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Neuronal Subtypes Revealed Using Single-Cell Methylation Sequencing

NEW YORK (GenomeWeb) – A Salk Institute for Biological Studies-led team has shown that it can cluster neuronal subtypes in mouse and human brain samples based on methylation and regulatory features, identifying a new subset of neurons in the human frontal cortex in the process.

The researchers used single-cell methylome sequencing to profile nearly 6,200 individual neurons from mouse and human frontal cortex brain samples, clustering known neuronal subtypes based on methylation and regulatory element profiles. But their findings, published online today in Science, also pointed to the presence of a previously unappreciated neuronal subtype in the human brain called "layer 6 excitatory neurons," which can now be delineated based on its distinctive methylation marks.

"Our research shows that we can clearly define neuronal types based on their methylomes," co-senior author Margarita Behrens, a computational neurobiology researcher at the Salk Institute, said in a statement. "This opens up the possibility of understanding what makes two neurons — that sit in the same brain region and otherwise look similar — behave differently."

For their analysis, Behrens and colleagues used a single-nucleus bisulfite conversion-based methylcytosine sequencing approach dubbed snmC-seq to profile combined 5-methyl- and 5-hydroxymethylcytosine patterns in 3,377 neurons isolated from the brain of an eight-week-old mouse and another 2,784 individual neurons isolated post-mortem from the frontal cortex region of a deceased 25-year-old man.

The library preparation method used for snmC-seq included approaches built from Swift Biosciences' Accel-NGS Adaptase module and Timothy Harkins, the company's president and CEO, was among the study co-authors. 

"Whereas single-cell RNA sequencing mainly yields information about highly expressed transcripts, single-neuron methylome sequencing assays any gene or non-gene region long enough to have sufficient coverage," the authors explained, noting that single-cell methylomics reveals "regulatory information from the vast majority of the genome not directly assessed by RNA sequencing."

The team's methylation clustering analyses led to 16 neuronal clusters for the mouse cells considered, along with 21 neuronal subtypes for the human frontal cortex cells tested. It also provided a look at methylation marks specific to certain excitatory or inhibitory neuronal cell types, while highlighting the distribution of such neurons across the human and mouse brain samples.

By folding in clues from their differential methylation analyses and transcription factor binding sequences in the underlying DNA, the researchers got a look at some of the regulatory features characterizing the neurons as well.

Though the authors cautioned that "anatomical, physiological, and functional experiments are needed to characterize the DNA methylation-based neuronal populations defined by our study," the identification of the newly described layer 6 excitatory neurons suggests that the methylation-based approach might help in delineating and characterizing neurons found in the brain.

"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," co-first author Chongyuan Luo, a research associate in co-senior author and Salk genomic analysis laboratory director Joseph Ecker's lab, said in a statement. "Our goal is to create a parts list of both mouse and human brains."