. Alzheimer's-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci. 2020 Aug;23(8):927-938. Epub 2020 Jun 8 PubMed.

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  1. This exciting new study by Andreone et al. examines the importance of PLCy2 activity downstream of TREM2 signaling for survival, myelin phagocytosis, lipid metabolism, and processing of neuronal debris by human microglia. This work also provides an elegant example of how iPSC-derived microglia can be used to examine the biology and interactions between AD risk genes. By combining CRISPR gene editing with iPS-microglia (iMG), Andreone et al. examine the impact of TREM2 or PLCG2 deletion on microglial gene expression, function, and lipid metabolism, providing important new insight into the role of and interactions between these two AD risk genes.

    As TREM2 signaling is known to be mediated at least in part by PLCy2 activity, the authors began by comparing the transcriptional changes that occur between TREM2 and PLCy2 isogenic knockout lines. As expected, many changes are shared between these two lines, further demonstrating the importance of PLCy2 in TREM2 signaling. However, the authors also uncover many additional genes that are downregulated only in PLCy2 KO microglia. Next, to determine whether TREM2 and PLCy2 deletion confers similar functional effects, the authors examine the microglial response to myelin, demonstrating that deletion in either gene leads to the accumulation of myelin and a failure to induce key lipid metabolism genes. By performing elegant lipidomic analysis, Andreone and colleagues further show that these impaired responses also lead to a highly similar accumulation of sterol species within TREM2 and PLCy2 knockout microglia. Importantly, this failed myelin response is paralleled by a similar failure in the clearance of neuronal debris in a co-culture axotomy model. But what about the AD-associated R47H TREM2 mutation? To determine whether this mutation confers similar effects to deletion, the authors performed rescue experiments to induce TREM2 R47H expression within TREM2 knockout microglia and reveal a similar accumulation of myelin and cholesterol esters as observed in TREM2 heterozygous knockout cells, suggesting that the R47H mutation acts as a hypomorph. Using an equivalent approach to examine the PLCy2 P522R protective AD variant, the authors demonstrate that P522R further reduces microglial lipid accumulation compared with wild-type PLCy2.

    So, do TREM2 and PLCy2 deletions simply phenocopy each other? As first alluded to in their initial RNA-sequencing experiment, PLCy2 deletion causes a downregulation of many additional genes that are not altered in TREM2 KO microglia. Indeed, in the periphery PLCy2 has been reported to signal downstream of multiple pro-inflammatory TLRs, thus the authors treated their iMGs with zymosan, a TLR ligand. This additional experiment showed that most zymosan-induced genes were shared between wild-type and TREM2 KO microglia, suggesting that TREM2 is not directly involved in TLR signaling. In contrast, ~50 percent fewer genes were upregulated in PLCy2 KOs treated with zymosan, supporting the notion that as in the periphery, microglial PLCy2 activity is also critical for TLR signaling. Notably, levels of IL1β, a proinflammatory cytokine that is strongly implicated in AD, were reduced by 50 percent in zymosan-treated PLCy2 KO cells, but conversely increased by 64 percent in zymosan-treated TREM2 KOs. Thus, PLCy2 signaling is required for TLR-mediated proinflammatory signaling, which under normal circumstances appears to be dampened by TREM2 activity. Additionally, NLRP3 inflammasome activation (which induces IL1β maturation) was attenuated upon PLCy2 loss, while TREM2 KOs secreted significantly more IL1β than wild-type cells. In this respect, the negative impact of microglial NLRP3 on both amyloid and tau pathology has been clearly shown (Venegas et al., 2017; Heneka et al., 2018; Ising et al., 2019). Thus, one must be cautious to explore therapies that simply increase PLCy2 signaling, as proper TREM2 function is likely also crucial to prevent an overactive hyperinflammatory state.

    Overall, this is an insightful and compelling study that greatly advances our understanding of TREM2 and PLCy2 signaling in human microglia. However, there are a couple of questions and caveats that future studies will hopefully address. The authors mention that loss of either TREM2 or PLCy2 reduces phagocytosis of both myelin and neurons and induced lipid accumulation. As TREM2 is not the only receptor responsible for the uptake of these ligands, one might hypothesize that the impaired phagocytosis of myelin or neuronal debris might also be exacerbated by defective degradation of these lipid-laden substrates which could in turn feed back to alter the expression of other relevant receptors. One technical caveat to also consider is that the rescue experiments were achieved through lentiviral transduction. Our lab has found in preliminary studies that lentiviral expression of GFP alone in iMGs changes thousands of genes. Thus, we need to be cautious with the interpretation of viral manipulations in a cell type that plays a role in viral defense. Another curious finding was the detection of reduced TREM2 mRNA expression in the R47H rescue experiment. This observation contrasts with the finding that TREM2 mRNA expression and splicing is unchanged in human R47H iMGs and patient brains (Xiang et al., 2018). However, the authors confirmed similar membrane localization of wild-type and R47H TREM2 protein and thus the observed effects of the R47H mutation are likely initially induced by altered ligand binding and reduced downstream signaling.

