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09 Jun 2017In the June 15 Cell, scientists led by Ido Amit and Michal Schwartz at the Weizmann Institute ofScience in Rehovot, Israel, report the molecular signature of thousands of individual immunecells in the brains of mice that model Alzheimer’s disease pathology. Using single‐celltranscriptomics, they identify a speciic subset of microglia that surround plaques. The cellsexpress proteins that break down lipids and gobble up Aβ deposits. The activation of thesedisease‐associated microglia (DAM), which may occur in two stages, requires TREM2, a riskgene for AD.

Scientists have been trying to igure out how immune cells—particularly microglia—contributeto Alzheimer’s and other neurodegenerative diseases for decades. They were limited byavailable methods, notably the use of commonly expressed cell‐surface markers to isolate andanalyze the cells. “Now we have single‐cell RNA sequencing technology that allows us to look atthese 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 ofindividual cells, he said. “This is like using a new microscope to get a higher resolution,” saidMarco Prinz, University of Freiburg, Germany. “It was not possible previously to analyzesubpopulations of microglial cells.”

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Single Cell Immune Array: Stochastic neighbor embedding (SNE) allows transcriptomicrelationships to be plotted in two dimensions (dim1 and dim2). The closer the dots, the moresimilar 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, withoutcapturing their diversity. To overcome this, Amit’s group has been working to apply single‐cellRNA sequencing (see Matcovitch‐Natan et al., 2016). The idea is to identify the molecularcharacteristics of subtypes of cells, Amit wrote to Alzforum.

First author Hadas Keren‐Shaul and colleagues used single‐cell RNA‐seq to compare microgliafrom wild‐type mice to those from 5xFAD animals, which express ive familial AD mutations.These mice develop plaques around two months of age, followed by neuronal loss and cognitivedeicits at six months. From the latter age group and age‐matched controls, the researchers irstisolated cells from whole brain tissue by capturing those that express the immune markerCD45+. They placed individual cells into separate wells and sequenced the messenger RNA(mRNA) from each. This gave them RNA ingerprints they used to group cells and pinpointdifferences between the diseased and healthy states.

The scientists found 10 distinct subtypes of immune cell, including monocytes, perivascularmacrophages, and a variety of lymphocytes. By far the largest group in both diseased andhealthy mice were resting‐state, or homeostatic, microglial cells. The researchers called thesegroup I (see image above). Diseased mice harbored two additional minor subsets—groups IIand III—that were absent from the healthy animals. These disease‐associated microglia, or

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DAMs, differed from group I microglia in that they turned down expression of certainhomeostatic genes, such as CX3CR1 and TMEM119, and dialed up genes for lipid metabolismand phagocytosis, such as CST7 and the AD risk genes APOE and LPL. They also expressed otherAD‐related genes at higher levels, including CTSD, TYROBP, and TREM2. Group II cells expressedmany of the same genes as group III microglia, bar the phagocytic ones, suggesting they may beintermediary between groups I and III. Li was surprised that the authors found no changes inexpression of inlammatory molecules, such as cytokines, which have been linked toactivated 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 ahomeostatic 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 inthese mice.

DAM Plaque Eaters. Microglia (red) surrounding Aβ plaques (gray) in the mouse brain expressCD11c (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 andCD11c to identify immune cells, then used single‐molecule luorescent in situ hybridization(smFISH) to label DAM‐associated genes such as CSF1 and LPL. The analysis revealed that DAMsconcentrated 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 ofmSOD1(G93A) mouse models of ALS, suggesting they clear general protease‐resistantaggregated 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 ADrisk gene TREM2. The 5xFAD mice that lack TREM2 had no group III DAMs. Rather, a largenumber of microglia seemed stalled in the intermediate group II state. “That was the mostexciting part,” Schwartz told Alzforum. “It means TREM2 is essential for the transition of thesemicroglia from normal to having robust phagocytic activity.” She is unsure what triggersmicroglia to enter the intermediate phase.

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The work its with data suggesting that microglia need TREM2 if they are to surround and eatplaques (Wang et al., 2015; May 2017 news). It also jibes with reports that the cells becomehyperactive 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 ofpostmortem tissue from human brain to look for DAMs in AD tissue, Schwartz said.

“This work conirms that these microglia are biologically distinct from those tiled [evenlyspread] through the parenchyma or in unaffected brain regions such as cerebellum,” wroteRichard Ransohoff, Biogen, Cambridge, Massachusetts, to Alzforum. That DAM cells appear inALS and aging suggests that they are nonspeciically triggered by altered brain homeostasis, hespeculated. 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 expressionphenotype, he added.

