A multidisciplinary team at the University of Illinois (U of I) Urbana-Champaign has devised a non-invasive MRI-based 3D imaging approach that directly captures DNA methylation, a key epigenetic change associated with regulating gene expression, and learning in the brain. The scientists say their proof-of-concept study, carried out in pigs, will easily translate to humans, as the method, which they’ve termed epigenetic MRI (eMRI), relies on standard MRI technology and biological markers that are already used in human medicine.
The researchers, co-led by King Li, PhD, a professor in the Carle Illinois College of Medicine at the U of I, that suggest eMRI will open up new avenues of research into how epigenetic changes mold the brain, allowing it to grow, learn and respond to stress. The technique also may be useful in the study of neurodegenerative processes like Alzheimer’s disease.
“Because DNA methylation is one of the major regulators of gene expression, eMRI promises to become a powerful tool to understand the molecular basis of brain function and disease,” concluded the researchers in their published paper in Proceedings of the National Academy of Sciences (PNAS), which is titled “Epigenetic MRI: Noninvasive imaging of DNA methylation in the brain,” U of I bioengineering professor Fan Lam, PhD, and Gene Robinson, PhD, the director of the Carl R. Woese Institute for Genomic Biology at Illinois, co-headed the work with Li, and a team of researchers at the University of Illinois Urbana-Champaign.
The brain is ever changing in structure and function as a result of development, aging, environmental influence, and disease, the authors explained. There are two control systems in the brain, operating at different time scales. “Both neuronal and genetic mechanisms regulate brain function.” Neurons and other brain cells respond to environmental cues within seconds or milliseconds, while changes in gene expression take longer. For example, when a honey bee experiences a threat, it must take action immediately. It relies on neurons to rapidly fire and allow it to act defensively. But the bee’s brain continues to respond even after the threat has lapsed, preparing itself for a potential future threat with changes in gene expression.
“Our DNA is the same from cell to cell and it doesn’t change,” Li said. “But tiny molecules, like methyl groups, are attached to the DNA backbone to regulate which genes are actively being transcribed into RNAs and translated into proteins. DNA methylation is a very important part of the control of gene functions.”
Previous research showed that DNA methylation is one of several epigenetic changes that occur in the brain when an animal responds to its environment, said Robinson, a professor of entomology at Illinois who studies the interplay of genomics, experience and behavior in honey bees. “Dynamic epigenetic activity is a fundamental mechanism underpinning how the brain changes its function during development and aging and in response to environmental and disease stimuli,” the authors further wrote. Robinson’s studies have shown that many genes in the brain are upregulated or downregulated in bees as they mature, change roles in the hive, encounter new food sources or respond to threats.
“We’re focusing on this second control system, the molecular control system, which relies on gene expression,” Robinson said. “These changes can take minutes to occur, but can last for hours, days or even longer.” But such changes can be hard to monitor, the authors pointed out. “While there are excellent methods to study neuronal activity in vivo, there are no nondestructive methods to measure global gene expression in living brains.”
Scientists have been unable to precisely capture the molecular changes that take place in the living brain over time. Earlier epigenetic studies of honey bees and other organisms required the removal of brain tissue or dissection of the animal for analysis. A previous research effort in the human brain imaged an enzyme involved in regulating one epigenetic change, but did not target the epigenetic change directly. The authors explained further, “PET imaging of histone deacetylases (HDACs) in the human brain was recently demonstrated, using a radioactive tracer that can pass the blood–brain barrier (BBB) and target HDAC isoform.” And while this form of imaging does probe histone acetylation, as another major form of epigenetic gene regulation, PET requires introducing radioactive materials into the body, and it only targets one of
the enzymes that regulates histone acetylation, rather than histone acetylation itself, they pointed out. “Moreover, PET lacks the specificity to distinguish between the target molecule and downstream metabolic products.”
