A spitting image of brain health

Topics to be covered

• Using glycoproteins as effective predictors of neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases
• Detecting glycoproteins predictors of traumatic brain injury

Thursday, March 21st, 2024 | 1:00 PM – 2:00 PM Eastern Time
This webinar will be available to view live and on demand.

Speaker

Yehia Mechref, PhD

Director, Center for Biotechnology and Genomics 
Texas Tech University

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Scientists once believed that the central nervous system was inaccessible to antibody therapeutics. With emerging evidence that antibodies cross the blood-brain barrier, researchers are now developing innovative treatments.

Download this poster from Drug Discovery News to learn about recent advances in antibody therapeutics for treating neurological diseases.

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Scientists once believed that the central nervous system was inaccessible to antibody therapeutics. With emerging evidence that antibodies cross the blood-brain barrier, researchers are now developing innovative treatments.

Download this poster from Drug Discovery News to learn about recent advances in antibody therapeutics for treating neurological diseases.

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Highlights in this article

Two soccer players collide as they try to head a ball. A soldier ducks near a detonating explosive. An elderly woman falls at home. All of these people are at risk of traumatic brain injury (TBI), where a blow to the head damages the brain’s delicate circuitry.

TBIs are infamous for their occurrences on sports fields, but their impact is much more widespread. In the United States, more than 2 million people suffer a TBI each year. Even mild TBIs — better known as concussions — can be debilitating, and severe TBIs can even be fatal. Timely medical tests and brain scans can ensure that people receive the appropriate TBI diagnosis and treatment.

But for many people, a bump to the head may not seem serious enough to warrant going to a doctor, especially if it happens in the middle of a high-stakes sporting event. In some situations — for example, on an active battlefield or in a remote, rural location — the nearest physician might be miles away. Antonio Belli, a neurosurgeon at the University of Birmingham, wondered whether there could be an easier way to diagnose TBI: a test that could be administered anywhere, without requiring specialized equipment or medical professionals.

One possible solution might be right at the tip of the tongue: Saliva, a substance best known for helping digest food, also holds clues about brain health, and is the key to a new TBI test in the works at the biotechnology company, Marker Diagnostics.

Biomarkers for the brain

Imagine a lump of Jell-O inside a bowl. Given a firm shake, the wobbling substance inside will compress and stretch as the impact ripples through it. This is what happens to the brain when it takes a blow. 

However, unlike an amorphous block of Jell-O, the brain has a precise structure of neural circuits and specialized regions that are essential to its function. When this structure gets disturbed by an outside force, it can interfere with the brain’s ability to carry out basic tasks. That’s why people with brain injuries report both cognitive symptoms including memory loss, difficulty sleeping, and changes in mood and physical symptoms such as headaches, vision changes, and dizziness.

Blood is obviously the fluid everybody goes to for diagnostics for many medical conditions, but it doesn’t really work terribly well when you look at athletes or soldiers or children. 
– Antonio Belli, University of Birmingham

When a person experiences a head injury, a TBI diagnosis requires immediate evaluation by a medical professional. Typically, they first conduct a neurological exam. The doctor asks the injured person questions about what they feel — a headache or tingling in their arms — and tests their memory. They also look for visible signs of concussion, such as an inability to balance or dilated pupils in the eyes.

These tests rely on external signs of TBI, which may be harder to spot in more subtle concussions or if patients don’t accurately report what they’re feeling — to avoid getting removed from an important game, for example. To see the internal effects of TBI, doctors often pair the neurological exam with computed tomography (CT) or magnetic resonance imaging (MRI) brain scans that reveal how the injury affected the brain’s structure.

But CT and MRI machines may not be available on the frontlines of a battle or the sidelines of a sporting event, and physicians may not be able to evaluate an injury immediately (1). However, studies have shown that when the brain is injured, the effects radiate throughout the body. This has spurred research into alternative tools to measure brain health with molecular biomarkers. For example, after a brain injury, the damaged neurons and supporting cells can shed proteins, such as neurofilament light (NfL) and S100 calcium-binding protein B, which can then be measured in the cerebrospinal fluid (CSF) (2). If the brain injury also disrupts the blood-brain barrier, these proteins can move into the blood. 