Alison Goate, Mount Sinai School of Medicine, New York, agreed that since these DAMs appear inaging, they might respond to more general neuronal damage, rather than protein aggregation. Ifthat is the case, then she would expect TREM2 to increase risk for other neurodegenerativediseases. 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 irst author TuanLeng Tay reported that microglia surrounding damaged brain tissue divide rapidly. They used amulticolored reporter that randomly labeled mouse microglia red, blue, yellow, or green. Understeady‐state conditions, adjacent microglia all blinked different colors, suggesting they wererelatively stable, with little division. A few days after severing the facial nerve, however, theauthors saw clusters of microglia of the same color near the damage, suggesting they wereclones derived from a dividing mother cell. It has been unclear up to now whether macrophagesare recruited to CNS damage from outside the region, or resident microglia divide to make morecells, Prinz said. His paper suggests the latter. Prinz hypothesizes that these dividing microgliaare active around Aβ plaques, and are likely the same DAMs described in the Amit study.—Gwyneth Dickey Zakaib

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Richard Ransohoff

Biogen

Posted: 12 Jun 2017

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There are points of real interest here, and it's potentially informative to take a step back andconsider what's been detected using the elegant scRNA‐Seq and computational modelingapproach. At a irst approximation, the investigators appear to have rediscovered andexpression‐proiled the plaque‐associated macrophages (in their view, derived from microglia),which have been known in AD since initial pathological descriptions. What's intriguing andprovocative is that they did so by identifying a subgroup deined through expression proiling,and subsequently localized this subset to the macrophages around plaques.

This work conirms that these cells are biologically distinct from those remaining tiled throughthe parenchyma or in unaffected brain regions such as the cerebellum. Temporal resolution ofthe plaque‐associated macrophage expression proile suggested that it emerges in stages, irstaccompanied by suppression of regulators of the microglial phenotype, such as Cx3cr1, and latermediated by signaling through TREM2 and TyroBP/DAP12.

The crucial unresolved question (inessed rather aggressively by the article’s title butnonetheless crucial and unresolved) concerns the proposed protective functions of thesemicroglia. Plaque‐associated macrophages are induced by effective anti‐amyloid passiveimmunization (Sevigny et al., 2016) and have been beautifully shown to limit nearby neuriticpathology (Condello et al., 2015), a function enhanced by genetic disruption of Cx3cr1. Amit andcolleagues document the pathophysiological downregulation of Cx3cr1 en route to the DAMphenotype, a inding of genuine interest. However, the eficiency of Cx3cr1‐deicient cells inplaque clearance (Liu et al., 2010; Lee et al., 2010) should by no means be interpreted asindicating their uniformly beneicial nature: in a model of tau pathology uncomplicated byexpression of mutant tau, Cx3cr1 deiciency markedly worsens pathology and cognition(Bhaskar et al., 2010). Therefore, these DAM cells may be regarded as exerting a highlytemporally restricted beneicial function in the initial phases of AD, but may become deleterioussubsequently. One salient corollary: application of what the authors term “checkpoint”therapeutics should be precisely timed as deined by objective biomarkers of a targetpathological process.

Other questions and research avenues opened by the present research report:

1. These DAM cells (it’s simply fun to write that phrase) emerge in other contexts, includingaging and SOD1‐G93A mutant mice. This observation suggests that the DAM expressionphenotype is rather non‐speciically triggered by altered brain homeostasis. It remainscompletely speculative whether the DAM‐like cells in ALS models (or disease) and in agingplay deleterious, helpful or neutral roles. The (probable) epigenetic basis for this expressionphenotype will be of considerable interest to decipher.

2. Genetic background of the control animals was not speciied. The 5xFAD models were on amixed background, so it will be important to determine to what extent the observationsreported here were related to strain background. Similarly, the sex of the mice was not

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reported (in the Methods section, at least) and gender dimorphism of microglia may play asigniicant role in deining transcriptional proiles. Both sexes need to be characterizedin detail.

3. CD11c+ microglia were previously characterized in detail as to morphology and localization(Prodinger et al., 2011). It would be useful to know the relationship between DAM cells andCD11c+ microglia in the healthy brain.

