Astrocytes play important roles in central nervous system (CNS) physiology and pathology for neurological diseases like multiple sclerosis (MS). Until recently, however, little was known about the stability of disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges.

New research from Francisco J. Quintana, PhD, professor of neurology at the Ann Romney Center for Neurologic Diseases at Brigham and Women’s Hospital, explores those issues and identifies an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. The research, published in Nature, is an early step toward the development of novel therapeutic approaches for MS and other neurological diseases.

“Astrocytes don’t work in isolation,” Dr. Quintana says. “They communicate with other cells in the brain and the spinal cord. Upsetting that communication drives disease pathology and progression. Identifying the nature of that upset is an important step when attempting to either re-establish or block this functional communication therapeutically.”

Non-immune Cells Can Keep Immune Memories

Central to the Nature study is the concept of epigenetic memory. Astrocytes are not immune cells but rather non-hematopoietic glial cells that reside in the brain. Like immune cells, though, their epigenome changes when they are exposed to inflammation. Even when the inflammatory stimuli disappear, astrocytes are left with an epigenetic signature that controls future astrocyte responses to restimulation.

Indeed, Dr. Quintana found that when those astrocytes are restimulated in the future, they will show stronger, faster, and broader pro-inflammatory responses. They also will be better at inducing neuronal death and maintaining their ability to support neuron metabolism.

“When we think of brain inflammation, we need to look beyond arresting the activity of immune system cells,” he says. “We also must learn to erase the memories left behind in the inflamed tissue.”

To illustrate the importance of doing this, Dr. Quintana pointed to examples of infections or concussions that precede the development of neurological diseases by years. These preceding events can leave immune inflammatory memory in astrocytes. A subsequent infection or concussion can activate those inflammatory memories and lead to exacerbated inflammation and CNS pathology.

Novel Screening Platforms Uncover Astrocyte Pathways

In the Nature study, Dr. Quintana identified a subset of memory astrocytes (ACLY⁺p300⁺) controlled by epigenetic programs induced in the context of autoimmune CNS inflammation. This deep knowledge was made possible through the Quintana Lab‘s development of several novel screening platforms designed to uncover regulators of astrocyte pathogenic activities.

One of the tools his lab developed is called FIND-seq (focused interrogation of cells by nucleic acid detection and sequencing). FIND-seq lets researchers isolate astrocytes and other cells based on the expression of marker genes and sequence them in depth to identify mechanisms of astrocyte regulation.

“We can do this single-cell sequencing based on the expression of just a few genes with no proteins required,” Dr. Quintana says. “Not only does this tool allow us to find CNS astrocytes of interest, interrogate them in detail, and identify mechanisms of disease pathogenesis and therapeutic targets, but we can also apply FIND-seq to any cell in the body.”

Another unique tool developed at the Quintana Lab is Smart-FISH. This single-cell sequencing tool combines novel zebrafish models of neurogeneration with small molecule screens and artificial intelligence to identify pathways that regulate astrocytes in the context of pathology as well as small molecules that could be used to modulate those pathways.

“We initially developed this technique to study astrocytes, but it also can be used to study dendritic cells or T cells,” Dr. Quintana says.

To study astrocyte cell interactions in vivo, Dr. Quintana and colleagues developed the rabies barcode interaction detection followed by sequencing (RABID-seq) technique, which combines barcoded viral tracing and single-cell RNA sequencing. Using RABID-seq on mice models enabled Dr. Quintana to identify mechanisms of communication that are perturbed in disease and develop a small molecule that can reestablish normal communication.

“We are currently working to optimize that small molecule for humans and developing a 2.0 version of the tool that works in clinical samples,” he says. “This work is now in the process of being published, describing its use to define mechanisms used by tumors to evade immune response and guide novel therapeutic strategies. The potential for developing novel immunotherapies for cancer may change the way we see cancer therapy.”

“When you run experiments on clinical samples in vivo, you don’t get a full dictionary of every single word that one cell can use to communicate with another cell,” he adds. “Some of those dictionary words are only used in specific regions or at specific timepoints.”

To address that challenge, the Quintana Lab developed a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell-cell interactions.

Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets, and microfluidic droplet sorting based on functional readouts determined by fluorescent reporter circuits to facilitate the unbiased discovery of interaction regulators. The technique overcomes limitations of traditional methods for the characterization of cell-cell communication, which require a priori knowledge of cellular interactions, are highly engineered, and lack functional readouts.

The Ability to Forget

A central concept in Dr. Quintana’s continuing research on astrocyte epigenetic memory is that “you also need to be able to forget.” To that end, he is exploring the signaling pathways that regulate astrocyte memories and how to therapeutically erase bad memories like inflammation.

“Communication is central to the control of astrocyte memory,” he says. “We’re actively working to identify the mechanisms of cell communications that establish and stabilize memory formation. For example, we’re looking at the role of the gut microbiome and the chemicals it produces, some of which can reach the brain and control astrocyte activity.”

Based on this knowledge, Dr. Quintana’s lab has identified several small molecules and engineered probiotics that have the potential to erase these bad memories. He and his team are in the process of evaluating and optimizing them.

“Conducting this research at Brigham and Women’s gives us wonderful access to the complementary expertise of medicinal chemists who are at the forefront of developing novel therapies,” he says. “As a bench scientist, it’s an exciting time to be doing this game-changing work at one of the top clinical centers for the study and treatment of neurological diseases.”