33,Cr1,Mef2c, members of the MS4A family, and the HLA locus, which are Alzheimer’s disease (AD) risk genes. These studies clearly point at microglia as leading actors in disease progression at very early stages of neurodegeneration [ 9 ]. From the broad array of immune-associated genes related to neurodegeneration, Trem2 certainly emerges as a critical factor in governing microglia activation states. In an effort to characterize molecular systems associated to AD, Zhang et al. [ 10 ] performed a whole-genome gene expression, profiling and genotyping data in hundreds of samples from late-onset AD (LOAD) patients and aged-matched controls subjects [ 10 ]. This study identified a significant number of modules ascribed to different functional categories and cellular phenotypes. Rank ordering of the most significant molecular networks identified the immune/microglia module, including tyro protein kinase binding protein (TYROBP; also known as DAP12), as the highest ranking in terms of regulatory strength and differential expression [ 10 ]. TYROBP is the adaptor protein binding partner of TREM2, which also binds a large number of immune receptors including TREM1, CLEC7A, SIRP β , PILR β , and NKp44 [ 9 , 11 ]. The study by Zhang et al. anticipated the importance of TREM2 in triggering microglia activation associated to neurodegenerative diseases. Since the cloning of TREM2, the attention on this receptor in microglia functioning under homeostatic and neurodegenerative conditions has been increased exponentially (for reviews see [ 12 , 13 ]). Two independent studies identified a rare variant of TREM2 (R47H) as a strong risk gene of AD [ 14 , 15 ]. Following this, another variant (R62H) was further identified, thus, supporting the important role of TREM2 in neurodegeneration. Since TREM2 is uniquely expressed by microglia in the CNS [ 13 ], further elucidation of TREM2-dependent roles of microglia under disease conditions has become a priority in the field. In this review, we will focus not just on TREM2 but on other critical receptors involved in microglia polarization such as toll-like receptors (TLRs), historically linked only in the proinflammatory activation of microglia (highly pro-oxidant). 2. Sensing the Disease-Associated Environment: TLRs and TREM2, Main Drivers of Microglia Polarization 2.1. Toll-Like Receptors (TLRs) The mammalian TLR family consists of 13 members that recognize specific patterns from different microbial components called pathogen-associated molecular patterns (PAMPs). Other endogenous molecules that are constitutively expressed and released upon injury, known as danger-associated molecular pattern molecules (DAMPs) [ 16 ], are also clearly involved. TLRs are constitutively expressed in different brain cell types, including microglia [ 17 ]. TLRs are either localized in the cell surface or in intracellular compartments such as the endoplasmic reticulum (ER) and endosome. Each member of the TLR family is constituted by three different structures: (i) an ectodomain with a horseshoe-like structure with leucine-rich repeats (LRRs) for PAMPs/DAMPs recognition; (ii) a transmembrane domain; and (iii) a cytoplasmic Toll/IL-1 receptor (TIR) domain that initiates downstream signaling. Upon recognition of their respective PAMPs/DAMPs, TLRs interact as a homo- or heterodimer, together J. Clin. Med. 2019,8, 1719 3 of 32 with a co-receptor. After that, TLRs recruit TIR domain-containing adaptor proteins such as MyD88 and TRIF, which trigger different signal transduction pathways that end with the activation of NF- κ B, IRFs, or MAP kinases [ 18 ]. Apart from the well-documented function for TLRs as the first barrier against deleterious stimuli of a different nature by triggering the innate immune response [ 19 ], several new roles have been assigned to TLRs [ 20 ]. In the context of neurodegenerative diseases, where a sterile inflammation occurs [ 16 ], different endogenous disease-related ligands may bind to and activate TLRs, such as amyloid β (A β ) [ 21 ], α -synuclein ( α -syn) [ 22 ], high-mobility group box protein 1 (HMGB1) [ 23 ], galectin-3 (GAL3; coded by Lgals3) [ 24 , 25 ], etc. After ligand recognition, TLRs assemble and activate different downstream signaling pathways, which activate several immune responses. For instance, TLR activation is necessary for priming inflammasome and to induce a second signal (e.g., Aβaggregates in AD) that will lead to the generation of the inflammasome complex [26]. In the context of ROS production, TLR stimulation does also “prim” the activation of NADPH oxidases (NOX) [ 27 ]. The NOX complex consists of several proteins that are recruited and activated in the cell membrane in response to TLR engagement [ 28 ]. For instance, p47phox is translocated into the membrane and phosphorylated at Ser345 via ERK pathway in lipopolysaccharide (LPS)-treated microglia [ 29 ]. The role of activated NOX2 has been proven in several models of neurodegenerative diseases. Thus, microglia in AD subjects displayed activated NOX2, resulting in the formation of ROS that are toxic to neighboring neurons [ 30 ]. Gene deletion of gp91phox confers protection in animal models of Parkinson ´ s disease (PD, MPTP model) [ 31 , 32 ] Huntington’s disease (HD) [ 33 ], and LPS-induced neuronal damage [ 34 ]. Furthermore, TLR activation induces the expression of the GTP/GDP exchange factor Vav, which promotes nucleotide exchange over Rac2, a pivotal protein during the oxidative burst in neutrophils and macrophages [35]. The high immune-related oxidative burst initiated by TLR is normally used as an “arsenal” to fight infection [ 36 ]; however, it may also play a signaling role in immune cells. For instance, the small acute oxidant burst induced by TLR activation mediates the rearrangements of TLRs into the lipid rafts in the membrane of macrophages [ 37 , 38 ]. Additionally, their role has been described during the formation of TRAF6 and ASK1 complex [39], both necessary for TLR-mediated inflammatory responses [40]. 2.2. TREM2 TREM2 is a fundamental receptor in sensing the environment under disease conditions. It is uniquely expressed in cells of the myeloid lineage. The extracellular domain of TREM2 belongs to the immunoglobulin superfamily and recognizes an extensive range of polyanionic molecules including LPS, glycosaminoglycans, phospholipid, and DNA [ 41 – 43 ]. Upon ligand binding, TREM2 is known to interact with two different adaptor proteins: DAP12 and DAP10. The intracellular domain of DAP12 activates spleen tyrosine kinase (SYK), activating downstream pathways that regulate survival, migration, proliferation, activation, and phagocytosis [ 13 ]. On the other hand, interaction of TREM2 with DAP10 leads to phatidylinositol 3-kinase (PI3K) recruitment, which triggers a phosphorylation cascade that promotes Ca 2+ mobilization, integrin activation, cytoskeleton rearrangement, mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) signaling, and activation of energetic metabolism [ 13 ]. How exactly each ligand activates different and specific signaling pathways of the TREM2 response remains unclear. Mutations and single nucleotide polymorphisms (SNPs) in the TREM2–DAP12 axis have been related to a variety of neurodegenerative disorders such as Nasu–Hakola disease, frontotemporal dementia, AD, PD, amyotrophic lateral sclerosis (ALS), and essential tremor (for reviews see [ 13 , 44 , 45 ]). Interestingly, the majority of the TREM2 disease-linked mutations occur in their extracellular Ig-like V-type domain [ 46 ]. These findings, together with the role of TREM2 in driving the disease-associated microglia (DAM) phenotype, strongly link the TREM2 signaling to the pathology of neurodegenerative diseases. Yet, the mechanism(s) where TREM2 is implicated in pathology progression has not been totally clarified. Indeed, it is unclear whether TREM2-related microglial signaling pathways are either protective or deleterious in neurodegeneration. This controversy may be related to the ambiguous role of TREM2 in modulating oxidative stress. J. Clin. Med. 2019,8, 1719 4 of 32 Uncovering the molecular pathways activated by TREM2 during microglia activation under disease conditions and how this microglia polarization may lead to harmful oxidative stress will help to develop novel therapeutic approaches to treat neurodegenerative diseases. 3. Microglia Subtypes Under Conditions of Neurodegeneration Historically, microglia polarization states were simplified in two opposite phenotypes termed proinflammatory M1 (classical activation) and immunosuppressive M2 (alternative activation; also known as tumor-supportive phenotype). However, recent massive transcriptomic studies have clearly demonstrated the oversimplification of the M1/M2 microglia polarization states and the existence of different microglia subtypes under disease conditions is now evident. 