4. The authors propose that downregulation of P2y12 may be a physiological adaptationenabling acquisition of the DAM phenotype. In contrast to the situation with Cx3cr1,evidence for transcriptional regulation downstream of P2y12 has not been presented.Instead, loss of this purinergic receptor will render microglia “blind” to ATP signaling, and islikely to be deleterious with regard to their physiological and wound‐limiting functions(Abiega et al., 2016; Davalos et al., 2005). Such lesions can include microvascularhemorrhage characteristic of AD (Lou et al., 2016).

5. It should also be noted that increased CSF‐soluble TREM2 was incisively studied indominantly inherited AD (Suarez‐Calvet et al., 2016), and shown conclusively to increasealong with CSF biomarkers of amyloid and tau pathology. As sTREM2 is shed from themembrane‐associated form, these data suggest that TREM2 upregulation in human ADoccurs relatively late in the pathogenic cascade, raising uncertainty whether the DAMphenotype is implicated in the response to preclinical AD pathology, the main focus ofpresent therapeutic efforts (Sperling et al., 2013).

In summary, DAM cells appear to constitute a distinct response of microglia to a number ofstates involving altered CNS homeostasis. This report provides a preliminary characterizationand points to a sequential acquisition of the phenotype. Their regulation and activities indisease states, for good or ill, remain undeined.

References:Sevigny J, Chiao P, Bussiere T, Weinreb PH, Williams L, Maier M, Dunstan R, Salloway S, Chen T,Ling Y, O'Gorman J, Qian F, Arastu M, Li M, Chollate S, Brennan MS, Quintero‐Monzon O,Scannevin RH, Arnold HM, Engber T, Rhodes K, Ferrero J, Hang Y, Mikulskis A, Grimm J, Hock C,Nitsch RM, Sandrock A. The antibody aducanumab reduces Aβ plaques in Alzheimer'sdisease. Nature. 2016 Aug 31;537(7618):50‐6. PubMed.

Condello C, Yuan P, Schain A, Grutzendler J. Microglia constitute a barrier that preventsneurotoxic protoibrillar Aβ42 hotspots around plaques. Nat Commun. 2015 Jan29;6:6176. PubMed.

Liu Z, Condello C, Schain A, Harb R, Grutzendler J. CX3CR1 in microglia regulates brainamyloid deposition through selective protoibrillar amyloid‐β phagocytosis. J Neurosci.2010 Dec 15;30(50):17091‐101. PubMed.

Bhaskar K, Konerth M, Kokiko‐Cochran ON, Cardona A, Ransohoff RM, Lamb BT. Regulation oftau pathology by the microglial fractalkine receptor. Neuron. 2010 Oct 6;68(1):19‐31.PubMed.

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Prodinger C, Bunse J, Kruger M, Schiefenhovel F, Brandt C, Laman JD, Greter M, Immig K,Heppner F, Becher B, Bechmann I. CD11c‐expressing cells reside in the juxtavascularparenchyma and extend processes into the glia limitans of the mouse nervous system.Acta Neuropathol. 2011 Apr;121(4):445‐58. Epub 2010 Nov 13 PubMed.

Abiega O, Beccari S, Diaz‐Aparicio I, Nadjar A, Laye S, Leyrolle Q, Gomez‐Nicola D, Domercq M,Perez‐Samartın A, Sanchez‐Zafra V, Paris I, Valero J, Savage JC, Hui CW, Tremblay ME , Deudero JJ,Brewster AL, Anderson AE, Zaldumbide L, Galbarriatu L, Marinas A, Vivanco Md, Matute C,Maletic‐Savatic M, Encinas JM, Sierra A. Neuronal Hyperactivity Disturbs ATPMicrogradients, Impairs Microglial Motility, and Reduces Phagocytic ReceptorExpression Triggering Apoptosis/Microglial Phagocytosis Uncoupling. PLoS Biol. 2016May;14(5):e1002466. Epub 2016 May 26 PubMed.

Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB. ATPmediates rapid microglial response to local brain injury in vivo. Nat Neurosci. 2005Jun;8(6):752‐8. PubMed.

Lou N, Takano T, Pei Y, Xavier AL, Goldman SA, Nedergaard M. Purinergic receptor P2RY12‐dependent microglial closure of the injured blood‐brain barrier. Proc Natl Acad Sci U S A.2016 Jan 26;113(4):1074‐9. Epub 2016 Jan 11 PubMed.

Suarez‐Calvet M, Araque Caballero MA , Kleinberger G, Bateman RJ, Fagan AM, Morris JC, Levin J,Danek A, Ewers M, Haass C, Dominantly Inherited Alzheimer Network. Early changes in CSFsTREM2 in dominantly inherited Alzheimer's disease occur after amyloid depositionand neuronal injury. Sci Transl Med. 2016 Dec 14;8(369):369ra178. PubMed.

