Brownjohn PW, Smith J, Solanki R, Lohmann E, Houlden H, Hardy J, Dietmann S, Livesey FJ. Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia. Stem Cell Reports, March 29, 2018.

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  1. Since it is notoriously difficult to investigate microglial function in human brains, and so-called microglial cell lines do not faithfully reproduce the characteristics of microglia isolated from brain (Butovsky et al., 2014), human microglia are an extremely important tool for understanding function and dysfunction of these cells in neurodegenerative disorders. Over the last 18 months, several protocols have been published describing the differentiation of human microglia-like cells from stem cells, such as iPSCs. The first report by the Jaenisch lab (Muffat et al., 2016) was followed by several similar approaches using altered protocols (Abud et al., 2017; Douvaras et al., 2017; Haenseler et al., 2017; Pandya et al., 2017). The major differences in these protocols are: the use of defined differentiation factors vs. coculture with human iPSC-derived brain cells, such as astrocytes; the use of embryoid bodies (EBs) vs. cell monolayers during the first differentiation phase yielding hematopoietic progenitor-like cells; and the base media and growth factors used for directed differentiation and specific time points. While this variability should help bring out the best possible protocol, it makes it quite hard for labs that are new to the field to decide which protocol to implement. In general, protocols using EBs and cocultures with other cell types are harder to transfer to a new lab, since small alterations in the handling of these crucial steps can have dramatic consequences on the fate and number of differentiated cells. At the current—still pioneering—state of the field, independent studies confirming transferability of protocols, validity of cellular fates/transcriptomics, as well as applicability for disease research are crucial.

    The recent paper by the Livesey lab, therefore, is a welcome addition, because it validates and expands the work of Sally Cowley’s lab (Haenseler et al., 2017) describing a very similar protocol, which itself was derived from earlier work on macrophage differentiation from Cowley’s lab (van Wilgenburg et al., 2013). However, Brownjohn et al. go beyond generating and characterizing human microglia-like cells by applying them to investigate TREM2 biology and AD-associated mutations. Nevertheless, one always needs to keep in mind that such iPSC-derived human microglia, which are kept outside of their natural environment under artificial culture conditions, may have several limitations that could affect their phenotypes in vitro.

    Livesey and colleagues demonstrate nicely that human microglia expressing the TREM2 variants T66M and W50C show reduced maturation of the full-length protein, which as a consequence results in reduced shedding and reduced formation of the TREM2 C-terminal fragment. This is fully consistent with previous findings in human CSF (Kleinberger et al., 2014), where no sTREM2 is found in Nasu-Hakola patients carrying the T66M mutation. Furthermore, very similar findings were also made upon overexpression of human TREM2 in cultured cells (Kleinberger et al., 2014; Kober et al., 2016; Park et al., 2015), and in a mouse model with the T66M variant knocked in via the CRISPR/Cas9 technology (Kleinberger et al., 2017). In contrast to these findings, Brownjohn et al. found no TREM2 genotype-specific differences in response to LPS. However, this compares short-term in vitro experiments using cultured microglia (Brownjohn et al.,) with long-term in vivo analyses in mice (Kleinberger et al., 2017). More surprising may be, however, the lack of a difference in phagocytosis and LDL uptake as we and others reported in mice and upon heterologous expression of TREM2 variants (Kleinberger et al., 2017; Yeh et al., 2016). As stated by the authors, this may be due to compensatory effects via changes in the expression of other phagocytic receptors. Indeed, we have shown that antibody-dependent plaque clearance is reduced upon loss of TREM2 (Xiang et al., 2016). However, increased antibody dose compensated for reduced plaque clearance. Compensation was obtained upon loss of TREM2 function via increased expression of Fcg-receptors and enhanced Syk signaling (Xiang et al., 2016). Therefore, loss-of-function phenotypes are likely masked by compensatory mechanisms, which, as the authors state, is compatible with the late onset of the disease. It would be interesting to see if genotype-dependent differences become more apparent when human microglia are investigated in an environment that drives the switch of microglia from a homeostatic state to a disease-associated state, since under these conditions genes involved in chemotaxis and phagocytosis are upregulated in wild-type TREM2 expressing cells, but blocked in T66M mutants (Butovsky et al., 2014; Krasemann et al., 2017; Mazaheri et al., 2017). Moreover, one needs to keep in mind that even in human microglia, the TREM2 variants failed to maturate and thus lost their biological function, although this may be difficult to quantitate under resting conditions.

