Hot DAM: Specific Microglia Engulf Plaques
In the June 15 Cell, scientists led by Ido Amit and Michal Schwartz at the Weizmann Institute of Science in Rehovot, Israel, report the molecular signature of thousands of individual immune cells in the brains of mice that model Alzheimer’s disease pathology. Using single-cell transcriptomics, they identify a specific subset of microglia that surround plaques. The cells express proteins that break down lipids and gobble up Aβ deposits. The activation of these disease-associated microglia (DAM), which may occur in two stages, requires TREM2, a risk gene for AD.
Scientists have been trying to figure out how immune cells—particularly microglia—contribute to Alzheimer’s and other neurodegenerative diseases for decades. They were limited by available methods, notably the use of commonly expressed cell-surface markers to isolate and analyze the cells. “Now we have single-cell RNA sequencing technology that allows us to look at these immune cells in an unbiased way on a single-cell level,” said Tristan Li, Stanford University, California, who was not involved in the study. What emerges is a clear picture of the states of individual cells, he said. “This is like using a new microscope to get a higher resolution,” said Marco Prinz, University of Freiburg, Germany. “It was not possible previously to analyze subpopulations of microglial cells.”
Single Cell Immune Array: Stochastic neighbor embedding (SNE) allows transcriptomic relationships to be plotted in two dimensions (dim1 and dim2). The closer the dots, the more similar the transcriptomes. Among immune cells from 5xFAD mice, resting microglia (yellow cloud) are most abundant, but two unique subsets emerge in diseased brains (orange and red). [Cell, Keren-Shaul et al. 2017.]
Using cell-surface markers to isolate microglia lumps a mixture of cells together, without capturing their diversity. To overcome this, Amit’s group has been working to apply single-cell RNA sequencing (see Matcovitch-Natan et al., 2016). The idea is to identify the molecular characteristics of subtypes of cells, Amit wrote to Alzforum.
First author Hadas Keren-Shaul and colleagues used single-cell RNA-Seq to compare microglia from wild-type mice to those from 5xFAD animals, which express five familial AD mutations. These mice develop plaques around two months of age, followed by neuronal loss and cognitive deficits at six months. From the latter age group and age-matched controls, the researchers first isolated cells from whole brain tissue by capturing those that express the immune marker CD45+. They placed individual cells into separate wells and sequenced the messenger RNA (mRNA) from each. This gave them RNA fingerprints they used to group cells and pinpoint differences between the diseased and healthy states.
The scientists found 10 distinct subtypes of immune cell, including monocytes, perivascular macrophages, and a variety of lymphocytes. By far the largest group in both diseased and healthy mice were resting-state, or homeostatic, microglial cells. The researchers called these group I (see image above). Diseased mice harbored two additional minor subsets—groups II and III—that were absent from the healthy animals. These disease-associated microglia, or DAMs, differed from group I microglia in that they turned down expression of certain homeostatic genes, such as CX3CR1 and TMEM119, and dialed up genes for lipid metabolism and phagocytosis, such as CST7 and the AD risk genes APOE and LPL. They also expressed other AD-related genes at higher levels, including CTSD, TYROBP, and TREM2. Group II cells expressed many of the same genes as group III microglia, bar the phagocytic ones, suggesting they may be intermediary between groups I and III. Li was surprised that the authors found no changes in expression of inflammatory molecules, such as cytokines, which have been linked to activated microglia.
To see how DAMs respond to disease, the scientists isolated cells from 5xFAD mice at one, three, six, and eight months. Prior to the onset of plaques, almost all microglia in the cortex were in a homeostatic state, i.e. group I. At three months, group II cells appeared, and by eight months, group III cells dominated. DAMs did not appear in cerebellum, which deposits no Aβ in these mice.
DAM Plaque Eaters. Microglia (red) surrounding Aβ plaques (gray) in the mouse brain express CD11c (green), a marker of disease-associated microglia. [Cell, Keren-Shaul et al. 2017.]
To pinpoint where in the cortex DAMS are, the researchers stained brain slices for plaques and CD11c to identify immune cells, then used single-molecule fluorescent in situ hybridization (smFISH) to label DAM-associated genes such as CSF1 and LPL. The analysis revealed that DAMs concentrated around Aβ plaques (see image above). Further, they contained Aβ particles inside. Postmortem brain slices from people with AD also contained microglia positive for the DAM-associated gene LPL around plaques. Interestingly, DAMs also appeared in the spinal cords of mSOD1(G93A) mouse models of ALS, suggesting they clear general protease-resistant aggregated proteins, not just Aβ, wrote the authors. DAMs appeared in very old wild-type mice, though fewer than in diseased ones.
