REVIEW ARTICLE

What is Microglia Neurotoxicity (Not)? Knut Biber,1,2 Trevor Owens,3 and Erik Boddeke2 Microglia most likely appeared early in evolution as they are not only present in vertebrates, but are also found in nervous systems of various nonvertebrate organisms. Mammalian microglia are derived from a specific embryonic, self-renewable myeloid cell population that is throughout lifetime not replaced by peripheral myeloid cells. These phylogenic and ontogenic features suggest that microglia serve vital functions. Yet, microglia often are described as neurotoxic cells, that actively kill (healthy) neurons. Since it is from an evolutionary point of view difficult to understand why an important and vulnerable organ like the brain should host numerous potential killers, we here review the concept of microglia neurotoxicity. On one hand it is discussed that most of our understanding about how microglia kill neurons is based on in vitro experiments or correlative staining studies that suffer from the difficulty to discriminate microglia and peripheral myeloid cells in the diseased brain. On the other hand it is described that a more functional approach by mutating, inactivating or deleting microglia is seldom associated with a beneficial outcome in an acute injury situation, suggesting that microglia are normally important protective elements in the brain. This might change in chronic disease or the aged brain, where; however, it remains to be established whether microglia simply lose their protective capacities or whether microglia become truly neurotoxic cells. GLIA 2014;00:000–000

Key words: activation states, microglia evolution, neuronal loss, neuroinflammation

Origin of Microglia Phylogeny t is well known that microglia are present in the nervous systems of “primitive” organisms like the leech, snails, mussels, insects and crustaceans (Sonetti et al., 1994). In fact, microglia research was first started in the leech as Pio del Rio-Hortega obtained his first microglia stainings in Hirudu Medicinalis (see for review: Kettenmann et al., 2011; Sieger and Peri, 2013). The nervous system of the leech is mainly composed of evenly distributed body ganglia. Each of these body ganglia contains around 400 neurons and 10.000 microglia, thus in the leech microglia by far outnumber neurons (Le Marrec-Croq et al., 2013). Although much remains to be learned from microglia in non-vertebrate organisms few studies would suggest striking similarities between nonvertebrate and vertebrate microglia, such as morphology, expression of inflammatory markers (cytokines) and receptors (TLR, cannabinoid, or chemoattractant receptors) (Kettenmann et al., 2011; Le Marrec-Croq et al., 2013; Sonnetti and Peruzzi, 2004; Tasiemski and Salzet, 2010). When and where microglia first appeared in nervous structures is not known,

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thus the evolutionary origin of microglia is an unsolved issue at the moment. The above described findings, however, point towards a rather early appearance of microglia in the animal kingdom, suggesting that the presence of microglia provides an advantage for the nervous structure of the host organism. Indeed, for the leech it is known that microglia are essential for its extraordinary nervous repair and regeneration capacity (Le Marrec-Croq et al., 2013). Ontogeny The last few years have seen the final settlement of a longstanding scientific debate about the ontological origin of microglia (see for excellent review: Ginhoux et al., 2013). Various incorrect ideas about the origin of microglia such as being neuroectodermally derived, or constituted of a mixed myeloid population have been discussed for almost one century. A combination of a variety of genetically modified mouse strains and other molecular tools has now shown that microglia are derived from primitive c-kit1 erythromyeloid yolk sac precursor cells that appear as early as embryonic Day 8 in the mouse (Ginhoux et al., 2010; Kierdorf et al., 2013).

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22654 Published online Month 00, 2014 in Wiley Online Library (wileyonlinelibrary.com). Received Nov 26, 2013, Accepted for publication Feb 14, 2014. Address correspondence to Knut Biber, Department of Psychiatry and Psychotherapy, University Hospital Freiburg, Hauptstrasse 5, 79104. E-mail: [email protected] From the 1Department of Psychiatry and Psychotherapy, University Hospital Freiburg, Hauptstrasse 5, 79104 Freiburg, Germany; 2Department of Neuroscience, University Medical Center Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands; 3Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.

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Importantly, only these cells invade the developing nervous tissue and mature into microglia. Microglia never exchange with cells that stem from fetal liver- or bone-marrow haematopoiesis, making microglia a myeloid cell population in the adult that is exclusively derived from primitive haematopoiesis (Ginhoux et al., 2010; Kierdorf et al., 2013; Schulz et al., 2012). Microglia therefore are a specialized and local cell population, that most likely display self-renewing capacities with no exchange with peripheral cells under physiological conditions (Ajami et al., 2007; Ginhoux et al., 2013). In other words, embryonic microglia mature in the brain and stay there throughout the lifetime of an organism, thus although microglia originally stem from the mesodermal compartment they become true brain cells. Why did it take so long to settle this scientific debate? Retrospectively, it seems that the mistaken ideas about the origin of microglia have been founded on experimental misinterpretations (Ginhoux et al., 2013). One likely reason for these misinterpretations is the fact that microglia are cells that are extremely difficult to target experimentally. For example, there always has been a difficulty to unequivocally distinguish morphologically “activated” microglia from macrophages in the inflamed brain (see below for a detailed discussion on this issue). As a result, there is yet no generally agreed definition of microglia, and very often microglia have been confused with blood-derived immigrants. These recent results on phylogeny and ontogeny concerning microglia suggest an important evolutionary benefit for having these cells in nervous tissue. Indeed numerous recent reviews have discussed our current understanding of the physiological role of microglia in nervous tissue (Hanisch, 2013; Kettenmann et al., 2013; Tremblay et al., 2011). On the other hand microglia are often described as neurotoxic cells, or the term “double-edged” sword is often used to describe beneficial and detrimental effects of microglia function (Block et al., 2007). From an evolutionary point of view, however, it is difficult to understand what benefit it might have for an important and predominantly post-mitotic organ like the brain to host numerous potential toxic cells. The aim of this review therefore is to critically examine the concept of microglia neurotoxicity. The Concept of Microglia Neurotoxicity: Where Does It Come From? IN VITRO STUDIES. The first direct evidence concerning

microglia as potential neurotoxic cells was derived from in vitro experiments published some 20 years ago (see for example: Boje and Arora, 1992; Chao et al., 1992). These reports used standard microglia cultures (shake-off microglia from cultured neonatal brain homogenate), stimulated with rather high concentrations of single or combined proinflammatory stimuli 2

such as LPS, IFNc, or TNFa. Subsequently, microglia (or the resulting supernatant) were transferred to cultured neurons before neuronal survival was assessed (Boje and Arora, 1992; Chao et al., 1992). Ever since these pioneering experiments were performed, numerous variations of this experimental design have identified an excess of toxic microglial (secretory) products and/or harmful microglia functions that have fuelled the notion that microglia are neurotoxic cells (see for recent examples: Burguillos et al., 2011; Gao et al., 2011; Lehnardt et al., 2008; Levesque et al., 2010; Pais et al., 2008). Standard cell culture experiments with microglia, however, should be approached carefully as they have at least three major disadvantages: First, since standard cultured microglia are derived from the neonatal brain, these cells have missed the potential maturation process that occurs in vivo. Second, cultured microglia are grown in serum-containing (usually 10%) medium, whereas in vivo microglia normally never come in contact with serum components. Third, nowadays it is also very well known that in vivo microglia are kept under constant restraint by a variety of inhibitory inputs such as CX3CL1, CD200, CD22, or CD172 (see for review: Biber et al., 2007; Hellwig et al., 2013; Prinz et al., 2011; Ransohoff and Cardona, 2010), which, of course, is not the case in culture. Indeed, various electrophysiological and genetic studies have provided evidence that standard cultured microglia are more closely related to activated peripheral macrophages, than to microglia in vivo (or ex vivo) (Beutner et al., 2013; Bouscein et al., 2000; Melief et al., 2012; Schmid et al., 2009). Thus cell culture experiments are useful to explore the principal capabilities of microglia, but the in vivo relevance of in vitro findings, however is at least questionable (Fig. 1). In other words, the role of microglia in the nervous tissue can only be investigated in vivo. Misconception of the Term Microglia “Activation” In Vivo Whereas microglia are often associated with neuroinflammation, in contrast to peripheral macrophages the basic state of microglia is tolerant and prohomeostatic (Graeber and Streit, 2010). Thus, essentially the innate immune response of the CNS and retina is weak. Yet, almost any type of brain injury or disease inevitably leads to a fast morphological response of microglia. Ramified microglia retract their fine processes, and acquire a morphology which resembles that of phagocytic macrophages, a transition originally described by RioHortega. This morphological reaction often is correlated with a migratory behaviour and/or proliferation of the cells. Because the morphological complexity of this transition is reduced (basically only one morphological phenotype is the result of this process) it has been defined as a stereotypic and graded process (Kreutzberg, 1996; Streit, 2002; van Rossum and Hanisch, 2004). On the basis of these morphological Volume 00, No. 00

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FIGURE 1: Pitfalls in microglia research and timely approached to study microglia. Because of the relative easiness of culturing microglia, in vitro microglia have been used for more than 2 decades. It is, however, meanwhile known that the culture condition itself has a tremendous influence and changes profoundly the properties of microglia. Thus cell culture experiments are useful to explore the principal capabilities of microglia but the in vivo relevance of in vitro findings is rather limited. A serious problem when addressing in vivo microglia function is their similarity with peripheral monocytes/macrophages that might also be present in the diseased brain, making it almost impossible to discriminate both cell types. Thus, staining experiments do not allow drawing conclusions about microglia function. The combination of in vivo staining and in vitro findings thus becomes a vicious circle of argumentation, often leading to the conclusion that microglia are neurotoxic cell. More modern experimental approaches should therefore be used to understand the function of these cells in the brain.

