Understanding the Language of Brain-Muscle Crosstalk

In 2020, as the COVID-19 pandemic escalated, many universities shut down or reduced the capacities of research laboratories in an attempt to limit the spread of the virus. At Washington University School of Medicine in St. Louis, developmental biologist Aaron Johnson was permitted one person in his lab to keep things running. Shuo Yang, then a postdoctoral researcher working on muscle developmental biology, stepped up.

An immunologist by training, Yang, now at Fudan University, was curious to learn more about the virus that was wreaking havoc in the world. Since his model organism—the fruit fly—was not naturally susceptible to the SARS-CoV-2 virus, Yang generated a fly line that expressed a coronavirus protein called open reading frame 3a, or ORF3a, in their brains to mimic infection.¹ He observed that this led to motor function impairment; the flies had lost the ability to climb upwards against gravity.

The researchers noted that this behavior paralleled some general symptoms of illness in humans. “[When] you get sick, your muscles are really tired and achy,” said Johnson. “We’ve all experienced this.” This prompted them to explore the phenomenon in more detail.

Almost four years later, their investigations demonstrated that viral and bacterial infection-induced cytokines interfered with mitochondrial activity in skeletal muscles in flies and mice, providing insights into why brain infections can cause motor dysfunction.² The results, published in Science Immunology, highlight a brain-muscle signaling axis that reveals potential therapeutic targets to relieve neuroinflammation-associated muscle distress.

After observing ORF3a-induced motor dysfunction in the fly line, Johnson’s team explored whether a bacterial infection that produces a similar immune response as a viral infection would have an identical effect on fly movement. When researchers infected the flies’ brains with Escherichia coli, the flies cleared the infection within 24 hours, but displayed motor dysfunction for up to nine days.

To identify the mechanisms by which a short-term central nervous system (CNS) infection caused long-lasting motor problems, the researchers assessed the structure and appearance of cells in the skeletal muscle, including mitochondria, which are important hubs of energy production. Both ORF3a and E. coli infection resulted in reduced mitochondrial activity in skeletal muscles.

Yang knew that infection could alter which chemicals—including signaling molecules like cytokines—are secreted from the affected tissue, so he dug into the literature to search for likely candidates.³ Unpaired-3 (Upd3), a fly counterpart of the mammalian interleukin-6 (IL-6), emerged as an important cytokine that stressed fly tissues secrete.⁴

When they analyzed gene expression in the brains of infected flies, they found increased upd3 levels. Getting a hit on testing the first candidate cytokine was “a little bit of luck and a lot of hard work,” said Johnson. “As they say, a day in the library will save you a week at the bench.”

In flies, Upd3 activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, which plays an essential role in development and immune responses in the fly.⁵ However, excessive activity of the pathway can impair normal mitochondrial activity in the skeletal muscle.⁶ The researchers investigated whether this pathway was involved in disrupting muscle mitochondrial activity in response to Upd3 secretion. They observed that infection increased expression of a gene in the JAK-STAT pathway in skeletal muscles, suggesting that this pathway is involved in mediating brain-muscle crosstalk.

Once they had established the link between Upd3 and muscle dysfunction, they began to investigate the mechanisms by which infection upregulated Upd3 production in the first place. Infection can trigger the production of reactive oxygen species (ROS), which induces cytokine expression in many cell types.⁷ When the researchers expressed ROS-reducing enzymes in the fly CNS, infected flies showed higher muscle mitochondrial activity.

These experiments led researchers to conclude that CNS infection-induced ROS trigger the production of cytokines, which exit the brain and travel to the skeletal muscles, where they suppress mitochondrial activity.

Next, the researchers investigated whether a similar pathway operated in mammals. They injected ORF3a into the CNS of mice, which resulted in elevated ROS, higher levels of cytokines—including IL-6—and induced cell death in their brains. These mice displayed fatigue during a routine treadmill-running experiment, and a closer look at their muscle cells revealed mitochondrial dysfunction.

These results were very interesting and exciting, said Johnson. “And when the mouse data started to recapitulate [observations in the] fly, it got even more exciting.”

A heart-shaped structure glowing red with blue boundaries.

COVID protein expression in the fly brain accumulated ROS (red), activating brain-muscle signaling.

Shuo Yang

Equipped with data from flies and mice, Johnson’s team wondered whether similar processes might be at play in humans and indeed, histological analyses revealed ORF3a in the brains of deceased SARS-CoV-2 patients.

Finally, the team explored whether similar players regulate the crosstalk between the brain and muscles in non-infectious diseases—like Alzheimer’s disease (AD)—that cause neuroinflammation and muscle weakness. The researchers performed a meta-analysis of previously published studies, which revealed that patients with AD had higher serum IL-6 levels than control participants.

To test whether modeling AD in flies would trigger a similar pathway, the researchers expressed amyloid beta—a neurotoxic protein involved in AD pathology—in the brain. This resulted in motor dysfunction accompanied by increased ROS and upd3 expression in the brain.

“This is a very elegant paper, very well done with multiple approaches and even more impressively with multiple model organisms,” said Fabio Demontis, a scientist working on inter-tissue signaling at St. Jude Children's Research Hospital who was not involved with the study. Since other research teams have found that people suffering from COVID-19 had dysfunction of the diaphragm, a skeletal muscle involved in breathing, Demontis said that investigating whether similar inflammatory pathways were involved in this impairment could help scientists do identify potential treatments for respiratory diseases.⁸

Overall, the results add a novel component to the field of inter-organ communication, said Demontis. They also suggest IL-6 and other players in the pathway as potential targets to prevent neuroinflammation-induced muscle fatigue.

“Hopefully, this paper will be a call to arms to start thinking about IL-6 as a target for treating people with serious chronic inflammation,” agreed Johnson. Next, his team plans to design and conduct clinical trials to test the effect of IL-6 antibodies, which are already approved by the Food and Drug Administration, on motor function in patients with AD or long-COVID.