Grubman A, Choo XY, Chew G, Ouyang JF, Sun G, Croft NP, Rossello FJ, Simmons R, Buckberry S, Landin DV, Pflueger J, Vandekolk TH, Abay Z, Zhou Y, Liu X, Chen J, Larcombe M, Haynes JM, McLean C, Williams S, Chai SY, Wilson T, Lister R, Pouton CW, Purcell AW, Rackham OJ, Petretto E, Polo JM. Transcriptional signature in microglia associated with Aβ plaque phagocytosis. Nat Commun. 2021 May 21;12(1):3015. PubMed.

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  1. In this study, Alexandra Grubman and colleagues leveraged an impressive set of experimental approaches and technologies to characterize a subpopulation of microglia with high phagocytic activities around amyloid plaques from the 5xFAD transgenic mouse model. To do so, the authors used an innovative flow cytometry approach to isolate two populations of microglia based on their labeled-fibrillar Aβ content; an approach that we have also successfully used to characterize phagocytic microglia in a novel APP KI mouse model (Xia et al., 2021). Grubman et al. provide important new insight into our understanding of how interaction with amyloid plaques impacts microglial biology and has the potential to inform therapeutic strategies targeting this cell type.

    The authors demonstrate that methoxy-XO4-positive (XO4-positive) microglia sorted from brains have a different gene expression signature compared to XO4-negative microglia in 5xFAD mice. A subset of the differentially expressed genes overlaps with those measured in XO4-negative microglia, but the magnitude of alteration is exacerbated in XO4-positive microglia. The upregulation of genes belonging to pathways involved in metabolic and phagolysosome functions and the increased phagocytic capacity of XO4-positive microglia supports the notion that plaque-associated microglia transition to a responsive state that may help them reduce Aβ aggregates and/or associated pathology (e.g., neuritic dystrophy).

    Those findings further add to our understanding of microglia phenotype at the site of amyloid plaques and partially confirm the “PIGs” signature defined by spatial transcriptomics (Jul 2020 news). Interestingly, we recently reported that XO4-positive microglia in the AppSAA knock-in mouse model (Xia et al., 2021), show not only exacerbated gene expression changes, notably in genes controlling lysosomal function, but also profound lipid alterations, which could indicate that those phagocytic microglia may struggle to regulate lipid metabolism as previously discussed (Lewcock et al., 2020). 

    Another interesting observation from this work is the discovery that XO4-negative microglia, potentially distal from amyloid plaques, show a relatively high level of non-fibrillar Aβ. This observation would suggest that not only the quantity of Aβ but its conformation may determine the transcriptomic and functional changes in microglia. This may have implications for amyloid-targeting antibodies that may bind to certain types of Aβ and drive internalization of the immunocomplex in microglia. However, it is unclear if this observation might be caused by the artificially high levels of Aβ production in the brain of this transgenic model. One alternative explanation is that XO4-positive microglia may become overwhelmed by the large amount of phagocytosed fibrillar Aβ and struggle to degrade those peptides. If this is the case, it would be important to determine if the deficit in lysosomal degradation could cause lysosomal lipid accumulation and eventually cellular dysfunction.

    This manuscript reports many other fascinating observations, including the ability of XO4-positive microglia to revert back to a homeostatic state when exposed to an environment deprived of amyloid plaques. State reversibility of microglia may have interesting implications for effective anti-amyloid therapies, since microglia in the vicinity of those plaques may revert back to a functional homeostatic state when the amyloid aggregates are cleared out.

    Overall, this study adds to our understanding of the complex and heterogeneous roles of microglia in the context of neurodegeneration. Continued work to examine whether alteration of the pathways described in this work is able to impact disease progression will be an exciting area for future study.

    References:

    Xia D, Lianoglou S, Sandmann T, Calvert M, Suh JH, Thomsen E, Dugas J, Pizzo ME, DeVos SL, Earr TK, Lin CC, Davis S, Ha C, Nguyen H, Chau R, Yulyaningsih E, Solanoy H, Masoud ST, Liang R, Lin K, Thorne RG, Garceau D, Whitesell JD, Sasner M, Harris JA, Scearce-Levie K, Lewcock JW, Di Paolo G, Sanchez PE. Fibrillar Aβ causes profound microglial metabolic perturbations in a novel APP knock-in mouse model. bioRxiv. January 20, 2021.

    Lewcock JW, Schlepckow K, Di Paolo G, Tahirovic S, Monroe KM, Haass C. Emerging Microglia Biology Defines Novel Therapeutic Approaches for Alzheimer's Disease. Neuron. 2020 Dec 9;108(5):801-821. Epub 2020 Oct 22 PubMed.