    One last caveat to consider is that in vitro iPS-microglia models most closely resemble cultured human microglia (Gosselin et al., 2017; Hasselmann et al., 2019). As significant differences exist between in vitro and in vivo microglial gene expression and perhaps then also microglial function, it will critical to follow up on this elegant work by examining similar questions in chimeric model systems (Hasselmann et al., 2019; Mancuso et. al., 2019). In this respect, ongoing studies in our lab are examining both P522R PLCy2 and TREM2 R47H mutant human microglia in chimeric AD mice. Thus far, our data indicates lipid accumulation and enrichment of many lipid-associated genes holds true in human DAMs in AD mice. Our ongoing lipidomic studies will hopefully further determine the extent to which these exciting findings hold true in AD chimeric mice in vivo.   

    Ongoing analysis of the impact of these mutations on microglia function, lipidomics, and AD pathology will hopefully reveal whether these risk genes influence microglia function in vivo as elegantly predicted by this important study.

    References:

    . Microglia-derived ASC specks cross-seed amyloid-β in Alzheimer's disease. Nature. 2017 Dec 20;552(7685):355-361. PubMed.

    . Inflammasome signalling in brain function and neurodegenerative disease. Nat Rev Neurosci. 2018 Oct;19(10):610-621. PubMed.

    . NLRP3 inflammasome activation drives tau pathology. Nature. 2019 Nov;575(7784):669-673. Epub 2019 Nov 20 PubMed.

    . The Trem2 R47H Alzheimer's risk variant impairs splicing and reduces Trem2 mRNA and protein in mice but not in humans. Mol Neurodegener. 2018 Sep 6;13(1):49. PubMed.

    . An environment-dependent transcriptional network specifies human microglia identity. Science. 2017 Jun 23;356(6344) Epub 2017 May 25 PubMed.

    . Development of a Chimeric Model to Study and Manipulate Human Microglia In Vivo. Neuron. 2019 Sep 25;103(6):1016-1033.e10. Epub 2019 Jul 30 PubMed.

    . 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.

    View all comments by Mathew Blurton-Jones
  2. This is a very important paper, because it supports the idea to therapeutically modulate TREM2 functions in microglia. Andreone and colleagues independently support this approach by demonstrating shared LOF phenotypes upon TREM2 or PLCγ2 knockout. This suggests that promoting TREM2/PLCγ2-dependent lipid metabolism may be protective in AD.

    This also nicely fits with our recent findings, together with Mikko Hiltunen, showing that the protective mutation stimulates exactly those functions, which are reduced by the TREM2 LOF. 

    However, as is always the case with microglia, any beneficial change may be associated with detrimental events. In this case, this is a TREM2-independent function downstream of toll-like receptors, which mediates an inflammatory response. Therefore, we need to understand in much greater detail the molecular signature of a protective microglia sub-population, and specifically how this population can be modulated/stimulated by therapeutic anti-TREM2 antibodies. 

    This paper also demonstrates very nicely the fantastic potential of iMG as models for functional analyses in a human background.

    View all comments by Christian Haass
  3. This study provides important new insights into the cell biological functions of PLCγ2 in (human) microglia. It has been known for some years that a coding variant in PLCγ2 (P522R) protects from Alzheimer’s disease and other dementias, but how PLCγ2 (a phospholipase C enriched in myeloid cells) connects to established AD pathogenic pathways remained unknown and difficult to foretell.

    Using an impressive array of experimental conditions in gene-edited iPSC-microglia, Andreone and Przybyla et al. demonstrate that PLCγ2 is a downstream effector of TREM2 and a regulator of lipid metabolism. This exciting discovery directly connects PLCγ2 to well-established AD pathways involving APOE, TREM2, and microglial activation. The authors show that PLCγ2 regulates microglial lipid homeostasis, lipid clearance, and is involved in lipid-dependent inflammatory signaling in human microglia, similar to what has been previously reported for TREM2 and APOE (Nugent et al., 2020). 

    Furthermore, the study confirms previous reports (Magno et al., 2019; Maguire et al., 2020) by showing that PLCγ2 P522R is a functional hypermorph. The authors extend these findings by showing that PLCγ2 P522R more potently reduces lipid accumulation in lipid-challenged microglia than wild-type PLCγ2. The overall findings of this paper fit with a broader body of recent literature reporting on altered cholesterol metabolism downstream of SAD-risk genes in astrocytes, microglia, and neurons. The current PLCγ2 finding is particularly exciting, as it is the first to demonstrate that AD protective factors may also play into this mechanism.