But measuring molecules in CSF or blood is invasive and requires a medical professional to insert the needle and collect the sample. “Blood is obviously the fluid everybody goes to for diagnostics for many medical conditions, but it doesn’t really work terribly well when you look at athletes or soldiers or children,” Belli said. “We’re looking for solutions that will be applicable in austere environments.”

A mirror reflecting the injured brain

This motivated Belli to investigate fluids that were easier to access, which brought him to saliva. Sometimes referred to as a “mirror of the body,” saliva is full of molecules that reflect the health of organs ranging from the brain to the liver (3,4). While scientists are still uncertain about how these molecules end up in saliva, one theory is that they pass between nerves or blood vessels and the salivary glands.

“The main advantage of using saliva is that you can collect it in any scenario, in any place, under any condition,” said Elizabeth Thomas, a neurology researcher at the University of California, Irvine who was not involved in Belli’s work. “It would be really difficult to imagine doing that with the blood draw.” 

Unlike CSF and blood, saliva doesn’t require needles or medical expertise: People just have to spit or swab their mouth. Thomas noted that this can be an advantage even beyond a warzone or football field because many people are hesitant about blood draws. Saliva is more palatable for many, especially now that people have experience with COVID-19 testing.

“[The COVID-19 pandemic] increased the public’s understanding that saliva is a biofluid that works really well,” said David Cohen, the chairman and director of Marker Diagnostics.

Importantly, saliva contains some of the same biomarkers seen in blood and CSF. In a 2022 study, Thomas and her colleagues measured concussion-associated proteins in the saliva of water polo players while tracking their head injuries (5). Players with more head injuries had higher levels of NfL, a neuronal protein that previous studies had labeled as a blood biomarker for concussion.

Proteins like NfL are shed by dying brain cells, but that can limit their utility in diagnosing less severe brain injuries, such as concussion. “There aren’t always many cells actually dying or structurally damaged, so it’s really difficult to detect protein signals,” Belli said. 

Luckily, proteins aren’t the only molecules in saliva. Saliva has very high levels of small non-coding RNAs (sncRNAs). These fragments, each just a few dozen nucleotides long, don’t encode the instructions to make proteins; instead, they themselves regulate the production of other RNA molecules or proteins, or they convey messages throughout the body. To detect these sncRNA molecules, “you don’t need profound damage,” Belli said.

To measure sncRNA biomarkers of concussion, Belli turned to a particularly high-impact sport: rugby. Belli followed more than 1,000 professional rugby players for two seasons, during which there were 156 head injuries (6). When a head injury occurred, medical staff determined whether the player had a concussion, and the player also provided three saliva samples by spitting into a tube: at the time of injury, after the game, and approximately two days later.

Belli’s team then measured the level of each sncRNA present in each sample and found that the levels of 32 sncRNA molecules differed between people with and without a concussion after a head injury. Some increased, while others decreased. The researchers narrowed this down to 14 sncRNA molecules that together differentiated people with a concussion from all other players.

Thomas appreciated that the researchers followed so many players and took samples at multiple timepoints, and she noted that the results convincingly support the use of salivary sncRNA molecules for diagnostics. “It’s becoming clear that small RNAs are pretty easy to measure in saliva, they’re pretty stable, and they can be preserved very easily — maybe more easily than protein,” Thomas said.

Testing for TBI

This study laid the foundation for a TBI diagnostic test that Belli began developing with Marker Diagnostics, for which he serves as the Chief Medical Officer. But Belli’s experience with the rugby players highlighted some issues that he would have to consider when designing the test. For example, spitting into a tube to collect sncRNA molecules wasn’t as easy as it might sound. Athletes were often dehydrated or used mouth rinses and gums that interfered with the sncRNA molecules in their saliva. He modified the approach to just require a 10-second cheek swab, rather than spitting into a tube.

