What happens when you replace a human gene with its Neanderthal version?

What are the key differences between modern humans and our closest relatives, the Neanderthals and Denisovans? For the Neanderthals, there doesn’t seem to be any sort of obvious difference. They used sophisticated tools, made art, and established themselves in some very harsh environments. But, as far as we can tell, their overall population was never particularly high. When modern humans arrived on the scene in Eurasia, our numbers grew larger, we spread even further, and the Neanderthals and Denisovans ended up displaced and eventually extinct.

With our ability to obtain ancient DNA, we’ve now gotten a look at the genomes of both Neanderthals and Denisovans, which allows us to ask a more specific question: could some of our differences be due to genetics?

The three species are close relatives, so the number of differences in our proteins are relatively small. But a large international research team has identified one and engineered it back into stem cells obtained from modern humans. And the researchers found that neural tissue made of these cells has notable differences from the same tissue grown with the modern human version of this gene.

A not-quite super NOVA

As the first step in their work, the researchers had to decide on a gene to target. As we mentioned above, the genomes of all three species are extremely similar. And the similarity only goes up when you look at those parts of the genome that encode proteins. An added complication is that some of the versions of genes found in Neanderthals are still found in a fraction of the modern human population. What the researchers wanted to do is find a gene where both Neanderthals and Denisovans had one version and nearly all modern humans had another.

Out of tens of thousands of genes, they found only 61 that passed this test. The one they chose to focus on was called NOVA1. Despite the explosive-sounding name, NOVA1 was simply named after having originally been found associated with cancer: Neuro-oncological ventral antigen 1. A look through the vertebrate family tree shows that Neanderthals and Denisovans share a version of NOVA1 with everything from other primates to chickens, meaning that it was present in the ancestor that mammals shared with dinosaurs.

Yet almost all humans have a different version of the gene (in a search of a quarter-million genomes in a database, the researchers were only able to identify three instances of the Neanderthal version). The difference is subtle—swapping in a closely related amino acid at a single location in the gene—but it is a difference. (For those who care, it’s isoleucine to valine.)

But NOVA1 is the sort of gene where small changes can potentially have a big impact. The RNAs that are used to make proteins are initially made of a mixture of useful parts separated by useless spacers that need to be spliced out. For some genes, the different parts can be spliced together in more than one way, allowing distinct forms of a protein to be made from the same starting RNA. NOVA1 regulates the splicing process and can determine which form of multiple genes gets made in cells where it’s active. For NOVA1, the cells where it’s active include many parts of the nervous system.

If that last paragraph was somewhat confusing, the short version is this: NOVA1 can change the types of proteins made in nerve cells. And, since behavior is one area where modern humans may have been different from Neanderthals, it’s an intriguing target of these sorts of studies.

On our nerves

Obviously, there are ethical issues with trying to see what the Neanderthal version would do in actual humans. But some technologies developed over the last decade or so now allow us to approach the question in a very different way. First, the researchers were able to take cells from two different people and convert them into stem cells, capable of developing into any cell in the body. Then, they used CRISPR gene-editing technology to convert the human version of the gene into the Neanderthal version. (Or, if you’re less charitable, you could call it the chicken version.)

After performing extensive checks that indicated that NOVA1 was the only gene altered by the editing, the researchers induced the stem cells to form the neurons typical of the brain’s cortex.

The clusters of neural cells that resulted were smaller when they were formed by cells with the Neanderthal version of NOVA1, although these clusters had a more complex surface shape. The cells with the Neanderthal version also grew more slowly and tended to undergo a process that ends in cell death more often. So it was clear that the Neanderthal version altered the stem cells’ behavior as they were converted into nerve cells.

Differences were apparent on the genetic level, as well. The research team looked for any genes that had altered activity (as measured by messenger RNA levels) in the cells with the Neanderthal NOVA1. There were quite a number of them, and they included some key regulators of neural development. And, as expected from a splicing regulator, there were hundreds of genes that saw changes to how their protein-coding RNAs were pieced together.

Many of these genes appear to be involved in the formation and activity of synapses, the individual connections among nerve cells that allow them to communicate with each other. Not surprisingly, this altered the behavior of those connections. Normally, nerve cells in culture form connections and coordinate their activity. In cells with the Neanderthal version of NOVA1, there was less coordination and a higher background of nerve cells firing off signals at random.

A matter of context

The results clearly show that having the Neanderthal version of NOVA1 is not a good thing for the nerve cells of modern humans. It will still take some more work, however, to determine whether all of the changes described here are the product of specific differences between the two forms of the protein or simply a consequence of the nerve cells being unhealthy due to the misregulation of genes.

But the researchers also caution against overinterpreting the results in general—while suggestive, these results are not a clear indication that gene changes make our brains fundamentally different from those of our closest relatives.

The evolution of the human version of this gene took place against a backdrop of many other subtle changes in human genes, either in their coding sequences or (more often) in the sequences that regulate their activity. Those changes could potentially offset any harmful effects caused by the differences in activity of the modern human version of NOVA1. Suddenly dropping in the original version of the gene again might only produce differences due to the mismatch between the gene and all of those compensations.

So, it’s going to take a while to sort out how much this one gene’s differences mean for human and Neanderthal brains. But the key thing is that it’s now possible to ask these questions at all. The technologies used to produce these results didn’t exist before this century—CRISPR gene editing is less than a decade old. So, the mere fact that we know this much is pretty astonishing.

Science, 2021. DOI: 10.1126/science.aax2537 (About DOIs).