    References:

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    Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, Koeglsperger T, Dake B, Wu PM, Doykan CE, Fanek Z, Liu L, Chen Z, Rothstein JD, Ransohoff RM, Gygi SP, Antel JP, Weiner HL. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat Neurosci. 2014 Jan;17(1):131-43. Epub 2013 Dec 8 PubMed.

    Douvaras P, Sun B, Wang M, Kruglikov I, Lallos G, Zimmer M, Terrenoire C, Zhang B, Gandy S, Schadt E, Freytes DO, Noggle S, Fossati V. Directed Differentiation of Human Pluripotent Stem Cells to Microglia. Stem Cell Reports. 2017 Jun 6;8(6):1516-1524. Epub 2017 May 18 PubMed.

    Haenseler W, Sansom SN, Buchrieser J, Newey SE, Moore CS, Nicholls FJ, Chintawar S, Schnell C, Antel JP, Allen ND, Cader MZ, Wade-Martins R, James WS, Cowley SA. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response. Stem Cell Reports. 2017 Jun 6;8(6):1727-1742. PubMed.

    Kleinberger G, Brendel M, Mracsko E, Wefers B, Groeneweg L, Xiang X, Focke C, Deußing M, Suárez-Calvet M, Mazaheri F, Parhizkar S, Pettkus N, Wurst W, Feederle R, Bartenstein P, Mueggler T, Arzberger T, Knuesel I, Rominger A, Haass C. The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism. EMBO J. 2017 Jul 3;36(13):1837-1853. Epub 2017 May 30 PubMed.

    Kleinberger G, Yamanishi Y, Suárez-Calvet M, Czirr E, Lohmann E, Cuyvers E, Struyfs H, Pettkus N, Wenninger-Weinzierl A, Mazaheri F, Tahirovic S, Lleó A, Alcolea D, Fortea J, Willem M, Lammich S, Molinuevo JL, Sánchez-Valle R, Antonell A, Ramirez A, Heneka MT, Sleegers K, van der Zee J, Martin JJ, Engelborghs S, Demirtas-Tatlidede A, Zetterberg H, Van Broeckhoven C, Gurvit H, Wyss-Coray T, Hardy J, Colonna M, Haass C. TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis. Sci Transl Med. 2014 Jul 2;6(243):243ra86. PubMed.

    Kober DL, Alexander-Brett JM, Karch CM, Cruchaga C, Colonna M, Holtzman MJ, Brett TJ. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms. Elife. 2016 Dec 20;5 PubMed.

    Krasemann S, Madore C, Cialic R, Baufeld C, Calcagno N, El Fatimy R, Beckers L, O'Loughlin E, Xu Y, Fanek Z, Greco DJ, Smith ST, Tweet G, Humulock Z, Zrzavy T, Conde-Sanroman P, Gacias M, Weng Z, Chen H, Tjon E, Mazaheri F, Hartmann K, Madi A, Ulrich JD, Glatzel M, Worthmann A, Heeren J, Budnik B, Lemere C, Ikezu T, Heppner FL, Litvak V, Holtzman DM, Lassmann H, Weiner HL, Ochando J, Haass C, Butovsky O. The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity. 2017 Sep 19;47(3):566-581.e9. PubMed.

    Mazaheri F, Snaidero N, Kleinberger G, Madore C, Daria A, Werner G, Krasemann S, Capell A, Trümbach D, Wurst W, Brunner B, Bultmann S, Tahirovic S, Kerschensteiner M, Misgeld T, Butovsky O, Haass C. TREM2 deficiency impairs chemotaxis and microglial responses to neuronal injury. EMBO Rep. 2017 Jul;18(7):1186-1198. Epub 2017 May 8 PubMed.

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    van Wilgenburg B, Browne C, Vowles J, Cowley SA. Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions. PLoS One. 2013;8(8):e71098. Epub 2013 Aug 12 PubMed.

    Xiang X, Werner G, Bohrmann B, Liesz A, Mazaheri F, Capell A, Feederle R, Knuesel I, Kleinberger G, Haass C. TREM2 deficiency reduces the efficacy of immunotherapeutic amyloid clearance. EMBO Mol Med. 2016 Sep 1;8(9):992-1004. PubMed.

