TREM2 Risk Variant Eggs on Clique of Microglia in AD Brain

The R47H variant in TREM2 works in mysterious ways. According to a study published December 1 in Science Translational Medicine, it may sharpen the edge of a maladaptive subset of microglia in people with AD. Among those who died with the disease, carriers of the AD risk variant had more microglia with a pro-inflammatory bent than did noncarriers. The work was led by Li Gan at Weill Cornell Medical College in New York.

  • Cantankerous subset of inflammatory microglia expands in R47H-TREM2 carriers with AD.
  • In tauopathy mice, R47H drove a similar shift, but only in females.
  • In these mice, suppressing the Akt kinase calmed inflammatory responses.

When using human TREM2 knock-in mice, the study came to similar conclusions. In tauopathy mice expressing a single copy of the R47H variant, pro-inflammatory microglia expanded and memory deficits worsened. Strikingly, the TREM2 risk variant made things more dire only in females. The scientists pegged much of the variant’s mischief to downstream activation of Akt, and were able to thwart the detrimental effects by blocking the kinase with a small molecule. In all, their findings paint a complex, sex-specific picture of how TREM2 influences microglial function and disease.

Alzforum covered some of the data at a Keystone meeting (Jul 2019 conference news). At that time, Gan shared initial findings from TREM2 knock-in mice, which expressed a single copy of either the common (TREM2-CV) or risk variant (TREM2-R47H) and were crossed onto either a wild-type or P301S-tau background. Gan reported that, in females, expression of a single copy of the R47H variant promoted the expansion of a subset of microglia that expressed some disease-associated microglia (DAM) signature genes. R47H also exacerbated memory deficits, again in the female tauopathy mice. Curiously, male tauopathy mice had similar numbers of DAM-esque microglia, and similar memory deficits, regardless of which TREM2 variant they expressed.

DAM Cluster. In mice, microglia (red) expressing both P301S tau and R47H TREM2 shift gene expression toward a DAM profile (green). [Courtesy of Sayed et al., Science Translational Medicine.]

In the paper, co-first authors Faten Sayed and Lay Kodama and colleagues expanded their microglial studies in the knock-in mice, and investigated how R47H-TREM2 influenced microglia in people with AD. Because only female mice were influenced by which TREM2 variant they expressed, the researchers used them for single-nucleus transcriptomic studies of microglia.

They isolated microglia from the hippocampi of mice expressing mouse TREM2 alone, or one copy of the R47H-TREM2 variant, both on the wild-type or P301S background. They detected two distinct transcriptional clusters of microglia. While cluster 1 was found in mice of all four genotypes, cluster 2 was predominantly in mice with tauopathy (P301S). This second cluster featured upregulation of several DAM genes, including ApoE, Itgax, Lpl, Axl, and Cst7, as well as a cadre of interferon response genes. This cluster was even larger in female tauopathy mice that expressed a single copy of R47H-TREM2 (see image above).

Further experiments showed that, in the environment of tauopathy, the R47H-TREM2 variant turned up downstream signaling through the Nfkβ and Akt pathways. Akt activation, in particular, was responsible for provoking many of the pro-inflammatory responses in microglia. In fact, chronic treatment of female R47H-TREM2 tauopathy mice with the Akt inhibitor MK-2206 abolished the DAM-like, inflammatory subset of microglia, and spared synapses (see image below). Merck had previously trialed MK-2206 in cancer, without success.

Together, the findings suggest that rather than simply hobbling TREM2 function, the R47H variant ramps up specific signaling pathways that enhance damaging pro-inflammatory responses in a subset of microglia.

Caught in the Akt. Mice expressing a single copy of the R47H TREM2 variant differentially express 318 genes (right). Blocking Akt with MK2206 reversed that gene expression pattern. [Courtesy of Sayed et al., Science Translational Medicine.]

Does R47H do the same in people? The researchers tackled this question using single-nucleus RNA sequencing of mid frontal cortical tissue of 46 people who had died with AD, including 24 R47H-TREM2 carriers. The samples were matched for age, tau burden, and, where available, the person’s degree of impairment. Data from 263,672 nuclei delineated clusters of major cell types in the brain.

First off, unlike in the mouse experiments, the R47H variant was associated with transcriptional differences in all cell types in both men and women. The scientists counted the most differentially expressed genes, and the greatest divergence was between the sexes, in microglia, astrocytes, and oligodendrocytes. Among the 20,461 microglial nuclei, the R47H variant induced more upregulation of immune activation pathways in women than men, and boosted more metabolic pathways in men than women.

The researchers identified seven transcriptional clusters of cells that expressed high levels of microglial markers. Only one of them—MG4—was clearly larger in R47H carriers (see image below). Notably, among the seven subclusters, MG4 mostly closely resembled the DAM signature. Multiple immune pathways were ramped up in MG4 cells, including TNF-α signaling via Nfkβ, other inflammatory response pathways—and Akt activation. The MG4 population was slightly expanded in both male and female R47H carriers, but while statistically significant, the difference in MG4 numbers between carriers and noncarriers was small. Gan told Alzforum that she considers this difference meaningful, especially since all samples came from people who died with AD.