    Importantly, the authors also note that PLCγ2 not only functions downstream of TREM2, but is also activated by other microglial receptors such as TLRs. To what extent protection by PLCγ2 P522R is conferred through its effects on lipid metabolism versus regulation of other immune pathways will be an interesting puzzle for coming studies. The strong overlap between lipid metabolic changes in PLCγ2 and TREM2 knockout microglia does, however, suggest that cholesterol metabolism is a common converging pathway downstream of these genes, as well as downstream of APOE risk variants.

    Overall, this paper takes a great stride forward toward understanding the function of PLCγ2 in human microglia and AD, and is of great value to the research community.

    References:

    . Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimers Res Ther. 2019 Feb 2;11(1):16. PubMed.

    . The Alzheimer’s disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function. bioRxiv, April 28, 2020

    . TREM2 Regulates Microglial Cholesterol Metabolism upon Chronic Phagocytic Challenge. Neuron. 2020 Mar 4;105(5):837-854.e9. Epub 2020 Jan 2 PubMed.

    View all comments by Rik van der Kant
  4. Andreone and colleagues have provided an extensive and compelling study on the roles of the PLCγ2 in human iPSC-derived microglia-like cells. A significant outcome of this study is the validation that the findings in the human iMG cells largely recapitulate those of murine microglia.

    The roles of TREM2 and PLCγ2 in microglia in  AD pathogenesis have commanded substantial attention by the identification that coding variants in TREM2 and PLCγ2 are associated with AD risk. The authors report that TREM2 signals through PLCγ2 to mediate cell survival, phagocytosis, processing of neuronal debris, and lipid metabolism in microglia. They demonstrate that TREM2 and PLCγ2 share common signaling pathways and that PLCγ2 act as a major regulator of microglial functional in AD. In this regard, the experimental outcomes serve largely to reinforce the current understanding of how PLCγ2 interacts with upstream and downstream signaling elements to elicit TREM2 and TLR-driven changes in microglial phenotypes.

    There are a few surprises, however. For example, TREM2 was shown to suppress proinflammatory responses in microglia in the context of TLR stimulation. These data also suggest that PLCγ2 represents the point of significant intersection of TLR and TREM2 signaling in AD. This would be consistent with an older literature on the involvement of microglial TLRs in AD pathogenesis.

    Recent studies provided data arguing that the protective P522R mutation in PLCγ2 promotes beneficial function in microglia through increased PLCγ2 activity, microglial activation, and enhanced endocytic clearance (Magno et al., 2019; Maguire et al., 2020; Takalo et al., 2020). The current report is consistent with these observations and broadens our understanding of the role of PLCγ2 in lipid metabolism. 

    Moreover, a recent study reported a novel missense variant (M28L) in PLCγ2 confers increased risk for LOAD. PLCγ2 expression was shown to be increased in several brain regions of LOAD patients and strongly correlated with brain amyloid burden in LOAD patients and AD animal models (Tsai et al., 2020). In AD, it is unclear whether the effects of genetic variants are related to alteration in enzymatic activity, the capacity of PLCγ2 to interact with other signaling elements, or topology. These findings highlight the importance of PLCγ2 biology in the brain, but also raise the question more broadly of whether increased PLCγ2 activity favors disease exacerbation or attenuation.

    References:

    . Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimers Res Ther. 2019 Feb 2;11(1):16. PubMed.

    . The Alzheimer’s disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function. bioRxiv, April 28, 2020

    . The Alzheimer's disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegener. 2020 Sep 11;15(1):52. PubMed.

    . PLCG2 as a Risk Factor for Alzheimer’s Disease. bioRxiv: 2020.2005.2019.104216

    View all comments by Andy Tsai
  5. This is a very interesting study. It linked TREM2 and PLCG2 signaling pathways in IPSC-derived macrophages/microglial cells to get insights into the mechanisms underlying the deleterious effect of TREM mutations and the protective effect of the PLCG2 P522R variant in the pathophysiology of AD.

    The authors show very elegantly (using corresponding KO IPSC macrophages and rescue experiments) that disruption of TREM2 and PLCG2, which is downstream of TREM2/DAP12 signaling pathway, lead to lipid processing dysregulation and lipid accumulation further validated by lipidomic profiling. This study also further highlights the power of using iPSC-derived macrophages/microglia as tools to better understand the biology of human macrophages and their contribution to disease.

