Uncovering the Mysteries of the Gut–Brain Connection

Links between the gut and the brain were discovered many years ago, but what role does the gut microbiome play in this relationship? Recent research suggests that disruption of or abnormal activity in the gut microbiome may increase our risk for neurological disease, but whether this knowledge can be harnessed to treat, diagnose, or monitor such conditions remains to be seen.

Research into the physiology of the gut–brain connection largely began in the 19th century. In the 1840s, William Beaumont, a surgeon in the U.S. army, showed that different emotional states affect digestion rates, leading to the concept of bi-directional communication between the gut and the brain, often called the “gut–brain axis.”

This two-way communication occurs through three main routes: circulating immune cells, neurological pathways, and soluble molecules. The gut has so many nerve cells (an estimated 168 million in the human enteric nervous system), that it is sometimes referred to as the “second brain.” This gut-based system can communicate with the brain via the vagus nerve, a long and branching nerve linking the two neural centers.

This gut–brain connection was first illustrated by Ivan Pavlov, of Pavlov’s dogs fame, who was awarded the Nobel Prize in Physiology or Medicine in 1904 for his work in this area, which expanded on Beaumont’s early research. However, it was not until much more recently that the role of the gut microbiome in brain and neurological function was recognized.

“In the last two decades, we’ve begun to appreciate that the microbiome is playing a key role in regulating this gut–brain axis, and that we really have a microbiota–gut–brain axis,” John Cryan, PhD, professor in the department of anatomy and neuroscience at University College Cork, told Inside Precision Medicine. Cryan’s research focuses on the interaction between the gut microbiome and the brain.

Immune cells in the gut also play a role in communicating with the brain. They are programmed in the gut and can carry information to the brain. From there, they can send signals via soluble molecules like cytokines into the brain to affect behavior.

Gut microbes also produce chemicals such as neurotransmitters, hormones, and metabolites that appear to protect or disrupt neurological function and can influence behavior. “These chemicals can go into systemic circulation, they can activate immune cells, gut cells, blood brain barrier cells, and the vagus nerve,” explained Cryan.

A recent area of emerging research focuses on neurodevelopment. “The actual development of the brain, both in utero and postnatally, seems to be subject to influence from the microbiome,” explained microbiota–gut–brain axis expert Sarkis Mazmanian, PhD, a professor at the California Institute of Technology.

Research findings show links between the gut microbiome and response to stress, attention-deficit hyperactive disorder (ADHD), Parkinson’s disease, autism, amyotrophic lateral sclerosis (ALS), and other neurological conditions including those linked with aging. Much of the research on the microbiota–gut–brain axis to date has been carried out in animals like mice, but researchers believe that these findings are likely transferable to humans and trials are now moving in that direction. “There’s a lot of correlative evidence, a lot of associations, and really some tantalizing clues, but rigorous validation still needs to be performed,” cautions Mazmanian.

Is Parkinson’s disease driven by the gut?

Parkinson’s disease is a neurodegenerative disease that is known as a synucleinopathy due to abnormal accumulation of the protein alpha-synuclein in the brains of people with the condition. This leads to the gradual loss of dopamine-producing neurons, resulting in movement problems and often subsequent dementia.

James Parkinson first described the condition in 1817 and speculated that the disease may be linked to the gut suggesting “a disordered state of the stomach and bowels may induce a morbid action in a part of the medulla spinalis.” Indeed, a common “precursor” symptom of Parkinson’s is severe constipation, occurring up to 20 years before motor symptoms start, and is thought to indicate a gut connection.

Mazmanian’s group has been researching links between the gut microbiome and Parkinson’s disease using a mouse model of the disease that overexpresses human alpha-synuclein. “We also showed that this mouse has gastrointestinal symptoms. It essentially has a constipation-like phenotype,” he explained.

Past research has shown an overabundance of Escherichia coli-like bacteria close to the gut wall in people with Parkinson’s disease. “That made us hypothesize that maybe there’s something about this group of organisms,” said Mazmanian. “They do lots of things, but one of the features is that Enterobacteriaceae, the family that includes E. coli, expresses a bacterial amyloid called curli.”

The researchers tested the effects of over-colonizing the gut of model mice with curli-producing E. coli. They found that alpha-synuclein aggregation was seen in the brain and gut of these animals, which had both gastric and motor symptoms similar to those seen in Parkinson’s patients.

“What we still don’t know is if the vagus nerve is involved. Those experiments are ongoing, but we do feel the vagus nerve is going to be required, because there are a number of studies showing in mice and rats that if you sever the vagus nerve, gut-induced pathology doesn’t travel to the brain,” explained Mazmanian. A similar lack of Parkinson’s disease symptoms has been observed in humans with a severed vagus nerve, a now defunct procedure that was carried out to treat peptic ulcers in the past.

“It seems like it’s the most likely explanation for how curli is leading to synuclein aggregation in the brain. We know it’s happening in gut and in the brain, we just don’t know the connection yet,” Mazmanian said.

Mitigating the symptoms of ALS

ALS is a progressive neurodegenerative disease that severely affects motor neurons. There is currently no cure for this condition and most people who are diagnosed die between two and four years after being diagnosed.