Cohen envisions that the test will be something people can buy from their local pharmacy, use the swab to collect a sample, and then mail it or drop it off at a certified lab to receive a concussion diagnosis electronically. Early versions of the test were relatively slow: Going from a sample to a result took as long as 12 hours, which was too slow to be used mid-sporting event. Technical advances in RNA sequencing — many of which arose from the need for fast COVID-19 tests to detect viral RNA — have since sped this up to around 30 to 40 minutes. But Belli still sees room for improvement. 

“There’s a whole world out there that wants the results to be available within two minutes, if you have to use it on a football pitch or even in a combat theater,” he said.

The test is not yet available for purchase, but Cohen anticipates that it will be available to consumers next year after more validation. “We’re not putting it on the market until we’re absolutely confident that every time it’s used, it’s fail-safe — even if somebody doesn’t follow the instructions properly,” Cohen said. 

Thomas agreed that releasing a test at this stage would be premature, and she hopes that the Marker Diagnostics team first shows that the test is accurate in predicting concussions with other causes and for other types of people. Belli has studied sncRNA molecules in elite soccer, skeleton, and boxing so far, and the team at Marker Diagnostics wants to validate the test in people who are not professional athletes.

The researchers at the company will get more insight into their test’s performance in the real world later this year when a professional rugby team will use it for real-time diagnostics. But while a TBI diagnostic test is Marker Diagnostics’ first step, it isn’t the end of their journey to improve TBI outcomes. 

Belli hopes to advance the technology to get a deeper understanding of head injuries. As a clinician, he often has patients or their coaches asking him when they can return to their sport. SncRNA molecules could also be used to predict how long it will take someone to recover from a concussion or to track someone’s recovery or response to treatment. A 2021 study from another team of researchers showed that 11 salivary ncRNA molecules and age jointly predicted recovery from a concussion as accurately as cognitive and balance tests (7).

This type of test would require clear guidance for consumers, Thomas said, so that they know exactly how to interpret the change over time. “Everybody would love to have this test,” she said. “They would love to have a way to determine post-concussion return to play.”

Ultimately, Belli doesn’t think there will be one comprehensive test that can fully describe a TBI case. “For some reason, traumatic brain injury lives in this illusion that you’re going to find something in a test tube that gives you all the answers,” he said. 

Instead, he looks to other conditions as a model for what might be possible. Take chest pain, for example. Patients undergo multiple tests that each address one aspect of their heart health: a troponin blood test for signs of a heart attack, an electrocardiogram to measure the heart’s electrical activity, a chest X-ray to check for other potential causes. 

“The beauty of sncRNAs is that they are a little bit more descriptive than other tests,” Belli said. “It’s almost like a piece of music, but you have to listen to all the notes being played together.”

References

1. Schmid, K.E. & Tortella, F.C. The diagnosis of traumatic brain injury on the battlefield. Front Neurol  3, 90 (2012).
2. Zetterberg, H. et al. Biomarkers of mild traumatic brain injury in cerebrospinal fluid and blood. Nat Rev Neurol  9, 201-10 (2013).
3. Orr, R. & Wang, C. The utility of salivary microRNA and proteins as potential diagnostic or prognostic biomarkers of concussion or mild traumatic brain injury: A systematic review. J Sci Med Sport  26 (Supp 2), S104-S105 (2023).
4. França de Lima, L.T. et al. A salivary biomarker panel to detect liver cirrhosis. iScience  26, 107015 (2023).
5. Monroe, D.C. et al. Salivary S100 calcium-binding protein beta (S100B) and neurofilament light (NfL) after acute exposure to repeated head impacts in collegiate water polo players. Sci Rep  12, 3439 (2022).
6. Di Pietro, V. et al. Unique diagnostic signatures of concussion in the saliva of male athletes: the Study of Concussion in Rugby Union through MicroRNAs (SCRUM). Br J Sports Med  55, 1395-1404 (2021).
7. Fedorchak, G. et al. Saliva RNA biomarkers predict concussion duration and detect symptom recovery: a comparison with balance and cognitive testing. J Neurol  268, 4349-4361 (2021).