3.1. Classical M1-Like and M2-Like Phenotypes The classical microglia M1 or proinflammatory phenotype is induced after LPS/(IFN)- γ treatments and upregulates inducible nitric oxide synthase (iNOS) and NF- κ B nuclear translocation [ 47 ]. This activation leads to strong activation of different proinflammatory cytokines, including tumor necrosis factor (TNF)- α , interleukin (IL)-1 β , NLRP3 (the best known inflammasome), FLIP (survival protein), etc. The M1 phenotype has been classically linked to oxidative and nitrosative stress generation [ 47 ]. It is noteworthy to mention that sustained proinflammatory activation of microglia has long been considered neurotoxic [48]. On the other side, alternative activation (M2-like) of microglia is achieved upon treatment with IL-4 or IL-13 and triggers upregulation of anti-inflammatory genes including arginase-1 (source of proline and polyamines), mannose receptor (CD206), YM1, and FIZZ. This activation pathway is considered to contribute to tissue repair and extracellular matrix reconstitution [49]. Since arginase-1 and iNOS compete for the same substrate (arginine; Arg), their equilibrium during disease will dictate nitric oxide (NO) production and subsequent oxidative stress. However, owing to the bigger complexity of the coexistence of a broad panel of microglial phenotypes during the progress of disease, the real existence of M1-like and M2-like phenotypes in microglial cells has already been a source of needed debate [ 50 ]. Since this review is devoted to discussing important aspects of microglia-associated oxidative stress and neurodegeneration, it is important to define particular microglial states that may be involved. Without any doubt, the microglia proinflammatory phenotype is pro-oxidant, and a strong induction of iNOS is a hallmark of this important phenotype [ 47 ] (see Section 4.2 of this review) (Figures 1and 2). Transcriptional profiles of isolated microglia in response to systemic LPS have been recently generated in an effort to understand the proinflammatory phenotype in vivo [ 51 , 52 ]. These studies highlight key differences between proinflammatory and DAM phenotypes. Findings showed that the LPS-induced microglia phenotype was not associated to DAM-like features such as TREM2/DAP12 or apolipoprotein E (APOE) upregulation, [ 51 , 52 ], although different DAM genes were upregulated (e.g., Itgax (CD11c), Axl,Clec7a, and Ccl2) (see Section 3.2). However, highly upregulated genes in response to LPS were associated with an oxidative stress response, especially Ptgs2 (COX2) and Cybb (NOX2) (major source of free radicals, see Section 4.2 and Figure 2) and classical proinflammatory genes like Il1β,Tnfα,and Ccl2 [51,52]. J. Clin. Med. 2019,8, 1719 5 of 32 J. Clin. Med. 2019, 8, 1719 5 of 32 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 dangerassociated molecular patterns (DAMPs), including aggregated proteins (amyloid β, Aβ, and αsynuclein, 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 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 , A β , and α -synuclein, α S); high-mobility group box protein 1 (HMGB1); nucleic acids (NA); and ATP. From these, A β 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; A β ; 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 A β , 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)-1 β , inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) may affect the DAM phenotype, which may thus evolve into a pro-oxidative DAM phenotype. J. Clin. Med. 2019,8, 1719 6 of 32 J. Clin. Med. 2019, 8, 1719 6 of 32 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. 3.2. Disease-Associated Microglia (DAM) The advent of massive transcriptomic analysis at the single cell level has helped to uncover different microglia states associated with controling homeostasis [51,53–55], aging [51], and diseaseassociated conditions [56–59]. Holtman et al. [56] analyzed transcriptional profiles of isolated microglia from different mouse models of aging and different diseases using weighted correlation network analysis (WGCNA)-based meta-analysis to determine the transcriptional signature and hub genes of different primed microglia phenotypes. In this study, after analyzing different diseaseassociated conditions like AD, ALS, and physiological and accelerated aging, they found a similar Figure 2. Pro-oxidant microglia under disease conditions. TLR signaling drives NF- κ B activation and transcription of proinflammatory and pro-oxidant molecules including iNOS, COX2, NADPH oxidase (NOX2), TNF- α , and pro-IL-1 β . 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 H 2 O 2 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- κ B 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-1 β into an active mature form (IL-1 β ). The figure also illustrates how different multivalent ligands, including A β , 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 A β and other DAMPS, may bind to and activate TLRs and trigger the microglia pro-oxidant response. 3.2. Disease-Associated Microglia (DAM) The advent of massive transcriptomic analysis at the single cell level has helped to uncover different microglia states associated with controling homeostasis [ 51 , 53 – 55 ], aging [ 51 ], and disease-associated conditions [ 56 – 59 ]. Holtman et al. [ 56 ] analyzed transcriptional profiles of isolated microglia from different mouse models of aging and different diseases using weighted correlation network analysis (WGCNA)-based meta-analysis to determine the transcriptional signature and hub genes of different primed microglia phenotypes. In this study, after analyzing different disease-associated conditions J. Clin. Med. 2019,8, 1719 7 of 32 like AD, ALS, and physiological and accelerated aging, they found a similar transcriptional network. Subsequent studies by Amit and colleagues using single-cell RNA analysis of brain immune cells identified a microglia subpopulation showing a unique molecular signature in 5xFAD mice (a transgenic mouse model of AD), which they named “disease-associated microglia” (DAM) [ 57 ]. In this study, two microglia clusters (groups II and III) were observed in 5xFAD mice but not in wild-type mice. They were characterized by (i) downregulation of key homeostatic genes including P2ry12, Cx3cr1, and Tmem119 and (ii) upregulation of selective genes (e.g., Apo3, Ctsd, Lpl, Itgax,Clec7a, Tyrobp and Trem2). Based on the degree of upregulation of DAM genes, the authors hypothesized that group II is an intermediate state between homeostatic microglia and group III (DAM), which was associated with microglia clustering A β plaques [ 57 ]. Mechanistically, the authors concluded that transition from group II microglia to DAM was TREM2-dependent, whilst transition from homeostatic microglia to group II was TREM2-independent. The relevance of this finding is the fact that many DAM genes, especially TREM2, have been found as risk genes of AD and other neurodegenerative conditions. In an independent study, Butovsky and colleagues also performed transcriptomes of isolated microgliaduringdifferentconditionsincludingaging, mouse modelsof AD(APP/PS1), ALS(SOD1 G93A ), and multiple sclerosis (MS) (experimental autoimmune encepahalomyelitis; EAE) [ 51 ]. Again, a similar microglia molecular response was found in the different models, exhibiting loss of homeostatic genes (e.g., P2ry12,Tmem119,Gpr34,Csf1r,Hexb, and Mertk), together with upregulation of inflammatory genes including Spp1,Itgax,Lgals3,Axl,Clec7a,Ccl2, and Apoe [ 51 ]. Although this microglia phenotype was named microglia neurodegenerative phenotype (MGnD), it shares many features with DAM [ 57 ], and hence, we will follow this nomenclature to avoid unnecessary confusion. Interestingly, microglia APOE upregulation correlated with disease progression in EAE, SOD1, and APP/PS1 models, thus raising the possibility that DAM could be neurotoxic [ 51 ]. Interestingly, APOE is believed to play a major role in the generation of the DAM phenotype, as Apoe deletion largely prevented upregulation of different DAM genes including Clec7a,Gpnmb, and Lgals3 [ 51 ]. Gene targeting of Trem2 overall prevented the switch from homeostatic to DAM microglia, and ameliorated the neuronal loss following facial nerve axotomy in SOD1 and APP/PS1 models [51]. In tau models, TREM2 is increased at late stages [ 60 , 61 ] and its inhibition protects against tau-induced brain neurodegeneration [ 61 ]. Since chronic neurodegenerative diseases are persistent in terms of neuronal cell death, the concept that microglia act as a double-edged sword in neurodegenerative disease has been lately a constant debate in the field [ 62 ]. Among the different DAM genes, glycoprotein nonmetastatic melanoma protein B (Gpnmb; also known as osteactivin) is a very interesting candidate in driving TREM2-dependent anti-inflammatory effects. Its importance is supported by the existence of Gpnmb gene mutations associated with increased risk of developing PD [ 63 ]. Regarding the inflammatory-related function of GPNMB, there