23 Aug 2019

With single-cell resolution, scientists now have a good idea of what amyloid plaques do to nearby cells in the brains of mice. Scientists led by Bart De Strooper, KU Leuven, Belgium, used a combination of spatial transcriptomics and in situ hybridization to build a detailed map of the gene-expression changes that occur around β-amyloid deposits. Coordinated changes of astrocyte and microglial transcriptomes indicated crosstalk between the cells. Oligodendrocytes did their own thing, ramping up expression of myelin-related genes soon after plaques deposited, only to temper that as plaque load increased. In their paper posted August 12 on bioRχiv, the authors conclude that plaques, rather than being innocuous sequela of pathology, induce strong and coordinated cellular responses. The work builds on the concept of a “cellular phase” of AD originally proposed by De Strooper and Eric Karran, then at Alzheimer Research U.K., Cambridge (see Alzforum Mar 2016 webinar).

In mice, amyloid plaques prompt nearby cells to shift transcription.

Astrocytes and microglia communicate intensely.

Oligodendrocytes overexpress, and later underexpress, genes needed for myelin repair.

“It’s a really fascinating study because it combines a whole-brain approach across the disease course with cell-specific analysis,” said Pieter Jelle Visser, VU University Medical Center, the Netherlands. He was not involved in the research. “Most previous studies use a more focused approach on cell type or region, but with these combined techniques, it’s possible to capture the larger picture.”

In recent years, researchers have found that microglia adopt a unique transcriptional profile in response to plaques (Jun 2017 news on Keren-Shaul et al., 2017; Srinivasan et al., 2019). How about other cell types? Researchers have tried to study astrocytes, oligodendrocytes, and neurons adjacent to plaques by isolating the cells, but such procedures both disrupt the cells and lack spatial resolution that might be critical to understand cellular responses to plaques.

Spatial Transcriptomics. From three adjacent brain slices, the outer two are stained for plaques, neurons, and astrocytes, while the inner yields a corresponding map of the transcriptome. [Courtesy of Chen et al., 2019.]

Co-first authors Wei-Ting Chen and Ashley Lu and colleagues combined a type of tissue-slice transcriptomics with in situ hybridization to try to preserve spatial information while still measuring RNA at the single-cell level. First, they used trios of adjacent coronal slices, each 10 μm thick, in a spatial-transcriptomics approach (Ståhl et al., 2016). The outer slices were used to identify cells and pathological changes, such as amyloid plaques, and to serve as a spatial reference for the inner slice used for transcriptomics (see image above). This slice was placed on a glass slide containing 1,000 reverse-transcriptase primer arrays, each capable of measuring complete transcriptomes. By referencing the transcriptomes with anatomical slides, the researchers were able to map expression changes in the vicinity of plaques.

Next, the researchers used in-situ hybridization to narrow down expression of individual genes to the specific cells that expressed them, tracking how they changed as plaques deposited. “The strength of this approach is clearly that spatial information is preserved and that transcriptomic changes can be comprehensively studied in defined brain regions with high resolution in an unbiased manner,” noted Annett Halle, German Center for Neurodegenerative Diseases, Bonn.

In this way, Chen and colleagues studied slices from 10 wild-type mice and 10 APPNL-G-F knock-ins, which carry the human APP gene with the Swedish, Iberian, and Artic mutations. Knock-in mice start accumulating diffuse plaques around three months of age, and they became positive for Thioflavin S around six months. By 18 months, they had extensive plaque.

The authors first compared transcriptomes of brain slices taken from young and old APPNL-G-F mice. In slices from 3-month-old animals, diffuse plaques were surrounded by transcripts related to myelination, which suggested to the authors that oligodendrocytes had begun to fix myelin damage. In slices from 18-month-old mice, however, cells near plaques barely expressed those genes. This fits with a recent report suggesting that oligodendrocyte precursors around plaques become senescent, stop differentiating into myelin-repairing oligodendrocytes, and instead release inflammatory molecules (Apr 2019 news).

What about other cell types? In slices from 18-month-old mice, the researchers identified a co-expression network of 57 genes that was upregulated around plaques. Most of these plaque-induced genes are known to be expressed by astrocytes or microglia and some, such as Trem2, Tyrobp, and ApoE, are well-known AD genes. Thirty-six, however, were not previously associated with plaques. These included genes involved in the classical-complement cascade, endocytosis, lysosomal degradation, antigen processing, immune responses, and oxidation-reduction responses.

To zero in on which cells in the slice expressed these genes, the authors used in situ hybridization (Ke et al., 2013). In a nutshell, they generated probes for each of the 57 genes, as well as for RNAs specific to microglia, astrocytes, neurons, and glia, and incubated them on a slide that was later stained for plaques. The patterns of co-localization revealed which cells expressed which transcripts (see image below).

Cell-Specific Expression. In a coronal section of the mouse brain (left), binding of probes for each of 57 plaque-induced genes, and for 27 cell-type-specific transcripts (bottom right), identify transcripts expressed by microglia, oligodendrocytes, astrocytes, and neurons, and which are up- or downregulated in the vicinity of plaques (white, top right). [Courtesy of Chen et al., 2019.]

Most plaque-induced genes were expressed by either microglia or astrocytes. Oligodendrocytes expressed only a few, including the complement-cascade protein C4 and some lysosomal genes. Interestingly, in the wild-type mice, the expression of these plaque-induced genes was not co-regulated. However, in the APPNL-G-F mice, their expression became coordinated at six months of age, and grew stronger as plaques accumulated, suggesting strong crosstalk between astrocytes and microglia around amyloid plaques.

The most intense RNA changes occurred within 10 μm from the edge of plaques. These were weaker in cells located farther away.

“The transcriptomic changes that are occurring in different cells surrounding plaques at various distances is intriguing,” wrote Shane Liddelow, New York University, to Alzforum. “Traditional sequencing efforts have largely missed these nuanced responses.” He was particularly interested in the localized response of astrocytes, which become reactive around plaques and secrete proinflammatory factors. “It suggests astrocytes’ negative effects on neurons are secondary to a positive response trying to destroy or remove plaque, or perhaps cells that the plaque has already damaged.”

The authors noted that while APPNL-G-F knock-in mice model Aβ deposition and inflammation, they offer little information about tau tangles, neurodegeneration, or memory deficits. The next steps will be to apply these techniques to human postmortem tissue to see if human cells respond similarly, De Strooper said. He also wants to see if transcriptomes differ between people whose cognition declines and those who remain healthy despite the presence of plaques. That could help explain why some people are resilient in the face of ongoing amyloid pathology, and might point to new therapeutic approaches. Liddelow added that spatial transcriptomics of human tissue will help identify the most appropriate rodent models for studying plaque-induced responses.

Michael Heneka, University of Bonn, Germany, cautioned that the gene-transcription data should be backed up by protein-level analysis, because only a small percentage of transcripts for inflammatory proteins are translated.—Gwyneth Dickey Zakaib