Not all microglia in the plaque-ridden brain are equal. Compared to those merely hanging out near plaques, mouse microglia that are actively consuming them express a distinct transcriptional signature, according to a study published May 21 in Nature Communications. Using a fluorescent dye to single out microglia with a belly full of fibrillary Aβ, researchers led by Enrico Petretto of Duke-National University of Singapore Medical School, and Jose Polo of Monash University in Clayton, Australia, reported that these plaque-eaters possessed a transcriptional profile akin to those identified previously in amyloid models, but with a wider array of genes involved. The profile was driven by the transcription factor HIF-1α, which also cropped up in some microglia in brain samples taken postmortem from people who had had Alzheimer’s disease. Curiously, microglia without Aβ in their innards aged faster in amyloid models, and were more likely to contain bits of synapses than were microglia gorging on Aβ.
- Aβ-laden microglia have a unique gene-expression signature.
- It includes TREM2, ApoE, other AD-associated genes.
- Moved to plaque-free environs, the microglia reverted to a homeostatic state.
- HIF-1α drove the transcriptional regime. It is up in microglia from AD postmortem brain.
“This work provides important new insight into our understanding of how interaction with amyloid plaques impacts microglial biology, and it has the potential to inform therapeutic strategies targeting this cell type,” wrote Joseph Lewcock and Pascal Sanchez of Denali Therapeutics in San Francisco.
As the resident immune cells in the brain, microglia, by definition, are poised to sense and rapidly respond to changes in their environment. In the case of amyloid, recent studies suggest that these cells not only surround plaques, but also build them by ingesting and regurgitating Aβ (Apr 2021 news). In mouse models of amyloidosis, the cells were found to ditch their homeostatic transcriptional signature for a disease-associated one, marked by more expression of TREM2, ApoE, and a handful of other genes (see Jun 2017 news; Sep 2017 news). The studies spotted microglia with this signature milling around plaques. However, these transcriptional studies did not determine whether active ingestion of plaques, as opposed to mere proximity to plaques, influenced the cells’ transcriptional profile.
To investigate, co-first authors Alexandra Grubman, Xin Yi Choo, and Gabriel Chew injected 5xFAD mice intraperitoneally with methoxy-XO4. A fluorescent dye, X04 binds Aβ fibrils. Grubman and colleagues extracted the animals’ brains two hours later, and used fluorescence-activated cell sorting to isolate microglia with and without the dye. They found that 13.5 and 15.8 percent of microglia were actively gobbling Aβ in the brains of 4- and 6-month-old mice, respectively. In the cerebellum, a region nearly devoid of plaques, only 4 percent of microglia did.
Caught in the Act. Two hours after injecting methoxy-XO4 (blue), the dye labeled plaques and co-localized with microglia (green) in the hippocampi of 5xFAD mice (right), but was absent from wild-type hippocampi (left). [Courtesy of Grubman et al., Nature Communications, 2021.]
Analyzing the transcriptomes of the microglia, the scientists identified 2,475 genes that were differentially expressed in fibrillar Aβ-positive versus fAβ-negative microglia. Featuring prominently in this profile were genes involved in ribosome function, oxidative phosphorylation, and phagolysosomal pathways. TREM2, ApoE, and interacting genes were among the most upregulated ones. The profile partly overlapped with the DAM and MGnD signatures previously reported in 5xFAD and APP/PS1 mice.
Compared with these prior signatures, however, the fibrillar Aβ (fAβ) profile encompassed more genes. They implicated a broader set of functions, including the HIF-1 signaling pathway, steroid biosynthesis, mitophagy, and endoplasmic reticulum protein processing. Genes linked to a range of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s, also cropped up among the plaque consumers. A principle component analysis indicated that plaque phagocytosis held sway over the microglial transcriptomes.
Lewcock and Sanchez noted that the fAβ trancriptome profile jibes with what they saw in XO4-labeled microglia in their APP knock-in mice, and partially confirms the “PIGs” signature defined by spatial transcriptomics (Feb 2021 news; Jul 2020 news). In addition, the Denali scientists reported that the Aβ-stuffed microglia had profound lipid alterations. A profile similar to the Aβ-phagocytosing one emerged when the researchers dissected transcriptomes using single-cell RNA sequencing instead of bulk sequencing of sorted microglia.
The scientists also compared the microglial transcriptomes from 6- or 24-month-old mice to look for age differences. They found that wild-type microglia from 24-month-old mice resembled fAβ-negative microglia from 6-month-old 5xFAD mice. This suggested, perhaps surprisingly, that in the plaque-laden brain, microglia that do not actively partake in plaque phagocytosis appear to age faster than do microglia in normal brain.
This aging signature was marked by an uptick in expression of α-defensin genes. These encode anti-microbial peptides with unknown functions in the brain (Selsted and Ouellette, 2005). Aβ also has antimicrobial properties (Mar 2010 news; May 2016 news).
Does something about the plaque environment trigger microglia to switch on the fAβ signature even before they take their first bite of a plaque, or does gobbling Aβ itself flip the switch? To address this, the researchers ran a series of ex vivo crossover experiments, in which they added microglia from 5xFAD or wild-type mice to hippocampal slice cultures from mice of either genotype. In a nutshell, they found that the fAβ-positive signature was induced by consumption of Aβ. Wild-type microglia transferred to a 5xFAD slice culture only turned on the transcriptional program once they engulfed Aβ, while those WT microglia that did not imbibe the peptide remained in homeostasis mode.
