Years before the muscle stiffness and tremors typical of Parkinson’s disease emerge, a seemingly innocuous symptom appears in most people who will eventually be diagnosed with the disease: constipation. 

Scientists have known about the connection between gut trouble and Parkinson’s disease for hundreds of years. In his initial description of the disease, James Parkinson himself wrote of a patient in the late stages of the disease, “The bowels, which had been all along torpid, now, in most cases, demand stimulating medicines of very considerable power” (1).

Constipation symptoms typically begin 20 or more years before Parkinson’s disease’s characteristic motor symptoms (2). This early onset of gut symptoms, which occur in about 80 percent of Parkinson’s disease patients, made many researchers wonder if the gut microbiome plays a role in the disease (3). An explosion of recent research points toward yes.

“What really struck me is the level of consistency between studies, between cohorts in Parkinson's,” said Sarkis Mazmanian, a gut microbiome researcher at the California Institute of Technology. “The same group of organisms is depleted. The same group of organisms is increased in [Parkinson’s disease] relative to controls. And I highlight this because this is not the norm in microbiome analysis. If you look across autism, you look across Crohn's disease, you look across any other disorder where the microbiome has been implicated, I personally haven't seen this level of consistency.”

As researchers probe this gut-Parkinson’s disease connection, new data continue to reveal the strength of this relationship. By using animal models and profiling human microbiomes, researchers uncover the molecular mechanisms and microbial species that may contribute to Parkinson’s disease, leading to potential gut-targeted therapies.

Nothing stays in vagus

In Parkinson’s disease, alpha-synuclein proteins form aggregates in neurons in the brain. Dopaminergic neurons are particularly vulnerable to alpha-synuclein build up, and their progressive loss is one of the hallmarks of the disease. While both genetics and environmental aspects contribute to Parkinson’s disease, no one combination of factors causes the disease.

In 2003, the neuropathologist Heiko Braak, then at the University of Frankfurt, put forward an intriguing hypothesis for a Parkinson’s disease cause (4). Because the gut connects to the brain via the vagus nerve, Braak wondered whether a pathogen originating in the gut could lead to the hallmark features of Parkinson’s disease: aggregation of alpha-synuclein proteins in dopaminergic neurons and their subsequent degeneration.

Since then, researchers reported that alpha-synuclein expressed in neurons that emanate from the gut — called enteric neurons — can travel via the vagus nerve to the brain in mice and rats (5,6). In humans, scientists saw that alpha-synuclein was expressed in colon tissue taken from three people two to five years before doctors diagnosed them with Parkinson’s disease, while healthy controls expressed none (7). Adding to that, people who had undergone a procedure called a truncal vagotomy — cutting off the connection between the vagus nerve and the stomach — had a significantly reduced risk for developing Parkinson’s disease compared to the general population (8). 

Numerous studies profiling the gut microbiome noted clear differences between the microbiome composition in healthy people versus people with Parkinson’s disease. But some of the strongest evidence linking the gut microbiome to Parkinson’s disease arose when Mazmanian and his team studied a mouse model of Parkinson’s disease that overexpresses alpha-synuclein in its neurons (9). When Mazmanian's team depleted the gut microbes from Parkinson’s disease mouse models, the animals’ motor functions improved, and they had decreased alpha-synuclein aggregates and neuroinflammation in their brains. Then when the researchers transplanted microbiomes from humans with Parkinson’s disease or heathy controls into their mouse models, the mice with the human Parkinson’s disease microbiome showed worse motor symptoms.

The results suggested that “the microbiome plays a role in the mouse model, but also much more tenuously, suggested that there may be a functional role for the human microbiome in Parkinson's,” added Mazmanian. But proving this functional role is no easy task.

A seed of gut alpha-synuclein

If Parkinson’s disease starts in the gut 20 or so years before motor symptoms begin, how can researchers determine who will eventually develop Parkinson’s disease and study those early gut stages? The answer that Yoon-Seong Kim, a Parkinson’s disease researcher at Rutgers University, landed on starts with giving mice an enema of rotenone — a pesticide outlawed for agricultural use in the United States since 2007.

