Olah M, Menon V, Habib N, Taga MF, Ma Y, Yung CJ, Cimpean M, Khairallah A, Coronas-Samano G, Sankowski R, Grün D, Kroshilina AA, Dionne D, Sarkis RA, Cosgrove GR, Helgager J, Golden JA, Pennell PB, Prinz M, Vonsattel JP, Teich AF, Schneider JA, Bennett DA, Regev A, Elyaman W, Bradshaw EM, De Jager PL. Single cell RNA sequencing of human microglia uncovers a subset associated with Alzheimer's disease. Nat Commun. 2020 Nov 30;11(1):6129. PubMed.

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  1. This paper describes the single-cell analysis of microglia from 17 individuals, 10 with AD, four MCI, and three surgical patients, consisting of 16,000 cells, yielding nine distinct microglial clusters. This work is of obvious importance, given the role of microglia in Alzheimer’s and other neurodegenerative diseases.

    A strong feature of this paper is that the authors used both postmortem and surgical resections, and subsequently confirmed their findings in a number of third-party datasets by integrating these datasets with their cells. I would be interested to see an even wider integration of human microglial datasets. For example, the authors mention the studies from Del-Aguila et al. and Habib et al., but do not investigate whether these microglia separate into their cluster structure (Del-Aguila et al., 2019; Habib et al., 2017). Also, the inclusion of human microglia from a chimeric mouse model recently published would have been informative (Mancuso et al., 2019). 

    The authors, when comparing results from the surgical resections and postmortem samples, chose to exclude a cluster of cells they associated with cell stress. In agreement with this, we and others have published studies showing that the microglial expression profiles can be influenced by experimental parameters and protocols (Thrupp et al., 2020; Marsh et al., 2020). This might (in part) be a reason for the somewhat limited consistency that most human microglia studies (including this one) find between their own data and other studies.

    In conclusion, this dataset will be very valuable for future understanding of the role of microglia in Alzheimer's disease. However, it does remain an open question as to why a number of excellent studies fail to converge on a consistent human-disease-associated microglial profile. Is this due to technicalities? Due to high variation between human samples? Or do microglia play a role much earlier in the pathogenesis of human AD that has been missed?

    References:

    Sankowski R, Böttcher C, Masuda T, Geirsdottir L, Sagar, Sindram E, Seredenina T, Muhs A, Scheiwe C, Shah MJ, Heiland DH, Schnell O, Grün D, Priller J, Prinz M. Mapping microglia states in the human brain through the integration of high-dimensional techniques. Nat Neurosci. 2019 Nov 18; PubMed. Correction.

    Del-Aguila JL, Li Z, Dube U, Mihindukulasuriya KA, Budde JP, Fernandez MV, Ibanez L, Bradley J, Wang F, Bergmann K, Davenport R, Morris JC, Holtzman DM, Perrin RJ, Benitez BA, Dougherty J, Cruchaga C, Harari O. A single-nuclei RNA sequencing study of Mendelian and sporadic AD in the human brain. Alzheimers Res Ther. 2019 Aug 9;11(1):71. PubMed.

    Thrupp N, Sala Frigerio C, Wolfs L, Skene NG, Fattorelli N, Poovathingal S, Fourne Y, Matthews PM, Theys T, Mancuso R, de Strooper B, Fiers M. Single-Nucleus RNA-Seq Is Not Suitable for Detection of Microglial Activation Genes in Humans. Cell Rep. 2020 Sep 29;32(13):108189. PubMed.

    Habib N, Avraham-Davidi I, Basu A, Burks T, Shekhar K, Hofree M, Choudhury SR, Aguet F, Gelfand E, Ardlie K, Weitz DA, Rozenblatt-Rosen O, Zhang F, Regev A. Massively parallel single-nucleus RNA-seq with DroNc-seq. Nat Methods. 2017 Oct;14(10):955-958. Epub 2017 Aug 28 PubMed.

    Mancuso R, Van Den Daele J, Fattorelli N, Wolfs L, Balusu S, Burton O, Liston A, Sierksma A, Fourne Y, Poovathingal S, Arranz-Mendiguren A, Sala Frigerio C, Claes C, Serneels L, Theys T, Perry VH, Verfaillie C, Fiers M, De Strooper B. Stem-cell-derived human microglia transplanted in mouse brain to study human disease. Nat Neurosci. 2019 Dec;22(12):2111-2116. Epub 2019 Oct 28 PubMed.