Some types of ALS are linked to genetics, but there is also thought to be a strong environmental component to disease onset. In model mice, it has been shown that animals have gut dysbiosis before the onset of symptoms and similar gut-linked abnormalities have been seen in people who develop the condition. Increasing evidence suggests that the gut microbiome may contribute to the symptoms of ALS.

Indeed, French biotech MaaT Pharma is currently testing a gut microbiome-based treatment, MaaT033 in patients with ALS in a Phase I trial. The idea of the treatment is to transplant a healthy gut microbiome into the guts of patients with ALS, who often have gut inflammation and abnormal microbiome composition, with a view to improving the symptoms and progression of the disease.

Hervé Jullien de Pommerol is a neurology drug developmemt expert working on MaaT’s ALS trial. “A French patient association contacted the CEO and co-founder Hervé Affagard. They came with a pretty solid scientific dossier to suggest the gut microbiome could be an interesting target to tackle this terrible disease,” he explained.

“We put together a group to run this first study. We have finished the recruitment, and we hope to have the results by the end of the year if everything goes well.”

The gut microbiome in ADHD and autism

Young people with autism spectrum disorder (ASD) are known to experience higher rates of bowel disorders than people without the condition. Mazmanian and colleagues published a study in 2013 that supported a role for the gut microbiome in ASD.

Building on earlier work that showed that mice with a model version of autism seemed to have thinner and more permeable gut walls than normal mice, Mazmanian and colleagues analyzed the gut microbiome composition in these mice and found abnormally low levels of a bacterium called Bacteroides fragilis.

They also found that mice with symptoms of ASD had 46 times as much 4-ethylphenylsulfate in their blood than control mice. Notably, a very similar chemical called para-cresol sulfate is found at high levels in the blood of people with ASD.

When the researchers injected non-ASD model mice with 4-ethylphenylsulfate, they found it appeared to induce autism-like behaviors. “We strongly believe it is only made by bacteria. It doesn’t come from the mouse’s diet. It’s not made by the mouse, to the best of our knowledge, it’s not made by flies or worms or any other organisms. It’s exclusively a product of microbial metabolism of certain bacteria,” explained Mazmanian.

“The bacteria produce 4-ethylphenyl, which is very rapidly sulfated in the liver … then 4-ethylphenylsulfate builds up in the circulation. In this case, we believe that this bacterial molecule, once modified with sulfate, then gets into the mouse brain.”

Supplementing the mice that had ASD-like symptoms with B. fragilis seemed to restore levels of 4-ethylphenylsulfate to normal and improve the behavior and gastrointestinal symptoms of the animals.

Mazmanian co-founded Axial Therapeutics in 2016 to try and bring microbiome-based treatments to people with ASD and conditions like Parkinson’s disease. The company’s lead candidate, now in Phase II trials, is an oral small molecule sequestrant called AB-2004 that is a potential treatment for irritability and anxiety in young people with ASD. It acts like a molecular sponge and soaks up potentially harmful bacterial metabolites like 4-ethylphenylsulfate as it travels through the gut before being excreted.

The candidate therapy showed good safety and efficacy results in a Phase Ib/IIa trial in 2022 and has now progressed to Phase IIb testing. “The caveat is that there was no placebo control,” cautioned Mazmanian about the first trial. “In neuropsychiatric conditions, there’s huge placebo effect so we’re not making any claims on behavior, but we’re making claims on safety, tolerability, and on target engagement, meaning that the goal of the drug is to reduce levels of this molecule [4-ethylphenylsulfate].”

Alejandro Arias Vásquez, PhD, is an assistant professor at Radboud university medical center in the Netherlands. He has a background in genetic epidemiology and is now researching links between gut bacteria and ADHD, as well as other conditions like depression.

He and his team carried out a study in 2020 in which feces from humans with ADHD were transplanted into young mice. Although carried out in mice, this study suggests that the gut microbiome also plays a role in ADHD. “Our hypothesis was that these animals are going to develop some sort of ADHD-like phenotype,” explained Arias Vásquez.

“The results were surprising in two ways. Their brain and their behavior developed differently. … They showed more anxious and fearful behavior and there was no ADHD. No hyperactive or aggressive behaviors.”

Arias Vásquez and colleagues are continuing to investigate how the gut microbiome influences people with ADHD. For example, by trying to narrow down which bacterial species may make ADHD behaviors better or worse.

Cryan and team are also planning to investigate the role of the microbiome in ADHD development. “It’s a new area for us, but it makes sense, because a lot of our data would support a role for the microbiome in shaping brain development and neurodevelopment,” he said.

Stress regulation, diet, and aging

Stress is known to have wide-ranging effects on human health, and this includes the gut microbiome. It can underlie conditions such as irritable bowel syndrome and is linked to many mental health disorders as well.

Cryan and colleagues at the APC Microbiome Institute at the University of Cork have already completed extensive research on the links between stress and the gut microbiome.

“We showed that if you stress animals, one of the things you do is change the microbiome. Stress changes the microbiome, and the microbiome is regulating stress,” he explained.

