16 Aug 2024

When confronting a threat, microglia rapidly splinter off into a menagerie of transcriptional states. What purpose do they serve? And who’s in charge of orchestrating them? These are difficult questions for scientists trying to understand these shifty cells, or to target them with therapeutics.

At the Alzheimer’s Association International Conference, held July 27 to August 1 in Philadelphia, researchers reported finding clues within short DNA sequences nestled within enhancer regions, which are fleetingly exposed in response to a microglial encounter. By investigating these unveiled sequences, scientists can infer which master transcription factors might be calling the shots. For example, in response to a TREM2-activating antibody, IRF3 and E2F emerged as potential master regulators of microglial transitions into interferon-producing and proliferative states, respectively. Yet, after a brief spell of transcriptional upheaval, the cells settled back into their calm, “undosed” state within days.

Because many AD risk genes are active in microglia, the tiny immune cells of the brain are thought to play an outsize role in AD. The cells are intimately involved in every aspect of AD pathogenesis. They often behave in seemingly contradictory ways, such as building and containing Aβ plaques, or curtailing tau pathology in one context while egging it on in another (Apr 2021 news; May 2023 conference news).

How the various transcriptional states of microglia relate to these different functions, and whether each is helpful or harmful, are hotly investigated questions at the present (Oct 2022 conference news). Making matters devilishly complex, microglia in one region of the brain might behave differently than microglia in another. Worse still, their states shift as the disease wears on (Apr 2024 conference news). This multidimensional diversity makes the cells tricky targets for drug developers, to say the least.

Christopher Glass of the University of California, San Diego, is taking an epigenetic approach to understanding what makes microglia tick (Jul 2016 conference news; reviewed in Balak et al., 2024). In Philadelphia, he told the audience that tools such as single-cell RNA sequencing have allowed scientists to identify microglial phenotypes with a high degree of resolution. “Yet, we still lack an understanding of how the cells achieve these different phenotypes,” he said. “We need to decipher those mechanisms to more effectively advance approaches for prevention and treatment that target these cells.”

To that end, Glass focuses on enhancer regions, where the epigenetic rubber hits the road. In these stretches of DNA, a combination of lineage- and signal-dependent transcription factors pile on to specific sequence motifs. This triggers the enhancer to loop itself into physical proximity to the promoter of the gene it’s about to turn on. By investigating which of these enhancer motifs are exposed before and after a given stimulus, scientists can infer—with the help of machine learning—which transcription factors are orchestrating gene expression changes (image below).

Glass’ group is looking at this so-called “enhancer landscape” under different conditions, including amyloidosis. In collaboration with Oleg Butovsky’s group at Brigham and Women’s Hospital in Boston, Glass lab scientist Johannes Schlachetzki identified 11 enhancer sequences that were differentially exposed in microglia from amyloid-ridden 5xFAD mice relative to those in wild-type animals. These motifs were predicted to host some 25-30 transcription factors. For example, several different transcription factors are able to bind the C/EBP motif, which is uniquely exposed in microglia in the presence of amyloid.

Still, Glass and other scientists have demonstrated that mouse and human microglia mount remarkably different transcriptional responses to amyloid. To zero in on the human-specific response, Glass teamed up with researchers in Matthew Blurton-Jones’ lab to make use of a xenotransplantation model (Aug 2019 news). When Glass analyzed the enhancer landscape of transplanted human microglia isolated from the brains of 5x-MITRG mouse hosts, he was in for quite a surprise. Nine of the 11 enhancer motifs implicated in the amyloid response in human microglia were the same as those found in their mouse counterparts (image below).

Same Regulators, Different Genes. Enhancer motif sequences identified in human (left) and mouse (right) microglia inferred a similar cast of transcription factors, ranked in order of significance. Lines indicate commonalities between human and mouse. [Courtesy of Christopher Glass, UCSD.]

How do these similarities square with the known differences between mouse and human microglia in their responses to amyloid? Glass reported that although similar enhancer motifs, and therefore a similar cast of transcription factors, orchestrated the microglial response to amyloid across the two species, the genes they targeted were mostly different. This makes sense, as many amyloid-activated enhancers identified in the mice had no functional counterpart in humans. “So the transcription factors may be the same, but their target regions in the genome are different,” Glass said.

