New data presented at the International Conference on Alzheimer’s and Parkinson’s Diseases, held March 28 to April 1 in Gothenburg, Sweden, further reinforced the field's growing recognition that microglia respond in myriad ways to different environmental stimuli (for a selection, see Part 13 of this series). How best to study brain cells of such astounding reactivity, at the levels of both transcription and function? This question was the focus of Beth Stevens’ talk at the conference (spoiler alert: lysosomes end up stealing the show, again).

  • In cell culture, human microglia mount a barrage of different transcriptional responses to disease-related stimuli.
  • Pooling microglial cells from different donors ties genetic variation to function.
  • This identified TMEM106b as an influencer of phagocytosis.
  • Scientists are injecting microglial “villages” into chimeric mice.

In their previous efforts to document microglial behavior across neurodegenerative diseases, Stevens, who is at the Broad Institute of MIT and Harvard, and others have collected massive genomic datasets detailing how expression of thousands of microglial genes change in response to various insults. Stevens wondered whether this diversity could be recapitulated in vitro, to better manipulate and study it. To find out, scientists in her lab generated iPSC-derived microglia, and exposed them to different triggers commonly encountered in the brain. Think aggregated Aβ, α-synuclein, tau, apoptotic neurons, myelin debris, and synapses.

Alzforum covered some findings from this study, which was completed in collaboration with the group of Evan Macosko, also at the Broad, when they appeared on bioRxiv (Oct 2022 news on Dolan et al., 2022). In essence, the scientists found that, before exposure, the iMGLs in these monocultures appeared identical in every way, but when confronted with a challenge, their diversity sprang to life. The cells transformed into a striking array of transcriptional states, including homeostatic, DAM-like, interferon-responsive, antigen-presenting, and proliferating clusters. The proportion of cells in these respective states changed depending on the specific exposure. For example, the researchers detected two DAM-like transcriptional clusters, one of which was provoked by all substrates except amyloid, the other only by amyloid fibrils and apoptotic neurons.

With this culture system in place, the scientists deployed omics techniques to decipher the master transcriptional regulators of these different states. One outcome was the discovery that the transcription factor MITF controls the shift into DAM-like states. Microglia in the AD brain were recently reported to upregulate this transcription factor in response to pathology (Smith et al., 2022). Then, wielding a lentiviral infection technique honed by postdoc Saša Jereb, the scientists understood a functional consequence of this transcriptional state. Overexpression of MITF in iMGLs not only switched them into the DAM state, it also quintupled their appetite for apoptotic neurons. The researchers are continuing to apply this method to connect multiple transcriptional regulators to different functional states, Stevens said.

At the same time, they started a new project that aims to decode the functional, disease-related consequences of genetic variation in microglia. “Imagine that instead of looking at one cell line at a time, we could compare 100 or more different lines from different patients, with high versus low disease risk, or resilience,” Stevens said at AD/PD. She described how her lab is doing just that.

She introduced the concept of a so-called “village culture system,” whereby iMGLs differentiated from multiple donors are pooled and grown together. Scientists can then expose these microglial “villages” to an array of conditions and probe their functional responses, such as changes in viability, proliferation, mitochondrial or lysosomal function, and phagocytosis, to name a few. Using single-cell barcode tags to track each microglial cell back to its original donor, the scientists are then able to connect the dots from genotype to phenotype, Stevens said.

The method, called Census-Seq, was initially developed by Broad/MIT’s Steve McCarroll and Kevin Eggan, who applied it in human neuronal cells (Mitchell et al., 2020; Wells et al., 2023). 

In Gothenburg, Stevens shared some early learnings from the application of this analysis technique in iMGL cultures. Postdoc Martine Therrien started with a pool of 27 donor lines, which came from people with a range of genetic risk for AD. She “fed” the cells fluorescently labeled, apoptotic neurons, and ranked the phagocytic prowess of each cell based on how much shiny debris it consumed. Using Census-seq, the researchers discovered that microglia that came from donors with a high polygenic risk score for AD—meaning they carried a heavy burden of risk variants—tended to be lackluster eaters of dying neurons. This suggested that AD risk variants may provoke disease by hobbling microglial phagocytosis, Stevens said.

Did any specific gene account for this effect more than others? By assessing which variants had the strongest association with phagocytosis, the researchers found TMEM106b—an endolysosomal protein with genetic ties to AD, ALS/FTD, and PD—sitting at the top of the list.

Dovetailing with other findings presented at AD/PD (see Part 13), the data underscore the importance of the microglial endolysosomal system in AD. By connecting genetics to function, the results go a step further, Stevens emphasized, suggesting that microglial digestive troubles are upstream drivers, rather than merely downstream consequences, of AD.

The scientists are starting to explore a next frontier of this line of work by taking cells from their in vitro system back in vivo. They are injecting such microglial “villages” into the brains of disease mice, using chimeric models as developed in the labs of Mathew Blurton-Jones and Bart De Strooper (Aug 2019 news and Oct 2022 news on Mancuso et al., 2022). After a while, they will remove the cells again and compare how the genetics of each microglial donor dictated their cells' differential response to the disease environment they encountered in the host brain.

The ultimate goal of these analyses is to tie genetics to transcriptional states and to function, Stevens said. In so doing, the scientists hope to illuminate essential disease mechanisms that could point to new therapeutic targets.—Jessica Shugart


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News Citations

  1. From Phagocytosis to Exophagy: Microglia's Digestive Tract Dissected
  2. Human Microglia Mount Multipronged Response to AD Pathology
  3. Human Microglia Make Themselves at Home in Mouse Brain

Paper Citations

  1. . A resource for generating and manipulating human microglial states in vitro. bioRxiv. May 2, 2022. bioRxiv
  2. . Diverse human astrocyte and microglial transcriptional responses to Alzheimer's pathology. Acta Neuropathol. 2022 Jan;143(1):75-91. Epub 2021 Nov 12 PubMed.
  3. . Mapping genetic effects on cellular phenotypes with "cell villages". 2020 Jun 29 10.1101/2020.06.29.174383 (version 1) bioRxiv.
  4. . Natural variation in gene expression and viral susceptibility revealed by neural progenitor cell villages. Cell Stem Cell. 2023 Mar 2;30(3):312-332.e13. Epub 2023 Feb 15 PubMed.
  5. . A multi-pronged human microglia response to Alzheimer’s disease Aβ pathology. bioRXiv. 7 Jul 2022 bioRxiv

Further Reading

No Available Further Reading