Scientists reveal single-neuron gene landscape of the human brain Newly identified neuronal diversity provides insights into brain function and disease

A team of scientists at The Scripps Research Institute (TSRI), University of California, San Diego (UC San Diego) and Illumina, Inc., has completed the first large-scale assessment of single neuronal "transcriptomes." Their research reveals a surprising diversity in the molecules that human brain cells use in transcribing genetic information from DNA to RNA and producing proteins.

The researchers accomplished this feat by isolating and analyzing single-neuronal nuclei from the human brain, allowing classification of 16 neuronal subtypes in the brain's cerebral cortex, the "gray matter" involved in thought, cognition and many other functions.

"Through a wonderful scientific collaboration, we found an enormous amount of transcriptomic diversity from cell to cell that will be relevant to understanding the normal brain and its diseases such as Alzheimer's, Parkinson's, ALS and depression," said TSRI Professor and neuroscientist Jerold Chun, who co-led the study with bioengineers Kun Zhang and Wei Wang of UC San Diego and Jian-Bing Fan of Illumina.

The study was published on June 24 in the journal Science.

All the Same

While parts of the cerebral cortex look different under a microscope--with different cell shapes and densities that form cortical layers and larger regions having functional roles called "Brodmann Areas"--most researchers treat neurons as a fairly uniform group in their studies.

"From a tiny brain sample, researchers often make assumptions that obtained information is true for the entire brain," said Chun.

But the brain isn't like other organs, Chun explained. There's a growing understanding that individual brain cells are unique, and a possibility has been that the microscopic differences among cerebral cortical areas may also reflect unique transcriptomic differences--i.e., differences in the expressed genes, or messenger RNAs (mRNAs), which carry copies of the DNA code outside the nucleus and determine which proteins the cell makes.

To better understand this diversity, the researchers in the new study analyzed more than 3,200 single human neurons--more than 10-fold greater than prior publications--in six Brodmann Areas of one human cerebral cortex.

With the help of newly developed tools to isolate and sequence individual cell nuclei (where genetic material is housed in a cell), the researchers deciphered the minute quantities of mRNA within each nucleus, revealing that various combinations of the 16 subtypes tended to cluster in cortical layers and Brodmann Areas, helping explain why these regions look and function differently.

Neurons exhibited anticipated similarities, yet also many differences in their transcriptomic profiles, revealing single neurons with shared, as well as unique, characteristics that likely lead to differences in cellular function.

"Now we can actually point to an enormous amount of molecular heterogeneity in single neurons of the brain," said Gwendolyn E. Kaeser, a UC San Diego Biomedical Sciences Graduate Program student studying in Chun's lab at TSRI. Kaeser was co-first author of the study with Blue B. Lake and Rizi Ai of UC San Diego and Neeraj S. Salathia of Illumina.

Many New Questions

Interestingly, some of these differences in gene expression have roots in very early brain development taking place before birth. The researchers found markers on some neurons showing that they originated from a specific region of fetal brain called the ganglionic eminence, which generates inhibitory neurons destined for the cerebral cortex. These neurons may have particular relevance to developmental brain disorders.

The enormous transcriptomic diversity of single neurons was predicted by earlier work from Chun's laboratory and others showing that the genomes--the DNA--of individual brain cells can be different from cell to cell. In future studies, the researchers hope to investigate how single-neuron DNA and mRNA differs in single neurons, groups and between human brains--and how these may be influenced by factors such as stress, medications or disease.

Additional authors of the study, "Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain," were Yun C. Yung, Julian Wong, Allison Chen and Xiaoyan Sheng of TSRI; Rui Liu, Andre Wildberg, Derek Gao, Ho-Lim Fung and Song Chen of UC San Diego; and Raakhee Vijayaraghavan, Fiona Kaper, Richard Shen and Mostafa Ronaghi of Illumina.

The study was supported by the National Institutes of Health Common Fund Single Cell Analysis Program (1U01MH098977-01) and a Neuroplasticity of Aging Training Grant (5T32AG000216-24).

First proposed human test of CRISPR passes initial safety review

A cancer study that would represent the first use of the red-hot gene-editing tool CRISPR in people passed a key safety review today. The proposed clinical trial, in which researchers would use CRISPR to engineer immune cells to fight cancer, won approval from the Recombinant DNA Advisory Committee (RAC) at the U.S. National Institutes of Health, a panel that has traditionally vetted the safety and ethics of gene therapy trials funded by the U.S. government and others.