Sperling RA, Karlawish J, Johnson KA. Preclinical Alzheimer disease‐the challenges ahead.Nat Rev Neurol. 2013 Jan;9(1):54‐8. Epub 2012 Nov 27 PubMed.

David Gosselin

Université Laval

Posted: 12 Jun 2017Work by Keren‐Shaul et al. provides a very compelling demonstration that microglia activationin the context of chronic diseases, and possibly aging, does not seem to operate in turns ofsimple “digital–on/off” states. Transition states are involved, and these may be also criticallyinvolved in brain pathologies. To my knowledge, this is the irst time such a state of “transition”has been described for microglia, at least in such a comprehensive manner. I also think that thedata here demonstrate the limitation of the paradigm of M1–M2 state of microglia (ormacrophage) 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 workhelps signiicantly reine our conceptualization of microglia activation phenotypes in the brain.

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In addition, there seems to be an important role for TREM2 in the transition of microglia fromthe 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 DAMstate is neuroprotective. It would have been very informative for the authors to show howabsence of TREM2 in their AD model affects cognitive functions in mice. Nonetheless, I think thehypothesis is reasonable. Thus, it could be that one key role of TREM2 is to promote an overallstate of microglia activation that enables these cells to perform better functions that helpneutralize disease mechanisms relevant to AD.

What triggers this microglia‐activating function of TREM2 is still not well understood, butevidence suggest that lipids and/or proteins involved in lipid biology like ApoE might have arole (Wang et al., 2015; Yeh et al., 2016). Also, what is it about the DAM state that helps microgliaprevent AD? Keren‐Shaul et al. note that many genes linked with the DAM state are related tolysosomal/phagocytosis functions and lipid metabolism. Thus, another very importantcontribution of that study is that it “nominates” genes whose activity might be critical to thatprotective state of DAM. This should prove very beneicial 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 Imicroglia to transition to the cluster II phenotype remain unknown. This is a point raised by theauthors, and one for which we still lack robust answers in 2017. Interestingly, some evidencefrom multiple studies suggests that a type I interferon signaling may be at play. One of the moreperipheral results from Keren‐Shaul et al. is that DAMs are also present in the brains of oldermice (Figure S4E). In addition, a previous study by the groups of Michal Schwartz and Ido Amitshowed 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 integratedwith this response. Nonetheless, putting the current study in the context of the previous one, itis tempting to hypothesize that the transition from cluster I to the cluster II and III state ofmicroglia may be driven, at least in part, by type I interferon signaling. This is a relativelystraightforward 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. Forone, the preliminary evidence from Keren‐Shaul et al. on Lpl mRNA expression, which is inducedin 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 atUniversity of Groningen, which reported that senile plaque‐associated microglia in the brains ofindividuals diagnosed with AD expressed high levels of APOE, AXL, TREM2, and TYROBP, whichwere all components of clusters II and/or DAM microglia (Yin et al., 2017). Lastly, the recent

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characterization of human microglia by Christopher Glass’ laboratory at UC San Diego showedthat key signaling pathways that are important to specify gene expression in mouse microgliaare relatively well conserved in human microglia (Gosselin et al., 2017).

Thus, there is certainly some early evidence suggesting that the mouse DAM phenotype may berelevant to human microglia in AD, but the full extent of these similarities needs to bethoroughly investigated. Given the huge progress we have made in our understanding ofmicroglia biology over the past 10 years, we expect that we will have an answer to that criticalquestion as well in the near future.

References:Guerreiro R, Wojtas A, Bras J, Carrasquillo M, Rogaeva E, Majounie E, Cruchaga C, Sassi C, KauweJS, Younkin S, Hazrati L, Collinge J, Pocock J, Lashley T, Williams J, Lambert JC, Amouyel P, GoateA, Rademakers R, Morgan K, Powell J, St George‐Hyslop P, Singleton A, Hardy J, . TREM2variants in Alzheimer's disease. N Engl J Med. 2013 Jan 10;368(2):117‐27. PubMed.

Wang Y, Ulland TK, Ulrich JD, Song W, Tzaferis JA, Hole JT, Yuan P, Mahan TE, Shi Y, Gilillan S,Cella M, Grutzendler J, DeMattos RB, Cirrito JR, Holtzman DM, Colonna M. TREM2‐mediatedearly microglial response limits diffusion and toxicity of amyloid plaques. J Exp Med.2016 May 2;213(5):667‐75. Epub 2016 Apr 18 PubMed.

