It is difficult to isolate large quantities of microglia from human brain. That’s why scientists still know little about the different ways these cells rear up in health and disease. A new single-cell—not single-nucleus—RNA-sequencing study led by Philip De Jager at Columbia University, New York, sheds some light on this. It analyzed the largest number of microglia yet, totaling more than 16,000, isolated from the brains of people with Alzheimer’s disease and normal cognition. In the November 30 Nature Communications, the authors describe nine distinct transcriptional states found in all brain samples. At least 80 percent of microglia fell into one of two homeostatic subtypes; that was true even in AD brains. Seven other subtypes comprised less than 5 percent of brain microglia each. Their proportion varied by individual and by disease state. One subtype, dubbed cluster 7, expressed numerous genes associated with AD; curiously, it was nearly gone from AD brain.
- Single-cell RNA-Seq reveals nine microglial subtypes in human brain.
- Most microglia are homeostatic, even in AD brain.
- A subtype involved in antigen presentation dwindles in AD.
“The microglia involved in disease are probably in these minor subsets,” De Jager told Alzforum. This implies therapeutic approaches will need to be precisely targeted to affect only the relevant subtype, he added.
Overall, the findings are broadly similar to previous microglial data from both mice and people. They offer a more refined view of different transcriptional states, and they reinforce differences in how humans and mice respond to amyloidosis. Some of the data were presented at a 2018 Keystone Symposium (Jul 2018 conference news).
“This is an important resource paper. Every human microglial project is a gold mine,” Oleg Butovsky at Brigham and Women’s Hospital, Boston, told Alzforum. Mark Fiers at KU Leuven, Belgium, agreed. “This dataset will be very valuable to future understanding of the role of microglia in Alzheimer's disease,” he wrote (full comment below).
Most previous studies of gene expression in human microglia relied on bulk tissue samples, and were unable to pick out subtypes (Jun 2017 news; Jul 2017 news; Feb 2018 news). Recently, groups led by Li-Huei Tsai at the Massachusetts Institute of Technology and Marco Colonna at Washington University in St. Louis turned to single-nuclei RNA-Seq of postmortem brain samples to zero in on activation states; alas, the number of microglia isolated from these mixed cortical cell samples was low, around 2,000 to 3,000 (May 2019 news; Jan 2020 news).
In addition, analyzing nuclei rather than whole cells may miss some cytoplasmic transcripts responsible for microglial activation (Oct 2020 news). Whole microglia can only be isolated from living tissue. Just one previous study has done this, when researchers led by Marco Prinz at the University of Freiburg in Germany analyzed 4,400 freshly isolated human microglia by single-cell RNA-Seq (Nov 2019 news).
De Jager and colleagues wanted to examine many more than that. To get living cells, joint first authors Marta Olah and Vilas Menon used fresh autopsy tissue from the dorsolateral prefrontal cortices of 14 participants in the Rush Memory and Aging Project who had had cognitive impairment or AD dementia. Researchers removed tissue samples from the skull within a few hours of death, placing them in a medium that keeps microglia alive (Olah et al., 2018). RNA was extracted within 24 to 48 hours. The authors rounded out this material with surgical resections of temporal cortex from three younger, cognitively healthy people undergoing treatment for epilepsy. From these samples, the authors isolated a total of 16,096 microglia.
Single-cell RNA-Seq defined nine transcriptional subtypes. More than 80 percent of the microglia fell into clusters 1 and 2, which seemed to represent homeostatic cells. These clusters expressed no unusual transcription factors or cell-surface markers. Other subtypes were more distinctive (see image at right). In cluster 3, genes involved in the cellular stress response were active, and these microglia were more abundant in AD brains than in the surgical samples. Cluster 4 turned on genes related to the interferon response, and could be identified by the gene ISG15, which encodes a ubiquitin-like protein induced by interferon. Clusters 5 and 6 sported anti-inflammatory genes, and the transmembrane protein CD83, an immunoglobulin. Cluster 7 expressed antigen-presenting genes and stood out through its high levels of CD74, part of the major histocompatibility complex class II (MHCII). Cluster 8 had no identifying marker, but expressed many metabolic genes. Cluster 9 seemed to contain proliferating microglia, which carried proliferating cell nuclear antigen (PCNA).