data it was widely assumed that ramified microglia in the healthy brain would be inactive or resting and that the morphological transition of microglia in response to neuronal injury sets these cells into action, thus it was described as “microglia activation”. Moreover, because the morphological transition is a stereotypic process it was more or less believed that “activated” microglia would respond in a stereotypic way with limited response variability. This general and simple concept of microglia “activation” is not valid anymore [see for recent reviews: Aguzzi et al., 2013; Colton, 2009; Graeber, 2010; Hanisch, 2013; Hellwig et al., 2013; Hanisch and Kettenmann, 2007; Kettenmann et al., 2011; London et al. 2013; Parkhurst and Gan, 2010; Prinz and Mildner, 2011; Ransohoff and Perry, 2009; Ransohoff and Cardona, 2010; Sierra et al., 2013; Tremblay et al., 2011; Yong and Rivest, 2009). Not only is it clear today that ramified microglia are by no means resting cells it moreover is also apparent now that microglia respond with a variety of different reactions by integrating multifarious inputs (Aguzzi et al., 2013; Graeber, 2010; Hanisch, 2013; Hellwig et al., 2013; Parkhurst and Gan, 2010; Kettenmann et al., 2011; Prinz and Mildner, 2011; Kettenmann et al., 2013; London et al., 2013; Ransohoff and Cardona, 2010; Sierra et al., 2013; Tremblay et al., 2011). Thus an overwhelming body of evidence from the last Month 2014

decade has convincingly shown that the simple discrimination between “resting” and “activated” microglia does not hold true. Since there are no inactive microglia (the moniker “never resting microglia” is quite popular among microgliaminded scientists at the moment (Karperien et al., 2013), the terms microglia “activation” or “activated” microglia are misleading (Graeber and Streit, 2010). Morphological Criteria of Microglia Are Poor Predictor of In Vivo Function It is known for more than a century that neuronal damage inevitably leads to the morphological transition of ramified microglia into a more macrophage-like phagocytic cell, thus neuronal damage and the morphological transition of microglia are strictly correlated. In other words there is no evidence that neuronal damage might occur without the morphological transition of microglia nearby, as this is the physiological response of microglia to neuronal death. This strict correlation has often been interpreted as sign of neurotoxic microglia function, especially when “activated” microglia were found to be positive for molecules or mechanisms that were identified as toxic in an in vitro setting (as discussed above) (Burguillos et al., 2011; Cunningham et al., 2005; Mount et al., 2007; Neher et al., 2013) (Fig. 1). Along the same 3

lines, microglia were discussed as being neurotoxic in reports of treatments that reduced both the morphological “activation” of microglia and neuronal loss (Fan et al., 2005; He et al., 2001; Hunter et al., 2004; Kriz et al., 2002; Tikka et al., 2001; Yrj€anheikki et al., 1998). On the other hand there are many examples where high numbers of morphologically “activated” microglia can be found in brain without any signs of neuronal loss. One example is peripheral or central LPS injections. Neuronal loss is not found after peripheral LPS and even central LPS injections lead to neuronal death only in the substantia nigra but not in other brain regions (Kim et al., 2000; Nadeau and Rivest, 2002, 2003). Yet the morphological “activation” of microglia is prominently induced in all cases of LPS injection, and surprisingly such “activated” microglia may also be positive for molecules that were identified as toxic in vitro, such as IL-1b or TNFa. It was recently shown in two myelin mutant mouse lines that microglia “activation” was uncoupled from axonal degeneration, thus morphologically “activated” microglia did not contribute to axonal degeneration, as had long been thought (Monasterio-Schrader et al., 2013). Other examples are rodent models for neuropathic pain (morphological “activation” of microglia in the spinal cord) or Alzheimer’s disease (morphological “activation” of microglia near amyloid-b plaques) where neuronal loss is not observable despite for example TNFa expression in microglia (Cho et al., 2011). The latter case is much to the disappointment of researchers because the lack of neuronal loss represents a major disconnect between most (if not all) mouse models of Alzheimer’s disease and clinical disease in humans (Radde et al., 2008). It can therefore be concluded that correlation is no evidence for causality (Fig. 1). The morphological transition of microglia can very well be a bystander reaction resulting from the death of neighbouring neurons (Hanisch, 2013), rather than the cause of it, as was shown in a model of MPTP and METH-induced neurotoxicity (O’Callaghan et al., 2008). Peripheral Monocytes vs. Microglia Discrimination A serious problem that researchers face when addressing in vivo microglia function is their similarity with peripheral monocytes/macrophages that might also be present in the diseased brain. Because of this similarity, microglia may often have been mistaken for blood-derived immigrants and vice versa. Two recent studies used bone-marrow transplantation or parabiosis to investigate the function of peripheral monocytes/macrophages vs. endogenous microglia in EAE (Ajami et al., 2011; Mildner et al., 2009). Both studies observed that peripheral myeloid cells utilize CCR2 in order to invade the diseased brain and that an inhibition of this invasion resulted in a significantly diminished EAE disease course, leading the 4

authors to conclude that peripheral monocytes/macrophages but not resident microglia cells are responsible for disease progression in EAE (Ajami et al., 2011; Mildner et al., 2009). Using bone-marrow transplantation and different genetic models it was further demonstrated that in acute spinal cord injury, endogenous microglia also differ functionally from invading peripheral monocytes/macrophages (see for review: Jung and Schwartz, 2012). These studies convincingly demonstrated that peripheral monocytes/macrophages have significantly different functions compared to endogenous microglia in the diseased brain, thus underlining the importance of discriminating between the two cell types in order to understand their roles. The difficulty to discriminate microglia from bloodderived immigrants may have added confusion to the question whether or not microglia in the diseased brain are neurotoxic cells: an example here is the chemokine receptor CCR2. On one hand there are various reports in which CCR2 expressing cells are suggested to be microglia (Abbadie et al., 2003; Fernandez-Lopez et al., 2012; Zhang et al., 2007) or described as microglia/macrophages (Yao and Tsirka, 2012) or referred to as amoeboid microglia cells (Deng et al., 2009). Often CCR2 is discussed to be an important receptor for the recruitment of microglia to injured brain areas (Deng et al., 2009; El Khoury et al., 2007; Raber et al., 2013; Zhang et al., 2007) and the inhibition or lack of CCR2 signaling is related to improved disease outcome (Abbadie et al., 2003; Dimitrijevic et al., 2007; Fernandez-Lopez et al., 2012; Zhang et al., 2007; Yao and Tsirka, 2012) implicating that CCR2-expressing microglia at least contribute to disease progression. On the other hand there is convincing evidence from mRNA expression studies, different transgenic mouse models and bone-marrow transplantation experiments that microglia do not express CCR2 or its mRNA in the healthy or diseased brain (Hickman et al., 2013; Jung et al., 2009; Mildner et al., 2007; Mizutani et al., 2012; Olah et al., 2012; Saederup et al., 2010; Zuurmann et al., 2003). In bone-marrow transplantation experiments it was shown that the response of endogenous microglia to stroke was not affected in CCR2 deficient animals, showing that CCR2 is not regulating microglia responses here (Schilling et al., 2009a,b). In these studies it was shown that the infiltration of peripheral monocytes into the brain was greatly reduced, which is in agreement with other reports clearly showing that peripheral monocytes require CCR2 in order to invade the diseased central nervous system (Mildner et al., 2007; Mitzutani et al., 2012; Prinz and Mildner, 2011; Schilling et al., 2009a,b). Thus, CCR2 in the brain should be regarded as marker of peripheral monocytes/macrophages making it questionable whether the published (mostly detrimental) effects of CCR2 Volume 00, No. 00

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expressing cells can be allocated to microglia. CCR2 most likely is not the only example in this respect, showing that without a proper identification it is not possible to draw conclusions about microglia function. From the above discussed it is concluded that in vivo evidence about microglia function (being detrimental or protective) that is solely based on staining and correlation is not standing on solid ground. Other methods are therefore required to understand the function of these cells in the brain (Fig. 1).

Addressing Microglia Function Mutations in Microglia It is clear today that microglia in the brain are under constant restraint, particularly because they specifically express receptors for a variety of inhibitory factors that are constitutively expressed in the brain, mostly by neurons (Biber et al., 2007; Ransohoff and Perry, 2009). The most prominent ligand– receptor pairs in this respect are CX3CL1-CX3CR1 and CD200-CD200r, and mutations of these ligand–receptors pairs in mice have revealed much about microglia. Regarding the CX3CR1-CX3CL1 axis, one of the most used mouse models in microglia research is the CX3CR1-EGFP mouse line in which all microglia are GFP-positive (Jung et al., 2000). The consequences of CX3CR1 deletion in microglia largely depends on the mouse model used (see for extensive review: (Prinz and Mildner, 2011; Ransohoff and Prinz, 2013; Wolf et al., 2013). However, the overall idea at the moment is that a lack of CX3CR1 leads to the “hyperactivity” of microglia in the diseased brain, thereby unleashing potential neurotoxic properties (Wolf et al., 2013). Accordingly, administration of CX3CL1 into the brain causes neuroprotection in experimental stroke and two models of Parkinsons disease (Cipriani et al., 2011; Morganti et al., 2012; Pabon et al., 2011). Similarly, removing the inhibitory input that is normally modulated by CD200 (i.e., as in CD200R knockout mice) reportedly promotes microglial morphological transition even in the healthy brain (Hoek et al., 2000) [this has not been reported in later studies of these animals (Broderick et al., 2002)] and leads to an exaggerated disease course both in EAE (Broderick et al., 2002) and retinal inflammation (Hoek et al., 2000). Thus muting the inhibitory input potentially sets free the neurotoxic potential of microglia. A not very well-understood issue with respect to these findings is the question whether or not these mutated mouse models reflect the disease situation. There are rather high levels of free CX3CL1 in the brain (around 300 pg/mL), which is suggestive of constitutive CX3CL1 release under normal physiological conditions (Cardona et al., 2006). In the acutely diseased (rodent or human) brain, the levels of CX3CL1 and/or CX3CR1 were either unchanged or Month 2014