What triggers homeostatic microglia to transform into DAMs? The researchers suspected the AD risk gene TREM2. The 5xFAD mice that lack TREM2 had no group III DAMs. Rather, a large number of microglia seemed stalled in the intermediate group II state. “That was the most exciting part,” Schwartz told Alzforum. “It means TREM2 is essential for the transition of these microglia from normal to having robust phagocytic activity.” She is unsure what triggers microglia to enter the intermediate phase.
The work fits with data suggesting that microglia need TREM2 if they are to surround and eat plaques (Wang et al., 2015; May 2017 news). It also jibes with reports that the cells become hyperactive and upregulate certain genes when they encounter plaques (see Yin et al., 2017; Kamphuis et al., 2016). The Israeli researchers plan to conduct the same single-cell analysis of postmortem tissue from human brain to look for DAMs in AD tissue, Schwartz said.
“This work confirms that these microglia are biologically distinct from those tiled [evenly spread] through the parenchyma or in unaffected brain regions such as cerebellum,” wrote Richard Ransohoff, Biogen, Cambridge, Massachusetts, to Alzforum. That DAM cells appear in ALS and aging suggests that they are nonspecifically triggered by altered brain homeostasis, he speculated. It is unclear whether the DAM-like cells in ALS models and in aging are deleterious, helpful, or neutral. It will be interesting to decipher the epigenetic basis for this expression phenotype, he added.
Alison Goate, Mount Sinai School of Medicine, New York, agreed that since these DAMs appear in aging, they might respond to more general neuronal damage, rather than protein aggregation. If that is the case, then she would expect TREM2 to increase risk for other neurodegenerative diseases. TREM2 has been linked to FTLD and ALS (Lill et al., 2015; Feb 2014 news).
In a related paper published online April 17 in Nature Neuroscience, Prinz and first author Tuan Leng Tay reported that microglia surrounding damaged brain tissue divide rapidly. They used a multicolored reporter that randomly labeled mouse microglia red, blue, yellow, or green. Under steady-state conditions, adjacent microglia all blinked different colors, suggesting they were relatively stable, with little division. A few days after severing the facial nerve, however, the authors saw clusters of microglia of the same color near the damage, suggesting they were clones derived from a dividing mother cell. It has been unclear up to now whether macrophages are recruited to CNS damage from outside the region, or resident microglia divide to make more cells, Prinz said. His paper suggests the latter. Prinz hypothesizes that these dividing microglia are active around Aβ plaques, and are likely the same DAMs described in the Amit study.—Gwyneth Dickey Zakaib
Research Models Citations
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Third Rock Ventures
There are points of real interest here, and it's potentially informative to take a step back and consider what's been detected using the elegant scRNA-Seq and computational modeling approach. At a first approximation, the investigators appear to have rediscovered and expression-profiled the plaque-associated macrophages (in their view, derived from microglia), which have been known in AD since initial pathological descriptions. What's intriguing and provocative is that they did so by identifying a subgroup defined through expression profiling, and subsequently localized this subset to the macrophages around plaques.
This work confirms that these cells are biologically distinct from those remaining tiled through the parenchyma or in unaffected brain regions such as the cerebellum. Temporal resolution of the plaque-associated macrophage expression profile suggested that it emerges in stages, first accompanied by suppression of regulators of the microglial phenotype, such as Cx3cr1, and later mediated by signaling through TREM2 and TyroBP/DAP12.
The crucial unresolved question (finessed rather aggressively by the article’s title but nonetheless crucial and unresolved) concerns the proposed protective functions of these microglia. Plaque-associated macrophages are induced by effective anti-amyloid passive immunization (Sevigny et al., 2016) and have been beautifully shown to limit nearby neuritic pathology (Condello et al., 2015), a function enhanced by genetic disruption of Cx3cr1. Amit and colleagues document the pathophysiological downregulation of Cx3cr1 en route to the DAM phenotype, a finding of genuine interest. However, the efficiency of Cx3cr1-deficient cells in plaque clearance (Liu et al., 2010; Lee et al., 2010) should by no means be interpreted as indicating their uniformly beneficial nature: in a model of tau pathology uncomplicated by expression of mutant tau, Cx3cr1 deficiency markedly worsens pathology and cognition (Bhaskar et al., 2010). Therefore, these DAM cells may be regarded as exerting a highly temporally restricted beneficial function in the initial phases of AD, but may become deleterious subsequently. One salient corollary: application of what the authors term “checkpoint” therapeutics should be precisely timed as defined by objective biomarkers of a target pathological process.