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

    View all comments by Dominik Paquet

  2. This paper describes a highly efficient method for generating microglia-like primitive macrophage precursors from wild-type, and TREM2-mutant (NHD-linked), fibroblasts. The wild-type PMPs have surface markers that we expect for macrophages and microglia (e.g., Iba1, CD45, and TREM2) and transcriptomic analysis, particularly when considering microglia-enriched transcripts, groups them with other preparations of primary cultured microglia. Thus, reassuringly, the stem cell-derived microglia are responding to the absence of the brain environment in the same way as primary cultured cells; furthermore, the cells are shown to be capable of migrating deep into brain organoids, eventually adopting a ramified, surveillance phenotype.

    The important impact of this study on translational neuroscience stems from the ability to make microglia from patients with neurological diseases and compare them with healthy controls. Here the team induced microglia from patients with hemizygous and homozygous destabilizing mutations in TREM2 and show a progressive failure of the protein to be delivered to the plasma membrane, essentially replicating the results of Kleinberger et al. (2014) and others. Accordingly, the normal regulated proteolytic degradation of surface-expressed wild-type TREM2, by ADAM10/17 followed rapidly by g-secretase, is disrupted for the abnormally trafficked variants. This probably indicates that an alternative pathway is available for microglia to clear immature TREM2 variants that are stalled on the secretory pathway.

    Things get even more exciting when Brownjohn and colleagues start to probe how the stem cell-derived microglia behave in innate immune system paradigms. It is known that the TLR4 and TREM2 signalling pathways exhibit reciprocal inhibition, however, in the context of the “nuclear option” of LPS agonism the pro-inflammatory pathway is maximally stimulated, likely rapidly downregulating TREM2 expression even in the wild-type cells. Of greater interest in neurodegenerative disease might be the interaction of the TREM2 pathway with weaker TLR4 agonist, such as amyloid containing debris (reviewed in Molteni et al., 2016), that may actually stimulate both pathways. The finding that induced microglia with destabilising TREM2 variants phagocytose E. coli normally, and show only minimal deficits in acetylated-LDL phagocytosis, goes against some literature for cultured microglia; however, as noted by the authors, this reflects the complexity of the system, where many proteins can detect innate immune triggers, and a possibly overlapping set of proteins may also directly mediate subsequent phagocytosis.

    These results go some way to informing a debate on whether TREM2 acts as the “eyes” of the microglia—allowing them to sense debris and pathogens and stimulate phagocytosis, but not necessarily taking part directly in the phagocytosis—or whether TREM2 may (also) be the “mouth” of the microglia directly mediating the uptake of material. NHD teaches us that signalling by the “eyes” is essential, because loss of the TREM2 co-receptor DAP12 is an alternative cause of the disease—imagine the microglia in NHD as being blindly active. So, failure to see differences in the phagocytosis assays between wild-type and TREM2-variant lines is less of a challenge to our understanding of NHD as the failure of the TREM2-variant cells to modulate innate immune interactions. The answer to a better understanding of the interactions of the TLR4 and TREM2 pathways may be in the choice of possible TLR4 ligands—those that are likely to be prevalent in neurodegenerative disease may be more informative.

    References:

    Kleinberger G, Yamanishi Y, Suárez-Calvet M, Czirr E, Lohmann E, Cuyvers E, Struyfs H, Pettkus N, Wenninger-Weinzierl A, Mazaheri F, Tahirovic S, Lleó A, Alcolea D, Fortea J, Willem M, Lammich S, Molinuevo JL, Sánchez-Valle R, Antonell A, Ramirez A, Heneka MT, Sleegers K, van der Zee J, Martin JJ, Engelborghs S, Demirtas-Tatlidede A, Zetterberg H, Van Broeckhoven C, Gurvit H, Wyss-Coray T, Hardy J, Colonna M, Haass C. TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis. Sci Transl Med. 2014 Jul 2;6(243):243ra86. PubMed.

    Molteni M, Gemma S, Rossetti C. The Role of Toll-Like Receptor 4 in Infectious and Noninfectious Inflammation. Mediators Inflamm. 2016;2016:6978936. Epub 2016 May 18 PubMed.

    View all comments by Damian Crowther

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