is deep evidence about its role as a negative regulator of inflammatory process [ 64 – 67 ]. In addition to its potential anti-inflammatory role, it has been described that GPNMB is related to the autophagic-lysosomal pathway, involved in lysosomal stress and in the degradation of cellular debris [ 68 , 69 ].The fact that GPNMB is overexpressed in a variety of neurodegenerative diseases, its key role as anti-inflammatory, and its relation with the autophagic-lysosomal pathway suggest that the normal function of this protein is essential for keeping homeostatic brain status. Therapeutic approaches modulating GPNMB function might raise potential treatments for neurodegenerative diseases. A key question is to understand the pro-oxidant role of DAM. It is paradoxical that absence of TREM2 may be protective in some paradigms [ 51 , 61 ] and exacerbate neuronal damage in others. Thus, absence of TREM2 in AD mice, which halts the switch from homeostatic microglia to DAM, leads to increased neuritic dystrophies associated with A β plaques [ 70 , 71 ]. The same is true in human carriers of the R47H TREM2 mutant form [ 12 ]. Similarly, the impairment of native TREM2 activity associated with its R47H variant seems to facilitate the formation of tau aggregates in the vicinity of neuritic plaques, owing to the reduction of microglia activation [72]. J. Clin. Med. 2019,8, 1719 8 of 32 The microglia polarization state relies on selective activation of microglia surface receptors by a broad nature of ligands. Historically, the microglia field has consistently used the terms PAMPs and DAMPs, which are recognized by pattern recognition receptors (PRRs) (Figure 1) [ 7 ]. Prototypical PRRs include the TLRs (see 2.1). Other examples of PRRs are the NOD-like receptors, C-type lectin receptors, and the receptor for advanced glycation end-products (RAGE) [ 73 ]. Recently, the term neurodegeneration-associated molecular pattern (NAMPs) has been suggested, which includes an array of danger signals commonly present in different neurodegenerative diseases and recognized by different receptors (including TREM2) to switch from homeostatic microglia to DAM (Figure 1) [ 74 ]. The family of NAMPS includes recently identified TREM2 ligands. The typical examples include phosphatidyl serine present in apoptotic cells and glycolipids sphingomyelin and sulfatide derived from damaged myelin (Figure 1). However, new interesting candidates could be products associated with A β plaques, including A β itself [ 75 , 76 ], several lipoproteins like APOE and CLU/APOJ [ 77 ], phosphatidylinositol, and phosphatidylcholine [ 43 , 78 ]. Besides, TREM2 has been found to bind different bacterial products, including those from Gram-negative, whose membrane contains LPS [ 45 ]. More recently, we have demonstrated an important role of GAL3, a carbohydrate-binding protein in AD pathology, and more important, its ability to bind to and stimulate TREM2 (Figure 1) [ 79 ]. However, GAL3 stimulates not just TREM2 but also other fundamental microglial receptors, including TLR4 [ 24 ] and insulin-like growth factor 1 receptor (IGFR1) [ 80 ]. GAL3 has been shown to be important in driving the proinflammatory activation of microglia in response to fibrillar A β [ 79 ]. Since GAL3 can be released by reactive microglia [ 24 , 79 ], there is a strong possibility that GAL3 may act as an endogenous candidate to drive both TLR- and TREM2-dependent signaling pathways (Figures 1and 2). Indeed, this feature is not restricted only to GAL3 since there are other exciting examples, including APOE and A β (Figure 2). Thus, Apoe ε 4allele, one of the strongest gene risk factors in AD, has been associated with strong proinflammatory stimulation of the human innate immune response through TLR activation [81]. In addition, microglia activation in response to A β has been previously shown to involve both TLR signaling and NOD-like receptors [ 26 , 82 , 83 ]. In fact, amyloid plaque compaction involves a chronic exposure of microglia to A β [ 83 , 84 ], which has been shown to induce the release of microglial proinflammatory and pro-oxidant factors that mediate neuroinflammation, a process that can be detrimental to the nervous tissue. Altogether, we could certainly expect that different microglia receptors play significant roles in driving microglia heterogeneous responses under disease conditions, including proinflammatory/pro-oxidant components (Figure 2). In the same vein, a recent study has applied WGCNA to existing transcriptomic datasets from CNS immune cells and analyzed three AD modules, including 5xFAD, 5xFAD/TREM2 knock-out (KO), and APP/PS1 [ 85 ]. This study identified two well-defined modules; a proinflammatory pro-oxidant module, including Ptgs2,Il12b,Tlr2,Hif1 α , Il1 β , and Irg1, and an anti-inflammatory module, including anti-inflammatory and phagocytic genes like Igf1,Dpp7,Apo3,Spp1, and Lpl [ 85 , 86 ]. Importantly, these two modules are closely related and are very different from the homeostatic microglia [ 85 ]. Both modules exhibited DAM genes and they were partly TREM2-dependent, thus, supporting the existence of at least two well-defined DAM subtypes with a common origin [ 43 ]. In addition, transcriptomic analysis in brains from 24-month-old aged wild-type and TREM2 KO mice revealed that most significant genes in response to TREM2 deficiency were associated to the complement system, microglia, and the oxidative stress response Cybb (NOX2) and Nos2 (iNOS) genes [87]. Lastly, different studies analyzing the presence of lipid peroxidation biomarkers, such as 4-hydroxynonenal (HNE), malondialdehyde, and intracellular ROS (see Box A1), revealed high levels in TREM2 KO mice, thus, supporting the view that TREM2 may also repress an oxidative stress response [ 87 , 88 ]. These studies are in line with those showing that activation of TREM2 modulates the TLR-induced inflammation, following a reduction in proinflammatory cytokine release and a decrease in ROS production (for review see [ 89 , 90 ]). However, the last referred studies were performed in vitro , J. Clin. Med. 2019,8, 1719 9 of 32 which have been shown not to mimic the prototypical DAM phenotype demonstrated under disease conditions in vivo. 3.3. Necroptotic Microglia Within the differentpotential pro-oxidant statesof microglia, necroptoticmicroglia deservesspecial attention since they have been described as highly proinflammatory and immunogenic. Necroptosis, a particular form of cell death, can be induced in response to different cellular stimuli, including TNF- α , FAS ligand, TRAIL, IFN γ , ischemia-reperfusion injury, and double-stranded RNA (dsRNA). Caspase-8 and receptor interacting protein kinases (RIPK1 and RIPK3) are essential to engage the necrosome assembly in order to activate necroptosis. Depending on the experimental settings, RIPK1 may repress or induce necroptosis [ 91 ]. At the same time, necroptosis can follow a dependent or independent RIPK1 pathway. In either case, activation of RIPK3 is always involved. RIPK3 activation phosphorylates and activates mixed lineage kinase domain-like pseudokinase (MLKL), a typical feature of necroptosis. Activated MLKL causes destabilization of plasma membrane and cell death [ 91 , 92 ]. It has long been assumed that necroptosis is highly proinflammatory because of the release of DAMPs. However, recent data support the view that necrosome formation may trigger a robust, cell death-independent, proinflammatory response [ 93 , 94 ]. Indeed, necroptosis has also been observed in primary microglia linked to TNF- α production [ 95 ]. The occurrence of physiological necroptopsis of microglia has been recently demonstrated [ 96 ]. Interestingly, the levels of RIPK1 are increased in brain samples from individuals with LOAD and positively correlate with Braak stages [ 97 , 98 ], thus, supporting a major role of necroptosis in neurodegenerative diseases. Evidence from these studies and those provided by AD mice models suggest necroptotic microglia as one of the key players leading to neuroinflammation and neurodegeneration [99]. 3.4. Dark Microglia Tremblay and colleagues identified a distinctive microglia subtype associated with the aging process, chronic stress, and AD and referred it as dark microglia (DM). This specific microglia subtype exhibits typical features of oxidative stress processes like condensed, electron-dense cytoplasm and nucleoplasm and endoplasmic reticulum dilation, giving them a “dark” appearance at the electron microscopy [ 100 ]. Although the physiological significance of DM has yet to be determined, they are thought to be extremely active, exhibiting highly ramified and extremely thin processes [ 100 ]. These processes are often associated with synaptic clefts, encircling axon terminals, dendritic spines, and entire excitatory synapses processes [ 100 ]. DM are also abundant during postnatal development, when microglia diversity is especially evident [ 101 ], and highly express Cd11b, which together with CD68, form the complement receptor 3 involved in synaptic pruning [ 101 ]. In the APP/PS1 mice, DM are associated with A β plaques and express TREM2, thus, raising the possibility that DAM and DM might be somehow related. Indeed, DM are associated to neuronal dystrophies [ 100 ]. Both DAM and DM downregulate homeostatic genes like Cx3cr1 and P2ry12. However, DM fail to express Cd11c, while DAM highly upregulate this marker [ 57 ]. Whether DM derive DM in APP/PS1 mice (about two-thirds of total microglia) [ 100 ], and the extremely high oxidative stress status of this subtype, highlights the potential contribution of microglia-derived free radicals in AD pathology. 