Rotenone is a potent insect and fish-killer, and it’s not great for humans either. In fact, researchers reported that farm workers exposed to rotenone had a higher incidence of Parkinson’s disease (10).

“I hypothesized that rotenone mediated some condition… that caused the microbiome change, or directly caused some gut condition that may feed back to affect the microbiome,” said Kim. “We want to investigate the molecular mechanism of how alpha synuclein… initially aggregates in the gut. What kind of cell is responsible for initial aggregate alpha synuclein in the gut?”

Kim and his team developed a rotenone-based mouse model of Parkinson’s disease, which they presented at the 2022 Society for Neuroscience meeting (11). After taking an initial gut microbiome sample, the researchers flushed the mouse colon with rotenone daily for six weeks and then took another microbiome sample. They let the mice live a normal life for seven months and then took a final sample of their gut microbes.

When Kim and his colleagues compared the six week and 28 week microbiome samples, they were shocked. “The microbiome change right after six weeks is minimal,” he said. “But when you leave the animal for an additional seven months, we can see the clear difference in the microbiome.”

The rotenone-treated mice accumulated alpha-synuclein aggregates in the gut mucosa and in the neurons that connect the gut to the brain. The composition of bacteria in their gut microbiomes also changed. Bacterial taxa that exist at increased levels in the guts of people with Parkinson’s disease — Lactobacillus and Bifidobacteria — both increased in rotenone-treated mice.

The detrimental effects did not just stay in the gut. The researchers tested the mice’s motor skills by having them walk on a rotating wheel. The rotenone-treated mice fell off the wheel much sooner than the controls. When the team dissected the mouse midbrains, the rotenone-treated mice had lost more dopaminergic neurons than control mice.

“We have some proof directly testing gut microbiome changes with this model,” said Kim. “That is the starting point.”

Now that they have a model that recapitulates the classic Parkinson’s disease characteristics, Kim and his team are ready to explore it. Their first goal is to identify the alpha-synuclein aggregate producing cells in the gut and to find a gut-targeted therapy to prevent it.

They are also following these animals for longer than seven months to see if they develop cognitive impairments associated with Parkinson’s disease, and since Parkinson’s disease is one of old age, they want to see how setting up the rotenone treatment model in older mice affects their microbiomes and Parkinson’s disease pathology.

“We know we can prevent Parkinson's disease pathogenesis, so that is the thing that everybody's working on. There is a reason why we need some good models to test,” he said.

The lactobacillus and the worm

Living on a solid diet of only bacteria, the roundworm Caenorhabditis elegans is an ideal model system for studying the gut microbiome and disease. A surprising challenge, though, is getting the worms to eat bacteria they’ve never seen before.

“They definitely have their preferences,” said Nicole Johnson, a graduate student studying the role of the gut microbiome in Parkinson’s disease in Danielle Mor’s laboratory at Augusta University.

Like Kim, Mor and Johnson want to use the their picky-eater worms to uncover how Parkinson’s disease begins.

“If we understand what's happening in the GI tracts of people who are going to then develop Parkinson's disease and have this unfortunate, devastating degeneration, we can maybe start to help people early on,” said Mor. “In the literature from Parkinson's patients, Lactobacillus species are increased [in the gut], and it's just not known if that's playing any direct role in the disease. So, we really want to investigate that.”

In a poster presentation at the 2022 Society for Neuroscience meeting, Johnson reported that when she fed the bacterial species Lactobacillus brevis — a species increased in the guts of people with Parkinson’s disease — to C. elegans expressing human alpha-synuclein in their muscles or neurons, a whole host of things went wrong (12). 

Compared to the worms fed a standard diet of E. coli, the L. brevis fed worms did not move as well, and they had reduced dopaminergic neuron function. Johnson and Mor also saw that cholinergic neuron function decreased in the L. brevis fed worms.

“There's a huge focus on dopamine neurons because they are absolutely an important neuron type in Parkinson's disease, and the loss of those cells causes motor defects. But actually, there are other cell types that die in Parkinson's disease,” explained Mor. The decreased signaling in cholinergic neurons may contribute to the decrease in motor function.