    View all comments by Pascal E. Sanchez

  2. The study, by sorting microglia on the basis of which ones had taken up fluorescently labeled Aβ (i.e., the cells become methoxy-XO4-positive), revealed a phagocytic population, which reflected some of the differentially expressed genes we have come to expect from other single-cell RNA-sequencing studies in the AD field (Trem2, Tyrobp, ApoE, Lpl and lysosomal proteases and protease inhibitors such as cathepsins and Cst7 respectively). However, a number of other pathways appeared to be induced, including HIF-1α-induced genes, indicating an activation of anaerobic glycolysis (Pkm, Ldha). This perhaps denies the citric-acid cycle and oxidative phosphorylation of the pyruvate that initiates those bioenergetic pathways. This is interesting in the context of the immunometabolic status of microglia-driving microglial phenotype.

    This signature appeared to be induced even in WT microglia following amyloid phagocytosis. This was shown in experiments using transplantation of Aβ-positive microglia onto organotypic hippocampal slice cultures prepared from WT or 5XFAD mice. However, these are complex experiments with many variables and are complex to interpret. For example, it did appear that some of the microglia that were not phagocytic (i.e., NIAD4-negative) still acquired the phagocytic gene signature and one wonders: Would simply presenting WT animals with fibrillar Aβ, in vivo, actually induce the phagocytic (XO4-positve) signature described here?

    There are fascinating data, suggesting that even though the XO4-positive microglia are clearly more phagocytic in ex vivo experiments (taking up more E. coli, PSD95 and fibrillar Aβ), it is actually the XO4-negative microglia that contain more PSD95 in the 5XFAD brain. Are the XO4-negative microglia, therefore, better equipped to phagocytose synaptic elements? Well, there are several interpretations of those data, but it may simply be a case of exposure: If XO4-positive microglia are most proximal to plaques and fully engaged in amyloid phagocytosis, it may be that those microglia that are a little more distal from the plaque are exposed to the synaptic loss that occurs in the vicinity of plaques.

    The authors do not dwell on one important finding: The XO4-positive signature, although present in microglia isolated from humans, was not enriched in microglia from brains of patients with Alzheimer’s disease.

    However, this is mitigated somewhat by the observation that patients who did have some XO4-positive microglial signatures tended to have upregulated genes from one particular cluster that, once again, expressed these HIF-1α-dependent genes. Possible upstream drivers of this HIF-1α-dependent signature are proinflammatory stimuli, including Pam3csk, which is an activator of Toll-like 1/2 receptors. TLR2 has been shown several years ago to recognize amyloids, including Aβ (Tükel et al., 2009). This could indicate that amyloid may activate pro-inflammatory pathways via TLR2, driving an HIF-1α response that is key in the induction of a phagocytic phenotype. It is intriguing that this is present in some AD cases but is not strongly associated with microglia from AD patients more generally. This could represent one level of differential susceptibility among individuals, perhaps affecting progression or patient-specific manifestations.

    References:

    Tükel C, Wilson RP, Nishimori JH, Pezeshki M, Chromy BA, Bäumler AJ. Responses to amyloids of microbial and host origin are mediated through toll-like receptor 2. Cell Host Microbe. 2009 Jul 23;6(1):45-53. PubMed.

    View all comments by Colm Cunningham

  3. This study by Grubman et al. is a bioinformatics tour de force that highlights the HIF-1α signaling pathway as a central mediator of the microglial response to Aβ plaque phagocytosis. These results are very interesting and match very well with our own previously published work, which highlighted HIF-1α signaling as an important microglial response to Aβ pathology (Wendeln et al., 2018). Moreover, in unpublished data on single-cell microglia from APP23 mice, we have also found that methoxy-XO4 (MX04)-positive microglia are characterized by increased HIF-1α signaling, fully in line with the current findings by Grubman et al., who demonstrate very nicely that this response is directly triggered by microglial interaction/uptake of aggregated Aβ.

    I am not completely convinced by their single-cell RNA-Seq analysis demonstrating that MX04-positive cells may be on a premature aging trajectory, because this dataset contains relatively few cells (sometimes from single animals per group or of different sex) and it does not demonstrate microglial heterogeneity within the same age/experimental groups, which is something that has now been shown repeatedly (e.g., Sala Frigerio et al., 2019; Hammond et al., 2019). Moreover, while the joint analysis of the different human datasets is commendable and important, I am a little concerned by the (sometimes almost complete) separation of the different datasets. One would at least expect to see an overlap of homeostatic microglial subtypes from all datasets, and this lack may indicate that there are still significant effects of batches/different methodologies that were not sufficiently compensated during data integration.