Other questions and research avenues opened by the present research report:
In summary, DAM cells appear to constitute a distinct response of microglia to a number of states involving altered CNS homeostasis. This report provides a preliminary characterization and points to a sequential acquisition of the phenotype. Their regulation and activities in disease states, for good or ill, remain undefined.
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Work by Keren-Shaul et al. provides a very compelling demonstration that microglia activation in the context of chronic diseases, and possibly aging, does not seem to operate in turns of simple “digital–on/off” states. Transition states are involved, and these may be also critically involved in brain pathologies. To my knowledge, this is the first time such a state of “transition” has been described for microglia, at least in such a comprehensive manner. I also think that the data here demonstrate the limitation of the paradigm of M1–M2 state of microglia (or macrophage) activation. As illustrated by the current work, microglia are incredibly plastic cells, and that quality of plasticity is poorly captured by this simple paradigm. Thus, the current work helps significantly refine our conceptualization of microglia activation phenotypes in the brain.
In addition, there seems to be an important role for TREM2 in the transition of microglia from the cluster II to the cluster III phenotype, or the DAM state. In human Alzheimer’s disease, TREM2 functions appear to be protective as the R47H mutation, which results in loss-of-function of TREM2, predisposes for the development of AD (Guerreiro et al., 2013). TREM2 loss-of-function studies in mice corroborated the human data (Wang et al., 2016).
Combining these studies/observations, Keren-Shaul et al. propose the hypothesis that the DAM state is neuroprotective. It would have been very informative for the authors to show how absence of TREM2 in their AD model affects cognitive functions in mice. Nonetheless, I think the hypothesis is reasonable. Thus, it could be that one key role of TREM2 is to promote an overall state of microglia activation that enables these cells to perform better functions that help neutralize disease mechanisms relevant to AD.
What triggers this microglia-activating function of TREM2 is still not well understood, but evidence suggest that lipids and/or proteins involved in lipid biology like ApoE might have a role (Wang et al., 2015; Yeh et al., 2016). Also, what is it about the DAM state that helps microglia prevent AD? Keren-Shaul et al. note that many genes linked with the DAM state are related to lysosomal/phagocytosis functions and lipid metabolism. Thus, another very important contribution of that study is that it “nominates” genes whose activity might be critical to that protective state of DAM. This should prove very beneficial to the AD research community. Finally, whether the DAM state can be promoted or induced pharmacologically needs to be studied.
A few outstanding points remain. First, the molecular mechanisms that trigger cluster I microglia to transition to the cluster II phenotype remain unknown. This is a point raised by the authors, and one for which we still lack robust answers in 2017. Interestingly, some evidence from multiple studies suggests that a type I interferon signaling may be at play. One of the more peripheral results from Keren-Shaul et al. is that DAMs are also present in the brains of older mice (Figure S4E). In addition, a previous study by the groups of Michal Schwartz and Ido Amit showed that aging is associated in the brain with an increase in type I interferon signaling (Baruch et al., 2014). This latter study, however, did not investigate how microglia are integrated with this response. Nonetheless, putting the current study in the context of the previous one, it is tempting to hypothesize that the transition from cluster I to the cluster II and III state of microglia may be driven, at least in part, by type I interferon signaling. This is a relatively straightforward hypothesis, and we expect that it will be the focus of a study in the near future.
Finally, there is some reason to think that DAM might also be seen in the human brain in AD. For one, the preliminary evidence from Keren-Shaul et al. on Lpl mRNA expression, which is induced in DAM in mice and is also seen with microglia associated with senile plaque in the human brain, is encouraging. It is also important to highlight recent work by Erik Boddeke’s group at University of Groningen, which reported that senile plaque-associated microglia in the brains of individuals diagnosed with AD expressed high levels of APOE, AXL, TREM2, and TYROBP, which were all components of clusters II and/or DAM microglia (Yin et al., 2017). Lastly, the recent characterization of human microglia by Christopher Glass’ laboratory at UC San Diego showed that key signaling pathways that are important to specify gene expression in mouse microglia are relatively well conserved in human microglia (Gosselin et al., 2017).
Thus, there is certainly some early evidence suggesting that the mouse DAM phenotype may be relevant to human microglia in AD, but the full extent of these similarities needs to be thoroughly investigated. Given the huge progress we have made in our understanding of microglia biology over the past 10 years, we expect that we will have an answer to that critical question as well in the near future.