increased (Hughes et al., 2002; Hulshof et al., 2003; Lindia et al., 2005; Tarozzo et al., 2002; Xu et al., 2012). This might change, however, in the aged brain (see below for discussion). Less is known about the regulation of CD200 or CD200R expression in disease. It was reported that in human MS patients, CD200 expression in neurons diminishes around the periphery and in the center of MS lesions (Koning et al., 2007); however, astrocytes in these lesions acquire CD200 expression (Koning et al., 2009). In a mouse model of hippocampal excitotoxicity, an increase in neuronal CD200 expression was observed (Yi et al., 2012), while a decrease in CD200 and CD200R expression was reported in a mouse model of Alzheimer’s disease (Walker et al., 2009). Thus at the moment it is unclear whether or not the inhibitory input for microglia provided by CX3CL1 and CD200 is generally diminished in the acutely diseased brain. In amyotrophic lateral sclerosis (ALS), mutations within the ubiquitously expressed enzyme superoxide dismutase 1 (SOD1) gene are responsible for about a quarter of the inherited disease cases. Accordingly, mice that express mutant human SOD1 exhibit motoneuron degeneration and a decreased life span [see for review: (Lobsiger et al., 2009)]. The role of microglia in this disease has been investigated in various elegant experiments in which mutated SOD1 was expressed in specific cell types. The conclusion that arose from these experiments was that microglia with mutated SOD1 do not initiate motor neuron degeneration but rather accelerate disease progression (Fendrick et al., 2007; Xiao et al., 2007) [see for review: (Lobsiger et al., 2009)]. A recent article has analyzed the gene expression pattern of SOD1 mutant microglia isolated from the SOD1 mouse brain (Chiu et al., 2013). It was found in this study that mutant SOD1 microglia displayed a characteristic and unique expression pattern, which discriminated these cells from wild type microglia, microglia isolated from LPS injected mice and M1/M2polarised peripheral macrophages (Chiu et al., 2013). SOD1 mutant microglia expressed potentially neurotoxic and neuroprotective factors at the same time, suggesting a double-edged role of these mutated cells in the disease course (Chiu et al., 2013). It should be kept in mind that the microglia analysed here were mutant for SOD1G93A. Since it was shown that the replacement of SOD1 mutated microglia with wild type cells slowed down disease progression and prolonged the life span of the animals (Beers et al., 2006), (an effect that required functional MyD88 signaling in microglia) (Kang and Rivest 2007), it would be of utmost interest to also analyse the gene expression pattern of the more protective wild type microglia in this model. TREM2 is another receptor expressed in microglia [for review see: (Linnartz et al., 2010)]. TREM2 belongs to the family of immunoreceptor tyrosine-based activation motif 5

(ITAM) receptors for which the ligand has yet not been identified. Activation of TREM2 stimulates phagocytic activity in microglia and downregulates TNFa and iNOS expression (Takahashi et al., 2005). TREM2 is thus an antiinflammatory receptor that at the same time promotes phagocytic activity. TREM2 is intracellularly coupled to the adapter protein DAP12 [for review see: (Linnartz et al., 2010)], and interestingly, loss of function mutations of either TREM2 or DAP12 lead to a rare chronic neurodegenerative disease known as Nasu-Hakola or polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL) (Colonna, 2003). Taken together, it can be concluded that an improved outcome in brain disease (models) is rarely seen when microglia function is impaired by mutations. It should be noted however, that the above-described mutations are not specific for microglia. To induce cell-specific mutations in microglia several Cre-lines (Cd11b-Cre, Emr1-Cre, and LysM-Cre) have been used (see for example Cho et al., 2008; Nijbor et al., 2010; Qin et al., 2012; Willemen et al., 2010). However, since other myeloid cells are also targeted with these promoters none of these Cre-lines hits microglia only. In a recent review on genetic targeting of glial cells it was concluded that there are so far no lines that specifically express (inducible) Cre in microglia (Prieger and Slezak, 2012). This shortcoming in microglia research has now been overcome. The group of Steffen Jung has recently generated a tamoxifen inducible CX3CR1-Cre line that for the first time allows the specific genetic targeting of microglia in the adult mouse brain (Yona et al., 2013). Although the CX3CR1-promotor is also active in some monocytes and subsets of dendritic cells, this mouse model benefits from the longevity and low proliferation rate of microglia in the healthy brain. Thus the recombinatory effects of tamoxifen-induced Cre activity fades away in peripheral cells, but remains long term in microglia in the brain (Goldmann et al., 2013; Wolf et al., 2013; Yona et al., 2013). Using this mouse model it was shown that the specific deletion of transforming growth factor-b-activated kinase 1 (TAK1) in microglia reduced disease course, axonal damage and inflammation in EAE (Goldmann et al., 2013). These data not only demonstrate the power of the CX3CR1Cre line, but strongly suggest that in autoimmune-driven brain inflammation microglia play a significant role for disease initiation corroborating earlier findings in a microglia depletion study (Heppner et al., 2005; see below for details). Microglia Depletion With Clodronate The bisphosphonate drug clodronate can be used to selectively deplete microglia in vivo and in vitro (Buiting and van Roojien, 1994; Drabek et al., 2012; Kohl et al., 2003; Lauro et al., 2010). Organotypic hippocampal slice cultures 6

(OHSC) are in vitro explant cultures that reflect many aspects of the hippocampus in vivo situation by maintaining a certain degree of intrinsic connectivity and lamination (see for example: Frotscher et al., 1995). With respect to microglia, it is known that after 10 days in culture these cells acquire a ramified morphology that is comparable to their in vivo counterparts (Hailer et al., 1996). OHSC have been used to address the function of microglia in NMDA-induced neuronal loss by depleting and replenishing microglia (Vinet et al., 2012). In agreement with earlier studies it was found that neuronal cell loss was prominently increased in the absence of microglia and that even neurons of the dentate gyrus were affected by the NMDA treatment when microglia cells were not present (Montero et al., 2009; Vinet et al., 2012; van Weering et al., 2011). In addition, it was shown that microglial replenishment of microglia-free OHSCs protected neurons from NMDA-induced toxicity to the same extent as in nondepleted control slices (Vinet et al., 2012). This effect was most prominently observed in the dentate gyrus, where the neuroprotective capacity was attributed to ramified microglia (Vinet et al., 2012). Although it is yet not clear whether similar processes also occur in vivo, it is tempting to speculate that numerous protective properties of microglia have simply gone unnoticed because ramified microglia were generally long considered to be inactive cells. Findings published in the neonatal brain subjected to middle cerebral artery occlusion (MCAO) in the absence of presence of ramified microglia (depletion by clodronate injection) may corroborate this speculation (Faustino et al., 2011). Mouse Lines For Microglia Depletion Transgenic mouse strains in which the herpes simplex virus thymidine kinase (HSVTK) is placed under the control of the CD11b promoter are a model to deplete microglia in vivo, because application of gancyclovir to these animals leads to the death of CD11b1 cells (Heppner et al., 2005; Gowing et al., 2006). The effects of gancyclovir on microglia in vivo are dependent on the application route of, the drug in these animals. If peripheral gancyclovir application (intraperitoneal injection or oral application) is used, transplantation of wild type bone-marrow is required to spare the peripheral myeloid compartment from gancyclovir treatment. In these resulting chimeric animals, gancyclovir application leads to the inhibition of morphological microglia activation in the case of EAE [microglial paralysis (Heppner et al., 2005)], or to the death of microglia undergoing proliferation after experimental stroke (Lalancette-Hebert et al., 2007). No effects of peripheral gancylcovir application on ramified microglia were found in these studies (Heppner et al., 2005; Lalancette-Hebert et al., 2007). Whereas the inhibition of morphological Volume 00, No. 00

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microglia activation (microglial paralysis) was protective in EAE (delayed disease onset and reduced clinical scores) (Heppner et al., 2005), the ablation of microglia proliferation in the stroke model led to a larger stroke lesion area and increased neuronal death (Lalancette-Hebert et al., 2007). The more recently used central application route of gancyclovir in these animals provides a two-fold benefit. First, the need for bone-marrow transplantation is circumvented, and secondly ramified microglia are also depleted (Gowing et al., 2008; Grathwohl et al., 2009; Mirrione et al., 2010; Varvel et al., 2012). In the corresponding studies it was shown that transient depletion of microglia by gancyclovir did not affect the development of beta amyloid plaques during the depletion period, in two different mouse models of Alzheimer’s disease (Grathwohl et al., 2009), nor did the absence of microglia change disease progression and motor neuron degeneration in the SOD mouse model of amyotrophic lateral sclerosis (Gowing et al., 2008). However, in the case of pilocarpine-induced seizures, the depletion of microglia prevented the protective effect of LPS preconditioning, indicating that the inflammatory capacity of microglia is beneficial in this mouse model (Mirrione et al., 2010). Another mouse model that allows the depletion of myeloid cells is the CD11b-DTR mouse line that expresses a diphtheria toxin receptor under the control of the CD11b promoter (Duffield et al., 2005). A recent study using this model showed that impaired microglial function or depletion of these cells by diphtheria toxin injection resulted in enhanced neuronal loss of layer V neurons. The authors furthermore provide evidence that microglia provide trophic support for layer V neurons through the synthesis and release of IGF1, a vital role that was attributed to amoeboid microglia (Ueno et al., 2013). Taken together, it can be concluded that deletion of microglia in vivo was only advantageous in EAE (Heppner et al., 2005), in agreement with the recent findings by Goldmann et al. (2013). All other reports either provided evidence for a beneficial role of microglial function in vivo (LalancetteHebert et al., 2007; Mirrione et al., 2010; Ueno et al., 2013) or showed no effect of blunting the microglial response (Gowing et al., 2008; Grathwohl et al., 2009). It should be noted here that the latter studies inhibited or depleted microglia for a limited time at rather late stages of chronic disease models (Gowing et al., 2008; Grathwohl et al., 2009), which may explain the surprising lack of effect. The inhibition or depletion of microglial function may have been too late or too short to unravel the role of these cells in mouse models of AD and ALS (Gowing et al., 2008; Grathwohl et al., 2009). Thus, inhibition or depletion of microglia for longer time periods may be required for chronic disease models. Month 2014

This, however, may be difficult to achieve since it was recently shown that following chemical depletion of microglia there is a rapid and efficient repopulation of the brain with CD45 high and CCR21 blood monocytes, which gradually engrafted into the microglia-free tissue with an overall distribution and morphology very similar to that of endogenous microglia (Varvel et al., 2012). Whether or not these invaded cells become true brain microglia remains to be established (Ginhoux et al., 2013). However, these results clearly show that there is a strong endogenous force towards maintaining the myeloid compartment of brain (Varvel et al., 2012).