Risk Cluster. In AD patients with the TREM2 R47H risk variant, the microglial cluster MG4 was slightly larger than in patients with the common variant. [Courtesy of Sayed et al., Science Translational Medicine.]

The findings offer an autopsy look back at how the R47H-TREM2 variant influences microglial function in people with AD. While the predominant view in the field holds that R47H hobbles TREM2's receptor function, Gan’s data suggest that the variant’s effects are more complicated. TREM2 acts as both a receptor for ligands such as Aβ and phospholipids, and as a ligand once it is shed from the cell surface. Gan believes the variant may affect these aspects of TREM2 activity differently.

In that same vein, previous studies have found that TREM2 deficiency exerts different effects in amyloidosis and tauopathy models, and at different stages of disease (Oct 2017 news; Jul 2019 news; Jun 2020 news). 

How does the variant hold sway differently in men than in women? Gan noted that especially with respect to immune cells such as microglia, men and women mount divergent transcriptional responses, hence how R47H-TREM2 modulates their responses to different insults, including Aβ and tau accumulation, would be expected to differ. Gan's group is both investigating how R47H influences microglial function differently in the sexes, and searching for common effects, such as stepped-up activation of Akt, as those could point to common therapeutic strategies.—Jessica Shugart

Comments

  1. Previous observations from our lab and others that TREM2 KO, or TREM2 R47H Tg expression on a TREM2 KO background, attenuates neurodegeneration in the P301S model of tauopathy that can be puzzling given that the R47H variant increases the risk of AD. This study identifies an enrichment of “DAM”-like microglia using hTREM2R47H/mTREM2 mice that may more closely model the extent of TREM2 function in human AD TREM2 variant carriers.

    As the authors note, several of the genes upregulated and pathways identified by GSEA were related to interferon signaling, which may be distinct from the DAM state that is generally linked with upregulation of things like cathepsins, ApoE, and tetraspanins such as Cd9 and Cd63. Distinctions in interferon states and so-called “activated response microglia (ARMs)” were elegantly demonstrated by Bart DeStrooper’s group and collaborators and by David Hansen’s group among others. Whether dysfunctional TREM2 favors one subdivision of microglial activation that corresponds with tissue damage over another that may limit or respond to damage will be interesting to investigate.

    References:

    Friedman BA, Srinivasan K, Ayalon G, Meilandt WJ, Lin H, Huntley MA, Cao Y, Lee SH, Haddick PC, Ngu H, Modrusan Z, Larson JL, Kaminker JS, van der Brug MP, Hansen DV. Diverse Brain Myeloid Expression Profiles Reveal Distinct Microglial Activation States and Aspects of Alzheimer's Disease Not Evident in Mouse Models. Cell Rep. 2018 Jan 16;22(3):832-847. 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.

    View all comments by Jason Ulrich

  2. The effect of sex on AD susceptibility is well established, but the molecular basis for it is not understood. This paper addressed whether an AD risk factor, the R47H allele of the immune receptor TREM2 expressed by microglia, manifests sex-specific molecular phenotypes. Single-nuclei RNA-Seq of R47H carriers with AD identified profound differences in affected gene networks between males and females. Using an elegant mouse model with engineered knock-ins of human TREM2, the common allele and the R47H variant, on the background of an established PS19 model of tauopathy, the study showed an exacerbated immune response in female mice, which was accompanied by memory and spatial learning deficits.

    The study corroborates our previous finding of the exaggerated immune response in the brains of AD patients with R47H, particularly an upregulation of pro-inflammatory cytokines and interferon response signatures (Korvatska et al., 2020). Importantly, the exaggerated immune response in the microglia was abolished by pharmacological inhibition of AKT kinase, a downstream effector of TREM2 signaling.

    Overall, the paper highlights the importance of analysis of both sexes when performing patient studies or modeling human neurodegenerative pathology in mice.

    References:

    Korvatska O, Kiianitsa K, Ratushny A, Matsushita M, Beeman N, Chien WM, Satoh JI, Dorschner MO, Keene CD, Bammler TK, Bird TD, Raskind WH. Triggering Receptor Expressed on Myeloid Cell 2 R47H Exacerbates Immune Response in Alzheimer's Disease Brain. Front Immunol. 2020;11:559342. Epub 2020 Sep 25 PubMed.

    View all comments by Olena Korvatska

  3. A large number of genetic risk factors have now been identified that alter risk for developing Alzheimer’s disease, however the mechanisms underlying their effects remain elusive in most cases. Cell-type contributions and sex-specific differences add additional layers of complexity. Heterozygous point mutations affecting TREM2 are well established to increase Alzheimer’s risk, and as TREM2 exhibits microglia-specific expression in the brain, provide a clear genetic and cell-type specific source of dysfunction. A large majority of the studies examining TREM2 functions in neurodegeneration, however, have utilized homozygous TREM2 loss-of-function mutations, which in humans cause distinct forms of neurodegeneration (Yeh et al., 2017). 

