In the presence of amyloid plaques, microglia in mouse and human brains react differently—or do they? In the September 29 Cell Reports, researchers led by Mark Fiers and Bart de Strooper at KU Leuven, Belgium, cast doubt on previous findings that human microglia do not activate the same genes in response to amyloidosis as do their mouse counterparts.
- Studies of AD brain use single-nuclei RNA-Seq to analyze microglial gene expression.
- But transcripts from activation genes are scarce in frozen microglial nuclei.
- This may cause snRNA-Seq to misread the microglial activation state.
Those previous expression studies were based on analyzing nuclei isolated from postmortem brain sections. Now, Fiers and colleagues report that transcripts of many microglial activation genes are relatively scarce in frozen microglial nuclei, but concentrated in the cytoplasm. Thus, those postmortem tissue studies easily could have missed an uptick in the expression level of these genes. The discrepancy might have masked microglial activation, the authors suggest. “At the moment, we don’t know what microglial activation in human brain looks like,” Fiers told Alzforum.
Other researchers agreed that this technical limitation could be a factor in the apparent difference between mouse and human microglial states. “This is an important study and makes a valid point,” said Oleg Butovsky at Brigham and Women’s Hospital, Boston. Likewise, Florent Ginhoux at Singapore’s Agency for Science, Technology and Research said the community should be aware of the limitations of existing methods. “[The data] stress the need to use complementary technologies, as well as the crucial need to validate as much as possible any conclusions drawn from such high-dimensional approaches,” he wrote to Alzforum (full comment below).
Numerous studies have documented characteristic expression changes in mouse microglia around plaques. This profile has been variously labeled as disease-associated microglia (DAM), microglial neurodegenerative phenotype (MGnD), or amyloid-response microglia (ARM) (Jun 2017 news; Sep 2017 news; Apr 2019 conference news). Alas, in microglial nuclei isolated from postmortem AD brains, only a small subset of DAM genes are activated, and the exact ones vary from study to study (May 2019 news; Nov 2019 news; Jan 2020 news).
In unpublished work on AD brain, Fiers and colleagues likewise found few activated DAM genes. Curiously, however, human microglia grafted into a mouse model of amyloidosis switched on a higher number of these genes (Apr 2019 conference news; Mancuso et al., 2019). Was this activation due to the new environment of the mouse brain, or was it there all along in the human brain but somehow overlooked? Tissue preparation varies in studies of mouse and human microglia. The former are typically isolated from fresh brains using fluorescence-activated cell sorting, allowing researchers to analyze whole cells by single-cell RNA sequencing. Human microglia typically come from frozen postmortem sections, meaning the best option for bulk studies is to isolate nuclei for single-nuclei RNA-Seq.
To find out if the method made a difference, first author Nicola Thrupp directly compared single-cell and single-nuclei RNA-Seq of microglia obtained from temporal cortex biopsies taken from four people during epilepsy surgery. The patients were otherwise healthy and did not have Alzheimer’s disease. The authors isolated 14,823 whole cells for single-cell RNA-Seq, and 3,940 microglial nuclei from frozen sections for single-nuclei RNA-Seq.
Overall, the expression pattern for more than 98 percent of genes was similar in both the cell and nuclei sets. However, about 1 percent, or 246 transcripts, were scarce in nuclei. This gene set was consistent across all four brains examined. In addition, analysis of eight publicly available datasets of single-cell or single-nuclei RNA-Seq turned up the same set of nuclear-depleted transcripts.
These DAM Genes
Crucially, many of these nuclear-depleted transcripts encoded proteins involved in microglial activation. These included APOE, SPP1, CST3, FTL, PLD3, B2M, and CD74. If transcripts of these DAM genes are scarce in the nucleus, then would it be harder to detect microglial activation in nuclei from frozen AD brain sections? In support of this reasoning, a previous snRNA-Seq study of human microglia from AD brains had reported finding a subset of these genes elevated around plaques, but did not detect activation of the full set of DAM genes (Mathys et al., 2019). Tellingly, Thrupp and colleagues found that the transcripts detected in that study tended to be those with the highest overall expression level, while DAM transcripts with lower expression, such as TREM2, did not show up (see graph above).