The Concept of M1/M2-Like Microglia In macrophage biology responses can be classified as an M1 (classical/pro-inflammatory activation), M2a (alternative activation/anti-inflammatory activation), and M2c (deactivation/ wound healing activation) response (Boche et al., 2013; Ginhoux et al., 2010; Jang et al. 2013; Mantovani et al., 2005). M1 activation is characterized by the up-regulation of inflammatory mediators (i.e., TNFa, IL-1b, COX2, and iNOS) and the production of reactive oxygen species (ROS, ex. H2O2, ONOO2) (Block et al., 2007). Critical for the regulation of the immune response, the initial M1 response is typically followed by a secondary M2 activation that is important for wound healing and resolving inflammation, which is marked by the expression of factors such as Arginase1, Ym1, and Fzz1 (Boche et al., 2013; Ginhoux et al., 2010; Jung et al., 2000). Microglia, being of myeloid origin, can express M1/ M2 markers and it is sometimes discussed that in analogy to macrophages M1 microglia may be proinflammatory (and potentially neurotoxic), whereas M2 microglia would take over more beneficial tasks (Cao and He, 2013; Goldmann and Prinz, 2013). However, microglia are highly plastic cells that may rapidly transit between different states (or phenotypes) and it is at the moment controversially debated whether or not the macrophage M1/M2 classification can be translated into the microglia field. For example there are numerous reports of microglia that express M1 and M2 markers at the same time (Crain et al., 2013; Olah et al., 2012). We here will use the terms M1-like and M2-like as descriptions for marker patterns that resemble the markers expressed by M1/M2 macrophages. The concept of there being different polarization states for microglia, as originally proposed for macrophages, at once reinforces that microglia are not intrinsically cytotoxic, as well as that both their “never-resting” and response statuses are potentially quite plastic. Broadly described, M1-like microglia express IL-1b, IL-6, TNFa, NOS2, and CD16/32, whereas M2-like microglia express lower levels of these but higher levels of IL10, BDNF, Arginase-1, CD206, to mention but a few of many 7

markers that are measured between these phenotypic extremes of what is likely a continuous spectrum of functional heterogeneity (Ajmone-Cat et al., 2013; Mantovani et al., 2005). Interestingly, microglia in early postnatal CNS have been reported to show a bias towards an M1-like phenotype that, however, also express the M2 marker Arginase 1 (Crain et al., 2013). This pattern changes as the animals mature and at 12 month of age higher levels of IL-1b and IL-6 mRNA were observed (Crain et al., 2013). In aging SOD1 overexpressing ALS mice M1-like microglia were observed that were neurotoxic in vitro (Liao et al., 2012). The noninflammatory clearance of apoptotic cells is more a functional characteristic of M2-like than of M1-like microglia. A number of studies have addressed mechanism for M1/M2 transitions. Heneka et al. showed that in mice deficient in NLRP3 inflammasome response, microglia were skewed towards an M2 profile, so identifying a mechanism controlling M1/M2 phenotypes, though without definitive resolution of the eventual role of these microglia in the AD mouse model that was being studied (Heneka et al., 2013). Another important player in controlling microglial M1/M2like phenotypes is the TREM2 receptor, which has been previously mentioned. Instructively for this review, Takahashi et al showed that microglia from mice lacking TREM2 were both inefficient in phagocytosis of apoptotic neurons and expressed elevated levels of M1-like phenotype associated TNFa and NOS2. Opposing effects were obtained by TREM2 over-expression (Takahashi et al., 2005). This represents to our opinion a more physiologically likely interpretation of the role of de-repressed microglia viz that they become proinflammatory, and inefficient at their normal job, but they were not reported to switch to becoming cytotoxic. A proregenerative functional outcome of M2-like microglial transition was shown by Miron et al who reported that oligodendrocyte differentiation in remyelination not only correlated with transition to M2-like microglia but was blocked by an inhibitor of Activin-A, an M2 marker (Miron et al., 2013). Correspondingly the M1-like phenotype correlated with severe EAE in rats, which could be alleviated by transfer of M2 cells (Mikita et al., 2011). The ability to promote an M2like transition using lentiviral IL25 was exploited to modulate macrophage/microglial phenotype in EAE and in a model of experimental hippocampal deafferentation, where CD206 was upregulated on microglia (Maiorino et al., 2013). An important point to be noted here is that all of the above-described phenotypic and functionally heterogeneous microglia would have been indistinguishable had only standard morphological criteria and “usual suspect” markers (such as Iba1, CD11b) been used. This reinforces the pitfall of lumping for instance all Iba11 microglia as “activated,” and then drawing conclusions from that. A second and equally 8

important point is that only in the extreme case of severe ALS-like disease in SOD1-overexpressing mice, where microglia needed to be explanted to an in vitro co-culture, was there any evidence for neurotoxicity of M1-like microglia (Liao et al., 2012).

Microglia in the Aging Brain The greatest risk factor for the development of neurodegenerative diseases and subsequent cognitive decline is age itself. Moreover, there is increasing evidence that the aged brain is more vulnerable with respect to infectious diseases, stroke, seizures, and mood disorders. The development of these brain pathologies must be therefore understood in the context of the aging process of the nervous system. Brain aging has accordingly been intensively studied in the last decade. Numerous subcellular processes like mitochondrial dysfunction, oxidative stress, epigenetic changes, or autophagy have been identified that all may contribute to the loss of synapses and neurons (Bishop et al., 2010; Burke and Barnes, 2010; Wong and Cuervo, 2011). Given the importance of glia cells for all aspects of brain function (Allen and Barres, 2009), it is very likely that aging related changes in glia cells are also of importance for the development of neurodegenerative disorders and other brain diseases (see for review: Copray et al., 2009; Hinman and Abraham, 2007; Rodrigeuz et al., 2009). Increasing evidence in models of infection, injury and neurodegeneration suggests that the aged brain sustains a chronically increased level of inflammation. Upon aging microglia show increased immunoreactivity for CD68, a lysosome associated phagocytic receptor, Toll-like-receptors (TLRs), MHC Class II antigen, Matrix remodeling enzyme MMP-12, Cd11b, and Cd11c integrins and cytokines (Luo et al., 2010). In the healthy aging brain, microglia acquire a hypersensitive phenotype resulting in exaggerated immune responsiveness called microglial “priming” (Godbout and Norden, 2012). The best characterized system for microglial priming is the response to peripheral infection. In vivo studies on intraperitoneal injection of lipopolysaccharide (LPS) or Escherichia coli (E. coli) caused a prolonged and exaggerated cytokine response resulting in enhanced sickness behavior in aged Balb/c mice compared with young adults (Godbout et al., 2005). Cytokine mRNA levels in microglia isolated ex-vivo from aged mice showed increased expression of proinflammatory mediators including IL-1b, TNFa, IL-6 (Henry et al., 2009; Sierra et al., 2007). On the basis of these results microglia from aged brain were suggested to be “primed” or “sensitized” for activation and may cause excessive injury to the aged brain (Godbout and Johnson, 2006; Perry et al., 2007). Microglia priming has been associated with secondary stimuli in an aging background. This has been shown for Volume 00, No. 00

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example in studies on 1-methyl- 4-phenyl-1, 2, 3, 6tetrahydropyridine (MPTP) mouse models, used to study Parkinson’s disease where aged animals were used (Ohashi et al., 2006; SugaMa et al., 2003). Intraperitoneal administration of MPTP to old C57BL6 mice (14- to 15-month-old) led to a remarkable loss of dopaminergic neurons with a marked decrease in dopamine levels (Phinney et al., 2006). Kainic acid-induced neurodegeneration and glial reactivity was also found to be more prominent in aged mice (Benkovic et al., 2006). These examples demonstrate that the inflammatory response of microglia is more pronounced in an aging background. Priming thus reflects a shift of microglia towards the pro-inflammatory state also known as “classically activated” or M1-like state. Accordingly, aged rats express increased amounts of IFN-c, an inducer of M1-like phenotype and decreased levels of IL-4, an inducer of the M2-like phenotype (Maher et al., 2006; Nolan et al., 2005). An age-dependent phenotypic shift from M2 to M1-like microglia might occur in mouse models of AD, such as APP/PS1, owing to accumulation of soluble Ab oligomers and neuronal loss (Jimenez et al., 2008). Additional evidence from APP mouse model has shown that M2 skewing of microglia facilitated Ab clearance (He et al., 2007; Yamamoto et al., 2007). Whereas microglia priming has been suggested to underlie age-related cognitive impairment and predisposition to neurodegenerative conditions (Norden and Godbout, 2012), another school of thought suggests age-related microglial dysfunction. This idea is predominantly based on morphological observations in human postmortem brain samples and suggests that a subpopulation of microglia in aged and diseased brain exhibit signs of deramification and cytoplasmic degeneration coined as “microglial dystrophy”. Dystrophic microglia show morphological characteristics of cytoplasmic spheroid formation containing phagocytic-intake material and have partially or completely broken processes (Streit, 2006). Indeed, presence of dystrophic microglia has been found to precede age-related neurodegeneration in senescence accelerated mice models of aging (Hasegawa-Ishii et al., 2010). Dystrophic microglia were also found to be associated with tau pathology preceding neurodegeneration in AD (Streit et al., 2009). In R6/2, a transgenic mouse model of Huntington’s disease presence of dystrophic microglia and a consequent decreased microglial density has been observed (Ma et al., 2003). These morphological observations thus suggest that microglia lose their neuronal supportive functions with age resulting in age-associated neurodegeneration (Streit et al., 2009). In addition, microglia from aged mice were shown to internalize less amyloid beta peptide (Ab) and were found to rather accumulate Ab than degrade it. Altogether, these studies suggest that microglial cell function changes with aging and that compromised functionality is a hallmark of microglial “dystrophy” (Njie et al., 2012). Month 2014

The concepts of “priming” and “dystrophy” have entirely different implications for microglial aging phenotype and function in terms of tissue support and inflammatory damage. Although entirely speculative, it may well be that microglia priming, as observed in mice represents an early coping response to aging, whereas microglial dystrophy could represent microglial degeneration at later stages of brain aging. The causes for microglia priming or the development of microglia dystrophy are currently not very well understood. Priming might reflect diminished inhibitory input, as it has for example been shown that CX3CL1 levels decrease in the aging brain (Wynne et al., 2010) and that aged microglia are more resistant for the inhibitory signals CX3CL1 and TGF-b (see for review: Norden and Godbout, 2012). Microglial dystrophy might also be the result of too many cellular divisions and subsequent loss of telomeres rendering these cells dysfunctional (Flamery et al., 2007). Anyway, it seems clear that microglia are affected by aging and that both primed and dystrophic microglia have diminished beneficial capacities. Whether or not microglia (due to their special location) may be affected by aging differently compared to peripheral myeloid cells is currently unknown. If so, however, it could mean that the evolutionary benefit of having a specialized and long living myeloid cell population in the brain that is not replaced by peripheral cells may turn with aging into a weakness. It should however be noted here that a direct proof that aged microglia actively kill neurons in vivo has not been reported.