4. Microglial Contribution to Oxidative Stress 4.1. The Basis: Free Radical Generation in Biological Systems Oxygen can be viewed as a double-edged sword as we need it for energy generation but oxygen-derived free radicals may harm cells too. The superoxide anion radical (O 2•− ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical ( • OH) are regular products of respiration and oxidation processes in an aerobic environment [ 102 ]. These reactive molecules and free radicals are named as ROS. At the same time, reactive nitrogen species (RNS) are a similar collective term that includes J. Clin. Med. 2019,8, 1719 16 of 32 Table 1. Clinical trials using anti-inflammatory/antioxidant drugs for the prevention or improvement of neurodegenerative diseases. Disease Compound Effects Reference Huntington´s disease Minocycline Trial in progress CN-01508775 Laquinimod Trial in progress CN-01364776 AZD3241 Trial in progress [188] Riluzole Trial in progress CN-01513126 Antioxidants Trials in progress CN-00120692;CN-00150820 Fenifibrate Trial in progress CN-01574117 Memantine Trial in progress CN-01533522 Creatin Trial in progress NCT00592995 Parkinson’s disease Green tea Trial in progress NCT00461942 Polyphenols Trial in progress NCT00461942 NSAIDs May reduce the risk of developing PD [189] Q10 coenzyme Trial in progress NCT00076492 Pramipexole Improved motor impairments and disability [190] Pergolide Improved motor impairments and disability [191] Bromocriptine No prevention in the onset of motor complications [192] Cabergoline Improved motor impairments and disability [193] Ropinirole Improved motor impairments and disability [194] MAO B inhibitors Reduced rate of motor fluctuations [195] Minocycline Phase II trial [196] Polyphenols Trials in progress NCT01001637; NCT00743743; NCT00164749 Vitamin E No evidence [197] Nicotine No results available [198] Piracetam No benefit was shown [199] Vitamins and minerals Evidence very limited [200] Masitinib Trial in progress CN-01867921 NSAIDs Sparing effect ranging between 36% and 80% [201–203] Minocycline Trial in progress CN-01797324; CN-01847636 Multiple sclerosis Minocycline Decreased risk of conversion from a clinically isolate syndrome to multiple sclerosis [204] Statins No convincing evidence [205] Dimethyl Fumarate Moderate-quality evidence of decreased relapses frequency [206] Laquinimod Low-level evidence as a disease-modifying therapy [207] Amantadine Poorly documented [208] Fingolimod Reduced inflammatory disease activity [209] Vitamin D No benefit [210] Carnitine Insufficient evidence [211] PUFAs Reduced relapses frequency [212]; CN-01579873 Lipoic acid Trials in progress CN-01518782; CN-01878397; CN-01266929 Epigallocatechin gallate Trial in progress CN-01794896 Q10 coenzyme Trials in progress CN-00866966; CN-01136521 Melatonin Trial in progress CN-01114089 Crocin Trial in progress CN-01836077 Curcumin Trial in progress N-01896018 Alpha-tocopherol Trial in progress CN-00912436 ω3 fatty acids Trial in progress CN-01650553 Fish oil Trial in progress CN-00982838 Creatin Trial in progress CN-01502285 NSAIDs Trial in progress CN-01010452 Amyotrophic lateral sclerosis Vitamin E Trial in progress CN-00442211 Creatin No effect on survival [213] Minocyclin Trials in progress CN-01616430; CN-01259752; CN-01381473 CN-00730827 Riluzole Probably prolongs survival [214] Celecoxib Trial in progress CN-00566124 Curcumin Might improve survival probability [215] Antioxidants Insufficient evidence of efficacy [216] Some of these compounds are specifically included in clinical trials due to their anti-inflammatory or antioxidative properties. Abbreviations: MAO B, monoamine oxidase B; NSAIDs, nonsteroidal anti-inflammatory drugs; PUFAs, polyunsaturated fatty acids. Currently, there are clinical trials using several natural antioxidants, such as resveratrol, vitamin D and E, polyphenols, lipoic acid, or Q10 coenzyme, among others. For instance, dietary regimes with high polyunsaturated fatty acid intake seem to reduce the frequency of relapses over two years in MS patients, although this intervention has no major effect on the main clinical outcome of the disease [212]. Another interesting compound is creatine. It is known that creatine improves oxidative stress, glutamatergic excitotoxicity, and apoptosis in both in vitro and in vivo models [ 217 ]. Since these J. Clin. Med. 2019,8, 1719 17 of 32 mechanisms are involved in several neurodegenerative diseases, creatine supplementation has received a lot of attention as a neuroprotective strategy, with several promising studies in animal models of neurodegenerative diseases. This has led to a number of randomized clinical trials with oral creatine supplementation in patients with PD, HD, MS, and ALS. Laquinimod is an orally active immunomodulator that downregulates proinflammatory cytokine production in peripheral blood mononuclear cells. In the brain, laquinimod supresses astrocytic and microglial activation by modulating NF κ B signaling [ 218 ]. It has been demonstrated that this compound has dual properties of immunomodulation and neuroprotection and is a potentially promising new oral disease-modifying therapy in clinical trials with MS and HD patients. Moreover, epidemiological studies have consistently demonstrated that incidence of idiopathic AD and PD is about 50% lower in chronic users of nonsteroidal anti-inflammatory drugs (NSAIDs) or COX inhibitors than in age-matched nonusers [219,220]. Unfortunately, most of the clinical trials are not showing encouraging results, either because of the limitations of the studies or because of the poor efficacy of the proposed treatments. This low effectiveness could also be explained because they are being used at late stages of the neurodegenerative diseases. Another compound with anti-inflammatory and antioxidant properties that deserves special attentionisminocycline(7-dimethylamino-6-dimethyl-6-deoxytetracycline). Thisisasecondgeneration, semi-synthetic tetracycline analogue active against a wide range of aerobic and anaerobic Gram-positive and Gram-negative bacteria [ 221 ]. This antibiotic has been mainly used for over 30 years in the treatment of acne vulgaris and some sexually transmitted diseases. Moreover, minocycline shows a good pharmacokinetic profile, a good safety record, and can easily pass through the blood–brain barrier due to its high lipophilic properties. Most interestingly, besides its antibioticeffect, minocycline hasbeen shownto have otherproperties including anti-inflammatory and antiapoptotic activities and inhibitory effects on proteolysis, tumor metastasis, and angiogenesis [ 222 ]. All these reasons have made minocycline a good candidate to treat diseases with an inflammatory basis, including rosacea, bullous dermatoses, neurophilic diseases, and sarcoidosis, among others [223–225]. Several proposed mechanisms of action of minocycline may be involved in its anti-inflammatory and antioxidant effects. These include: (i) inhibitory effects on the activities of key enzymes like iNOS and PLA2; (ii) reduction of protein tyrosine nitration because of its peroxynitrite-scavenging properties; (iii) inhibition of caspase-1 and caspase-3 activation; (iv) enhancement of BCL-2-derived effects, thus protecting the cells against apoptosis; (v) reduction of p38 MAPK phosphorylation; and (vi) inhibition of PARP-1 activity. The well-known ability of tetracycline to bind Ca 2+ and Mg 2+ may account for some of these biological activities, via the chelation of these cations and their transport into intracellular compartments (reviewed in [222]). Taking into account the above mentioned anti-inflammatory/antioxidant properties of minocycline and its capacity to cross the blood–brain barrier, it could be useful in the treatment of neurodegenerative diseases in which inflammation and oxidative stress play a central role, such as PD, AD, MS, HD, or ALS. Therefore, minocycline has been used in several in vivo and in vitro models of these diseases with promising results [ 226 – 228 ], which has made this drug emerge as an attractive strategy to treat these neurological disorders. Many clinical trials have been then performed in an effort to prove the efficacy of this treatment in the clinic (see Table 1). Hence, the potential neuroprotective activity of minocycline is currently undergoing phase II or III trial testing in AD, HD, PD, MS, and ALS patients [ 229 ]. Patients suffering these neurological disorders receive doses ranging from 200 to 400 mg/day; effectiveness, tolerability, and safety of these treatments are being studied for 6 to 24 months. These clinical trials are showing some promising results. Hence, the risk of conversion from a clinically isolated syndrome to MS was significantly lower with minocycline than with placebo, suggesting that minocycline will be considered for definitive phase III trials to determine if it alters the long-term progression of neurodegenerative J. Clin. Med. 2019,8, 1719 18 of 32 diseases. For all these reasons, minocycline seems to be one of the most promising molecules for the treatment of neurodegenerative diseases, expected to give good results in the near future. However, factors such as safety, tolerability, activity, cost, and availability of this drug must be considered when comparing minocycline with other agents currently used in neurodegenerative diseases. 