    To further our understanding of TREM2-based Alzheimer’s disease, Sayed and colleagues performed a comprehensive examination of both human Alzheimer’s disease tissue and model mice carrying heterozygous TREM2 R47H mutations. They perform the first large-scale, single-nucleus RNA-Seq experiment from TREM2 R47H Alzheimer’s disease patients, identifying sex-specific transcriptional changes and a pro-inflammatory microglia signature.

    To complement these postmortem transcriptional studies, the authors also generate heterozygous, humanized TREM2 R47H mice, which they combine with the well-characterized tau P301S model. Surprisingly, Sayed and colleagues find that in contrast to homozygous TREM2 R47H or knockout mutations (Leyns et al., 2017; Gratuze et al., 2020), heterozygous TREM2 R47H mutations exacerbate cognitive deficits and neuron loss in P301S. Moreover, these disease-enhancing effects were observed specifically in female mice, highlighting both sex-specific differences and important differences between heterozygous and homozygous mutations in TREM2.

    These latter findings are consistent with an earlier study by the Gan group, showing stronger effects due to heterozygous versus homozygous TREM2 knockout mutations, also in the P301S background (Sayed et al., 2018). Sayed and colleagues further find that heterozygous TREM2 R47H, P301S mice, and microglia isolated from them, exhibit a pro-inflammatory phenotype consistent with their human and mouse sequencing datasets.

    The authors also identified a gene-expression signature in human and mouse samples consistent with activation of AKT signaling, which they validated at the protein level in mouse brain tissue. Importantly, they find that inhibition of AKT signaling is capable of reducing inflammation in tau-treated microglia in culture, as well as largely preventing TREM2 R47H-dependent cognitive deficits, synapse loss and gene-expression changes in P301S mice.

    Thus, this work suggests a possible node for therapeutic intervention in TREM2 R47H-mediated Alzheimer’s disease, as well as adding further support to the conclusion that TREM2 R47H mutations have unique effects. Indeed, these and other findings suggest that differences likely exist between TREM2 knockout and R47H mutations, between heterozygous and homozygous TREM2 mutations, and between the effects that these mutations have on male versus female mice and humans.

    References:

    Gratuze M, Leyns CE, Sauerbeck AD, St-Pierre MK, Xiong M, Kim N, Serrano JR, Tremblay MÈ, Kummer TT, Colonna M, Ulrich JD, Holtzman DM. Impact of TREM2R47H variant on tau pathology-induced gliosis and neurodegeneration. J Clin Invest. 2020 Sep 1;130(9):4954-4968. PubMed.

    Leyns CE, Ulrich JD, Finn MB, Stewart FR, Koscal LJ, Remolina Serrano J, Robinson GO, Anderson E, Colonna M, Holtzman DM. TREM2 deficiency attenuates neuroinflammation and protects against neurodegeneration in a mouse model of tauopathy. Proc Natl Acad Sci U S A. 2017 Oct 24;114(43):11524-11529. Epub 2017 Oct 9 PubMed.

    Sayed FA, Telpoukhovskaia M, Kodama L, Li Y, Zhou Y, Le D, Hauduc A, Ludwig C, Gao F, Clelland C, Zhan L, Cooper YA, Davalos D, Akassoglou K, Coppola G, Gan L. Differential effects of partial and complete loss of TREM2 on microglial injury response and tauopathy. Proc Natl Acad Sci U S A. 2018 Oct 2;115(40):10172-10177. Epub 2018 Sep 19 PubMed.

    Yeh FL, Hansen DV, Sheng M. TREM2, Microglia, and Neurodegenerative Diseases. Trends Mol Med. 2017 Jun;23(6):512-533. Epub 2017 Apr 22 PubMed.

    View all comments by Li-Huei Tsai View all comments by Jay Penney

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References

Mutations Citations

  1. TREM2 R47H

News Citations

  1. Down to Sex? Boy and Girl Microglia Respond Differently
  2. Changing With the Times: Disease Stage Alters TREM2 Effect on Tau
  3. TREM2, Microglia Dampen Dangerous Liaisons Between Aβ and Tau
  4. Boost or Block TREM2? Either Way, Therapy May Need Careful Timing

Research Models Citations

  1. Tau P301S (Line PS19)

Further Reading

No Available Further Reading

Primary Papers

  1. Sayed FA, Kodama L, Fan L, Carling GK, Udeochu JC, Le D, Li Q, Zhou L, Wong MY, Horowitz R, Ye P, Mathys H, Wang M, Niu X, Mazutis L, Jiang X, Wang X, Gao F, Brendel M, Telpoukhovskaia M, Tracy TE, Frost G, Zhou Y, Li Y, Qiu Y, Cheng Z, Yu G, Hardy J, Coppola G, Wang F, DeTure MA, Zhang B, Xie L, Trajnowski JQ, Lee VM, Gong S, Sinha SC, Dickson DW, Luo W, Gan L. AD-linked R47H-TREM2 mutation induces disease-enhancing microglial states via AKT hyperactivation. Sci Transl Med. 2021 Dec;13(622):eabe3947. PubMed.

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