Guerreiro R, Wojtas A, Bras J, Carrasquillo M, Rogaeva E, Majounie E, Cruchaga C, Sassi C, Kauwe JS, Younkin S, Hazrati L, Collinge J, Pocock J, Lashley T, Williams J, Lambert JC, Amouyel P, Goate A, Rademakers R, Morgan K, Powell J, St George-Hyslop P, Singleton A, Hardy J, Alzheimer Genetic Analysis Group. TREM2 variants in Alzheimer's disease. N Engl J Med. 2013 Jan 10;368(2):117-27. Epub 2012 Nov 14 PubMed.
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Wang Y, Cella M, Mallinson K, Ulrich JD, Young KL, Robinette ML, Gilfillan S, Krishnan GM, Sudhakar S, Zinselmeyer BH, Holtzman DM, Cirrito JR, Colonna M. TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model. Cell. 2015 Mar 12;160(6):1061-71. Epub 2015 Feb 26 PubMed.
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Baruch K, Deczkowska A, David E, Castellano JM, Miller O, Kertser A, Berkutzki T, Barnett-Itzhaki Z, Bezalel D, Wyss-Coray T, Amit I, Schwartz M. Aging-induced type I interferon response at the choroid plexus negatively affects brain function. Science. 2014 Aug 21; PubMed.
Yin Z, Raj D, Saiepour N, Van Dam D, Brouwer N, Holtman IR, Eggen BJ, Möller T, Tamm JA, Abdourahman A, Hol EM, Kamphuis W, Bayer TA, De Deyn PP, Boddeke E. Immune hyperreactivity of Aβ plaque-associated microglia in Alzheimer's disease. Neurobiol Aging. 2017 Jul;55:115-122. Epub 2017 Mar 27 PubMed.
Gosselin D, Skola D, Coufal NG, Holtman IR, Schlachetzki JC, Sajti E, Jaeger BN, O'Connor C, Fitzpatrick C, Pasillas MP, Pena M, Adair A, Gonda DD, Levy ML, Ransohoff RM, Gage FH, Glass CK. An environment-dependent transcriptional network specifies human microglia identity. Science. 2017 Jun 23;356(6344) Epub 2017 May 25 PubMed.
The DAM microglia reported by Keren-Shaul et al. share several markers with "dark microglia,” a phenotype we recently characterized at the ultrastructural level (Bisht et al., 2016). Dark microglia are rarely present under steady-state conditions, in the hippocampus, cerebral cortex, amygdala, and hypothalamus, but become prevalent upon chronic stress, aging, fractalkine signaling deficiency (CX3CR1 KOs), and in the APP-PS1 model of Alzheimer’s disease.
Dark microglia show reduced expression of CX3CR1 (but also of IBA1, contrary to the DAM microglia), and were not found to co-localize with P2RY12. They are strongly positive for CD11b (but not for CD11c using immunoEM), 4D4, and TREM2, when they associa Aβ te with Aβ plaques. Besides their phagocytosis of Aβ (we very frequently observed engulfments), dark microglia appear extremely active at synapses, even more than normal microglia, suggesting their implication in the pathological/traumatic remodeling of neuronal circuits.
Regarding the DAM microglia, I agree with David Gosselin that it would be extremely important to determine their consequences on cognitive function by conducting behavioral experiments.
Bisht K, Sharma KP, Lecours C, Sánchez MG, El Hajj H, Milior G, Olmos-Alonso A, Gómez-Nicola D, Luheshi G, Vallières L, Branchi I, Maggi L, Limatola C, Butovsky O, Tremblay MÈ. Dark microglia: A new phenotype predominantly associated with pathological states. Glia. 2016 May;64(5):826-39. Epub 2016 Feb 5 PubMed.
Universitat Autònoma de Barcelona
The existence of plaque-associated microglia as a distinct molecular entity was previously documented by the labs of Elly Hol and Javier Vitorica. The current paper is a very elegant dissection and analysis of microglia subtypes, but there is no demonstration that the DAM microglia engulf plaques or restrict neurodegeneration, as respectively stated in the titles of the Alzforum piece and the article itself. Clearly, the mice have plaques galore.
My view is that DAM microglia might represent a maladaptive response of surveillant microglia, which, in the presence of excessive amounts of Aβ, suffer a phenotypical involution such that vestigial pathways from the macrophage precursors that give rise to microglia become activated in an aberrant manner. The resulting DAM microglia might be a defective microglia that neglects regulation of neuronal circuits, and a defective macrophage that has no capacity to efficiently phagocytose plaques.
With regard to the role of TREM2 in the phenotypical transformation of microglia, I find quite on target recent studies from the Haass lab, which have shown alterations in microglia motility in a model of FTD caused by a TREM2 mutation. I posit that impaired microglia surveillance might alter synaptic scaling, thereby causing neuronal hyperexcitation and ensuing “burn out,” which may explain the striking decrease in brain metabolism shown in the mice.
