“Previous publications from our lab have mainly focused on neuronal protection,” said Li-Huei Tsai, Picower Professor in The Picower Institute for Learning and Memory and the Department of Brain and Cognitive Sciences at MIT and senior author of the new study in Nature Communications. Tsai also lead’s MIT’s Aging Brain Initiative. “But this study shows that it’s not just the gray matter, but also the white matter that’s protected by this method.”

This year Cognito Therapeutics, the spin-off company that licensed MIT’s sensory stimulation technology, published phase II human trial results in the Journal of Alzheimer’s Disease indicating that 40Hz light and sound stimulation significantly slowed the loss of myelin in volunteers with Alzheimer’s. Also this year Tsai’s lab published a study showing that gamma sensory stimulation helped mice withstand neurological effects of chemotherapy medicines, including by preserving myelin. In the new study, members of Tsai’s lab led by former postdoc Daniela Rodrigues Amorim used a common mouse model of myelin loss — a diet with the chemical cuprizone — to explore how sensory stimulation preserves myelination.

Amorim and Tsai’s team found that 40Hz light and sound not only preserved myelination in the brains of cuprizone-exposed mice, it also appeared to protect oligodendrocytes (the cells that myelinate neural axons), sustain the electrical performance of neurons, and preserve a key marker of axon structural integrity. When the team looked into the molecular underpinnings of these benefits, they found clear signs of specific mechanisms including preservation of neural circuit connections called synapses; a reduction in a cause of oligodendrocyte death called “ferroptosis;” reduced inflammation; and an increase in the ability of microglia brain cells to clean up myelin damage so that new myelin could be restored.

“Gamma stimulation promotes a healthy environment,” said Amorim who is now a Marie Curie Fellow at the University of Galway in Ireland. “There are several ways we are seeing different effects.”

The findings suggest that gamma sensory stimulation may help not only Alzheimer’s disease patients but also people battling other diseases involving myelin loss, such as multiple sclerosis, the authors wrote in the study.

Maintaining myelin

To conduct the study, Tsai and Amorim’s team fed some male mice a diet with cuprizone and gave other male mice a normal diet for six weeks. Halfway into that period, when cuprizone is known to begin causing its most acute effects on myelination, they exposed some mice from each group to gamma sensory stimulation for the remaining three weeks. In this way they had four groups: completely unaffected mice, mice that received no cuprizone but did get gamma stimulation, mice that received cuprizone and constant (but not 40Hz) light and sound as a control, and mice that received cuprizone and also gamma stimulation.

After the six weeks elapsed, the scientists measured signs of myelination throughout the brains of the mice in each group. Mice that weren’t fed cuprizone maintained healthy levels, as expected. Mice that were fed cuprizone and didn’t receive 40Hz gamma sensory stimulation showed drastic levels of myelin loss. Cuprizone-fed mice that received 40Hz stimulation retained significantly more myelin, rivaling the health of mice never fed cuprizone by some, but not all, measures.

The researchers also looked at numbers of oligodendrocytes to see if they survived better with sensory stimulation. Several measures revealed that in mice fed cuprizone, oligodendrocytes in the corpus callosum region of the brain (a key point for the transit of neural signals because it connects the brain’s hemispheres) were markedly reduced. But in mice fed cuprizone and also treated with gamma stimulation, the number of cells were much closer to healthy levels.

Electrophysiological tests among neural axons in the corpus callosum showed that gamma sensory stimulation was associated with improved electrical performance in cuprizone-fed mice who received gamma stimulation compared to cuprizone-fed mice left untreated by 40Hz stimulation. And when researchers looked in the anterior cingulate cortex region of the brain, they saw that MAP2, a protein that signals the structural integrity of axons, was much better preserved in mice that received cuprizone and gamma stimulation compared to cuprizone-fed mice who did not.

Molecular mechanisms

A key goal of the study was to identify possible ways in which 40Hz sensory stimulation may protect myelin.

To find out, the researchers conducted a sweeping assessment of protein expression in each mouse group and identified which proteins were differentially expressed based on cuprizone diet and exposure to gamma frequency stimulation. The analysis revealed distinct sets of effects between the cuprizone mice exposed to control stimulation and cuprizone-plus-gamma mice.

A highlight of one set of effects was the increase in MAP2 in gamma-treated cuprizone-fed mice. A highlight of another set was that cuprizone mice who received control stimulation showed a substantial deficit in expression of proteins associated with synapses. The gamma-treated cuprizone-fed mice did not show any significant loss, mirroring results in a 2019 Alzheimer’s 40Hz study that showed synaptic preservation. This result is important, the researchers wrote, because neural circuit activity, which depends on maintaining synapses, is associated with preserving myelin. They confirmed the protein expression results by looking directly at brain tissues.