    Marsh SE, Kamath T, Walker AJ, Dissing-Olesen L, Hammond TR, Young AM, Abdulraouf A. Single Cell Sequencing Reveals Glial Specific Responses to Tissue Processing & Enzymatic Dissociation in Mice and Humans. BioRxiv, December 3, 2020

    Thrupp N, Sala Frigerio C, Wolfs L, Skene NG, Fattorelli N, Poovathingal S, Fourne Y, Matthews PM, Theys T, Mancuso R, de Strooper B, Fiers M. Single-Nucleus RNA-Seq Is Not Suitable for Detection of Microglial Activation Genes in Humans. Cell Rep. 2020 Sep 29;32(13):108189. PubMed.

    View all comments by Mark Fiers

  2. The paper by Olah et al. evaluates the microglial signature through scRNA-seq analyses of live microglia cells from 10 Alzheimer’s disease (AD), four mild cognitive impairment (MCI) and three temporal lobe epilepsy (TLE) patients and compares these data with microglial signatures from two mouse models of neurodegeneration (CKp25 and 5XFAD), as well as with human microglial signatures previously delineated through scRNA-seq and snRNA-seq (Sankowski et al., 2019; Mathys et al., 2019). 

    The study characterizes four major human microglia populations involved in, respectively, homeostasis, interferon response, antigen presentation, and proliferation. This is in line with the four subsets that were previously found in mouse models of AD. However, the frequencies of the non-homeostatic subsets were significantly reduced in AD/MCI patients compared to those with TLE. Notably, the frequency of the antigen presentation cluster is diminished in AD samples, although immunohistochemical studies have shown an increase in HLA-DR expression. Overall, the functional impact of these microglial subsets remains obscure.

    Furthermore, Olah et al. showed that the disease-associated microglia (DAM) signature identified in mouse models of AD is not confined to one subset of human microglia, but rather is distributed among all microglia populations, confirming the idea that the mouse DAM signature does not entirely recapitulate the microglial response in human AD. Earlier this year we published a study in Nature Medicine in which we performed snRNA-seq of AD and control patients using brain specimens obtained through ROSMAP. We also reached the conclusion that microglia from human AD patients exhibit a signature partially distinct from that in mouse models: AD-associated risk factors, as well as “homeostatic” genes were upregulated in microglia from AD patients but not in those from controls (Zhou et al., 2020). 

    Unfortunately, our paper and conclusions have been ignored in the analyses carried out by Olah et al., although the data have been broadly available to the community since February 2020 from the ROSMAP portal. Moreover, the conceptual difference between human AD and mouse models of AD was clearly highlighted in our paper. Our data would have corroborated some of the conclusions reached by Olah et al., but also yielded some important differences that may be due to heterogeneity of patients analyzed, brain regions processed, or technical procedures used to perform snRNA-seq. Evaluation and discussion of these differences would have been helpful for advancing the field of Alzheimer’s disease and neurodegeneration in general.

    References:

    Sankowski R, Böttcher C, Masuda T, Geirsdottir L, Sagar, Sindram E, Seredenina T, Muhs A, Scheiwe C, Shah MJ, Heiland DH, Schnell O, Grün D, Priller J, Prinz M. Mapping microglia states in the human brain through the integration of high-dimensional techniques. Nat Neurosci. 2019 Nov 18; PubMed. Correction.

    Mathys H, Davila-Velderrain J, Peng Z, Gao F, Mohammadi S, Young JZ, Menon M, He L, Abdurrob F, Jiang X, Martorell AJ, Ransohoff RM, Hafler BP, Bennett DA, Kellis M, Tsai LH. Author Correction: Single-cell transcriptomic analysis of Alzheimer's disease. Nature. 2019 Jul;571(7763):E1. PubMed.