Wang Y, Cella M, Mallinson K, Ulrich JD, Young KL, Robinette ML, Gilillan S, Krishnan GM,Sudhakar S, Zinselmeyer BH, Holtzman DM, Cirrito JR, Colonna M. TREM2 lipid sensingsustains the microglial response in an Alzheimer's disease model. Cell. 2015 Mar12;160(6):1061‐71. Epub 2015 Feb 26 PubMed.

Yeh FL, Wang Y, Tom I, Gonzalez LC, Sheng M. TREM2 Binds to Apolipoproteins, IncludingAPOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid‐Beta by Microglia.Neuron. 2016 Jul 20;91(2):328‐40. PubMed.

Baruch K, Deczkowska A, David E, Castellano JM, Miller O, Kertser A, Berkutzki T, Barnett‐ItzhakiZ, Bezalel D, Wyss‐Coray T, Amit I, Schwartz M. Aging‐induced type I interferon response atthe 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, Moller T, Tamm JA,Abdourahman A, Hol EM, Kamphuis W, Bayer TA, De Deyn PP, Boddeke E. Immunehyperreactivity of Aβ plaque‐associated microglia in Alzheimer's disease. NeurobiolAging. 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 DG, Levy ML, Ransohoff RM, Gage FH, Glass CK.An environment‐dependent transcriptional network speciies human microgliaidentity. Science. 2017 May 25; PubMed.

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Marie­Eve Tremblay

Université Laval

Posted: 12 Jun 2017The DAM microglia reported by Keren‐Shaul et al. share several markers with "dark microglia,” aphenotype we recently characterized at the ultrastructural level (Bisht et al., 2016). Darkmicroglia are rarely present under steady‐state conditions, in the hippocampus, cerebral cortex,amygdala, and hypothalamus, but become prevalent upon chronic stress, aging, fractalkinesignaling deiciency (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 DAMmicroglia), 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 Aβplaques. Besides their phagocytosis of Aβ (we very frequently observed engulfments), darkmicroglia appear extremely active at synapses, even more than normal microglia, suggestingtheir implication in the pathological/traumatic remodeling of neuronal circuits.

Regarding the DAM microglia, I agree with David Gosselin that it would be extremely importantto determine their consequences on cognitive function by conducting behavioral experiments.

References:Bisht K, Sharma KP, Lecours C, Sanchez MG, El Hajj H, Milior G, Olmos‐Alonso A, Gomez‐Nicola D,Luheshi G, Vallieres L, Branchi I, Maggi L, Limatola C, Butovsky O, Tremblay ME . Darkmicroglia: A new phenotype predominantly associated with pathological states. Glia.2016 May;64(5):826‐39. Epub 2016 Feb 5 PubMed.

Elena Galea

Universitat Autònoma de Barcelona

Posted: 12 Jun 2017The existence of plaque‐associated microglia as a distinct molecular entity was previouslydocumented by the labs of Elly Hol and Javier Vitorica. The current paper is a very elegantdissection and analysis of microglia subtypes, but there is no demonstration that the DAMmicroglia engulf plaques or restrict neurodegeneration, as respectively stated in the titles of theAlzforum 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 thatvestigial pathways from the macrophage precursors that give rise to microglia become activatedin an aberrant manner. The resulting DAM microglia might be a defective microglia that neglectsregulation of neuronal circuits, and a defective macrophage that has no capacity to eficientlyphagocytose plaques.

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With regard to the role of TREM2 in the phenotypical transformation of microglia, I ind quite ontarget recent studies from the Haass lab, which have shown alterations in microglia motility in amodel of FTD caused by a TREM2 mutation. I posit that impaired microglia surveillance mightalter synaptic scaling, thereby causing neuronal hyperexcitation and ensuing “burn out,” whichmay explain the striking decrease in brain metabolism shown in the mice.

We recently published an opinion piece proposing a revision of the notion of"neuroinlammation": Masgrau et al., 2017.

References:Masgrau R, Guaza C, Ransohoff RM, Galea E. Should We Stop Saying 'Glia' and'Neuroinlammation'?. Trends Mol Med. 2017 Jun;23(6):486‐500. Epub 2017 May 9 PubMed.