8. Conclusions The identification of multiple microglia subtypes has certainly changed our view about their protective/deleterious roles in neurodegenerative diseases. The recent identification of DAM microglia shouldopen newresearchstrategiesaimedatidentifyingdifferent(evencontradictory)rolesofmicroglia under disease conditions. Two microglia receptors, TLRs and TREM2, drive main polarization states, proinflammatory and DAM. Ligands of these receptors fit well with the classical DAMPs, supported by the recent discovery of Lgals3 (GAL3), a DAM gene highly upregulated in microglia clustering A β plaques. Other examples of multivalent ligands are APOE and A β itself through its different conformational states. Consequently, a cross-talk between these two main microglia polarization states is expected to increase heterogeneity. Different DAM subtypes are actually supported by WGCNA, including the existence of pro-oxidant DAM. In summary, the unique plasticity of microglia upon different kinds of stimuli has become a great challenge in the field. To uncover the broad panel of polarization states of microglia that may exist and/or coexist during the onset and progression of different neurodegenerative disorders is an unmet clinical need that may help to develop novel immunotherapeutic strategies. Author Contributions: Conceptualization, J.L.V., R.M.P., M.S., M.A.B., R.R., A.J.H., and J.G.-R.; writing, review and editing, I.A.A.-B., A.M.E.-O., L.-C.-H., I.-G.-D., M.A.R.-C., J.A.R.-G., M.S.S.; supervision, J.L.V. All the authors have read and approved the final version of the manuscript. Funding: This research and APC were funded by the Spanish Ministerio de Ciencia, Innovaci ó n y Universidades/FEDER/UE RTI2018-098645-B-100. J.G.-R. and I.G.-D. were funded by predoctoral fellowships from Spanish Government. M.A.B was funded by the Spanish Ministerio de Economia y Competitividad (Programa Ram ó n y Cajal: RYC-2017-21804). MSS was funded by a Marie-Sklodowska Curie Fellowship H2020-MSCA-IF-2017-795695. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Box A1. Tools for studying oxidative stress in vivo/in vitro. Thecriticalrelevanceofoxidativestressinneurodegenerativediseasesexposestheneedforreliablebiomarkers of redox status to monitor disease stages and progression [230]. Biomarkers and methods for redox status monitorization can be divided in two groups: (a) DIRECT METHODS The direct quantification of ROS/RNS may reflect the pathological process in some diseases, but the natural high reactivity and short lifespan of the species make its direct measurement extremely difficult in the pathogenic focus [231] and is currently limited to in vitro applications. Direct methods include electron spin resonance, fluorescence magnetic resonance, flow cytometry, mass spectrometry techniques, or chemiluminiscent probes [232–234]. (b) INDIRECT METHODS Indirect methods are based on the different products of the reactions of ROS/RNS with the main biomolecules: proteins, lipids, and nucleic acids. (i) Protein oxidation 3. Nitrotyrosine 3-nitrotyrosine (3-NT) can be obtained mainly from tyrosine residues in proteins through the chemical conversion by tetranitromethane and peroxynitrite [235,236]. J. Clin. Med. 2019,8, 1719 19 of 32 Box A1. Cont. The methods used for detection include immunochemical and chromatographic procedures, including liquid chromatography using ultraviolet, fluorescence, and electrochemical detection and variants like liquid chromatography–mass spectrometry (LC–MS) or gas chromatography–mass spectrometry (GC–MS) [236]. Currently, the most used methods for measuring 3-NT are immunochemical methods including ELISA because it is a cheap, simple, and suitable assay [237]. Glutathione The tripeptide GSH is synthesized in two steps from the amino acids glutamate, cysteine, and glycine, catalyzed by the enzyme glutamate cysteine ligase. GSH is used as a biomarker because it is the most abundant endogenous intracellular antioxidant, so any condition associated with excessive ROS will decrease GSH levels or the GSH/GSSG ratio [238]. In addition, GSH is able to react with a reactive cysteine residue and form a disulphide bridge, giving rise to GSSG, which is also used as a biomarker [239]. Although there are several widely used methods to measure GSH and GSSG levels, like Western blotting, MS techniques, ELISA, or enzyme assays, the most sensitive method is high performance liquid chromatography (HPLC) [240,241]. Protein carbonyls This biomarker can be detected after its derivatization, resulting in a stable product (DNP) [ 242 ]. To measure this biomarker, HPLC, proteomic techniques [ 243 ], and, for more specificity, techniques based on the use of anti-DNP antibodies, like ELISA or Western blot [244] could be used. Advanced glycation end products (AGEs): The AGEs are proteins or lipids that become glycated as a result of exposure to sugars [ 244 ]. The most used methods to identify these adducts are mass spectrometry-based techniques [ 245 ], immune-based methods, or HPLC [246]. Advanced oxidation protein products (AOPP) AOPPs are created during oxidative stress through the reaction of plasma proteins with chlorinated oxidants. 3-chloro-tyrosine is one of the main products of the reaction [ 231 ] and could be detected by mass spectrometry [247,248]. (ii) Lipid oxidation Malondialdehyde and 4-hydroxy-2-nonenal Malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) are the best-known end products of lipid oxidation [ 249 ]. To detect HNE directly, HPLC, GC-MS, or immunological techniques using specific anti-HNE antibodies [ 249 ] could be used. On the other hand, MDA could react with thiobarbituric acid to give an adduct, which can be measured by colorimetric or fluorimetric assays. However, HPLC combined with UV or fluorescence is normally selected for its measure [250]. F2 isoprostanes and isolevuglandins Isoprostanes (IsoPs) are a family of stable prostaglandin-like compounds generated by the free radical-induced peroxidation of arachidonic acid [ 251 ]. The most useful as biomarkers are F2-IsoPs. HPLC/GC-MS is the most reliable method for its detection [252]. Isolevuglandins (IsoLGs) are products from the oxidation of arachidonic acid. They can be measured owing to structural modifications through liquid chromatography/tandem mass spectrometry methods (LC/MS/MS) [ 253 ]. (iii) Nucleic Acids oxidation Nucleic acids are highly susceptible to oxidation and by this process oxidizing agents generate many modifications in DNA and RNA. If these modifications are not repaired, they could potentially lead to mutagenesis [ 254 ]. The main mutagenic lesions of nucleic acids caused by free radical are the 8-oxo-7, 8-dihydroguanine (8-oxoGua), and 8-oxo-7, 8-dihydroguanosine (8-oxodG) [231]. The levels of 8-oxoGua and 8-oxodG can be measured by HPLC-MS/MS, but limitations of this technique make the evaluation of low damage levels not reliable [ 254 , 255 ]. In contrast, ELISA assays are useful for measuring low levels of damage, but these methods are less specific compared to HPLC/MS-MS [ 256 , 257 ]. Overall, a more reliable method to measure low levels of damage relies on enzymatic methodology, which uses excision DNA repair enzymes formamidopyrimidine DNA glycosylase (Fpg), endonuclease III (EndoIII), or 8-hydroxyguanine glycosylase 1 (hOGG1) [230,255]. J. Clin. Med. 2019,8, 1719 20 of 32 References 1. Del Rio-Hortega, P. El tercer elemento de los centros nerviosos. Bol. Soc. Esp. Biol. 1919,9, 69–120. 2. Del Rio-Hortega, P. The microglia. Lancet 1939,1, 1023–1026. [CrossRef] 3. Alliot, F.; Godin, I.; Pessac, B. Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain. Dev. Brain Res. 1999,117, 145–152. [CrossRef] 4. Kierdorf, K.; Erny, D.; Goldmann, T.; Sander, V.; Schulz, C.; Perdiguero, E.G.; Wieghofer, P.; Heinrich, A.; Riemke, P.; Hölscher, C.; et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat. Neurosci. 