Conclusions Microglia are the endogenous myeloid cells of the central nervous system. They are derived from a specific embryonic myeloid cell population and invade the developing brain very early. Microglia become true brain cells in the sense that they do not leave the brain, and are a self-renewable population that is not replaced by peripheral myeloid cells. Microglia are important protective elements in the brain, because mutating, inactivating or deleting them is seldom associated with a beneficial outcome in an acute injury situation. This might change in chronic disease or the aged brain, where it, however, remains to be established whether microglia simply lose their protective capacities or whether microglia become truly neurotoxic cells that actively kill neurons. It is argued here that based on correlative histological data and corresponding in vitro experiments, it is not trustworthy to conclude about microglia function in vivo. Additionally, it was shown in the last decade that under specific circumstances peripheral myeloid cells (CCR21, Lys6high inflammatory monocytes) may enter the brain and develop into cells even with a microglia-like ramified morphology. 9

Thus, peripheral myeloid cells and endogenous microglia can morphologically become “look-a-likes”. Since, it is at the moment highly questionable whether peripheral myeloid cells can take over the elaborate repertoire of brain specific functions of endogenous microglia (see for recent review: Ginhoux et al., 2013; Neumann and Weckerle, 2013) the presence of peripheral monocytes/macrophages has distorted the picture concerning the role of microglia. An often used minimalism; namely to refer to macrophages/microglia (for example in the stroke brain) as microglia (in order to increase the readability of the manuscript) (see for recent example: Neher et al., 2013) is not very helpful to clear up the confusion between the two cell types and should no longer be utilized. To define microglia (and their numerous disease specific phenotypes) in order to discriminate them from peripheral monocytes/macrophages and to elucidate the functional spectrum of microglia in the (healthy and diseased) brain will be a major challenge. Recently new mouse models have emerged that will be helpful to address this task and to clarify the question whether microglia may become truly toxic cells that actively kill neurons (Goldmann et al., 2013; Mizutani et al., 2012; Varvel et al., 2013; Yona et al., 2013).

Acknowledgment Grant sponsor: DFG; Grant numbers: DFG BI 668/5-1; BI 668/2-2; Grant sponsor: KNDD.; Grant sponsor: Danish Council for Independent Research.; Grant sponsor: the Lundbeck Foundation, the Region of Southern Denmark, Danish MS Society. The authors thank Dr. Hilmar van Weering for graphical support.

References Abbadie C, Lindia JA, Cumiskey AM, Peterson LB, Mudgett JS, Bayne EK, DeMartino JA, MacIntyre DE, Forrest MJ. 2003. Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Proc Natl Acad Sci USA 100:7947–7952. Aguzzi A, Barres BA, Bennett ML. 2013. Microglia: scapegoat, saboteur, or something else? Science 339:156–161. Ajami B, Bennett JL, Krieger C, McNagny KM, Rossi FM. 2011. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat Neurosci 14:1142–1149. Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FM. 2007. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci 10:1538–1543. Ajmone-Cat MAM, Mancini R, De Simone PC, Minghetti L. 2013. Microglial polarization and plasticity: Evidence from organotypic hippocampal slice cultures. Glia 61:1698–1711. Allen NJ, Barres BA. 2009. Neuroscience: Glia - more than just brain glue. Nature 457:675–677. Beers DR, Henkel JS, Xiao Q, Zhao W, Wang J, Yen AA, Siklos L, McKercher SR, Appel SH. 2006. Wild-type microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 103: 16021–16026.

10

Benkovic SA, O’Callaghan JP, Miller DB. 2004. Sensitive indicators of injury reveal hippocampal damage in C57BL/6J mice treated with kainic acid in the absence of tonic–clonic seizures. Brain Res 1024:59–76. Beutner C, Linnartz-Gerlach B, Schmidt SV, Beyer M, Mallmann MR, Staratschek-Jox A, Schultze JL, Neumann H. 2013. Unique transcriptome signature of mouse microglia. Glia 61:1429–1442. Biber K, Neumann H, Inoue K, Boddeke HW. 2007. Neuronal ’On’ and ’Off’ signals control microglia. Trends Neurosci 30:596–602. Bishop NA, Lu T, Yankner BA. 2010. Neural mechanisms of ageing and cognitive decline. Nature 464:529–535. Block ML, Zecca L, Hong JS. 2007. Microglia-mediated neurotoxicity: Uncovering the molecular mechanisms. Nat Rev Neurosci 8:57–69. Boche D, Perry VH, Nicoll JA. 2013. Review: Activation patterns of microglia and their identification in the human brain. Neuropathol Appl Neurobiol 39: 3–18. Boill ee S, Yamanaka K, Lobsiger CS, Copeland NG, Jenkins NA, Kassiotis G, Kollias G, Cleveland DW. 2006. Onset and progression in inherited ALS determined by motor neurons and microglia. Science 312:1389–1392. Boje KM, Arora PK. 1992. Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res 587:250–256. Boucsein C, Kettenmann H, Nolte C. 2000. Electrophysiological properties of microglial cells in normal and pathologic rat brain slices. Eur J Neurosci 12: 2049–2058. Broderick C, Hoek RM, Forrester JV, Liversidge J, Sedgwick JD, Dick AD. 2002. Constitutive retinal CD200 expression regulates resident microglia and activation state of inflammatory cells during experimental autoimmune uveoretinitis. Am J Pathol 161:1669–1677. Buiting AM, Van Rooijen N. 1994. Liposome mediated depletion of macrophages: An approach for fundamental studies. J Drug Target 2:357–362. Burguillos MA, Deierborg T, Kavanagh E, Persson A, Hajji N, GarciaQuintanilla A, Cano J, Brundin P, Englund E, Venero JL, Joseph B. 2011. Caspase signalling controls microglia activation and neurotoxicity. Nature 472:319–324. Burke SN, Barnes CA. 2010. Senescent synapses and hippocampal circuit dynamics. Trends Neurosci 33:153–161. Cao L, He C. 2013. Polarization of macrophages and microglia in inflammatory demyelination. Neurosci Bull 29:189–198. Cardona AE, Pioro EP, Sasse ME, Kostenko V, Cardona SM, Dijkstra IM, Huang D, Kidd G, Dombrowski S, Dutta R, Lee JC, Cook DN, Jung S, Lira SA, Littman DR, Ransohoff RM. 2006. Control of microglial neurotoxicity by the fractalkine receptor. Nat Neurosci 9:917–924. Chao CC, Hu S, Molitor TW, Shaskan EG, Peterson PK. 1992. Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J Immunol 149:2736–2741. Chiu IM, Morimoto ET, Goodarzi H, Liao JT, O’Keeffe S, Phatnani HP, Muratet M, Carroll MC, Levy S, Tavazoie S, Myers RM, Maniatis T. 2013. A neurodegeneration-specific gene-expression signature of acutely isolated microglia from an amyotrophic lateral sclerosis mouse model. Cell Rep 4:385–401. Cho IH, Hong J, Suh EC, Kim JH, Lee H, Lee JE, Lee S, Kim CH, Kim DW, Jo EK, Lee KE, Karin M, Lee SJ. 2008. Role of microglial IKKbeta in kainic acidinduced hippocampal neuronal cell death. Brain 131:3019–3033. Cho SH, Sun B, Zhou Y, Kauppinen TM, Halabisky B, Wes P, Ransohoff RM, Gan L. 2011. CX3CR1 protein signaling modulates microglial activation and protects against plaque-independent cognitive deficits in a mouse model of Alzheimer disease. J Biol Chem 286:32713–32722. Cipriani R, Villa P, Chece G, Lauro C, Paladini A, Micotti E, Perego C, De Simoni MG, Fredholm BB, Eusebi F, Limatola C. 2011. CX3CL1 is neuroprotective in permanent focal cerebral ischemia in rodents. J Neurosci 31: 16327–16335. Colonna M. 2003. DAP12 signaling: From immune cells to bone modeling and brain myelination. J Clin Invest 111:313–314.