Notably, many of the activated enhancer regions that Glass uncovered in the human microglia are known to activate AD risk genes, such as HLADR8 and ApoE.

Glass noted that this initial work may point to master regulators of microglial responses to pathology. His group is also looking at enhancer landscapes in hopes of inferring the controllers of individual signaling pathways, such as TREM2 signaling.

Along those lines, Kathryn Monroe of Denali Therapeutics in Philadelphia presented findings from a collaboration with Glass to dissect how microglia respond to treatment with a TREM2-activating antibody, ATV:4D9. The molecule is an engineered antibody with Fab regions that recognize mouse TREM2, and an Fc region that adheres to the human transferrin receptor. When infused into transgenic mice expressing the human transferrin receptor, the antibody binds receptors that line the blood-brain border, gaining access into the parenchyma, where its target awaits on the surface of microglia. When bound, the antibody encourages TREM2 clustering on the microglial surface, which triggers microglial proliferation within the mouse brain. 

In Philadelphia, Monroe first reported single-nuclei RNA sequencing data on microglia isolated from hTfR mice at one, seven, 14, and 28 days after treatment with ATV:4D9. Already on the day after dosing, microglia had undergone a dramatic shift in transcriptional states relative to untreated mice, ditching homeostatic signatures in favor of a cycling/proliferative profile, two disease associated microglia (DAM) states, and an interferon-producing state.

By day seven, the cells had largely returned to their control state. The exception? A persistent “pDAM” state, which was still detectable at 14 and 28 days. These findings demonstrated that TREM2 agonism triggers a rapid, yet largely reversible, transformation of microglial states.

There and Back Again. Relative to microglia in mice treated with a control antibody (top), microglia from mice injected with a TREM2-activating antibody transformed from homeostatic states to interferon, cycling, and DAM profiles within 24 hours. Some DAM persisted (pDAM) at later time points, but most cells returned to control state. [Courtesy of Kathryn Monroe, Denali.]

Monroe also presented preliminary snRNA-Seq findings in microglia from APP knock-in mice treated with the antibody. So far, this data comes from only two time points—before dosing and 24 hours later. Monroe reported that in these amyloid-burdened mice, the antibody instigated a massive shift away from the homeostatic state, and toward metabolic, interferon, and cell cycling states. Unlike its effect in mice without amyloid, the antibody reduced numbers of microglia in a DAM-like state.

Shifting Flocks. Relative to APP knock-in/hTfR mice treated with a control antibody (left), those dosed with a TREM2-activating antibody (right), shifted states dramatically. (Teal=homeostatic; pink=DAM/immune activity; purple=metabolic; yellow=interferon; green=cycling.)

Why would the TREM2 antibody shape microglial responses differently in the presence of amyloid? Monroe said that because TREM2 is an immunomodulatory receptor, how it steers microglia likely varies based on the state of the cells upon TREM2 agonism. Case in point, microglia were found to respond differently to antibody treatment depending on how much TREM2 they expressed at the time of agonism, according to a recent study that Denali scientists did in collaboration with Christian Haass at the German Center for Neurodegenerative Diseases in Munich (Feiten et al., 2024). “We interpret that to mean that TREM2 levels and, accordingly, microglial state, can be toggled in different directions based on the condition in which they exist,” she told Alzforum. More on this study below.

To find out what controls these dynamic responses, Monroe teamed up with Glass, who mapped the enhancer landscapes of the cells one and seven days after hTfR mice were treated with ATV:4D9. At 24 hours post-dose, the researchers detected 1,166 activated and 340 repressed enhancers in microglia from mice treated with ATV:4D9 relative to a control antibody. Among the most significantly exposed enhancer sequences in response to the antibody treatment were a combination of microglia-specific TFs, including PU.1 and AP-1, as well as signal-determining TFs. These included IRF3, which Monroe suspects might dictate the microglial shift into the interferon-responsive state, and E2F, which could push the cells into a cell cycling state.

Which master regulators are responsible for pushing microglia into an enhanced metabolic state is still unanswered, Monroe said. She suspects coordination among different transcription factors may turn out to push microglia into various states, rather than a single one for each. NFkB-p65—another TF that cropped up from the analysis—could be one of these coordinators.

Remarkably, seven days after treatment with the antibody, these revealing changes to the enhancer landscape had all but vanished. “The microglia had relaxed back to their naïve, undosed state,” Monroe said.

