Neurons are thought to harbor differences that cannot be detected just by looking at them, or even by analyzing their RNA on a cell-by-cell basis. Neuron structure, connectivity, and electrophysiology, though varied, can tell us only so much. And RNA transcripts in individual cells tend to fluctuate, complicating efforts to establish stable neuron identities. Yet neuron types are being distinguished from each other with a new technique—single-cell methylomics. In a new study, this technique has already been used to identify new neuron subtypes. Going forward, the technique could dramatically improve our understanding about brain development and dysfunction.
Single-cell methylomics is a way of profiling chemical modifications of DNA molecules in individual neurons. Each cell's methylome—the pattern of chemical markers made up of methyl groups that stud its DNA—give a distinct readout of how its genetic switches are set.
Over 6000 methylomes were examined by a scientific team that included researchers from the Salk Institute and the University of California, San Diego (UCSD). These scientists report that they were able to identify 16 mouse and 21 human neuronal subpopulations in the frontal cortex. The scientists also found greater complexity of excitatory neurons in deep brain layers than in superficial layers.
Additional details appeared August 10 in the journal Science, in an article entitled “Single-Cell Methylomes Identify Neuronal Subtypes and Regulatory Elements in Mammalian Cortex.”
“CG and non-CG methylation exhibited cell type–specific distributions, and we identified regulatory elements with differential methylation across neuron types,” wrote the article’s authors. “Methylation signatures identified a layer 6 excitatory neuron subtype and a unique human parvalbumin-expressing inhibitory neuron subtype.”
“We think it's pretty striking that we can tease apart a brain into individual cells, sequence their methylomes, and identify many new cell types along with their gene regulatory elements, the genetic switches that make these neurons distinct from each other,” said co-senior author Joseph Ecker, Ph.D., professor and director of Salk's Genomic Analysis Laboratory and an investigator of the Howard Hughes Medical Institute.
In the past, to identify what sets different types of neurons apart from each other, researchers have studied levels of RNA molecules inside individual brain cells. But levels of RNA can rapidly change when a cell is exposed to new conditions, or even throughout the day. So, the Salk/UCSD team turned instead to the cells' methylomes, which are generally stable throughout adulthood.
The team began their work on both mouse and human brains by focusing on the frontal cortex, the area of the brain responsible for complex thinking, personality, social behaviors, and decision making, among other things. They isolated 3377 neurons from the frontal cortex of mice and 2784 neurons from the frontal cortex of a deceased 25-year-old human.
The researchers then used a new method they recently developed, called snmC-seq, to sequence the methylomes of each cell. Unlike other cells in the body, neurons have two types of methylation, so the approach mapped both types—called CG methylation (for DNA sequence containing the nucleotides cytosine and guanine) and non-CG methylation.
Neurons from the mouse frontal cortex, they found, clustered into 16 subtypes based on methylation patterns, while neurons from the human frontal cortex were more diverse and formed 21 subtypes. Inhibitory neurons—those that provide stop signals for messages in the brain—showed more conserved methylation patterns between mice and humans compared to excitatory neurons. The study also identified unique human neuron subtypes that had never been defined before. These results open the door to a deeper understanding of what sets human brains apart from those of other animals.
“This study opens a new window into the incredible diversity of brain cells,” commented Eran Mukamel, Ph.D., of the UC San Diego Department of Cognitive Science, a co-senior author of the work.
Next, the researchers plan to expand their methylome study to look at more parts of the brain, and more brains.
“There are hundreds, if not thousands, of types of brain cells that have different functions and behaviors and it's important to know what all these types are to understand how the brain works,” noted Chongyuan Luo, Ph.D., a Salk research associate and co-first author of the new paper. “Our goal is to create a parts list of both mouse and human brains.”
Once that “parts list” is complete, Ecker says they'd also like to begin studying whether the methylomes of neurons in people with brain diseases are different than those from healthy people. “If there's a defect in just 1% of cells, we should be able to see it with this method,” he asserted. “Until now, we would have had no chance of picking something up in that small a percentage of cells.”