    Zhou Y, Song WM, Andhey PS, Swain A, Levy T, Miller KR, Poliani PL, Cominelli M, Grover S, Gilfillan S, Cella M, Ulland TK, Zaitsev K, Miyashita A, Ikeuchi T, Sainouchi M, Kakita A, Bennett DA, Schneider JA, Nichols MR, Beausoleil SA, Ulrich JD, Holtzman DM, Artyomov MN, Colonna M. Author Correction: Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease. Nat Med. 2020 Jun;26(6):981. PubMed.

    View all comments by Yingyue Zhou

  3. Cluster 13 is rather curious. Is there any possibility that scRNA-seq could pick up RNA from cells that microglia have phagocytosed? RNA has the reputation of being exquisitely labile, and I'm sure that's true if your objective is a complete, intact strand. But some of my colleagues are picking up sequence-able RNA from the insoluble aggregates that accrue in the aging brain.

    View all comments by Steve Barger

  4. We thank our colleagues for their interest in our work and their comments. In a rapidly moving field—such as the study of human microglia in Alzheimer’s disease—it is difficult to have a manuscript fully up to date, in terms of incorporating recently published studies, by the time it is posted online. Drs. Colonna, Fiers, and Zhou highlight excellent recent work that needs to be considered as we interpret our results.

    The last major revision of our manuscript occurred at the beginning of 2020, prior to the publication of many of these papers, and they were therefore not incorporated into our article. Our manuscript had a protracted review process, since it was posted publicly on bioRxiv on June 11, 2018 (Olah et. al., 2018). Though the paper has gone through multiple revisions in two different journals since that date, and was presented at several international conferences, the basic message of the study did not change. It includes the discoveries that in the aged brain, the vast majority of microglia are in a state that can be best described as homeostatic, and that one subset (in the bioRxiv version the antigen presentation related subset was named cluster 4) has a negative association with AD dementia. Additionally, we also reported in the preprint that the murine disease-associated microglial (DAM) signature was distributed across a number of human microglia clusters, with the cluster depleted in AD showing the strongest enrichment in DAM genes.

    Since the observation regarding the prevalence of the homeostatic microglia phenotype in AD, and the discordance between mouse and human in terms of DAM, both represent a major paradigm shift in the field—and accordingly attracted a significant amount of criticism from reviewers—we were delighted to see consistent results replicating our findings emerging in the literature before the printed version of our manuscript became available. In particular, Dr. Colonna and Dr. Zhou’s manuscript is important in this regard, and the observed differences probably arise from the reported reduced ability of single-nucleus RNA sequencing approaches to capture microglial transcriptional activation states when compared to live-cell based approaches (Thrupp et. al., 2020). Larger datasets that are emerging should eventually converge on a more definitive model of microglial population structure that we can apply across studies and platforms, but it is clear that, in humans, there is no single microglial subtype that causes disease.

    There are a large number of different transcriptional programs that can be pathogenic in different conditions. In response to Dr. Barger’s comment, we agree that cluster 13 is an intriguing subset of cells that deserve further evaluation. They are quite distinct from the other CD45+ cells that we purified; it is possible that they represent phagocytic cells that have absorbed RNA from other cell types. However, this is speculative at this time, and it is possible that they represent a form of technical artifact. Further work will hopefully provide more answers to this question.

    References:

    Olah M, Menon V, Habib N, Taga M, Yung C, Cimpean M, Khairalla A, Dionne D, Hopp S, Frosch MP, Hyman BT, Beach TG, Sarkis R, Cosgrove GR, Helgager J, Golden JA, Pennell PB, Schneider JA, Bennett DA, Regev A, Elyaman W, Bradshaw EM, De Jager PL. A single cell-based atlas of human microglial states reveals associations with neurological disorders and histopathological features of the aging brain. bioRχiv. June 11, 2018 BioRxiv.

    Thrupp N, Sala Frigerio C, Wolfs L, Skene NG, Fattorelli N, Poovathingal S, Fourne Y, Matthews PM, Theys T, Mancuso R, de Strooper B, Fiers M. Single-Nucleus RNA-Seq Is Not Suitable for Detection of Microglial Activation Genes in Humans. Cell Rep. 2020 Sep 29;32(13):108189. PubMed.

    View all comments by Philip De Jager

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  1. Most Detailed Look Yet at Activation States of Human Microglia