    The analyses performed by Grubman et al. regarding the role of HIF-1α in altering microglial effector functions rely heavily on in vitro assays and (as pointed out by the authors themselves) a chronic microglial-plaque interaction in vivo may result in very different responses. In this regard, a recent study has shed more light on the role of microglial HIF-1α activation in response to plaque pathology (March-Diaz et al., 2021). It not only corroborates the crucial role of HIF-1α in the microglial response to Aβ plaque deposition, but also shows that, in vivo, stabilization of HIF-1α (leading to enhanced HIF-1α signaling) induces “microglial quiescence,” reducing microglial association with plaques and increasing neuritic damage. This is reminiscent of the microglial phenotype in Trem2 knockout animals and TREM2 mutation carriers as described by the Colonna group, and it raises the question of how these pathways are interrelated.

    Grubman et al. speculate that MX04-positive microglia may represent a subset of DAM microglia and it will be interesting to see whether this holds up in future work, or if HIF-1α may actually be required for inducing the DAM phenotype. 

    References:

    Wendeln AC, Degenhardt K, Kaurani L, Gertig M, Ulas T, Jain G, Wagner J, Häsler LM, Wild K, Skodras A, Blank T, Staszewski O, Datta M, Centeno TP, Capece V, Islam MR, Kerimoglu C, Staufenbiel M, Schultze JL, Beyer M, Prinz M, Jucker M, Fischer A, Neher JJ. Innate immune memory in the brain shapes neurological disease hallmarks. Nature. 2018 Apr;556(7701):332-338. Epub 2018 Apr 11 PubMed.

    Sala Frigerio C, Wolfs L, Fattorelli N, Thrupp N, Voytyuk I, Schmidt I, Mancuso R, Chen WT, Woodbury ME, Srivastava G, Möller T, Hudry E, Das S, Saido T, Karran E, Hyman B, Perry VH, Fiers M, De Strooper B. The Major Risk Factors for Alzheimer's Disease: Age, Sex, and Genes Modulate the Microglia Response to Aβ Plaques. Cell Rep. 2019 Apr 23;27(4):1293-1306.e6. PubMed.

    Hammond TR, Dufort C, Dissing-Olesen L, Giera S, Young A, Wysoker A, Walker AJ, Gergits F, Segel M, Nemesh J, Marsh SE, Saunders A, Macosko E, Ginhoux F, Chen J, Franklin RJ, Piao X, McCarroll SA, Stevens B. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity. 2019 Jan 15;50(1):253-271.e6. Epub 2018 Nov 21 PubMed.

    March-Diaz R, Lara-Ureña N, Romero-Molina C, Heras-Garvin A, Ortega-de San Luis C, Alvarez-Vergara MI, Sanchez-Garcia MA, Sanchez-Mejias E, Davila JC, Rosales-Nieves AE, Forja C, Navarro V, Gomez-Arboledas A, Sanchez-Mico MV, Viehweger A, Gerpe A, Hodson EJ, Vizuete M, Bishop T, Serrano-Pozo A, Lopez-Barneo J, Berra E, Gutierrez A, Vitorica J, Pascual A. Hypoxia compromises the mitochondrial metabolism of Alzheimer’s disease microglia via HIF1. Nature Aging. 1, 2021, pp.385–99. Nat. Aging.

    View all comments by Jonas Neher

  4. The work by Grubman et al. is a nice step toward understanding the complex transcriptional responses observed in Aβ-plaque-associated microglia (AβAM). Using an elegant approach, they confirm a trend observed in other works: The closer to Aβ deposits, the stronger/more complex the induced microglial transcriptional responses. Previous studies have shown that the HIF-1 pathway is highly active in microglia from AD mouse models (Baik et al., 2019; March-Diaz et al., 2021; Ulland et al., 2017; Wendeln et al., 2018) and HIF-1 itself is included in the DAM signature (Keren-Shaul et al., 2017). This new work is a direct confirmation of the relevance of the hypoxic/HIF-1 pathway in AD microglia. However, what role HIF-1 plays in AD microglia, and what the therapeutic consequences of modulating the pathway could be, are open questions.