    View all comments by Florent Ginhoux
  6. This study eloquently consolidates Magno et al., 2019, and Maguire et al., 2020, and it adds to our understanding of the interaction of TREM2 and PLCγ2 as well as the independent role of PLCγ2 in iMG. The authors present a clear hypothesis as to how these AD-associated loci may act in AD etiology and highlight the complex interplay of AD risk loci in disease.

    The authors use CRISPR to knock out rather than manipulate disease-specific variation. The findings correlate with the suggested loss of function of TREM2 and the gain of function of PLCγ2, as conferred by R47H and R62H in TREM2 and the protective P522R mutation in PLCG2. However, the recent identification of the M28L risk variant in PLCG2 (Tsai et al. 2020), and the observation that PLCG2 expression levels are increased in several brain regions of individuals living with AD, suggest that elevation of PLCγ2 activity could have both beneficial and detrimental effects.

    Of great interest is the articulately presented utility of an iMG model to recapitulate human disease in vitro. CRISPR targeting of specific risk variants in disease-relevant models such as iMG will hopefully further unpack the complex interplay of these and other multifaceted disease loci.

    This valuable research further highlights microglia and cholesterol metabolism in AD, validating the extensive genetic and epidemiological evidence. It is exciting to observe the convergence of evidence across numerous scientific fields identifying valid therapeutic targets for AD but also emphasizing the complexity of targeting multifunctional proteins.

    References:

    . Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimers Res Ther. 2019 Feb 2;11(1):16. PubMed.

    . The Alzheimer’s disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function. bioRxiv, April 28, 2020

    . PLCG2 as a Risk Factor for Alzheimer’s Disease. bioRxiv: 2020.2005.2019.104216

    View all comments by Rebecca Sims
  7. This paper provides an exciting and important step forward in the understanding of the multiple functions of the PLCγ2 enzyme in microglia.

    Although previous studies have described a key role for this PLC isoform in the regulation of peripheral immune system responses, ideas on its contribution to the brain’s innate immune system have so far remained speculative. This is partly due to PLCγ2 being only recently recognized as playing a role in central neurodegenerative conditions, and to this enzyme’s involvement in complex and not fully characterized microglia signaling pathways, many of which display crosstalk with each other. Thus, it is conceivable that activation of PLCγ2 via different membrane receptors, but also via intracellular enzymes such as Rac, might contribute to several and diverse microglia functions.

    Interestingly, the authors show that PLCγ2 signals not only downstream of Trem2, to support cholesterol metabolism, but also—and independently—downstream of TLR, to elicit inflammatory responses. Therefore, as discussed by the authors, activation of PLCγ2 could have opposing consequences in terms of beneficial and detrimental effects in neurodegenerative disorders, such as late-onset Alzheimer’s disease.

    The DAM phenotype, which depends on the ApoE-Trem2 pathway and shows a transcriptomic profile associated with increased expression of inflammatory factors, has been associated with both beneficial and detrimental effects (Keren-Shaul et al., 2017; Krasemann et al., 2017). This might be both cell-status-dependent and dosage-dependent, as exemplified by the effect of the P522R PLCγ2 variant (Magno et al., 2019; Maguire et al., 2020; Takalo et al., 2020; this paper). Therefore, a potentially more nuanced and less dualistic interpretation of these mechanisms in AD would be needed.

    Also, further investigations would be required to define the interplay between lipid metabolism and neuroinflammation, and how the key enzyme PLCγ2 is wired in the signalling networks supporting those functions.

    Furthermore, it would be interesting to understand:

    • how the pathways are regulated in different scenarios and at different stages of disease progression, including as a substrate of an aging immune system, with altered metabolism/bioenergetics and declined immune function;
    • how this model fits with the activation of other immune receptors, including Fc receptors, which have been shown to activate PLCγ2 in innate immune cells;
    • how this network is modulated in different cell states, to support microglia function.

    A selective pharmacological tool for PLCγ2 would help address some of these questions, and help define further roles of this enzyme in innate immune cell signalling pathways.

    Dysregulation of lipid metabolism in the brain has been linked to Alzheimer’s disease. Andreone and colleagues neatly show that potentiation of the Trem2-PLCγ2 pathway should be further explored as a potential avenue for the development of disease-modifying therapies.

    References:

    . 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.

    . The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity. 2017 Sep 19;47(3):566-581.e9. PubMed.

    . The Alzheimer’s disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function. bioRxiv, April 28, 2020

    . Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimers Res Ther. 2019 Feb 2;11(1):16. PubMed.

    . The Alzheimer's disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegener. 2020 Sep 11;15(1):52. PubMed.

    . PLCG2 as a Risk Factor for Alzheimer’s Disease. bioRxiv: 2020.2005.2019.104216

    View all comments by Lorenza Magno

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