2013,16, 273–280. [CrossRef] 5. Ginhoux, F.; Lim, S.; Hoeffel, G.; Low, D.; Huber, T. Origin and differentiation of microglia. Front. Cell. Neurosci. 2013,7, 45. [CrossRef] 6. Wolf, S.A.; Boddeke, H.; Kettenmann, H. Microglia in Physiology and Disease. Annu. Rev. Physiol. 2017 ,79, 619–643. [CrossRef] 7. Shen, X.; Venero, J.L.; Joseph, B.; Burguillos, M.A. Caspases orchestrate microglia instrumental functions. Prog. Neurobiol. 2018,171, 50–71. [CrossRef] 8. Verheijen, J.; Sleegers, K. Understanding Alzheimer Disease at the Interface between Genetics and Transcriptomics. Trends Genet. 2018,34, 434–447. [CrossRef] 9. Hammond, T.R.; Marsh, S.E.; Stevens, B. Immune Signaling in Neurodegeneration. Immunity 2019 ,50, 955–974. [CrossRef] 10. Zhang, B.; Gaiteri, C.; Bodea, L.G.; Wang, Z.; McElwee, J.; Podtelezhnikov, A.A.; Zhang, C.; Xie, T.; Tran, L.; Dobrin, R.; et al. Integrated systems approach identifies genetic nodes and networks in late-onset Alzheimer’s disease. Cell 2013,153, 707–720. [CrossRef] 11. Hamerman, J.A.; Ni, M.; Killebrew, J.R.; Chu, C.L.; Lowell, C.A. The expanding roles of ITAM adapters FcRgamma and DAP12 in myeloid cells. Immunol. Rev. 2009,232, 42–58. [CrossRef] [PubMed] 12. Gratuze, M.; Leyns, C.E.G.; Holtzman, D.M. New insights into the role of TREM2 in Alzheimer’s disease. Mol. Neurodegener. 2018,13, 66. [CrossRef] [PubMed] 13. Ulland, T.K.; Colonna, M. TREM2—A key player in microglial biology and Alzheimer disease. Nat. Rev. Neurol. 2018,14, 667–675. [CrossRef] [PubMed] 14. Guerreiro, R.; Wojtas, A.; Bras, J.; Carrasquillo, M.; Rogaeva, E.; Majounie, E.; Cruchaga, C.; Sassi, C.; Kauwe, J.S.; Younkin, S.; et al. TREM2 variants in Alzheimer’s disease. N. Engl. J. Med. 2013 ,368, 117–127. [CrossRef] [PubMed] 15. Jonsson, T.; Stefansson, H.; Steinberg, S.; Jonsdottir, I.; Jonsson, P.V.; Snaedal, J.; Bjornsson, S.; Huttenlocher, J.; Levey, A.I.; Lah, J.J.; et al. Variant of TREM2 associated with the risk of Alzheimer’s disease. N. Engl. J. Med. 2013,368, 107–116. [CrossRef] [PubMed] 16. Kumar, V. Toll-like receptors in the pathogenesis of neuroinflammation. J. Neuroimmunol. 2019 ,332, 16–30. [CrossRef] [PubMed] 17. Fiebich, B.L.; Batista, C.R.A.; Saliba, S.W.; Yousif, N.M.; De Oliveira, A.C.P. Role of Microglia TLRs in Neurodegeneration. Front. Cell. Neurosci. 2018,12, 329. [CrossRef] 18. Kawasaki, T.; Kawai, T. Toll-like receptor signaling pathways. Front. Immunol. 2014,5, 461. [CrossRef] 19. Lehnardt, S. Innate immunity and neuroinflammation in the CNS: The role of microglia in Toll-like receptor-mediated neuronal injury. Glia 2010,58, 253–263. [CrossRef] 20. Okun, E.; Griffioen, K.J.; Mattson, M.P. Toll-like receptor signaling in neural plasticity and disease. Trends Neurosci. 2011,34, 269–281. [CrossRef] 21. Jana, M.; Palencia, C.A.; Pahan, K. Fibrillar amyloid-beta peptides activate microglia via TLR2: Implications for Alzheimer’s disease. J. Immunol. 2008,181, 7254–7262. [CrossRef] [PubMed] 22. Kim, C.; Ho, D.H.; Suk, J.E.; You, S.; Michael, S.; Kang, J.; Lee, S.J.; Masliah, E.; Hwang, D.; Lee, H.J.; et al. Neuron-released oligomeric α -synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia. Nat. Commun. 2013,4, 1562. [CrossRef] [PubMed] 23. Agalave, N.M.; Larsson, M.; Abdelmoaty, S.; Su, J.; Baharpoor, A.; Lundbäck, P.; Palmblad, K.; Andersson, U.; Harris, H.; Svensson, C.I. Spinal HMGB1 induces TLR4-mediated long-lasting hypersensitivity and glial activation and regulates pain-like behavior in experimental arthritis. Pain 2014 ,155, 1802–1813. [CrossRef] [PubMed] J. Clin. Med. 2019,8, 1719 21 of 32 24. Burguillos, M.A.; Svensson, M.; Schulte, T.; Boza-Serrano, A.; Garcia-Quintanilla, A.; Kavanagh, E.; Santiago, M.; Viceconte, N.; Oliva-Martin, M.J.; Osman, A.M.; et al. Microglia-Secreted Galectin-3 Acts as a Toll-like Receptor 4 Ligand and Contributes to Microglial Activation. Cell Rep. 2015 ,10, 1626–1638. [CrossRef] 25. Yip, P.K.; Carrillo-Jimenez, A.; King, P.; Vilalta, A.; Nomura, K.; Chau, C.C.; Egerton, A.M.S.; Liu, Z.H.; Shetty, A.J.; Tremoleda, J.L.; et al. Galectin-3 released in response to traumatic brain injury acts as an alarmin orchestrating brain immune response and promoting neurodegeneration. Sci. Rep. 2017 ,7, 41689. [CrossRef] 26. Heneka, M.T.; Kummer, M.P.; Stutz, A.; Delekate, A.; Schwartz, S.; Vieira-Saecker, A.; Griep, A.; Axt, D.; Remus, A.; Tzeng, T.C.; et al. NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature 2013,493, 674–678. [CrossRef] 27. DeLeo, F.R.; Renee, J.; McCormick, S.; Nakamura, M.; Apicella, M.; Weiss, J.P.; Nauseef, W.M. Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J. Clin. Investig. 1998 ,101, 455–463. [CrossRef] 28. El-Benna, J.; Dang, P.M.C.; Gougerot-Pocidalo, M.A.; Elbim, C. Phagocyte NADPH oxidase: A multicomponent enzyme essential for host defenses. Arch. Immunol. Ther. Exp. 2005,53, 199–206. 29. Qian, L.; Wei, S.J.; Zhang, D.; Hu, X.; Xu, Z.; Wilson, B.; El-Benna, J.; Hong, J.S.; Flood, P.M. Potent anti-inflammatory and neuroprotective effects of TGF-beta1 are mediated through the inhibition of ERK and p47phox-Ser345 phosphorylation and translocation in microglia. J. Immunol. 2008 ,181, 660–668. [CrossRef] 30. Shimohama, S.; Tanino, H.; Kawakami, N.; Okamura, N.; Kodama, H.; Yamaguchi, T.; Hayakawa, T.; Nunomura, A.; Chiba, S.; Perry, G.; et al. Activation of NADPH Oxidase in Alzheimer’s Disease Brains. Biochem. Biophys. Res. Commun. 2000,273, 5–9. [CrossRef] 31. Wu, D.C.; Teismann, P.; Tieu, K.; Vila, M.; Jackson-Lewis, V.; Ischiropoulos, H.; Przedborski, S. NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease. Proc. Natl. Acad. Sci. USA 2003,100, 6145–6150. [CrossRef] [PubMed] 32. Zhang, W.; Wang, T.; Qin, L.; Gao, H.M.; Wilson, B.; Ali, S.F.; Hong, J.S.; Liu, B. Neuroprotective effect of dextromethorphan in the MPTP Parkinson’s disease model: Role of NADPH oxidase. FASEB J. 2004 ,18, 589–591. [CrossRef] [PubMed] 33. Valencia, A.; Sapp, E.; Kimm, J.S.; McClory, H.; Reeves, P.B.; Alexander, J.; Ansong, K.A.; Masso, N.; Frosch, M.P.; Kegel, K.B.; et al. Elevated NADPH oxidase activity contributes to oxidative stress and cell death in Huntington’s disease. Hum. Mol. Genet. 2013,22, 1112–1131. [CrossRef] 34. Ch é ret, C.; Gervais, A.; Lelli, A.; Colin, C.; Amar, L.; Ravassard, P.; Mallet, J.; Cumano, A.; Krause, K.H.; Mallat,M.NeurotoxicActivationofMicrogliaIsPromotedbyaNox1-DependentNADPHOxidase. J. Neurosci. 2008,28, 12039–12051. [CrossRef] 35. Miletic, A.V.; Graham, D.B.; Montgrain, V.; Fujikawa, K.; Kloeppel, T.; Brim, K.; Weaver, B.; Schreiber, R.; Xavier, R.; Swat, W. Vav proteins control MyD88-dependent oxidative burst. Blood 2006 ,109, 3360–3368. [CrossRef] 36. Bedard, K.; Krause, K.H. The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology. Physiol. Rev. 2007,87, 245–313. [CrossRef] [PubMed] 37. Nakahira, K.; Kim, H.P.; Geng, X.H.; Nakao, A.; Wang, X.; Murase, N.; Drain, P.F.; Wang, X.; Sasidhar, M.; Nabel,E.G.; etal. CarbonmonoxidedifferentiallyinhibitsTLRsignalingpathwaysbyregulating ROS-induced trafficking of TLRs to lipid rafts. J. Exp. Med. 2006,203, 2377–2389. [CrossRef] [PubMed] 38. Wong, S.W.; Kwon, M.J.; Choi, A.M.K.; Kim, H.P.; Nakahira, K.; Hwang, D.H. Fatty Acids Modulate Toll-like Receptor 4 Activation through Regulation of Receptor Dimerization and Recruitment into Lipid Rafts in a Reactive Oxygen Species-dependent Manner. J. Biol. Chem. 2009,284, 27384–27392. [CrossRef] [PubMed] 39. Matsuzawa, A.; Saegusa, K.; Noguchi, T.; Sadamitsu, C.; Nishitoh, H.; Nagai, S.; Koyasu, S.; Matsumoto, K.; Takeda, K.; Ichijo, H. ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat. Immunol. 2005,6, 587–592. [CrossRef] [PubMed] 40. Lao, Y.; Yang, K.; Wang, Z.; Sun, X.; Zou, Q.; Yu, X.; Cheng, J.; Tong, X.; Yeh, E.T.H.; Yang, J.; et al. DeSUMOylation of MKK7 kinase by the SUMO2/3 protease SENP3 potentiates lipopolysaccharide-induced inflammatory signaling in macrophages. J. Biol. Chem. 2018,293, 3965–3980. [CrossRef] 41. Cannon, J.P.; O’Driscoll, M.; Litman, G.W. Specific lipid recognition is a general feature of CD300 and TREM molecules. Immunogenetics 2012,64, 39–47. [CrossRef] [PubMed] J. Clin. Med. 2019,8, 1719 22 of 32 42. Daws, M.R.; Sullam, P.M.; Niemi, E.C.; Chen, T.T.; Tchao, N.K.; Seaman, W.E. Pattern recognition by TREM-2: Binding of anionic ligands. J. Immunol. 2003,171, 594–599. [CrossRef] [PubMed] 43. Wang, Y.; Cella, M.; Mallinson, K.; Ulrich, J.D.; Young, K.L.; Robinette, M.L.; Gilfillan, S.; Krishnan, G.M.; Sudhakar, S.; Zinselmeyer, B.H.; et al. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 2015,160, 1061–1071. [CrossRef] [PubMed] 44. Zhou, S.L.; Tan, C.C.; Hou, X.H.; Cao, X.P.; Tan, L.; Yu, J.T. TREM2 Variants and Neurodegenerative Diseases: A Systematic Review and Meta-Analysis. J. Alzheimer’s Dis. 2019,68, 1171–1184. [CrossRef] 45. Jay, T.R.; Von Saucken, V.E.; Landreth, G.E. TREM2 in Neurodegenerative Diseases. Mol. Neurodegener. 