    HIF-1 is a well-known cell response to metabolic stress, which adapts cellular metabolism to low oxygen levels by increasing anaerobic glycolysis (glucose to lactate) and decreasing aerobic respiration (OXPHOS). Paradoxically, AβAM from AD mouse models and human samples upregulate OXPHOS (March-Diaz et al., 2021). Of note, the MX-04-positive microglia described by Grubman et al. and the human amyloid-responsive microglia also upregulated both OXPHOS and HIF-1(Nguyen et al., 2020). We also showed OXPHOS activation in all DAM we analyzed, suggesting that mitochondrial activity is a requirement for microglial activity, whereas HIF-1 activation was only observed in Aβ models, and could be an unwanted side response (March-Diaz et al., 2021). Our in vivo conditional genetic experiments to stabilize HIF-1 against degradation, or systemic hypoxia exposure, demonstrated that overactivation of HIF-1 reduces the microglial defensive responses against Aβ and aggravates neuropathology, phenocopying Trem2-deficient mutants (March-Diaz et al., 2021; see also Jonas Neher comment above). Further in vivo work with conditional microglial mutations of HIF-1 or HIF-1 transcriptional targets will be key to define the role of this pathway in AD progression.

    As stated in other comments, the in vitro analysis of the role of HIF-1 in microglial responses to Aβ should be taken with caution, as the optimal nutrient and oxygen conditions in which we culture microglia are far from those bathing microglia in vivo. We have recently shown that Aβ plaques constitute ischemic foci lacking blood vessels, shedding some light on the activation of HIF-1 observed around Aβ plaques (Alvarez-Vergara et al., 2021). Indeed, microglia are almost the only cells able to survive under those stressful conditions. Perhaps they pay a toll for it?

    References:

    Alvarez-Vergara MI, Rosales-Nieves AE, March-Diaz R, Rodriguez-Perinan G, Lara-Ureña N, Ortega-de San Luis C, Sanchez-Garcia MA, Martin-Bornez M, Gómez-Gálvez P, Vicente-Munuera P, Fernandez-Gomez B, Marchena MA, Bullones-Bolanos AS, Davila JC, Gonzalez-Martinez R, Trillo-Contreras JL, Sanchez-Hidalgo AC, Del Toro R, Scholl FG, Herrera E, Trepel M, Körbelin J, Escudero LM, Villadiego J, Echevarria M, de Castro F, Gutierrez A, Rabano A, Vitorica J, Pascual A. Non-productive angiogenesis disassembles Aß plaque-associated blood vessels. Nat Commun. 2021 May 25;12(1):3098. PubMed.

    Baik SH, Kang S, Lee W, Choi H, Chung S, Kim JI, Mook-Jung I. A Breakdown in Metabolic Reprogramming Causes Microglia Dysfunction in Alzheimer's Disease. Cell Metab. 2019 Sep 3;30(3):493-507.e6. Epub 2019 Jun 27 PubMed.

    Keren-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 Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell. 2017 Jun 15;169(7):1276-1290.e17. Epub 2017 Jun 8 PubMed.

    March-Diaz R, Lara-Ureña N, Romero-Molina C, Heras-Garvin A, Ortega-de San Luis C, Alvarez-Vergara MI, Sanchez-Garcia MA, Sanchez-Mejias E, Davila JC, Rosales-Nieves AE, Forja C, Navarro V, Gomez-Arboledas A, Sanchez-Mico MV, Viehweger A, Gerpe A, Hodson EJ, Vizuete M, Bishop T, Serrano-Pozo A, Lopez-Barneo J, Berra E, Gutierrez A, Vitorica J, Pascual A. Hypoxia compromises the mitochondrial metabolism of Alzheimer’s disease microglia via HIF1. Nature Aging. 1, 2021, pp.385–99. Nat. Aging.

    Nguyen AT, Wang K, Hu G, Wang X, Miao Z, Azevedo JA, Suh E, Van Deerlin VM, Choi D, Roeder K, Li M, Lee EB. APOE and TREM2 regulate amyloid-responsive microglia in Alzheimer's disease. Acta Neuropathol. 2020 Oct;140(4):477-493. Epub 2020 Aug 25 PubMed. Correction.

    Ulland TK, Song WM, Huang SC, Ulrich JD, Sergushichev A, Beatty WL, Loboda AA, Zhou Y, Cairns NJ, Kambal A, Loginicheva E, Gilfillan S, Cella M, Virgin HW, Unanue ER, Wang Y, Artyomov MN, Holtzman DM, Colonna M. TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease. Cell. 2017 Aug 10;170(4):649-663.e13. PubMed.

    Wendeln AC, Degenhardt K, Kaurani L, Gertig M, Ulas T, Jain G, Wagner J, Häsler LM, Wild K, Skodras A, Blank T, Staszewski O, Datta M, Centeno TP, Capece V, Islam MR, Kerimoglu C, Staufenbiel M, Schultze JL, Beyer M, Prinz M, Jucker M, Fischer A, Neher JJ. Innate immune memory in the brain shapes neurological disease hallmarks. Nature. 2018 Apr;556(7701):332-338. Epub 2018 Apr 11 PubMed.

    View all comments by Javier Vitorica

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