She told Alzforum that she interprets this finding as a good thing, as it suggests that treatment with a TREM2 antibody won’t irreversibly alter microglia, for better or worse. In Philadelphia, an audience member asked what the fleeting nature of this response might imply about how often people would need to be dosed with a TREM2 activating antibody. Monroe considers this an important question. She said answering it will require a deeper understanding of how well the persistence of changes at the epigenetic and transcriptional levels translate into staying power of functional effects.

Along those lines, Monroe and other Denali researchers teamed up with Haass and Astrid Feiten in his lab to investigate how levels of TREM2 expression might steer the downstream effects of TREM2 activation in microglia. To that end, the researchers generated a TREM2 reporter mouse, in which the mKate2 fluorophore reflects TREM2 expression. They were able to identify—and sort—microglia expressing low, medium, or high levels of TREM2 under different conditions. In the absence of amyloid, most microglia expressed low to medium amounts of TREM2, but in APP/PS1 mice with amyloid, TREM2-high microglia emerged; they were primarily spotted huddled around plaques.

When the researchers sorted microglia based on TREM2 expression levels and disease condition and compared their transcriptomes, they uncovered both TREM2- and amyloid-induced signatures. In short, the findings indicated differential transcriptional responses as a result of TREM2 expression level that were further shaped by exposure to amyloid plaques, the authors wrote. What’s more, they discovered TREM2 expression correlated with an uptick in glucose uptake and energy metabolism, as well as improved cholesterol handling in microglia, regardless of the presence of amyloid.

How would a TREM2 agonist antibody change this picture? To find out, the researchers administered monthly intravenous doses of ATV:4D9 to their TREM2 reporter, amyloidosis mice for four months. Then, a month after the last dose, they compared metabolomic and lipidomic profiles of microglia along with their levels of TREM2 expression. Based on these profiles, microglia expressing low levels of TREM2 mounted minuscule responses, whereas cells expressing intermediate or high levels of TREM2 changed dramatically. Strikingly, cells expressing moderate amounts of TREM2 were most responsive to the antibody, revving up metabolites related to glucose uptake as well as antioxidants. Some of these metabolites plummeted among cells expressing the most TREM2, suggesting a ceiling effect.

“These findings may have important consequences for the current design of clinical trials using TREM2 agonists,” the authors wrote. “Based on our findings, one must carefully monitor TREM2 expression in patients, to determine the optimal time point for interference.” They suggested that soluble TREM2 in the CSF as a potential biomarker for this purpose.

At this point in this fast-moving research area, modulating microglia via TREM2 looks like it comes with much complexity. In Philadelphia, Monroe interpreted the body of data collected so far to indicate that even a brief transcriptional shake-up might be capable of promoting a more durable functional responses in microglia.

For an example of how epigenetic changes to microglia can steer the role of ApoE isoforms in AD pathogenesis, see next story.—Jessica Shugart

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References

News Citations

1. Microglia Build Plaques to Protect the Brain 21 Apr 2021
2. Microglia Conflicted: To Help, or to Hinder, Tau’s March Across the Brain? 2 May 2023
3. Human Microglia Mount Multipronged Response to AD Pathology 27 Oct 2022
4. Over the Span of AD, Roles of Astrocytes and Microglia Change 12 Apr 2024
5. When a Microglia Is No Longer a Microglia 15 Jul 2016
6. Human Microglia Make Themselves at Home in Mouse Brain 1 Aug 2019
7. Microglial Epigenetics Hints at How ApoE Toggles Alzheimer’s Risk 16 Aug 2024

Paper Citations

1. Balak CD, Han CZ, Glass CK. Deciphering microglia phenotypes in health and disease. Curr Opin Genet Dev. 2024 Feb;84:102146. Epub 2024 Jan 3 PubMed.
2. Feiten AF, Dahm K, vanLengerich B, Suh JH, Reifschneider A, Wefers B, Bartos LM, Wind-Mark K, Schlepckow K, Ulas T, De-Domenico E, Becker M, Khalin I, Davis SS, Wurst W, Plesnila N, Neher JJ, Brendel M, Lewcock JW, DiPaolo G, Capell A, Monroe KM, Schultze JL, Haass C. TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism. 2024 Jul 22 10.1101/2024.07.18.604115 (version 1) bioRxiv.

Further Reading

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