Michal Schwartz

Weizmann Institute of Science

Marco Colonna

Washington University School of Medicine

Ido Amit

Weizmann Institute of Science

Posted: 16 Jun 2017We 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 pathologicaldescriptions. However, this is the irst study that describes the disease‐associated microgliapopulation in precise molecular terms. Previous attempts over the last decade to identify thesecells classiied an entire zoo of different myeloid populations, and as such overlooked theimportant pathways and genes at play in DAM, while attributing inaccurate (and sometimesdeleterious) functions to these cells. We believe the cells and pathways we describe are animportant stepping‐stone to move the ield forward.

2. The genetic background of the control mice is identical to the 5xFAD. They are raised in thesame facility and cages to avoid any unrelated genetic or environmental effects. We fully agreethat both sexes need to be characterized in detail. We reported the sex of the mice in both theigure legends and methods section. Our conclusions are based on single‐cell proiling of morethan 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 theresolution that we proile. It is important to note that while all DAM cells are CD11c+, manyCD11c+ cells in Alzheimer’s disease brains are not DAM. This may cause artifactual results, as

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seen in the many papers that reported on such mixtures of cell populations. See, for example,the following igure plotting CD11c intensity (measured with index sorting on the single celllevel) and the DAM program.

(A) Scatter plot showing the correlation to the average DAM transcription program and index‐sorting intensities of CD11c. (B) tSNE plot of CD11b+ cells from both wild‐type and AD mouse. Cellcolor based on cluster association as shown in A. All DAM cells (red) show high levels (3.5E3) ofCD11c but not all CD11c cells are DAM.

4. A note regarding the statement in the Alzforum news article “The epigenetic basis for thisexpression phenotype will be of considerable interest to decipher.” We show in the paper thatthe epigenetic proile of DAM and homeostatic microglia are almost identical (S2F and S2G).Focusing on DAM‐speciic genes, we observed active H3K4me2 regions in both the microglia andDAM, demonstrating that the DAM program is already primed in homeostatic microglia (FiguresS2F and S2G). This inding is in line with our single‐cell proiling of WT and TREM2 KO. TheDAM program is highly anticipated by the microglia and regulated; these are not cells losingcontrol in the face of neuronal damage.

5. The inding that myeloid cells around plaques are brain‐resident microglia, not bone marrow‐derived macrophages, has been deinitively demonstrated through recent parabiosisexperiments in two distinct models of AD, including that presented in our study. The detailedmolecular characterization of DAM, and the two stages, strongly support these studies, settingthese cells aside from conventional macrophages. In fact, previous attempts to characterize the

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myeloid cells around plaques based on cellular markers have generated opposite resultsregarding TREM2‐expressing myeloid cells. This can be explained by the impurity of the myeloidcells due to the markers used.

6. Genetic studies have shown that TREM2 polymorphisms impairing TREM2 function increasethe risk of AD three‐ to ivefold. Our identiication of a TREM2‐dependent stage in the activationof 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, isan important question for future studies. It can be addressed with the cutting‐edge approach wehave developed and reported.

Ido Amit of the Weizmann Institute of Science also contributed to this comment.

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News

PRIMARY PAPERSKeren‐Shaul H, Spinrad A, Weiner A, Matcovitch‐Natan O, Dvir‐Szternfeld R, Ulland TK, David E,Baruch K, Lara‐Astaiso D, Toth B, Itzkovitz S, Colonna M, Schwartz M, Amit I. A UniqueMicroglia Type Associated with Restricting Development of Alzheimer's Disease. Cell. 2017Jun 15;169(7):1276‐1290.e17. Epub 2017 Jun 8 PubMed.

Tay TL, Mai D, Dautzenberg J, Fernandez‐Klett F, Lin G, Sagar, Datta M, Drougard A, Stempl T,Ardura‐Fabregat A, Staszewski O, Margineanu A, Sporbert A, Steinmetz LM, Pospisilik JA, Jung S,Priller J, Grun D, Ronneberger O, Prinz M. A new fate mapping system reveals context‐dependent random or clonal expansion of microglia. Nat Neurosci. 2017 Jun;20(6):793‐803.Epub 2017 Apr 17 PubMed.

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Colonna M, Butovsky O. MicrogliaFunction in the Central Nervous SystemDuring Health and Neurodegeneration.Annu Rev Immunol. 2017 Apr 26;35:441­468. Epub 2017 Feb 9 PubMed.

What Makes a Microglia? Tales from theTranscriptome 5 Jun 2017

Do Perivascular Macrophages Mediate AβPathology? 26 May 2017

Brain Inflammation Drastically ChangesBehavior and Lifespan in Animals 5 May2017

New Evidence Confirms TREM2 Binds Aβ,Drives Protective Response 19 Apr 2017