Mor and Johnson’s biggest surprise came when they looked at alpha-synuclein aggregation in the L. brevis-fed worms.

“There's actually a decrease in the number of large aggregates,” Johnson said. “That was definitely really shocking, but also really interesting because it went the opposite direction from what we had anticipated. So, then we went back, [and] we're like, well why?”

When they looked more closely, Mor and Johnson saw that the L. brevis fed worms had an increase in soluble alpha-synuclein. This soluble fraction could contain nontoxic alpha-synuclein monomers or small aggregates or oligomers of alpha-synuclein, which can be quite damaging to the body.

“There's a big field of oligomer research showing that those small aggregates are pretty toxic, and there's kind of a debate as to whether or not oligomers are the aggregate species that we should be targeting for therapy,” said Mor. “This is part of the larger question of what protein aggregation is even doing in these diseases,” she added. “Across diseases — Alzheimer's, Parkinson's, Huntington's, ALS — everybody is trying to understand the role of protein aggregates in neuron death, and it's just not clear.”

Johnson and Mor plan to use liquid chromatography to determine whether there is an increase in alpha-synuclein monomers or toxic oligomers in the worms fed L. brevis and to better understand how different sized alpha-synuclein aggregates may contribute to Parkinson’s disease.

“If we had seen what we had expected, we probably wouldn't have gone down the route of trying to understand oligomers,” said Mor. Johnson added, “That was just a really fun science moment, just in general, because it did something completely opposite of the initial expectation but still gave really promising data on another lead to follow.”

Some fiber a day keeps Parkinson’s disease away

Eating a high fiber diet has all kinds of beneficial effects. Fiber promotes good bowel health, helps maintain blood sugar levels, and reduces cholesterol levels, just to name a few. It turns out that a high fiber diet also associates with a decreased risk for Parkinson’s disease (13).

Mazmanian wondered if increasing dietary fiber might have beneficial effects in a mouse model of Parkinson’s disease.

“Fiber can be broken into many things, one of which is short chain fatty acids,” he said. “One of the things they do is really modulate the immune system in the gut in ways that appear to be quite protective.”

Signals from the gut travel via the enteric nervous system and communicate with microglia, which are immune cells in the brain. In neurodegenerative diseases, including Parkinson’s disease, researchers hypothesize that microglial activity becomes improperly activated, leading to increased neuroinflammation which is characteristic of neurodegenerative diseases.

Mazmanian and his team fed mice overexpressing alpha-synuclein a high fiber diet for 17 weeks. When they compared the microbiomes, the alpha-synuclein overexpressing mice fed the high fiber diet  had microbiomes that looked more similar to healthy ones (14). The mice also had improved motor abilities and reduced alpha-synuclein aggregates and microglial activation in the brain. In particular, they saw an increased expression of anti-inflammatory genes in microglia of mice fed a high fiber diet, suggesting that the diet shifted microglia into a more neuroprotective state.

“Even though we got a beneficial effect, an exciting result, we wanted to know, is it because of microglia?” Mazmanian said.

He and his team then depleted microglia from the mice. Even when they fed these mice a high fiber diet, the mice without microglia showed none of the beneficial effects, indicating that microglia are necessary for the good effects of the high fiber diet.

“What the study did not reveal is how. What are microglia doing differently in the context of dietary fiber that makes them more protective than microglia that are not exposed to short chain fatty acids?” Mazmanian asked. He and his team plan to investigate that mechanism in follow up studies.

For now, this study is the first that shows that a dietary intervention proffers clear benefits in a mouse model of Parkinson’s disease, adding to the other potential benefits of eating a high fiber diet.

“There are just so many benefits to dietary fiber, and just in your normal everyday life, let alone staving off a disease that may be 20, 30 years away,” Mazmanian said. But getting people to stick with a high fiber diet is hard.

“People like to take pills. They don't like to change their dietary habits over long periods of time,” he said. “But again, it introduces another way that one can intervene through the microbiome, and it's not by fecal transplants or by probiotics, but by diet — using diet as the actual medicine.”

References

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