Volume 00, No. 00

Biber et al.: Microglia Neurotoxicity? Colton CA. 2009. Heterogeneity of microglial activation in the innate immune response in the brain. J Neuroimmune Pharmacol 4:399–418. Copray S, Huynh JL, Sher F, Casaccia-Bonnefil P, Boddeke E. 2009. Epigenetic mechanisms facilitating oligodendrocyte development, maturation, and aging. Glia 15:1579–1587. Crain JM, Nikodemova M, Watters JJ. 2013. Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice. J Neurosci Res 91:1143–1151. Cunningham C, Wilcockson DC, Campion S, Lunnon K, Perry VH. 2005. Central and systemic endotoxin challenges exacerbate the local inflammatory response and increase neuronal death during chronic neurodegeneration. J Neurosci 25:9275–9284. de Monasterio-Schrader P, Patzig J, M€ obius W, Barrette B, Wagner TL, Kusch K, Edgar JM, Brophy PJ, Werner HB. 2013. Uncoupling of neuroinflammation from axonal degeneration in mice lacking the myelin protein tetraspanin-2. Glia 61:1832–47. doi: 10.1002/glia.22561. Deng YY, Lu J, Ling EA, Kaur C. 2009. Monocyte chemoattractant protein-1 (MCP-1) produced via NF-kappaB signaling pathway mediates migration of amoeboid microglia in the periventricular white matter in hypoxic neonatal rats. Glia 156:604–621. Dimitrijevic OB, Stamatovic SM, Keep RF, Andjelkovic AV. 2007. Absence of the chemokine receptor CCR2 protects against cerebral ischemia/reperfusion injury in mice. Stroke 38:1345–1353. Drabek T, Janata A, Jackson EK, End B, Stezoski J, Vagni VA, JaneskoFeldman K, Wilson CD, van Rooijen N, Tisherman SA, Kochanek PM. 2012. Microglial depletion using intrahippocampal injection of liposomeencapsulated clodronate in prolonged hypothermic cardiac arrest in rats. Resuscitation 83:517–526. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. 2005. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 115:56–65. Dunston CR, Griffiths HR, Lambert PA, Staddon S, Vernallis AB. 2011. Proteomic analysis of the anti-inflammatory action of minocycline. Proteomics 11: 42–51. El Khoury J, Toft M, Hickman SE, Means TK, Terada K, Geula C, Luster AD. 2007. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nat Med 13:432–438. Fan LW, Pang Y, Lin S, Rhodes PG, Cai Z. 2005. Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain. Neuroscience 133:159–168. Faustino JV, Wang X, Johnson CE, Klibanov A, Derugin N, Wendland MF, Vexler ZS. 2011. Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke. J Neurosci 31:12992–13001. Fendrick SE, Xue QS, Streit WJ. 2007. Formation of multinucleated giant cells and microglial degeneration in rats expressing a mutant Cu/Zn superoxide dismutase gene. J Neuroinflammation 4:9. Fern andez-L opez D, Faustino J, Derugin N, Wendland M, Lizasoain I, Moro MA, Vexler ZS. 2012. Reduced infarct size and accumulation of microglia in rats treated with WIN 55,212-2 after neonatal stroke. Neuroscience 207:307–315. Flanary BE, Sammons NW, Nguyen C, Walker D, Streit WJ. 2007. Evidence that aging and amyloid promote microglial cell senescence. Rejuvenation Res 10:61–74. Frotscher M, Zafirov S, Heimrich B. 1995. Development of identified neuronal types and of specific synaptic connections in slice cultures of rat hippocampus. Prog Neurobiol 45:vii–xxviii. Gao HM, Zhou H, Zhang F, Wilson BC, Kam W, Hong JS. 2011. HMGB1 acts on microglia Mac1 to mediate chronic neuroinflammation that drives progressive neurodegeneration. J Neurosci 31:1081–1092. Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M. 2010. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330:841–845.

Month 2014

Godbout JP, Chen J, Abraham J, Richwine AF, Berg BM, Kelley KW, Norden DM. 2005. Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system. FASEB J 19:1329–1331. Godbout JP, Johnson RW. 2009. Age and neuroinflammation: A lifetime of psychoneuroimmune consequences. Immunol Allergy Clin North Am 29:321– 337. Goldmann T, Wieghofer P, M€ uller PF, Wolf Y, Varol D, Yona S, Brendecke SM, Kierdorf K, Staszewski O, Datta M, Luedde T, Heikenwalder M, Jung S, Prinz M. 2013. A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation. Nat Neurosci 16:1618–1626. Gowing G, Philips T, Van Wijmeersch B, Audet JN, Dewil M, Van Den Bosch L, Billiau AD, Robberecht W, Julien JP. 2008. Ablation of proliferating microglia does not affect motor neuron degeneration in amyotrophic lateral sclerosis caused by mutant superoxide dismutase. J Neurosci 28:10234–10244. Gowing G, Vallie`res L, Julien JP. 2006. Mouse model for ablation of proliferating microglia in acute CNS injuries. Glia 53:331–337. Graeber MB. 2010. Changing face of microglia. Science 330:783–788. Graeber MB, Streit WJ. 2010. Microglia: Biology and pathology. Acta Neuropathol 119:89–105. Grathwohl SA, K€ alin RE, Bolmont T, Prokop S, Winkelmann G, Kaeser SA, Odenthal J, Radde R, Eldh T, Gandy S, Aguzzi A, Staufenbiel M, Mathews PM, Wolburg H, Heppner FL, Jucker M. 2009. Formation and maintenance of Alzheimer’s disease beta-amyloid plaques in the absence of microglia. Nat Neurosci 12:1361–1363. Hailer NP, Jarhult JD, Nitsch R. 1996. Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures. Glia 18:319–331. Hanisch UK. 2013. Functional diversity of microglia—How heterogeneous are they to begin with? Front Cell Neurosci 14;7:65. Hanisch UK, Kettenmann H. 2007. Microglia: Active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10:1387–1394. Hasegawa-Ishii S, Takei S, Chiba Y, Furukawa A, Umegaki H, Iguchi A, Kawamura N, Yoshikawa K, Hosokawa M, Shimada A. 2011. Morphological impairments in microglia precede age-related neuronal degeneration in senescence-accelerated mice. Neuropathology 31:20–28. He Y, Appel S, Le W. 2001. Minocycline inhibits microglial activation and protects nigral cells after 6-hydroxydopamine injection into mouse striatum. Brain Res 909:187–193. Hellwig S, Heinrich A, Biber K. 2013. The brain’s best friend: Microglial neurotoxicity revisited. Front Cell Neurosci 16;7:71. Heneka MT, Kummer MP, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A, Griep A, Axt D, Remus A, Tzeng TC, Gelpi E, Halle A, Korte M, Latz E, Golenbock DT. 2013. NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature 493:674–678. Henry CJ, Huang Y, Wynne AM, Godbout JP. 2009. Peripheral lipopolysaccharide (LPS) challenge promotes microglial hyperactivity in aged mice that is associated with exaggerated induction of both pro-inflammatory IL-1beta and anti-inflammatory IL-10 cytokines. Brain Behav Immun 23:309–317. Heppner FL, Greter M, Marino D, Falsig J, Raivich G, H€ ovelmeyer N, Waisman A, R€ ulicke T, Prinz M, Priller J, Becher B, Aguzzi A. 2005. Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat Med 11:146–152. Hickman SE, Kingery ND, Ohsumi TK, Borowsky ML, Wang LC, Means TK, El Khoury J. 2013. The microglial sensome revealed by direct RNA sequencing. Nat Neurosci. doi: 10.1038/nn.3554. Hinman JD, Abraham CR. 2007. What’s behind the decline? The role of white matter in brain aging. Neurochem Res 32:2023–2031. Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, Blom B, Homola ME, Streit WJ, Brown MH, Barclay AN, Sedgwick JD. 2000. Downregulation of the macrophage lineage through interaction with OX2 (CD200). Science 290:1768–1771.

11

Hughes PM, Botham MS, Frentzel S, Mir A, Perry VH. 2002. Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, during acute and chronic inflammation in the rodent CNS. Glia 37:314–327. Hulshof S, van Haastert ES, Kuipers HF, van den Elsen PJ, De Groot CJ, van der Valk P, Ravid R, Biber K. 2003. CX3CL1 and CX3CR1 expression in human brain tissue: Noninflammatory control versus multiple sclerosis. J Neuropathol Exp Neurol 62:899–907.

and microglial cells mediate CX3CL1-induced protection of hippocampal neurons against Glu-induced death. Neuropsychopharmacology 35:1550–1559. Le Marrec-Croq F, Drago F, Vizioli J, Sautie`re PE, Lefebvre C. 2013. The leech nervous system: A valuable model to study the microglia involvement in regenerative processes. Clin Dev Immunol 2013:274019.

Hunter CL, Bachman D, Granholm AC. 2004. Minocycline prevents cholinergic loss in a mouse model of Down’s syndrome. Ann Neurol 56:675–688.

Lehnardt S, Schott E, Trimbuch T, Laubisch D, Krueger C, Wulczyn G, Nitsch R, Weber JR. 2008. A vicious cycle involving release of heat shock protein 60 from injured cells and activation of toll-like receptor 4 mediates neurodegeneration in the CNS. J Neurosci 28:2320–2331.

Jang E, Lee S, Kim JH, Kim JH, Seo JW, Lee WH, Mori K, Nakao K, Suk K. 2013. Secreted protein lipocalin-2 promotes microglial M1 polarization. FASEB J 27:1176–1190.

Levesque S, Wilson B, Gregoria V, Thorpe LB, Dallas S, Polikov VS, Hong JS, Block ML. 2010. Reactive microgliosis: Extracellular micro-calpain and microglia-mediated dopaminergic neurotoxicity. Brain 133:808–821.

Jimenez S, Baglietto-Vargas D, Caballero C, Moreno-Gonzalez I, Torres M, Sanchez-Varo R, Ruano D, Vizuete M, Gutierrez A, Vitorica J. 2008. Inflammatory response in the hippocampus of PS1M146L/APP751SL mouse model of Alzheimer’s disease: Age-dependent switch in the microglial phenotype from alternative to classic. J Neurosci 28:11650–11661.

Liao B, Zhao W, Beers DR, Henkel JS, Appel SH. 2012. Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS. Exp Neurol 237:147–152.

