Early-phase trials in patients with Alzheimer’s disease and studies in mouse models of the disease have suggested positive effects on pathology and symptoms from exposure to light and sound in the 40 Hz “gamma” band frequency. A new study focuses on how 40 Hz sensory stimulation helps maintain an essential process in which the signal-sending branches of neurons, called axons, are wrapped in fatty insulation called myelin. Often called the “white matter” of the brain, myelin protects axons and ensures better transmission of electrical signals in brain circuits.
“Previous publications from our lab have focused primarily on neuronal protection,” said Li-Huei Tsai, the 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 CommunicationsTsai also heads MIT’s Aging Brain Initiative. “But this study shows that it’s not just gray matter, but also white matter that is protected by this approach.”
This year, Cognito Therapeutics, the spin-off company that licensed MIT’s sensory stimulation technology, published results from Phase II human trials in Alzheimer’s disease journal The results indicate that light and sound stimulation at 40 Hz significantly slowed myelin loss in volunteers with Alzheimer’s. Also this year, Tsai’s lab published a study showing that gamma sensory stimulation helped mice withstand the neurological effects of chemotherapy drugs, even preserving myelin. In the new study, members of Tsai’s lab, led by former postdoctoral researcher Daniela Rodrigues Amorim, used a common model of myelin loss in mice — a diet containing the chemical cuprizone — to explore how sensory stimulation preserves myelination.
Amorim and Tsai’s team found that light and sound at 40 Hz not only preserved myelination in the brains of mice exposed to cuprizone, but also appeared to protect oligodendrocytes (the cells that myelinate neuronal axons), maintain the neurons’ electrical performance, and preserve a key marker of axon structural integrity. When the team investigated the molecular basis of these benefits, they found clear signals for specific mechanisms, including preservation of neuronal 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 currently a Marie Curie Fellow at the University of Galway in Ireland. “We are seeing different effects in different ways.”
The findings suggest that gamma sensory stimulation may help not only Alzheimer’s patients but also people battling other diseases that involve myelin loss, such as multiple sclerosis, the authors wrote in the study.
Maintenance of myelin
To conduct the study, Tsai and Amorim’s team fed some male mice a cuprizone-containing diet and gave other male mice a normal diet for six weeks. Halfway through that period, when cuprizone is known to begin causing its most acute effects on myelination, they exposed some mice in each group to gamma sensory stimulation for the remaining three weeks. This left them with four groups: completely healthy mice, mice that did not receive cuprizone but did receive gamma stimulation, mice that received cuprizone and constant light and sound (but not 40 Hz) as a control, and mice that received cuprizone and also gamma stimulation.
After the six weeks were up, the scientists measured signs of myelination throughout the brains of the mice in each group. Mice that were not fed cuprizone maintained healthy levels, as expected. Mice that were fed cuprizone and did not receive 40 Hz gamma sensory stimulation showed dramatic levels of myelin loss. Cuprizone-fed mice that received 40 Hz stimulation retained significantly more myelin, rivaling the health of mice that were never fed cuprizone in some, but not all, measurements.
The researchers also looked at the number of oligodendrocytes to see if they survived better with sensory stimulation. Several measurements 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 was much closer to healthy levels.
Electrophysiological testing between neuronal axons in the corpus callosum showed that gamma sensory stimulation was associated with better electrical performance in cuprizone-fed mice that received gamma stimulation compared to cuprizone-fed mice that did not receive 40 Hz stimulation treatment. And when the researchers looked at 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 both cuprizone and gamma stimulation compared to cuprizone-fed mice that did not receive it.
Molecular mechanisms
A key goal of the study was to identify possible ways in which 40 Hz sensory stimulation may protect myelin.
To find out, the researchers performed a comprehensive assessment of protein expression in each group of mice and identified which proteins were expressed differently depending on the cuprizone diet and exposure to gamma-frequency stimulation. The analysis revealed distinct sets of effects between cuprizone mice exposed to control stimulation and cuprizone plus gamma mice.
A highlight of one set of effects was the increase in MAP2 in cuprizone-fed mice treated with gamma rays. A highlight of another set was that cuprizone-fed mice that received control stimulation showed a substantial deficit in the expression of proteins associated with synapses. Cuprizone-fed mice treated with gamma rays showed no significant loss, mirroring the results of a 2019 40 Hz Alzheimer’s study that showed synaptic preservation. This result is important, the researchers wrote, because neural circuit activity, which depends on maintaining synapses, is associated with myelin preservation. 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 erratic iron metabolism 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. However, gamma stimulation reduced HMGB1 levels.
Further studying 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 highly inflammatory in cuprizone control mice, but gamma stimulation calmed that response. Fewer cells became inflammatory, and direct tissue observations showed that microglia became more competent at removing myelin debris, a key step in effecting repairs.
The team also learned more about how oligodendrocytes from mice fed cuprizone and exposed to 40 Hz sensory stimulation managed to survive better. The expression of protective proteins such as HSP70 increased, as did the expression of GPX4, a master regulator of processes that limit ferroptosis.
In addition to Amorim and Tsai, the other authors of the article 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.
Funding for the study was provided by the la Caixa Banking Foundation, 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.