2017 , 12, 56. [CrossRef] 46. Yeh, F.L.; Hansen, D.V.; Sheng, M. TREM2, Microglia, and Neurodegenerative Diseases. Trends Mol. Med. 2017,23, 512–533. [CrossRef] 47. Tang, Y.; Le, W. Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases. Mol. Neurobiol. 2016,53, 1181–1194. [CrossRef] 48. Block, M.L.; Zecca, L.; Hong, J.S. Microglia-mediated neurotoxicity: Uncovering the molecular mechanisms. Nat. Rev. Neurosci. 2007,8, 57–69. [CrossRef] 49. Cherry, J.D.; Olschowka, J.A.; O’Banion, M.K. Neuroinflammation and M2 microglia: The good, the bad, and the inflamed. J. Neuroinflamm. 2014,11, 98. 50. Ransohoff, R.M. A polarizing question: Do M1 and M2 microglia exist? Nat. Neurosci. 2016 ,19, 987–991. [CrossRef] 51. Krasemann, S.; Madore, C.; Cialic, R.; Baufeld, C.; Calcagno, N.; El Fatimy, R.; Beckers, L.; O’Loughlin, E.; Xu, Y.; Fanek, Z.; et al. The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity 2017,47, 566–581. [CrossRef] [PubMed] 52. Sousa, C.; Golebiewska, A.; Poovathingal, S.K.; Kaoma, T.; Pires-Afonso, Y.; Martina, S.; Coowar, D.; Azuaje, F.; Skupin, A.; Balling, R.; et al. Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures. EMBO Rep. 2018,19, e46171. [CrossRef] [PubMed] 53. Butovsky, O.; Jedrychowski, M.P.; Moore, C.S.; Cialic, R.; Lanser, A.J.; Gabriely, G.; Koeglsperger, T.; Dake, B.; Wu, P.M.; Doykan, C.E.; et al. Identification of a unique TGF- β -dependent molecular and functional signature in microglia. Nat. Neurosci. 2014,17, 131–143. [CrossRef] [PubMed] 54. Hammond, T.R.; Dufort, C.; Dissing-Olesen, L.; Giera, S.; Young, A.; Wysoker, A.; Walker, A.J.; Gergits, F.; Segel, M.; Nemesh, J.; et al. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity 2019,50, 253–271. [CrossRef] [PubMed] 55. Masuda, T.; Sankowski, R.; Staszewski, O.; Böttcher, C.; Amann, L.; Sagar; Scheiwe, C.; Nessler, S.; Kunz, P.; Van Loo, G.; et al. Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution. Nature 2019,566, 388–392. [CrossRef] 56. Holtman, I.R.; Raj, D.D.; Miller, J.A.; Schaafsma, W.; Yin, Z.; Brouwer, N.; Wes, P.D.; Möller, T.; Orre, M.; Kamphuis, W.; et al. Induction of a common microglia gene expression signature by aging and neurodegenerative conditions: A co-expression meta-analysis. Acta Neuropathol. Commun. 2015 ,3, 31. [CrossRef] 57. Keren-Shaul, H.; Spinrad, A.; Weiner, A.; Matcovitch-Natan, O.; Dvir-Szternfeld, R.; Ulland, T.K.; David, E.; Baruch,K.; Lara-Astaiso,D.; Toth, B.; etal. A UniqueMicroglia Type AssociatedwithRestricting Development of Alzheimer’s Disease. Cell 2017,169, 1276–1290. [CrossRef] 58. Mathys, H.; Adaikkan, C.; Gao, F.; Young, J.Z.; Manet, E.; Hemberg, M.; De Jager, P.L.; Ransohoff, R.M.; Regev, A.; Tsai, L.H. Temporal Tracking of Microglia Activation in Neurodegeneration at Single-Cell Resolution. Cell Rep. 2017,21, 366–380. [CrossRef] 59. Jord ã o, M.J.C.; Sankowski, R.; Brendecke, S.M.; Sagar; Locatelli, G.; Tai, Y.H.; Tay, T.L.; Schramm, E.; Armbruster, S.; Hagemeyer, N.; et al. Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 2019,363. [CrossRef] 60. Matarin, M.; Salih, D.A.; Yasvoina, M.; Cummings, D.M.; Guelfi, S.; Liu, W.; Solim, M.A.N.; Moens, T.G.; Paublete, R.M.; Ali, S.S.; et al. A Genome-wide Gene-Expression Analysis and Database in Transgenic Mice during Development of Amyloid or Tau Pathology. Cell Rep. 2015,10, 633–644. [CrossRef] 61. Leyns, C.E.G.; Ulrich, J.D.; Finn, M.B.; Stewart, F.R.; Koscal, L.J.; Serrano, J.R.; Robinson, G.O.; Anderson, E.; Colonna, M.; Holtzman, D.M. TREM2 deficiency attenuates neuroinflammation and protects against J. Clin. Med. 2019,8, 1719 23 of 32 neurodegeneration in a mouse model of tauopathy. Proc. Natl. Acad. Sci. USA 2017 ,114, 11524–11529. [CrossRef] [PubMed] 62. Hickman, S.; Izzy, S.; Sen, P.; Morsett, L.; El Khoury, J. Microglia in neurodegeneration. Nat. Neurosci. 2018, 21, 1359–1369. [CrossRef] [PubMed] 63. Nalls, M.A.; Pankratz, N.; Lill, C.M.; Do, C.B.; Hernandez, D.G.; Saad, M.; DeStefano, A.L.; Kara, E.; Bras, J.; Sharma, M.; et al. Large-scale meta-analysis of genome-wide association data identifies six new risk loci for Parkinson’s disease. Nat. Genet. 2014,46, 989–993. [CrossRef] 64. Ripoll, V.M.; Irvine, K.M.; Ravasi, T.; Sweet, M.J.; Hume, D.A. Gpnmb Is Induced in Macrophages by IFN- γ and Lipopolysaccharide and Acts as a Feedback Regulator of Proinflammatory Responses. J. Immunol. 2007 , 178, 6557–6566. [CrossRef] [PubMed] 65. Yu, B.; Alboslemy, T.; Safadi, F.; Kim, M.H. Glycoprotein Nonmelanoma Clone B Regulates the Crosstalk between Macrophages and Mesenchymal Stem Cells toward Wound Repair. J. Investig. Dermatol. 2017 ,138, 219–227. [CrossRef] [PubMed] 66. Zhou, L.; Zhuo, H.; Ouyang, H.; Liu, Y.; Yuan, F.; Sun, L.; Liu, F.; Liu, H. Glycoprotein non-metastatic melanoma protein b (Gpnmb) is highly expressed in macrophages of acute injured kidney and promotes M2 macrophages polarization. Cell. Immunol. 2017,316, 53–60. [CrossRef] [PubMed] 67. Budge, K.M.; Neal, M.L.; Richardson, J.R.; Safadi, F.F. Glycoprotein NMB: An Emerging Role in Neurodegenerative Disease. Mol. Neurobiol. 2017,55, 5167–5176. [CrossRef] [PubMed] 68. Li, B.; Castano, A.P.; Hudson, T.E.; Nowlin, B.T.; Lin, S.L.; Bonventre, J.V.; Swanson, K.D.; Duffield, J.S. The melanoma-associated transmembrane glycoprotein Gpnmb controls trafficking of cellular debris for degradation and is essential for tissue repair. FASEB J. 2010,24, 4767–4781. [CrossRef] [PubMed] 69. Metz, R.L.; Patel, P.S.; Hameed, M.; Bryan, M.; Rameshwar, P. Role of human HGFIN/nmb in breast cancer. Breast Cancer Res. 2007,9, R58. [CrossRef] 70. Zhong, L.; Chen, X.F.; Wang, T.; Wang, Z.; Liao, C.; Wang, Z.; Huang, R.; Wang, D.; Li, X.; Wu, L.; et al. Soluble TREM2 induces inflammatory responses and enhances microglial survival. J. Exp. Med. 2017 ,214, 597–607. [CrossRef] 71. Yuan, P.; Condello, C.; Keene, C.D.; Wang, Y.; Bird, T.D.; Paul, S.M.; Luo, W.; Colonna, M.; Baddeley, D.; Grutzendler, J. TREM2 Haplodeficiency in Mice and Humans Impairs the Microglia Barrier Function Leading to Decreased Amyloid Compaction and Severe Axonal Dystrophy. Neuron 2016 ,92, 252–264. [CrossRef] [PubMed] 72. Leyns, C.E.G.; Gratuze, M.; Narasimhan, S.; Jain, N.; Koscal, L.J.; Jiang, H.; Manis, M.; Colonna, M.; Lee, V.M.Y.; Ulrich, J.D.; et al. TREM2 function impedes tau seeding in neuritic plaques. Nat. Neurosci. 2019 , 22, 1217–1222. [CrossRef] [PubMed] 73. Kigerl, K.A.; Vaccari, J.P.D.R.; Dietrich, W.D.; Popovich, P.G.; Keane, R.W. Pattern recognition receptors and central nervous system repair. Exp. Neurol. 2014,258, 5–16. [CrossRef] [PubMed] 74. Deczkowska, A.; Keren-Shaul, H.; Weiner, A.; Colonna, M.; Schwartz, M.; Amit, I. Disease-Associated Microglia: A Universal Immune Sensor of Neurodegeneration. Cell 2018,173, 1073–1081. [CrossRef] 75. Zhao, Y.; Wu, X.; Li, X.; Jiang, L.L.; Gui, X.; Liu, Y.; Sun, Y.; Zhu, B.; Piña-Crespo, J.C.; Zhang, M.; et al. TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function. Neuron 2018,97, 1023–1031. [CrossRef] 76. Zhong, L.; Wang, Z.; Wang, D.; Martens, Y.A.; Wu, L.; Xu, Y.; Wang, K.; Li, J.; Huang, R.; Can, D.; et al. Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2). Mol. Neurodegener. 2018,13, 15. [CrossRef] 77. Yeh, F.L.; Wang, Y.; Tom, I.; Gonzalez, L.C.; Sheng, M. TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia. Neuron 2016 ,91, 328–340. [CrossRef] 78. Poliani, P.L.; Wang, Y.; Fontana, E.; Robinette, M.L.; Yamanishi, Y.; Gilfillan, S.; Colonna, M. TREM2 sustains microglial expansion during aging and response to demyelination. J. Clin. Investig. 2015 ,125, 2161–2170. [CrossRef] 79. Boza-Serrano, A.; Ruiz, R.; Sanchez-Varo, R.; Garc í a-Revilla, J.; Yang, Y.; Jimenez-Ferrer, I.; Paulus, A.; Wennström, M.; Vilalta, A.; Allendorf, D.; et al. Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease. Acta Neuropathol. 2019. [CrossRef] J. Clin. Med. 2019,8, 1719 24 of 32 80. Lalancette-Hebert, M.; Swarup, V.; Beaulieu, J.M.; Bohacek, I.; Abdelhamid, E.; Weng, Y.C.; Sato, S.; Kriz, J. Galectin-3 Is Required for Resident Microglia Activation and Proliferation in Response to Ischemic Injury. J. Neurosci. 2012,32, 10383–10395. [CrossRef] 81. Gale, S.C.; Gao, L.; Mikacenic, C.; Coyle, S.M.; Rafaels, N.; Dudenkov, T.M.; Madenspacher, J.H.; Draper, D.W.; Ge, W.; Aloor, J.J.; et al. APO ε 4 is associated with enhanced in vivo innate immune responses in human subjects. J. Allergy Clin. Immunol. 2014,134, 127–134. [CrossRef] [PubMed] 82. Doens, D.; Fernandez, P.L. Microglia receptors and their implications in the response to amyloid β for Alzheimer’s disease pathogenesis. J. Neuroinflamm. 2014,11, 48. [CrossRef] [PubMed] 83. Heneka, M.T.; Golenbock, D.T.; Latz, E. Innate immunity in Alzheimer’s disease. Nat. Immunol. 2015 ,16, 229–236. [CrossRef] [PubMed] 84. El Khoury, J.B.; Moore, K.J.; Means, T.K.; Leung, J.; Terada, K.; Toft, M.; Freeman, M.W.; Luster, A.D. CD36 Mediates the Innate Host Response to β-Amyloid. J. Exp. Med. 2003,197, 1657–1666. [CrossRef] 85. Rangaraju, S.; Dammer, E.B.; Raza, S.A.; Rathakrishnan, P.; Xiao, H.; Gao, T.; Duong, D.M.; Pennington, M.W.; Lah, J.J.; Seyfried, N.T.; et al. Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer’s disease. Mol. Neurodegener. 2018,13, 24. [CrossRef] 86. Landel, V.; Baranger, K.; Virard, I.; Loriod, B.; Khrestchatisky, M.; Rivera, S.; Benech, P.; F é ron, F. Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer’s disease. Mol. Neurodegener. 2014,9, 33. [CrossRef] 87. Linnartz-Gerlach, B.; Bodea, L.G.; Klaus, C.; Ginolhac, A.; Halder, R.; Sinkkonen, L.; Walter, J.; Colonna, M.; Neumann, H. TREM2 triggers microglial density and age-related neuronal loss. Glia 2019 ,67, 539–550. [CrossRef] 88. Klesney-Tait, J.; Turnbull, I.R.; Colonna, M. The TREM receptor family and signal integration. Nat. Immunol. 2006,7, 1266–1273. [CrossRef] 89. Painter, M.M.; Atagi, Y.; Liu, C.C.; Rademakers, R.; Xu, H.; Fryer, J.D.; Bu, G. TREM2 in CNS homeostasis and neurodegenerative disease. Mol. Neurodegener. 2015,10, 43. [CrossRef] 90. Li, J.T.; Zhang, Y. TREM2 regulates innate immunity in Alzheimer’s disease. J. Neuroinflamm. 2018 ,15, 107. [CrossRef] 91. Silke,J.; Rickard,J.A.; Gerlic,M.The diverserole ofRIPkinasesinnecroptosisandinflammation. Nat. Immunol. 2015,16, 689–697. [CrossRef] [PubMed] 92. Weinlich, R.; Oberst, A.; Beere, H.M.; Green, D.R. Necroptosis in development, inflammation and disease. Nat. Rev. Mol. Cell Biol. 2016,18, 127–136. [CrossRef] [PubMed] 93. Najjar, M.; Saleh, D.; Zelic, M.; Nogusa, S.; Shah, S.; Tai, A.; Finger, J.N.; Polykratis, A.; Gough, P.J.; Bertin, J.; et al. RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor. Immunity 2016,45, 46–59. [CrossRef] 94. Buchrieser, J.; Oliva-Martin, M.J.; Moore, M.D.; Long, J.C.D.; Cowley, S.A.; Perez-Sim ó n, J.A.; James, W.; Venero, J.L. RIPK1 is a critical modulator of both tonic and TLR-responsive inflammatory and cell death pathways in human macrophage differentiation. Cell Death Dis. 2018,9, 973. [CrossRef] [PubMed] 95. Fricker, M.; Vilalta, A.; Tolkovsky, A.M.; Brown, G.C. Caspase inhibitors protect neurons by enabling selective necroptosis of inflamed microglia. J. Biol. Chem. 2013,288, 9145–9152. [CrossRef] [PubMed] 96. Lloyd, A.F.; Davies, C.L.; Holloway, R.K.; Labrak, Y.; Ireland, G.; Carradori, D.; Dillenburg, A.; Borger, E.; Soong, D.; Richardson, J.C.; et al. Central nervous system regeneration is driven by microglia necroptosis and repopulation. Nat. Neurosci. 2019,22, 1046–1052. [CrossRef] 97. Caccamo, A.; Branca, C.; Piras, I.S.; Ferreira, E.; Huentelman, M.J.; Liang, W.S.; Readhead, B.; Dudley, J.T.; Spangenberg, E.E.; Green, K.N.; et al. Necroptosis activation in Alzheimer’s disease. Nat. Neurosci. 2017 ,20, 1236–1246. [CrossRef] 98. Ofengeim, D.; Mazzitelli, S.; Ito, Y.; DeWitt, J.P.; Mifflin, L.; Zou, C.; Das, S.; Adiconis, X.; Chen, H.; Zhu, H.; et al. RIPK1 mediates a disease-associated microglial response in Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 2017,114, E8788–E8797. [CrossRef] 99. Yang, S.H.; Lee, D.; Shin, J.; Lee, S.; Baek, S.; Kim, Y. Nec-1 Alleviates Cognitive Impairment with Reduction of Aβand Tau Abnormalities in App/Ps1 Mice. EMBO Mol. Med. 2017,9, 61–77. [CrossRef] 100. Bisht, K.; Sharma, K.P.; Lecours, C.; S á nchez, M.G.; El Hajj,H.; Milior,G.; Olmos-Alonso, A.; G ó mez-Nicola, D.; Luheshi, G.; Valli è res, L.; et al. Dark microglia: A new phenotype predominantly associated with pathological states. Glia 2016,64, 826–839. [CrossRef] J. Clin. Med. 2019,8, 1719 25 of 32 101. Stratoulias, V.; Venero, J.L.; Tremblay, M.; Joseph, B. Microglial subtypes: diversity within the microglial community. EMBO J. 2019,38, e101997. [CrossRef] 102. Lass è gue, B.; Mart í n, A.S.; Griendling, K.K. Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system. Circ. Res. 2012,110, 1364–1390. [CrossRef] 103. Ishimoto, Y.; Tanaka, T.; Yoshida, Y.; Inagi, R. Physiological and pathophysiological role of reactive oxygen species and reactive nitrogen species in the kidney. Clin. Exp. Pharmacol. Physiol. 2018 ,45, 1097–1105. [CrossRef] 104. Kalogeris, T.; Bao, Y.; Korthuis, R.J. Mitochondrial reactive oxygen species: A double edged sword in ischemia/reperfusion vs preconditioning. Redox Biol. 2014,2, 702–714. [CrossRef] 105. Miro´nczuk-Chodakowska, I.; Witkowska, A.M.; Zujko, M.E. Endogenous non-enzymatic antioxidants in the human body. Adv. Med. Sci. 2018,63, 68–78. [CrossRef] 106. Cherubini, A.; Ruggiero, C.; Morand, C.; Lattanzio, F.; Dell’aquila, G.; Zuliani, G.; Di Iorio, A.; Andres-Lacueva, C. Dietary antioxidants as potential pharmacological agents for ischemic stroke. Curr. Med. Chem. 2008,15, 1236–1248. [CrossRef] 107. He, L.; He, T.; Farrar, S.; Ji, L.; Liu, T.; Ma, X. Antioxidants Maintain Cellular Redox Homeostasis by Elimination of Reactive Oxygen Species. Cell Physiol. Biochem. 2017,44, 532–553. [CrossRef] 108. Zhu, X.; Smith, M.A.; Perry, G.; Aliev, G. Mitochondrial failures in Alzheimer’s disease. Am. J. Alzheimer’s Dis. Dement. 2004,19, 345–352. [CrossRef] 109. Barnham, K.J.; Masters, C.L.; Bush, A.I. Neurodegenerative diseases and oxidative stress. Nat. Rev. Drug Discov. 2004,3, 205–214. [CrossRef] 110. Carri, M.T.; Ferri, A.; Cozzolino, M.; Calabrese, L.; Rotilio, G. Neurodegeneration in amyotrophic lateral sclerosis: The role of oxidative stress and altered homeostasis of metals. Brain Res. Bull. 2003 ,61, 365–374. [CrossRef] 111. Giordano, G.; White, C.C.; McConnachie, L.A.; Fernandez, C.; Kavanagh, T.J.; Costa, L.G. Neurotoxicity of Domoic Acid in Cerebellar Granule Neurons in a Genetic Model of Glutathione Deficiency. Mol. Pharmacol. 2006,70, 2116–2126. [CrossRef] 112. Duan, Y.; Sahley, C.L.; Muller, K.J. ATP and NO dually control migration of microglia to nerve lesions. Dev. Neurobiol. 2009,69, 60–72. [CrossRef] 113. Weidinger, A.; Kozlov, A.V. Biological Activities of Reactive Oxygen and Nitrogen Species: Oxidative Stress versus Signal Transduction. Biomolecules 2015,5, 472–484. [CrossRef] 114. Freudenberg, F.; Alttoa, A.; Reif, A. Neuronal nitric oxide synthase (NOS1) and its adaptor, NOS1AP, as a genetic risk factors for psychiatric disorders. Genes Brain Behav. 2015,14, 46–63. [CrossRef] 115. Necchi, D.; Virgili, M.; Monti, B.; Contestabile, A.; Scherini, E. Regional alterations of the NO/NOS system in the aging brain: A biochemical, histochemical and immunochemical study in the rat. Brain Res. 2002 ,933, 31–41. [CrossRef] 116. Min, K.J.; Pyo, H.K.; Yang, M.S.; Ji, K.A.; Jou, I.; Joe, E.H. Gangliosides activate microglia via protein kinase C and NADPH oxidase. Glia 2004,48, 197–206. [CrossRef] 117. Bal-Price, A.; Matthias, A.; Brown, G.C. Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production. J. Neurochem. 2002,80, 73–80. [CrossRef] 118. Mander, P.; Brown, G.C. Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: A dual-key mechanism of inflammatory neurodegeneration. J. Neuroinflamm. 2005,2, 20. [CrossRef] 119. Trujillo, M.; Ferrer-Sueta, G.; Radi, R. Peroxynitrite Detoxification and Its Biologic Implications. Antioxid. Redox Signal. 2008,10, 1607–1620. [CrossRef] 120. David, K.K.; Andrabi, S.A.; Dawson, T.M.; Dawson, V.L. Parthanatos, a messenger of death. Front. Biosci. 2009,14, 1116–1128. [CrossRef] 121. Kim,H.J.; Lee,K.W.; Kim,M.S.; Lee, H.J.Piceatannolattenuateshydrogen-peroxide-andperoxynitrite-induced apoptosis of PC12 cells by blocking down-regulation of Bcl-XL and activation of JNK. J. Nutr. Biochem. 2007 , 19, 459–466. [CrossRef] 122. Franco, M.C.; Ricart, K.C.; Gonzalez, A.S.; Dennys, C.N.; Nelson, P.A.; Janes, M.S.; Mehl, R.A.; Landar, A.; Estevez, A.G. Nitration of Hsp90 on Tyrosine 33 Regulates Mitochondrial Metabolism. J. Biol. Chem. 2015 , 290, 19055–19066. [CrossRef] J. Clin. Med. 2019,8, 1719 32 of 32 252. Milne, G.L.; Sanchez, S.C.; Musiek, E.S.; Morrow, J.D. Quantification of F2-isoprostanes as a biomarker of oxidative stress. Nat. Protoc. 2007,2, 221–226. [CrossRef] 253. Li, W.; Laird, J.M.; Lu, L.; Roychowdhury, S.; Nagy, L.E.; Zhou, R.; Crabb, J.W.; Salomon, R.G. Isolevuglandins covalently modify phosphatidylethanolamines in vivo : Detection and quantitative analysis of hydroxylactam adducts. Free Radic. Biol. Med. 2009,47, 1539–1552. [CrossRef] [PubMed] 254. Dizdaroglu, M.; Jaruga, P.; Birincioglu, M.; Rodriguez, H. Free radical-induced damage to DNA: Mechanisms and measurement. Free Radic. Biol. Med. 2002,32, 1102–1115. [CrossRef] 255. Weimann, A.; Belling, D.; Poulsen, H.E. Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography–electrospray tandem mass spectrometry. Nucleic Acids Res. 2002,30, e7. [CrossRef] 256. Cooke, M.; Olinski, R.; Loft, S. Measurement and Meaning of Oxidatively Modified DNA Lesions in Urine. Cancer Epidemiol. Biomark. Prev. 2008,17, 3–14. [CrossRef] 257. Weimann, A.; Broedbaek, K.; Henriksen, T.; Stovgaard, E.S.; Poulsen, H.E. Assays for urinary biomarkers of oxidatively damaged nucleic acids. Free Radic. Res. 2012,46, 531–540. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).