Jung H, Bhangoo S, Banisadr G, Freitag C, Ren D, White FA, Miller RJ. 2009. Visualization of chemokine receptor activation in transgenic mice reveals peripheral activation of CCR2 receptors in states of neuropathic pain. J Neurosci 29:8051–8062. Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, Littman DR. 2000. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20:4106–4114. Jung S, Schwartz M. 2012. Non-identical twins—Microglia and monocytederived macrophages in acute injury and autoimmune inflammation. Front Immunol 3:89. Kang J, Rivest S. 2007. MyD88-deficient bone marrow cells accelerate onset and reduce survival in a mouse model of amyotrophic lateral sclerosis. J Cell Biol 179:1219–1230. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. 2011. Physiology of microglia. Physiol Rev 91:461–553. Kettenmann H, Kirchhoff F, Verkhratsky A. 2013. Microglia: New roles for the synaptic stripper. Neuron 77:10–18. Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, Wieghofer P, Heinrich A, Riemke P, H€ olscher C, M€ uller DN, Luckow B, Brocker T, Debowski K, Fritz G, Opdenakker G, Diefenbach A, Biber K, Heikenwalder M, Geissmann F, Rosenbauer F, Prinz M. 2013. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat Neurosci 16:273–280.

Lindia JA, McGowan E, Jochnowitz N, Abbadie C. 2005. Induction of CX3CL1 expression in astrocytes and CX3CR1 in microglia in the spinal cord of a rat model of neuropathic pain. J Pain 6:434–438. Linnartz B, Wang Y, Neumann H. 2010. Microglial immunoreceptor tyrosinebased activation and inhibition motif signaling in neuroinflammation. Int J Alzheimers Dis doi:pii: 587463. 10.4061/2010/587463. Lobsiger CS, Boillee S, McAlonis-Downes M, Khan AM, Feltri ML, Yamanaka K, Cleveland DW. 2009. Schwann cells expressing dismutase active mutant SOD1 unexpectedly slow disease progression in ALS mice. Proc Natl Acad Sci USA 106:4465–4470. doi: 10.1073/pnas.0813339106. London A, Cohen M, Schwartz M. 2013. Microglia and monocyte-derived macrophages: Functionally distinct populations that act in concert in CNS plasticity and repair. Front Cell Neurosci 8;7:34. Luo XG, Ding JQ, Chen SD. 2010. Microglia in the aging brain: Relevance to neurodegeneration. Mol Neurodegen 5:12. Ma L, Morton AJ, Nicholson LF. 2003. Microglia density decreases with age in a mouse model of Huntington’s disease. Glia 43:274–280. Maher FO, Martin DS, Lynch MA. 2004. Increased IL-1beta in cortex of aged rats is accompanied by downregulation of ERK and PI-3 kinase. Neurobiol Aging 25:795–806. Maiorino C, Khorooshi R, Ruffini F, Lobner M, Bergami A, Garzetti L, Martino G, Owens T, Furlan R. 2013. Lentiviral-mediated administration of IL-25 in the CNS induces alternative activation of microglia. Gene Therapy 20:487–496. Mantovani A, Sica A, Locati M. 2005. Macrophage polarization comes of age. Immunity 23:344–346.

Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B, Hong JS. 2000. Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: Role of microglia. J Neurosci 20:6309–6316.

Melief J, Koning N, Schuurman KG, Van De Garde MD, Smolders J, Hoek RM, Van Eijk M, Hamann J, Huitinga I. 2012. Phenotyping primary human microglia: Tight regulation of LPS responsiveness. Glia 60:1506–1517.

Kohl A, Dehghani F, Korf HW, Hailer NP. 2003. The bisphosphonate clodronate depletes microglial cells in excitotoxically injured organotypic hippocampal slice cultures. Exp Neurol 181:1–11.

Mikita J, Dubourdieu-Cassagno N, Deloire MS, Vekris AM, Biran G, Raffard B, Brochet MH, Canron JM, Franconi C, Boiziau, Petry KG. 2011. Altered M1/ M2 activation patterns of monocytes in severe relapsing experimental rat model of multiple sclerosis. Amelioration of clinical status by M2 activated monocyte administration. Mult Scler 17:2–15.

Koning N, B€ o L, Hoek RM, Huitinga I. 2007. Downregulation of macrophage inhibitory molecules in multiple sclerosis lesions. Ann Neurol 62:504–514. Koning N, Swaab DF, Hoek RM, Huitinga I. 2009. Distribution of the immune inhibitory molecules CD200 and CD200R in the normal central nervous system and multiple sclerosis lesions suggests neuron-glia and glia-glia interactions. J Neuropathol Exp Neurol 68:159–167. Kreutzberg GW. 1996. Microglia: A sensor for pathological events in the CNS. Trends Neurosci 19:312–318. Kriz J, Nguyen MD, Julien JP. 2002. Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis. Neurobiol Dis 10:268–278. Lalancette-H ebert M, Gowing G, Simard A, Weng YC, Kriz J. 2007. Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain. J Neurosci 27:2596–2605. Lauro C, Cipriani R, Catalano M, Trettel F, Chece G, Brusadin V, Antonilli L, van Rooijen N, Eusebi F, Fredholm BB, Limatola C. 2010. Adenosine A1 receptors

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Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Br€ uck W, Priller J, Prinz M. 2007. Microglia in the adult brain arise from Ly-6ChiCCR21 monocytes only under defined host conditions. Nat Neurosci 10:1544–1553. Mildner A, Mack M, Schmidt H, Br€ uck W, Djukic M, Zabel MD, Hille A, Priller J, Prinz M. 2009. CCR21Ly-6Chi monocytes are crucial for the effector phase of autoimmunity in the central nervous system. Brain 132:2487–2500. Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, van Wijngaarden P, Wagers AJ, Williams A, Franklin RJ, ffrench-Constant C. 2013. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat Neurosci 16:1211–1218. Mirrione MM, Konomos DK, Gravanis I, Dewey SL, Aguzzi A, Heppner FL, Tsirka SE. 2010. Microglial ablation and lipopolysaccharide preconditioning affects pilocarpine-induced seizures in mice. Neurobiol Dis 39:85–97.

Volume 00, No. 00

Biber et al.: Microglia Neurotoxicity? Mizutani M, Pino PA, Saederup N, Charo IF, Ransohoff RM, Cardona AE. 2012. The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood. J Immunol 188:29–36.

Prinz M, Priller J, Sisodia SS, Ransohoff RM. 2011. Heterogeneity of CNS myeloid cells and their roles in neurodegeneration. Nat Neurosci 14:1227– 1235.

Montero Domınguez M, Gonz alez B, Zimmer J. 2009. Neuroprotective effects of the anti-inflammatory compound triflusal on ischemia-like neurodegeneration in mouse hippocampal slice cultures occur independent of microglia. Exp Neurol 218:11–23.

Qin H, Yeh WI, De Sarno P, Holdbrooks AT, Liu Y, Muldowney MT, Reynolds SL, Yanagisawa LL, Fox TH III, Park K, Harrington LE, Raman C, Benveniste EN. 2012. Signal transducer and activator of transcription-3/suppressor of cytokine signaling-3 (STAT3/SOCS3) axis in myeloid cells regulates neuroinflammation. Proc Natl Acad Sci USA 109:5004–5009.

Montero M, Gonzalez B, Zimmer J. 2009. Immunotoxic depletion of microglia in mouse hippocampal slice cultures enhances ischemia-like neurodegeneration. Brain Res 1291:140–152. Morganti JM, Nash KR, Grimmig BA, Ranjit S, Small B, Bickford PC, Gemma C. 2012. The soluble isoform of CX3CL1 is necessary for neuroprotection in a mouse model of Parkinson’s disease. J Neurosci 32:14592–14601. Nadeau S, Rivest S. 2002. Endotoxemia prevents the cerebral inflammatory wave induced by intraparenchymal lipopolysaccharide injection: Role of glucocorticoids and CD14. J Immunol 169:3370–3381.

Raber J, Allen AR, Rosi S, Sharma S, Dayger C, Davis MJ, Fike JR. 2013. Effects of 56Fe radiation on hippocampal function in mice deficient in chemokine receptor 2 (CCR2). Behav Brain Res doi:pii: S0166–4328:00136–00138. Radde R, Duma C, Goedert M, Jucker M. 2008. The value of incomplete mouse models of Alzheimer’s disease. Eur J Nucl Med Mol Imaging Suppl 1: S70–S74. doi: 10.1007/s00259-007-0704-y. Ransohoff RM, Cardona AE. 2010. The myeloid cells of the central nervous system parenchyma. Nature 468:253–262.

Nadeau S, Rivest S. 2003. Glucocorticoids play a fundamental role in protecting the brain during innate immune response. J Neurosci 23:5536–5544.

Ransohoff RM, Perry VH. 2009. Microglial physiology: Unique stimuli, specialized responses. Annu Rev Immunol 27:119–145.

Neher JJ, Emmrich JV, Fricker M, Mander PK, Th ery C, Brown GC. 2013. Phagocytosis executes delayed neuronal death after focal brain ischemia. Proc Natl Acad Sci USA 110:E4098–E4107. doi: 10.1073/pnas.1308679110.

Ransohoff RM, Prinz M. 2013. Editors’ preface: Microglia—A new era dawns. Glia 61:1–2. doi: 10.1002/glia.22439.

Neumann H, Wekerle H. 2013. Brain microglia: Watchdogs with pedigree. Nat Neurosci 16:253–255. Nijboer CH, Heijnen CJ, Willemen HL, Groenendaal F, Dorn GW II, van Bel F, Kavelaars A. 2010. Cell-specific roles of GRK2 in onset and severity of hypoxicischemic brain damage in neonatal mice. Brain Behav Immun 24:420–426. Njie EG, Boelen E, Stassen FR, Steinbusch HW, Borchelt DR, Streit WJ. 2012. Ex vivo cultures of microglia from young and aged rodent brain reveal agerelated changes in microglial function. Neurobiol Aging 33:195.e1–e12.

Rodrıguez JJ, Olabarria M, Chvatal A, Verkhratsky A. 2009. Astroglia in dementia and Alzheimer’s disease. Cell Death Differ 16:378–385. Rodrıguez JJ, Olabarria M, Chvatal A, Verkhratsky A. 2009. Astroglia in dementia and Alzheimer’s disease. Cell Death Differ 16:378–385. Saederup N, Cardona AE, Croft K, Mizutani M, Cotleur AC, Tsou CL, Ransohoff RM, Charo IF. 2010. Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice. PLoS One doi: 10.1371/journal.pone.0013693.