Another set of protein expression results hinted at another important mechanism: ferroptosis. This phenomenon, in which errant metabolism of iron leads to a lethal buildup of reactive oxygen species in cells, is a known problem for oligodendrocytes in the cuprizone mouse model. Among the signs was an increase in cuprizone-fed, control stimulation mice in expression of the protein HMGB1, which is a marker of ferroptosis-associated damage that triggers an inflammatory response. Gamma stimulation, however, reduced levels of HMGB1.

Looking more deeply at the cellular and molecular response to cuprizone demyelination and the effects of gamma stimulation, the team assessed gene expression using single-cell RNA sequencing technology. They found that astrocytes and microglia became very inflammatory in cuprizone-control mice but gamma stimulation calmed that response. Fewer cells became inflammatory and direct observations of tissue showed that microglia became more proficient at clearing away myelin debris, a key step in effecting repairs.

The team also learned more about how oligodendrocytes in cuprizone-fed mice exposed to 40Hz sensory stimulation managed to survive better. Expression of protective proteins such as HSP70 increased and as did expression of GPX4, a master regulator of processes that constrain ferroptosis.

In addition to Amorim and Tsai, the paper’s other authors are Lorenzo Bozzelli, TaeHyun Kim, Liwang Liu, Oliver Gibson, Cheng-Yi Yang, Mitch Murdock, Fabiola Galiana-Meléndez, Brooke Schatz, Alexis Davison, Md Rezaul Islam, Dong Shin Park, Ravikiran M. Raju, Fatema Abdurrob, Alissa J. Nelson, Jian Min Ren, Vicky Yang and Matthew P. Stokes.

Fundacion Bancaria la Caixa, The JPB Foundation, The Picower Institute for Learning and Memory, the Carol and Gene Ludwig Family Foundation, Lester A. Gimpelson, Eduardo Eurnekian, The Dolby Family, Kathy and Miguel Octavio, the Marc Haas Foundation, Ben Lenail and Laurie Yoler, and the National Institutes of Health provided funding for the study.

“I ignored it for years because I thought ‘I am a limnologist, methane is in lakes,'” she said.

But when a local reporter contacted Walter Anthony, who is a research professor at the Institute of Northern Engineering at University of Alaska Fairbanks, to inspect the waterbed-like ground at a nearby golf course, she started to pay attention. Like others in Fairbanks, they lit “turf bubbles” on fire and confirmed the presence of methane gas.

Then, when Walter Anthony looked at nearby sites, she was shocked that methane wasn’t just coming out of a grassland. “I went through the forest, the birch trees and the spruce trees, and there was methane gas coming out of the ground in large, strong streams,” she said.

“We just had to study that more,” Walter Anthony said.

With funding from the National Science Foundation, she and her colleagues launched a comprehensive survey of dryland ecosystems in Interior and Arctic Alaska to determine whether it was a one-off oddity or unforeseen concern.

Their study, published in the journal Nature Communications this July, reported that upland landscapes were releasing some of the highest methane emissions yet documented among northern terrestrial ecosystems. Even more, the methane consisted of carbon thousands of years older than what researchers had previously seen from upland environments.

“It’s a totally different paradigm from the way anyone thinks about methane,” Walter Anthony said.

Because methane is 25 to 34 times more potent than carbon dioxide, the discovery brings new concerns to the potential for permafrost thaw to accelerate global climate change.

The findings challenge current climate models, which predict that these environments will be an insignificant source of methane or even a sink as the Arctic warms.

Typically, methane emissions are associated with wetlands, where low oxygen levels in water-saturated soils favor microbes that produce the gas. Yet methane emissions at the study’s well-drained, drier sites were in some cases higher than those measured in wetlands.

This was especially true for winter emissions, which were five times higher at some sites than emissions from northern wetlands.

Digging into the source

“I needed to prove to myself and everyone else that this is not a golf course thing,” Walter Anthony said.

She and colleagues identified 25 additional sites across Alaska’s dry upland forests, grasslands and tundra and measured methane flux at over 1,200 locations year-round across three years. The sites encompassed areas with high silt and ice content in their soils and signs of permafrost thaw known as thermokarst mounds, where thawing ground ice causes some parts of the land to sink. This leaves behind an “egg carton” like pattern of conical hills and sunken trenches.