The Illinois team instead turned to MRI, to see if this technology could be used to directly image epigenetic changes in live subjects. For the new approach, the researchers relied on a key insight. The carbon isotope carbon-13 occurs naturally in the body, but its sister isotope, carbon-12, is much more abundant, and about 99% of the carbon in living tissues is carbon-12. Li realized that the essential amino acid, methionine, could carry carbon-13 into the brain, where it could donate the carbon-13-labeled methyl group needed for DNA methylation. This process would then mark the DNA with a rare isotope of carbon.
Methionine must be obtained through the diet, so the tested the idea that feeding the carbon-13-labeled methionine (13C-Met) to their test subjects would allow it to pass into the brain and label those regions undergoing methylation. “We chose 13C-Met because Met is an essential amino acid and the major methyl group donor for DNA methylation. Met is also commonly used as a nutritional supplement and approved for human use,” the investigators noted. “To validate our labeling approach, we designed a special diet that replaced all protein with free amino acids in the proper proportions and substituted all Met with enriched 13C-Met.” Lam, who worked with Illinois chemistry professor Scott Silverman to develop a method to distinguish between methylated DNA and other methylated molecules in the brain, noted, “When we started this project, we thought it might fail, but the potential was so exciting that we had to try.”
Previous studies had already shown that MRI can image carbon-13, and orally administered carbon-13 has been in use in human subjects for decades. But the carbon-13 signal from living animals is weak, so Lam and U of I electrical and computer engineering professor Zhi-Pei Liang, PhD, relied on their expertise in MRI and MR spectroscopy to significantly enhance the eMRI signal.
The team first tried the method in rodents, then switched to working in piglets, whose larger brains are more like human brains. “For this, they relied on the expertise of co-author Ryan Dilger, PhD, a professor of animal sciences at Illinois who studies the factors that influence neurodevelopment in pigs.
“This project is highly multidisciplinary,” Lam said. “We have on the team engineers, imaging and radiology experts, and people with very strong backgrounds in clinical applications. We also have scientists with expertise in nutrition science, animal science, chemistry and genomics.”
In the experiments in piglets fed a diet that included carbon-13-labeled methionine, the researchers found that MRI could detect an increasing signal from carbon-13-labeled methyl groups in the brain. Further analyses allowed them to differentiate methyl groups on DNA from other methylated molecules.
The piglets had more new DNA methylation in the brain a few weeks after birth than they did at birth, and the increase was much greater than expected based on changes in size alone.
“This finding is very encouraging because it reflects what we expect to see if this signal is environmentally responsive,” Li said. “It is known from animal studies that brain regions that are most involved in learning and memory experience more epigenetic changes. There also were regional differences in DNA methylation across the pig brain, just like there are regional differences in classical MRI studies. We now expect to apply this technique in humans. Getting this label into the brain is easy and does no harm to the body. We’ll give it to people through the diet and then we can detect the signal.”
The team further pointed out that eMRI is able to provide information on the turnover of DNA methylation in the brain in vivo. In the piglets, which have growing brains, the 13C label can be incorporated into the DNA of brain cells through both new cell formation and turnover of DNA methylation, but in in adult animals and humans, brain growth is not a major factor, so incorporation of 13C labels into the DNA of adult brain cells is primarily through turnover of DNA methylation. “No previously known in vivo technique can measure this turnover, and doing so with eMRI may shed light on how the dynamics of brain DNA methylation contribute to the regulation of higher brain functions.”
Their first application of the approach will likely occur in studies comparing the brains of people with and without neurodegenerative disease, Li suggested. But as the authors concluded, “Given the noninvasive nature of eMRI, our results pave the way for a DNA-methylation imaging paradigm for living human brains … Just as functional MRI measurements of regional neuronal activity have had a transformational effect on neuroscience, we expect that the eMRI signal, both as a measure of regional epigenetic activity and as a possible surrogate for regional gene expression, will enable many new investigations of human brain function, behavior, and disease.”