This makes sense, Fiers said, because high-expressing genes might produce enough transcripts for single-nuclei RNA-Seq to reveal a boost in activation, even if those transcripts are relatively depleted in nuclei. Low-expressing genes, on the other hand, provide less statistical power to detect differential expression. “We think this is part of the reason why a number of studies have had a hard time seeing the full extent of microglial activation in the human AD brain,” Fiers said.
If so, how can researchers better detect DAM gene expression? Butovsky said that single-nuclei RNA-Seq remains the most viable option for expression studies of human brain. He believes isolating larger numbers of nuclei would boost statistical power, and speculated that if the authors had isolated as many microglial nuclei for analysis as they did whole cells, they might have seen fewer discrepancies between the expression profiles.
Fiers agrees that more nuclei and deeper RNA sequencing might help. He sees an alternative in spatial transcriptomics, which combines transcriptome analysis with in situ hybridization to reveal expression changes in individual cells in tissue slices. De Strooper’s lab has used this method to examine gene-expression changes in multiple cell types around amyloid plaques (Aug 2019 news; Chen et al., 2020).
Marco Colonna and Yingyue Zhou at Washington University in St. Louis agreed that technical limitations of single-nuclei RNA-Seq could cause false-negative results. “Validation of the differences identified by single-nuclei RNA-Seq at the protein level, such as by immunohistochemistry, becomes important,” they wrote to Alzforum (full comment below).
That said, they noted that Fiers and colleagues used only non-AD brain tissue for their study. In Alzheimer’s, the boost in DAM gene expression in activated microglia may make it easier to detect these transcripts, Colonna and Zhou suggested. For his part, Fiers wonders if amyloidosis might affect the distribution of DAM transcripts between nucleus and cytoplasm. He plans to compare the expression profiles of activated and homeostatic microglia using transgenic and chimeric mouse models.—Madolyn Bowman Rogers
- Hot DAM: Specific Microglia Engulf Plaques
- ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
- Parsing How Alzheimer’s Genetic Risk Works Through Microglia
- When It Comes to Alzheimer’s Disease, Do Human Microglia Even Give a DAM?
- Single-Cell Expression Atlas Charts Changes in Alzheimer’s Entorhinal Cortex
- Human and Mouse Microglia React Differently to Amyloid
- Chimeric Mice: Can They Model Human Microglial Responses?
- Spatial Transcriptomics Uncovers Coordinated Cell Responses to Amyloid
- Mancuso R, Van Den Daele J, Fattorelli N, Wolfs L, Balusu S, Burton O, Liston A, Sierksma A, Fourne Y, Poovathingal S, Arranz-Mendiguren A, Sala Frigerio C, Claes C, Serneels L, Theys T, Perry VH, Verfaillie C, Fiers M, De Strooper B. 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.
- Mathys H, Davila-Velderrain J, Peng Z, Gao F, Mohammadi S, Young JZ, Menon M, He L, Abdurrob F, Jiang X, Martorell AJ, Ransohoff RM, Hafler BP, Bennett DA, Kellis M, Tsai LH. Single-cell transcriptomic analysis of Alzheimer's disease. Nature. 2019 Jun;570(7761):332-337. Epub 2019 May 1 PubMed.
- Chen WT, Lu A, Craessaerts K, Pavie B, Sala Frigerio C, Corthout N, Qian X, Laláková J, Kühnemund M, Voytyuk I, Wolfs L, Mancuso R, Salta E, Balusu S, Snellinx A, Munck S, Jurek A, Fernandez Navarro J, Saido TC, Huitinga I, Lundeberg J, Fiers M, De Strooper B. Spatial Transcriptomics and In Situ Sequencing to Study Alzheimer's Disease. Cell. 2020 Aug 20;182(4):976-991.e19. Epub 2020 Jul 22 PubMed.
- Thrupp N, Sala Frigerio C, Wolfs L, Skene NG, Fattorelli N, Poovathingal S, Fourne Y, Matthews PM, Theys T, Mancuso R, de Strooper B, Fiers M. Single-Nucleus RNA-Seq Is Not Suitable for Detection of Microglial Activation Genes in Humans. Cell Rep. 2020 Sep 29;32(13):108189. PubMed.