We recently published an opinion piece proposing a revision of the notion of "neuroinflammation": Masgrau et al., 2017.
Masgrau R, Guaza C, Ransohoff RM, Galea E. Should We Stop Saying 'Glia' and 'Neuroinflammation'?. Trends Mol Med. 2017 Jun;23(6):486-500. Epub 2017 May 9 PubMed.
Weizmann Institute of Science
Washington University School of Medicine
Weizmann Institute of Science
We thank Richard Ransohoff for his insightful comments. We would like to clarify several points:
1. Indeed, plaque-associated macrophages have been known in AD since initial pathological descriptions. However, this is the first study that describes the disease-associated microglia population in precise molecular terms. Previous attempts over the last decade to identify these cells classified an entire zoo of different myeloid populations, and as such overlooked the important pathways and genes at play in DAM, while attributing inaccurate (and sometimes deleterious) functions to these cells. We believe the cells and pathways we describe are an important stepping-stone to move the field forward.
2. The genetic background of the control mice is identical to the 5xFAD. They are raised in the same facility and cages to avoid any unrelated genetic or environmental effects. We fully agree that both sexes need to be characterized in detail. We reported the sex of the mice in both the figure legends and methods section. Our conclusions are based on single-cell profiling of more than 30 independent mice replicates, both male and female. As can be seen in Figure 1b, aged-matched 5xFAD females tend to have slightly more DAM cells than males.
3. We do not identify any DAM cells in healthy mice at six months of age, at least to the resolution that we profile. It is important to note that while all DAM cells are CD11c+, many CD11c+ cells in Alzheimer’s disease brains are not DAM. This may cause artifactual results, as seen in the many papers that reported on such mixtures of cell populations. See, for example, the following figure plotting CD11c intensity (measured with index sorting on the single cell level) and the DAM program.
Left, scatter plot showing the correlation to the average DAM transcription program and index-sorting intensities of CD11c. Right, tSNE plot of CD11b+ cells from both wild-type and AD mouse. Cell color based on cluster association as shown in A. All DAM cells (red) show high levels (3.5E3) of CD11c but not all CD11c cells are DAM.
4. A note regarding the statement in the Alzforum news article “The epigenetic basis for this expression phenotype will be of considerable interest to decipher.” We show in the paper that the epigenetic profile of DAM and homeostatic microglia are almost identical (S2F and S2G). Focusing on DAM-specific genes, we observed active H3K4me2 regions in both the microglia and DAM, demonstrating that the DAM program is already primed in homeostatic microglia (Figures S2F and S2G). This finding is in line with our single-cell profiling of WT and TREM2 KO. The DAM program is highly anticipated by the microglia and regulated; these are not cells losing control in the face of neuronal damage.
5. The finding that myeloid cells around plaques are brain-resident microglia, not bone marrow-derived macrophages, has been definitively demonstrated through recent parabiosis experiments in two distinct models of AD, including that presented in our study. The detailed molecular characterization of DAM, and the two stages, strongly support these studies, setting these cells aside from conventional macrophages. In fact, previous attempts to characterize the myeloid cells around plaques based on cellular markers have generated opposite results regarding TREM2-expressing myeloid cells. This can be explained by the impurity of the myeloid cells due to the markers used.
6. Genetic studies have shown that TREM2 polymorphisms impairing TREM2 function increase the risk of AD three- to fivefold. Our identification of a TREM2-dependent stage in the activation of DAM in models of Aβ accumulation support a protective function of DAM in this type of lesion. Whether DAM have protective functions in other lesions associated with AD, such as taupathy, is an important question for future studies. It can be addressed with the cutting-edge approach we have developed and reported.
Ido Amit of the Weizmann Institute of Science also contributed to this comment.
University of Southern California
In addition to the certainty in life of death and taxes, we also have the guarantee of scientific advancements—although sometimes met with intermittent setbacks. This rings true when considering the back-and-forth play with respect to the role of TREM2 in AD. Amit’s team elegantly uses single-cell RNAseq supplemented by unbiased algorithms to illustrate the transition of microglial activation states from homoeostatic to neurodegenerative phenotypes.
Although at face value this resembles the M1-M2 nomenclature, it’s a step forward because the authors acknowledge a dynamic shift in microglial activation states. Interestingly, they report on a heterogeneous CD11c myeloid population within the CNS. A logical next step would be to tease apart central versus peripherally infiltrating myeloid cells in the brain.
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