Although other forms of gene editing have already been used to treat disease in people, the CRISPR trial would break new ground by modifying three different sites in the genome at once, which has not been easy to do until now. The study has also grabbed attention because—as first reported by the MIT Technology Review—tech entrepreneur Sean Parker’s new $250 million Parker Institute for Cancer Immunotherapy will fund the trial.

“It’s an important new approach. We’re going to learn a lot from this. And hopefully it will form the basis of new types of therapy,” says clinical oncologist Michael Atkins of Georgetown University in Washington, D.C., one of three RAC members who reviewed the protocol.

The proposed CRISPR trial builds off the pioneering efforts of Carl June and others at University of Pennsylvania (UPenn) to genetically modify a cancer patient’s own immune cells, specifically a class known as T cells, to treat leukemia and other cancers. For the CRISPR trial, a UPenn-led team wants to remove T cells from patients and use a harmless virus to give the cells a receptor for NY-ESO-1, a protein that is often present on certain tumors but not on most healthy cells. The modified T cells are then reinfused back into a patient and, if all goes well, attack the person’s NY-ESO-1–displaying tumors. The UPenn team has already tested this strategy in a small clinical trial for multiple myeloma. But although most patients’ tumors initially shrank, the reintroduced T cells eventually became less effective and stopped proliferating.

To boost the staying power of the engineered T cells, the UPenn group wants to use CRISPR to disrupt the gene for a protein called PD-1. The protein sits on the surface of T cells and helps dampen the activity of the cells after an immune response, but tumors have found ways to hide from T cell attack by flipping on the PD-1 switch themselves. (Drugs that block PD-1 eliminate this immune suppression and have proven to be a promising immunotherapy cancer treatment.)

June’s team also wants to knock out two gene segments that encode different portions of the protein that makes up a T cell’s primary receptor so that the engineered NS-ESO-1 receptor will be more effective. To do this, they will introduce into the T cells so-called guide RNAs, which tell CRISPR’s DNA-snipping enzyme, Cas9, where to cut the genome.

The 2-year trial will treat 18 people with myeloma, sarcoma, or melanoma who have stopped responding to existing treatments at three sites that are members of the Parker Institute—UPenn; the University of California, San Francisco; and the University of Texas MD Anderson Cancer Center in Houston. June pointed out to RAC that his team already has experience with gene editing. They have used a different technique, called zinc finger nucleases, to disrupt a gene on T cells that HIV uses to enter the cells. In a small trial, this strategy appeared to be safe and has shown promise for helping HIV patients. Those data suggest that CRISPR gene editing should be safe in humans, June said.

To confirm that, researchers conducting the CRISPR trial will look for signs of an immune reaction to the Cas9 enzyme, which comes from a bacterium. They will also look for evidence that it has made cuts in wrong places, potentially creating or triggering a cancer gene. When the UPenn team recently used CRISPR to edit T cells from healthy donors as a test run, they checked the 148 genes they most feared Cas9 would mistakenly slice and only found one cut in a harmless location. For the CRISPR trial, the team will do various tests to watch for uncontrolled growth of the modified T cells. Because they are cutting the genome in three places, one RAC member also noted, the team should watch for large swapped chunks of chromosomes.

Another concern raised by several RAC members is that June, who would not treat the cancer patients but would serve as the trial’s scientific adviser, and UPenn have a financial interest in the trial. (June has patents on using engineered T cells to treat cancer and has advised companies developing these treatments.) Some on the panel suggested they were particularly sensitive about such concerns given that it was at UPenn in 1999 that a young man, Jessie Gelsinger, died in a gene therapy trial, setting the field back for years. “Penn does have an infamous history in this regard,” says biomedical ethicist and RAC member Lainie Ross of the University of Chicago in Illinois.

However, others on the panel noted that the university could take various steps to mitigate the conflict of interest, for example by recusing June from specific tasks. UPenn itself should decide whether it can directly treat patients or merely supply the modified T cells to other sites for the trial, RAC concluded. Ultimately, RAC members voted unanimously (with one abstention) to approve the trial.

Although RAC endorsement is a big step, the researchers must now seek approval from their own institutions’ ethics boards and the U.S. Food and Drug Administration. Others are likely nipping at their heels. Many thought the Cambridge, Massachusetts–based biotech company Editas Medicine would conduct the first CRISPR clinical trial—it has announced plans to use CRISPR to treat an inherited eye disease in 2017—but RAC has not yet reviewed a proposal from the company.