Cellular culprits that contribute to pathogenesis in multiple sclerosis include a rare and heterogenous form of star-shaped glial cells in the brain and spinal cord called astrocytes. Technical difficulties in identifying, isolating, and analyzing astrocyte subtypes pose a major roadblock in identifying therapeutic targets for this chronic, and progressively debilitating inflammatory disease where the immune system attacks the myelin sheath that protects neuronal projections.
An article published in Nature Identification of astrocyte regulators by nucleic acid cytometry” reports a new high-throughput microfluidic cytometry method that involves encapsulating cells in droplets, detecting target nucleic acids using PCR and deep transcriptomics following droplet sorting to analyze and identify single cells.
The method, called FIND-seq (focused interrogation of cells by nucleic acid detection and sequencing), was developed by a team led by Francisco Quintana, PhD, a professor of neurology at Harvard University and associate scientist at the Brigham and Women’s Hospital. “These findings identify novel targets for therapeutic intervention in neurologic diseases such as MS,” said Quintana.
Conventionally, identifying rare cell types, such as pathogenic astrocyte populations that cause MS, involves the detection of unique cell surface markers that can conclusively distinguish these cellular culprits from other related cell types. However, few unique cell surface markers are available for the isolation of astrocyte subsets, which poses a technical challenge in studying these cells. Single-cell RNA sequencing can help identify cells, even in the absence of distinguishing cell surface markers, but the technique can be quite expensive.
To solve this problem, Quintana and his team combined nucleic acid cytometry, microfluidics, droplet sorting, and digital droplet PCR to develop FIND-seq. The new method can isolate and analyze rare cells of interest based on the expression of mRNA biomarkers.
“We applied FIND-seq to study the regulation of astrocytes,” the authors noted.
A key component in the pathological mechanism of MS in patients and in EAE (experimental autoimmune encephalomyelitis) models used to study MS, is RNA splicing which drives the activation of the transcription factor XBP1. To determine the role of XBP1 in MS pathology, the authors used FIND-Seq in conditional knock-out mice models, in CRISPR/Cas9-driven genetic perturbation studies, as well as in bulk and single-cell RNA-sequencing studies of mouse EAE and human MS samples.
“We identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses,” the authors noted. Using FIND-seq, the investigators demonstrated the ability to identify a therapeutic target mechanism that restricts XBP1-driven pathogenic mechanisms in astrocytes.
In another study published in the same issue of Nature, researchers used FIND-seq to identify mechanisms used by HIV to remain hidden in immune cells in patients treated with anti-retroviral therapies (“HIV silencing and cell survival signatures in infected T cell reservoirs”).
The application of FIND-Seq can have a broad impact in accessing previously inaccessible cells involved in various diseases, using unique signatures of gene expression. Cell identification no longer needs to be limited to cell surface markers.