Further, the fAβ-positive signature was reversible. Fibril-containing microglia all reverted to a homeostatic state when they were added to wild-type cultures. Together, the ex vivo data suggest that internalized Aβ fibrils set a transcriptional program in motion and, when Aβ is no longer around, the cells revert back to normal.
Lewcock and Sanchez found this fascinating. “State reversibility of microglia may have interesting implications for effective anti-amyloid therapies, since microglia in the vicinity of those plaques may revert back to a functional homeostatic state when the amyloid aggregates are cleared out,” they wrote.
Microglia can also acquire a taste for synapses, and have indeed been found to prune them rather zealously in mouse models of amyloidosis (Apr 2016 news). Do the plaque eaters also prune synapses? Perhaps; however, Grubman et al. found more synaptic material within fAβ-negative microglia than within fAβ-positive cells, at least in 6-month-old 5xFAD mice. Still, when they isolated microglia from the 5xFAD mouse brain and fed them synaptosomes, the fAβ-positive cells engulfed more hungrily, in keeping with their more phagocytic transcriptional profile.
Why did fAβ-positive microglia nosh on synaptosomes fed to them in culture, but not on synapses within the brain? The answer is unclear. One possibility is that in the brain, fAβ-positive microglia simply contain less synaptic material than do fibril-negative microglia because synapses are sparser around plaques.
On that note, the authors found that the HIF-1α transcription factor appeared to drive a large proportion of the fAβ-positive signature. It also promoted phagocytosis of synaptosomes. The researchers went on to demonstrate that HIF-1α signaling could be partially induced by Pam3csk, a toll-like receptor 2 ligand, and repressed by rapamycin, an inducer of autophagy.
But How About People?
Do any microglia in the human brain adopt an Aβ phagocytosis transcriptional signature? The researchers tackled this important question by integrating single-nucleus transcriptomic data from four independent postmortem cohorts (Zhou et al., 2020; Mathys et al., 2019; Grubman et al., 2019; Leng et al., 2021). Combined, the dataset included nearly 12,000 microglial nuclei from entorhinal and prefrontal cortex samples taken postmortem from 102 people, some of whom had died of Alzheimer’s. These nuclei split into 21 clusters based on their transcriptomes; some clusters only appeared in one dataset.
Two transcriptional clusters, dubbed 10 and 11, had profiles that resembled the mouse fibrillar Aβ signature. These clusters were identified in every dataset, but not in every sample. Among the samples that contained cluster 10 microglia, those that came from people with AD had a higher proportion of these cells than did samples from people without AD. The microglial genes in cluster 10 overlapped with roughly 20 percent of genes in the mouse Aβ signature, compared with 10 percent of genes in the previously reported DAM signature. Notably, genes controlled by HIF-1α were among the genes differentially expressed in cluster 10 microglia.
Cluster 10 was not found in all AD samples. This intrigued Colm Cunningham of Trinity College Dublin. “This could represent one level of differential susceptibility among individuals, perhaps affecting progression or patient-specific manifestations,” he wrote (comment below).
All told, the researchers propose that as microglia age, their transcriptomes change in a way that is accelerated in the AD brain, as exemplified by the fAβ-negative signature in the 6-month-old 5xFAD mice. Upon Aβ plaque internalization, the HIF-1α program switches on, sparking a feed-forward loop that promotes even more gorging on Aβ. The authors suspect this same transcriptional program could also exacerbate the destruction of synapses by microglia, highlighting the delicate balance of microglial functions that can both help and hurt the brain.
Polo and Grubman said the Aβ signature is likely beneficial in the early stages of amyloidosis. How the signature changes throughout disease, and in the face of other stressors including tau pathology and age-related comorbidities, remains to be tested.
“This study is a bioinformatics tour de force that highlights the HIF-1α signaling pathway as a central mediator of the microglial response to Aβ plaque phagocytosis,” commented Jonas Neher of the German Center for Neurodegenerative Diseases in Tübingen. Neher added that the results align well with previously published work from his lab, which highlighted HIF-1α signaling as a critical microglial response to Aβ pathology (Apr 2018 news).—Jessica Shugart
- Microglia Build Plaques to Protect the Brain
- Hot DAM: Specific Microglia Engulf Plaques
- ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
- Striking Microgliosis in New APP Knock-in Mice
- Paper Alert: Those PIGs! Spatial Transcriptomics Add Human Data
- Paper Alert: Aβ’s Day Job—Slayer of Microbes?
- Like a Tiny Spider-Man, Aβ May Fight Infection by Cocooning Microbes
- Paper Alert: Microglia Mediate Synaptic Loss in Early Alzheimer’s Disease
- Stuck in the Past? Microglial Memories Dictate Response to Aβ
Research Models Citations
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- Grubman A, Choo XY, Chew G, Ouyang JF, Sun G, Croft NP, Rossello FJ, Simmons R, Buckberry S, Landin DV, Pflueger J, Vandekolk TH, Abay Z, Zhou Y, Liu X, Chen J, Larcombe M, Haynes JM, McLean C, Williams S, Chai SY, Wilson T, Lister R, Pouton CW, Purcell AW, Rackham OJ, Petretto E, Polo JM. Transcriptional signature in microglia associated with Aβ plaque phagocytosis. Nat Commun. 2021 May 21;12(1):3015. PubMed.