The findings dovetail with other microglial studies. Prinz’s whole-cell RNA-Seq analysis of human microglia identified eight of the same clusters, but not cluster 9 proliferating microglia. Data from postmortem brain matches less closely. Both Tsai and Colonna found fewer subtypes, but did implicate interferon in the microglial response to amyloidosis. Colonna reported that microglia in AD brain dial up interferon regulatory factor 8, similar to De Jager’s cluster 4 (Zhou et al., 2020). Meanwhile, in a mouse model, Tsai found an interferon-rich microglial subtype active in the late stage of neurodegeneration. In the same mouse study, Tsai identified a microglial subtype distinguished by MHCII, and a proliferating subtype that appeared early in neurodegeneration (Oct 2017 news).
“It was reassuring that the authors found these same microglial subsets in human brain,” Tsai told Alzforum. She noted that she was unable to identify these subsets in her own snRNA-Seq study of 2,000 microglia from postmortem human brain, probably because of the small number of cells analyzed. Intriguingly, a recent mouse study implicated interferon in the microglial response to tangles, as well (Dec 2020 news).
On the other hand, De Jager and colleagues did not find a single microglial subtype that matched the disease-associated microglia (DAM) seen in mouse models of amyloidosis (Jun 2017 news). Rather, DAM genes were highly expressed among four human microglial subtypes: 4, 5, 7, and 8, with cluster 7 the most enriched (see image at left). De Jager noted that amyloidosis in human brain is much slower, and less intense, than in mouse models. “The kinetics are different, and the fundamental microglial biology is probably quite different,” he suggested.
Because cluster 7 expressed many genes associated with Alzheimer’s in addition to the DAM genes, the authors took a closer look at how it changed in AD brain. Immunohistochemistry on sections from eight AD and 11 control brains showed that this microglial subtype made up about 3 percent of all microglia in healthy brain, but only 1 percent in AD. Likewise, an RNA-Seq analysis of bulk postmortem frontal cortex sections from 541 older adults found that only cluster 7 gene expression was dampened in AD brain. Could a dearth of these MHCII-expressing cells hasten AD progression? A recent mouse study hints it could. In it, 5XFAD mice lacking the complex had more plaques and worse inflammation than 5xFAD controls (Mittal et al., 2019).
De Jager is expanding this microglial RNA-Seq data set to delve deeper into specific subtypes. His group has now analyzed more than 200,000 microglia isolated from different brain regions of 75 donors. Samples came from people with different neurological diseases, including AD, multiple sclerosis, frontotemporal dementia, and amyotrophic lateral sclerosis. De Jager believes these data will clarify how microglial activation relates to disease, which could yield clues on how to intervene therapeutically. Because the majority of microglia are homeostatic even in the face of disease, De Jager cautioned against approaches that kill off or suppress microglia en masse (Mar 2018 news). Instead, disease progression might slow if scientists found ways to subtly shift the proportion of microglia in a given activation state.—Madolyn Bowman Rogers
- A Delicate Frontier: Human Microglia Focus of Attention at Keystone
- What Makes a Microglia? Tales from the Transcriptome
- Human and Mouse Microglia Look Alike, but Age Differently
- Microglial Transcriptome Hints at Shortcomings of AD Model
- When it Comes to Alzheimer’s Disease, Do Human Microglia Even Give a DAM?
- Human and Mouse Microglia React Differently to Amyloid
- Single-nucleus RNA Sequencing Misses Activation of Human Microglia
- The Human Brain Hosts a Menagerie of Microglia
- Changing With the Times: Disease Stage Alters TREM2 Effect on Tau
- Microglia in Tauopathy: Not Just Homeostatic Versus DAM
- Hot DAM: Specific Microglia Engulf Plaques
- Wiping Out Microglia Prevents Neuritic Plaques
Research Models Citations
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