The researchers found all but three sites were emitting methane.

The research team, which included scientists at UAF’s Institute of Arctic Biology and the Geophysical Institute, combined flux measurements with an array of research techniques, including radiocarbon dating, geophysical measurements, microbial genetics and directly drilling into soils.

They found that unique formations known as taliks, where deep, expansive pockets of buried soil remain unfrozen year-round, were likely responsible for the elevated methane releases.

These warm winter havens allow soil microbes to stay active, decomposing and respiring carbon during a season that they normally wouldn’t be contributing to carbon emissions.

Walter Anthony said that upland taliks have been an emerging concern for scientists because of their potential to increase permafrost carbon emissions. “But everyone’s been thinking about the associated carbon dioxide release, not methane,” she said.

The research team emphasized that methane emissions are especially high for sites with Pleistocene-era Yedoma deposits. These soils contain large stocks of carbon that extend tens of meters below the ground surface. Walter Anthony suspects that their high silt content prevents oxygen from reaching deeply thawed soils in taliks, which in turn favors microbes that produce methane.

Walter Anthony said it’s these carbon-rich deposits that make their new discovery a global concern. Even though Yedoma soils only cover 3% of the permafrost region, they contain over 25% of the total carbon stored in northern permafrost soils.

The study also found through remote sensing and numerical modeling that thermokarst mounds are developing across the pan-Arctic Yedoma domain. Their taliks are projected to be formed extensively by the 22nd century with continued Arctic warming.

“Everywhere you have upland Yedoma that forms a talik, we can expect a strong source of methane, especially in the winter,” Walter Anthony said.

“It means the permafrost carbon feedback is going to be a lot bigger this century than anybody thought,” she said.

The study, published in the journal Physiology & Behavior, shows that cutting calories by 20% did not significantly reduce the distance that mice voluntarily chose to run each day.

The researchers set out to understand what happens to mice when the amount of food available to them is reduced. The findings, they hoped, would be relevant to wild animals that do not always get as much food as they want on a given day, and also to humans, whose doctors often prescribe dieting.

It is somewhat difficult to obtain accurate data on the amount of voluntary exercise that humans engage in. Though it is easy to categorize what people recognize as voluntary exercise, like a trip to the gym, there is much gray area that’s hard to quantify, such as walking to a cafeteria to purchase lunch instead of eating a meal from a nearby lunch box.

Tracking what lab mice choose to do is much easier, and lab mice generally like to run on wheels for many hours per day. In this study, researchers saw the mice chose to run at similar levels, regardless of how much they ate.

“Voluntary exercise was remarkably resistant to reducing the amount of food by 20% and even by 40%,” said UCR biologist and corresponding study author Theodore Garland, Jr. “They just kept running.”

The researchers spent three weeks getting a baseline level of running activity for the mice, then a week with calories reduced by 20%, and another week at minus 40%. This experiment was done both with regular mice as well as “high runner” mice bred to enjoy running.

Though the high runners reduced their total distance slightly with 40% calorie restriction, the distance was only an 11% reduction. As they started out running three times farther per day than normal mice, the reduction is considered slight. “They’re still running at extremely high levels,” Garland said. The regular mice did not reduce their daily distance, even at 40% calorie reduction.

Because running gives a “runners high,” in part by increasing dopamine and cannabinoid levels in the brain, the researchers believe the mice were motivated to keep going even with less food. “Wheel running is a self-rewarding behavior,” Garland said.

In addition, the researchers were surprised to find that body mass was not significantly affected by the 20% reduction in calories in either the regular or high-runner mice. Although there was some drop in body mass with a 40% reduction, it was not as high as predicted.

“People often lose about 4% of their body mass when they’re dieting. That’s in the same range as these mice,” Garland said.

This study contributes to our understanding of why some people like to exercise and others don’t. In the future, the researchers are planning additional studies to understand why both the amount of voluntary exercise and body mass are so resistant to calorie restriction.

“There has to be some type of compensation going on if your food goes down by 40% and your weight doesn’t go down very much,” Garland said. “Maybe that’s reducing other types of activities, or becoming metabolically more efficient, which we didn’t yet measure.”

As habitat destruction causes food shortages for wild animals, this type of information could be instrumental for people trying to preserve species. And for the many people interested in improving their health, the implications could be similarly significant.

“We don’t want people on diets to say, ‘I don’t have enough energy, so I’ll make up for it by not exercising.’ That would be counterproductive, and now we know, it doesn’t have to be this way,” Garland said.