Nolan Y, Maher FO, Martin DS, Clarke RM, Brady MT, Bolton AE, Mills KH, Lynch MA. 2005. Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus. J Biol Chem 280:9354–9362.

Schilling M, Strecker JK, Ringelstein EB, Sch€ abitz WR, Kiefer R. 2009. The role of CC chemokine receptor 2 on microglia activation and blood-borne cell recruitment after transient focal cerebral ischemia in mice. Brain Res 1289:79–84.

Norden DM, Godbout JP. 2013. Review: Microglia of the aged brain: Primed to be activated and resistant to regulation. Neuropathol Appl Neurobiol 39: 19–34.

Schilling M, Strecker JK, Sch€ abitz WR, Ringelstein EB, Kiefer R. 2009. Effects of monocyte chemoattractant protein 1 on blood-borne cell recruitment after transient focal cerebral ischemia in mice. Neuroscience 161:806–812.

O’Callaghan JP, Sriram K, Miller DB. 2008. Defining "neuroinflammation". Ann NY Acad Sci 1139:318–330.

Schmid CD, Melchior B, Masek K, Puntambekar SS, Danielson PE, Lo DD, Sutcliffe JG, Carson MJ. 2009. Differential gene expression in LPS/IFNgamma activated microglia and macrophages: In vitro versus in vivo. J Neurochem 109:117–125.

Ohashi S, Mori A, Kurihara N, Mitsumoto Y, Nakai M. 2006. Age-related severity of dopaminergic neurodegeneration to MPTP neurotoxicity causes motor dysfunction in C57BL/6 mice. Neurosci Lett 401:183–187. Olah M, Amor S, Brouwer N, Vinet J, Eggen B, Biber K, Boddeke HW. 2012. Identification of a microglia phenotype supportive of remyelination. Glia 60: 306–321. Pabon MM, Bachstetter AD, Hudson CE, Gemma C, Bickford PC. 2011. CX3CL1 reduces neurotoxicity and microglial activation in a rat model of Parkinson’s disease. J Neuroinflammation 25;8:9. Pais TF, Figueiredo C, Peixoto R, Braz MH, Chatterjee S. 2008. Necrotic neurons enhance microglial neurotoxicity through induction of glutaminase by a MyD88-dependent pathway. J Neuroinflammation 9;5:43. Parkhurst CN, Gan WB. 2010. Microglia dynamics and function in the CNS. Curr Opin Neurobiol 20:595–600. Perry VH. 2007. Stress primes microglia to the presence of systemic inflammation: Implications for environmental influences on the brain. Brain Behav Immun 21:45–46. Pfrieger FW, Slezak M. 2012. Genetic approaches to study glial cells in the rodent brain. Glia 60:681–701. Phinney AL, Andringa G, Bol JG, Wolters EC, Muiswinkel FL, Dam AM, Drukarch B. 2006. Enhanced sensitivity of dopaminergic neurons to rotenoneinduced toxicity with aging. Parkinsonism Relat Disord 12:228–238. Prinz M, Mildner A. 2011. Microglia in the CNS: Immigrants from another world. Glia 59:177–187.

Month 2014

Schulz C, Gomez Perdiguero E, Chorro L, Szabo-Rogers H, Cagnard N, Kierdorf K, Prinz M, Wu B, Jacobsen SE, Pollard JW, Frampton J, Liu KJ, Geissmann F. 2012. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336:86–90. Sieger D, Peri F. 2013. Animal models for studying microglia: the first, the popular, and the new. Glia 61:3–9. Sierra A, Abiega O, Shahraz A, Neumann H. 2013. Janus-faced microglia: Beneficial and detrimental consequences of microglial phagocytosis. Front Cell Neurosci 30;7:6. Sierra A, Gottfried-Blackmore AC, McEwen BS, Bulloch K. 2007. Microglia derived from aging mice exhibit an altered inflammatory profile. Glia 55:412–424. Sonetti D, Peruzzi E. 2004. Neuron-microglia communication in the CNS of the freshwater snail Planorbarius corneus. Acta Biol Hung 55:273–285. Sonetti D, Ottaviani E, Bianchi F, Rodriguez M, Stefano ML, Scharrer B, Stefano GB. 1994. Microglia in invertebrate ganglia. Proc Natl Acad Sci USA 91:9180–9184. Streit WJ. 2006. Microglial senescence: Does the brain’s immune system have an expiration date? Trends Neurosci 29:506–510. Streit WJ. 2002. Microglia as neuroprotective, immunocompetent cells of the CNS. Glia 40:133–139. Streit WJ, Braak H, Xue QS, Bechmann I. 2009. Dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely

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precede neurodegeneration in Alzheimer’s disease. Acta Neuropathol 118: 475–485. Sugama S, Yang L, Cho BP, DeGiorgio LA, Lorenzl S, Albers DS, Beal MF, Volpe BT, Joh TH. 2003. Age-related microglial activation in 1-methyl-4phenyl- 1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in C57BL/6 mice. Brain Res 964:288–294. Takahashi K, Rochford CD, Neumann H. 2005. Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2. J Exp Med 201:647–657. Tarozzo G, Campanella M, Ghiani M, Bulfone A, Beltramo M. 2002. Expression of fractalkine and its receptor, CX3CR1, in response to ischaemiareperfusion brain injury in the rat. Eur J Neurosci 15:1663–1668. Tasiemski A, Salzet M. 2010. Leech immunity: From brain to peripheral responses. Adv Exp Med Biol 708:80–104. Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J. 2001. Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 21:2580–2588.  Stevens B, Sierra A, Wake H, Bessis A, Nimmerjahn A. 2011. Tremblay ME, The role of microglia in the healthy brain. J Neurosci 31:16064–16069. Ueno M, Fujita Y, Tanaka T, Nakamura Y, Kikuta J, Ishii M, Yamashita T. 2013. Layer V cortical neurons require microglial support for survival during postnatal development. Nat Neurosci doi: 10.1038/nn.3358. van Rossum D, Hanisch UK. 2004. Microglia. Metab Brain Dis 19(3-4):393– 411. van Weering HR, Boddeke HW, Vinet J, Brouwer N, de Haas AH, van Rooijen N, Thomsen AR, Biber KP. 2011. CXCL10/CXCR3 signaling in glia cells differentially affects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus. Hippocampus 21:220–232. Varvel NH, Grathwohl SA, Baumann F, Liebig C, Bosch A, Brawek B, Thal DR, Charo IF, Heppner FL, Aguzzi A, Garaschuk O, Ransohoff RM, Jucker M. 2012. Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells. Proc Natl Sci Acad USA 109:18150–18155.

Willemen HL, Eijkelkamp N, Wang H, Dantzer R, Dorn GW II, Kelley KW, Heijnen CJ, Kavelaars A. 2010. Microglial/macrophage GRK2 determines duration of peripheral IL-1beta-induced hyperalgesia: Contribution of spinal cord CX3CR1, p38 and IL-1 signaling. Pain 150:550–560. Wolf Y, Yona S, Kim KW, Jung S. 2013. Microglia, seen from the CX3CR1 angle. Front Cell Neurosci 7:26. Wong E, Cuervo AM. 2010. Autophagy gone awry in neurodegenerative diseases. Nat Neurosci 7:805–811. Wynne AM, Henry CJ, Huang Y, Cleland A, Godbout JP. 2010. Protracted downregulation of CX3CR1 on microglia of aged mice after lipopolysaccharide challenge. Brain Behav Immun 24:1190–1201. Xu Y, Zeng K, Han Y, Wang L, Chen D, Xi Z, Wang H, Wang X, Chen G. 2012. Altered expression of CX3CL1 in patients with epilepsy and in a rat model. Am J Pathol 180:1950–1962. Yamamoto M, Kiyota T, Horiba M, Buescher JL, Walsh SM, Gendelman HE, Ikezu T. 2007. Interferon-gamma and tumor necrosis factor-alpha regulate amyloid-beta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice. Am J Pathol 170:680–692. Yao Y, Tsirka SE. 2012. The CCL2-CCR2 system affects the progression and clearance of intracerebral hemorrhage. Glia 60:908–918. Yi MH, Zhang E, Kang JW, Shin YN, Byun JY, Oh SH, Seo JH, Lee YH, Kim DW. 2012. Expression of CD200 in alternative activation of microglia following an excitotoxic lesion in the mouse hippocampus. Brain Res 1481:90–96. Yona S, Kim KW, Wolf Y, Mildner A, Varol D, Breker M, Strauss-Ayali D, Viukov S, Guilliams M, Misharin A, Hume DA, Perlman H, Malissen B, Zelzer E, Jung S. 2013. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38:79–91. Yong VW, Rivest S. 2009. Taking advantage of the systemic immune system to cure brain diseases. Neuron 64:55–60. Yrj€ anheikki J, Kein€ anen R, Pellikka M, H€ okfelt T, Koistinaho J. 1998. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci USA 95:15769–15774.

Vinet J, Weering HR, Heinrich A, K€ alin RE, Wegner A, Brouwer N, Heppner FL, Rooijen Nv, Boddeke HW, Biber K. 2012. Neuroprotective function for ramified microglia in hippocampal excitotoxicity. J Neuroinflammation 9:27.

Zhang J, Shi XQ, Echeverry S, Mogil JS, De Koninck Y, Rivest S. 2007. Expression of CCR2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain. J Neurosci 27:12396–12406.

Walker DG, Dalsing-Hernandez JE, Campbell NA, Lue LF. 2009. Decreased expression of CD200 and CD200 receptor in Alzheimer’s disease: A potential mechanism leading to chronic inflammation. Exp Neurol 215:5–19.

Zuurman MW, Heeroma J, Brouwer N, Boddeke HW, Biber K. 2003. LPSinduced expression of a novel chemokine receptor (L-CCR) in mouse glial cells in vitro and in vivo. Glia 41:327–336.

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