Methods to study interacting cells and their transcriptomes are difficult to apply in living organisms. To facilitate such in vivo studies, Francisco Quintana from Harvard Medical School in Boston and the Broad Institute in Cambridge, Massachusetts, and his team came up with the idea of rabies barcode interaction detection followed by sequencing (RABID-seq). “We’ve been interested in astrocytes and their roles in health and disease,” says Quintana, explaining that astrocyte responses are controlled by multiple factors, such as metabolism and environment. “But one of the most important factors is literally cell–cell interactions,” says Quintana. He explains that RABID-seq allows studying these interactions in a comprehensive and unbiased fashion.
In RABID-seq, astrocytes are infected with a rabies-virus-based library encoding barcoded mCherry. Specific infection of astrocytes is achieved by using rabies virus pseudotyped with the envelope protein EnvA and by transgenically expressing the EnvA receptor TVA in astrocytes only, so that the initial infection is limited to astrocytes. The rabies virus used is the RabΔG variant, which lacks a crucial gene for a structural protein. This protein is transgenically expressed in astrocytes, which allows astrocytes to produce infectious virus particles, while other cell types, after infection due to their interactions with astrocytes, cannot produce functional virus particles and therefore do not continue a chain of infection.
Once astrocyte-interacting cells are infected, the tissue is dissociated, astrocytes and their interaction partners are enriched by sorting for mCherry expression, and their transcriptomes are analyzed with single-cell RNA sequencing. The aforementioned barcodes are inserted in the 3′ untranslated region of the mCherry transcripts and are read out by single-cell RNA sequencing as well. Cells that interacted with each other will harbor the same barcodes.
Quintana says that establishing RABID-seq has been a highly collaborative efforts and gives special credit to Iain Clark, Cristina Gutiérrez-Vázquez and Michael Wheeler, who are joint first authors of the publication. While RABID-seq may appear to be a straightforward combination of existing technologies, the team had to overcome hurdles to make the technology work. For instance, replication of the libraries was a challenge, as was the optimization of a computational pipeline to analyze the data. “The single-cell RNA-seq dataset allows you to establish cell types, cell subsets and activation status,” says Quintana, and the data can be mined for specific astrocyte populations and their interaction partners, ligands and receptors that might mediate these interactions, as well as signaling pathways that are upregulated.
Quintana and his team applied RABID-seq to study the interactions of astrocytes in experimental autoimmune encephalomyelitis (EAE) mice, which serve as a model for multiple sclerosis. In control mice, astrocytes interacted with other astrocytes, microglia and a few other cell types. In contrast, astrocytes in the EAE model also interacted with immune cells such as T cells, dendritic cells, monocytes and macrophages, which is consistent with the inflammation observed in the central nervous system of this mouse model. The researchers then focused on microglia–astrocyte interactions. They analyzed potential ligand–receptor interactions and identified the semaphorin–plexin pathway as a promising candidate for microglia–astrocyte communication. The researchers also found a role for EphB3 in the proinflammatory activity of astrocytes via its ligand ephrin-B3 in microglia.
One concern about rabies virus is its potential for causing deleterious effects in infected cells. “We didn’t see significant neurotoxicity,” says Quintana. In fact, he was more concerned “whether you would induce an immune response to the virus.” This was not a substantial problem in their studies, but Quintana cautions that care must be exercised when studying subtle effects.
RABID-seq has proven a useful tool in the hands of Quintana and his team. Now they are working on a second generation of RABID-seq. So far, RABID-seq relies on transgenic components, which complicates experiments with mouse lines that have a complex genetic background or prevents experiments with ex vivo human tissue samples. To overcome these hurdles, the team is establishing a RABID-seq version that makes use exclusively of viral tools to deliver the different components.
Research paper
Clark, I.C. et al. Barcoded viral tracing of single-cell interactions in central nervous system inflammation. Science 372, 360 (2021).
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