“That led us to think: could we activate the microbiome in some way to alleviate the effects of stress? That allowed us to start testing various interventions, both specific strains of bacteria, probiotics, or specific dietary substances that feed the bacteria, known as prebiotics.”

Cryan and colleagues have now completed several human studies in this area. For example, they recently carried out a dietary intervention trial where they tested a “psychobiotic” diet rich in prebiotic and fermented foods against a standard diet in healthy volunteers to assess the impact on perceived stress. The intervention took place over four weeks and those consuming the psychobiotic diet reported a 32% reduction in stress compared with 17% in the control group. This work is now continuing over a longer time period.

Julia Rode, PhD, is a researcher and associate senior lecturer at Örebro University in Sweden. She is also working on the impact of probiotic supplementation on mental health. She is currently involved in setting up a new study to evaluate whether probiotic use in an aging population can help maintain good mental health and cognitive function.

“What we have done in the past is assessed healthy populations. What we want to do now is to move more and more into compromised populations. This could be to treat a disease state, but it could also be before you have reached a disease state, for example, we are interested in aging,” she explained.

“Those compromised populations will always come in a more heterogeneous state than a very homogeneous, healthy study group. But they also allow us hopefully to improve something and to counteract a potential ceiling effect that we might have on healthy subjects.”

Diet and the microbiome may also play a role in conditions such as the brain cancer glioblastoma. More research is needed in this area, but it is thought that microbes in the gut may indirectly influence brain tumor metabolism and the surrounding immune cells through bacterial metabolite signaling.

Khalid Shah, PhD, is a professor at Harvard University and specializes in developing cell-based therapies for brain cancers. “We’ve been doing the same thing [in brain cancer] for the last 30 years now. The key is, how do we change that? I think that has to be a bold initiative, knowing that that there is a gut–brain axis, there is communication between the gut and the brain,” he said.

“We should go in at the time of surgery with therapeutic drugs, and maybe at the same time, put patients on a ketogenic diet. We know that many people have gone onto a ketogenic diet, and it has been shown to alleviate effects of chemotherapy or radiotherapy. These patients feel much better, they have less pains. It’s well documented, particularly in brain tumors.”

Challenges and future directions

The gut microbiome field is slowly moving forward in terms of research and the development of medicinal products, but many challenges remain to be overcome before gut microbiome-based therapeutics can be approved on a large scale to treat neurological diseases.

In November 2022, the first two therapies based on the gut microbiome, both for treatment of recurrent Clostridium difficile infections, were approved. BiomeBank’s Biomictra donor-derived fecal microbiota transplant (FMT) product in Australia was approved by local regulators and Ferring Pharmaceuticals’ donor-derived rectally administered FMT product Rebyota was approved in the U.S. by the FDA. In April 2023, Seres Therapeutics’ oral capsule FMT treatment for the same indication, developed in collaboration with Nestlé Health Science, was also given the green light by the FDA.

Although these approvals were big steps forward for the overall microbiome field, research into the gut microbiome and how it can be targeted or used to treat disease is still at an early stage. No gut microbiome-based therapeutics for neuropsychiatric or neurodegenerative disorders have yet been approved by the FDA or other regulators and most clinical trials are at Phase I/II.

“It’s still early in man, because we’re not at Phase III, but we are closer than 15 years ago,” said de Pommerol. “The academics, drug developers, clinicians, and patient associations need to keep doing their jobs and continuing to work together, and this will come.”

One big challenge that continues to affect the field is study heterogeneity, which makes replication and comparisons difficult. “We need to replicate our studies. When you have replication from an independent study with a different sample done a year later, then you can say there is something in that signal. It’s a true signal,” said Arias Vásquez, who emphasizes that excessive heterogeneity also applies to bioinformatics pipelines that analyze bacterial sequences. “I have a student who did a review of the literature, and she found that there are over 60 bioinformatics pipelines currently being used, which is a lot.”

Another potential problem for researchers trying to develop therapeutic products is that probiotic supplements mostly come under food and not drug regulatory controls, so there is limited pressure for companies developing these products to undergo rigorous scientific testing and verify functional claims.

“Probiotics are not used as therapeutics in most cases, and there are discussions that suggest the probiotic definition should only be used to describe supplements instead of as a medical term,” explained Rode, who is hoping to see more standardized studies in the future. “What I would love to see, but what I think will still take some time, is if we could compare different interventions head-to-head and see if it truly is a specific effect of those interventions,” she added.

Increased data collection for different gut microbiome samples linked to many disorders is already underway. This will be needed in the future to integrate the gut microbiome into precision healthcare. For example, Arias Vásquez and his team are compiling a database of gut microbiome samples linked to brain disorders. “We’re trying to collect as many samples as possible, which has been a tremendous effort,” he emphasized.

Looking to the future, Cryan believes that diagnostics will become increasingly important. “People will start monitoring their microbiome and linking it to their mental health,” he speculated.

“I think with various technologies and wearables, people will be able to see how their microbiome is shifting with their own brain function. I think in healthy brain aging, we’re going to see a lot of exciting things coming up. I think supporting your microbiome as you age will be a really important part of the development of this. It’s something that I am quite excited to follow and see how it matures.”