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Scientists Insert Neanderthal Gene Into Lab-Grown "Mini Brains"

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Scientists have used genome-editing technology to replace a modern gene involved in neurodevelopment with its ancestor that was expressed in Neanderthal/Denisovan populations. The gene has been expressed in brain organoids to study its function. The study findings – published in the journal Science – bring us one step closer to understanding how the modern human brain has evolved.

The ability to sequence ancient DNA extracted from fossils has gifted humanity with exceptional insights into the molecular biology of our ancestors. We can study what the Neanderthals and the Denisovans ate, where they lived and how they spent their time on Earth.

But wouldn't it be wonderful to take a peek inside their brains? To understand how their neuronal hardwiring compares to our own? To explore how the modern human brain has evolved into the complex and sophisticated machinery that it is today? Indeed, there have been many major advances in the field of neuroscience in the last few decades but magicking the existence of archaic brain tissue is not one of them. Soft materials do not fossilize, so they cannot be studied.

But we can edit the genetic code, thanks to
CRISPR-Cas9 genome-editing technology. We can also model brain tissue in a laboratory dish using brain organoids – sometimes known as "mini brains" – that are constructed from induced pluripotent stem cells (iPSCs).

A team of scientists led by Cleber Trujillo, former researcher at the University of California San Diego (UCSD), utilized these scientific advances to address the longstanding question "What makes us human?" by exploring the role of an archaic gene in neurodevelopment.

What is so special about NOVA1?

There are 61 genes that differ between Homo sapiens and the Neanderthals/ Denisovans,
Alysson Muotri, professor in the Departments of Pediatrics and Cellular and Molecular Medicine at UCSD, and corresponding author of the study, explained. For the purpose of this experiment, the scientists focused on one particular gene: NOVA1.

"NOVA1 is the only gene that is active in early brain development, has been previously implicated in neurological conditions and is a master regulator in the developing nervous system," Muotri says. Such genes earn their title of "master genes" because they reside at the top of the gene regulation hierarchy, controlling the expression of hundreds of downstream genes via alternative splicing.

The archaic form of the NOVA1 gene is different to the modern allele as it possesses a single-nucleotide substitution at position 200, which results in an
amino acid change of isoleucine to valine. The scientists wanted to study the functional significance of this substitution. "We want to catalogu the genomic changes that make up the modern human brain," said Muotri. "Our hypothesis was that during evolution, a key set of mutations, brought us advantages that allow our species do survive were other humans didn’t."

A toolbox of novel scientific techniques

The scientists used CRISPR-Cas9 technology to insert the archaic variant of NOVA1 into the genome of iPSCs that had been derived from two human individuals. Expression of the gene variant was confirmed through genomic sequencing, and off-target edits – a common limitation to the use of CRISPR-Cas9 genome-editing – were searched for. Next, functional cortical organoids were derived from the iPSC cell lines. As control measures, cortical organoids were also generated that expressed the modern variant.

On the emergence of brain organoids as a research tool, Muotri said, "Brain organoids mimic the development of the human brain in several aspects, including gene expression, cell types and more recently, organized neural activity."

Brain organoids are not a perfect model. They have limitations including their small size, lack of vascularization and the fact they are not physically connected to a body. "However, being reductionist also has advantages; it allows us to determine even subtle alterations more specifically," Muotri stressed.

The researchers gathered and sequenced RNA from the cortical organoids at two timepoints – one and two months of development – to investigate whether any differences in gene expression could be detected. Comparing cortical organoids that were homozygous for the archaic allele with organoids that were homozygous for the modern human allele, they identified 277 differentially expressed genes – many of which are known to be involved in different stages of neurodevelopment.

"Because we cannot experiment with live human embryos, we need a model to recreate the brain outside the womb," – Muotri.

"Several of these downstream genes were implicated in synaptic connectivity," said Muotri. One example is the
HOMER3 gene, which encodes a member of the HOMER protein family found in the postsynaptic density.

The scientists also explored how the connections of the different organoids formed and found that the neurons of the archealized organoids matured faster than those carrying the modern NOVA1 allele. This is a key finding from the study, as it implies that the modern NOVA1 allele may slow down neurodevelopment. "By having a slow neurodevelopment, our brains might achieve a higher level of complexity," Muotri explained, he also suggested that this might be an evolutionary trade-off: "We need to take care of our infants until they become independent, and consequently, they will develop more complex brains. A baby chimpanzee can outsmart a human brain, but it does not reach the same complexity. This is true for most species. We humans are an outlier in this sense."

An important event in the evolution of the neural phenotype

The collective results of the study led the researchers to hypothesize that the genetic change in NOVA1 was an important event in the evolution of the human neural phenotype. As such, it warrants further study. "We want to further investigate this hypothesis. Are the neural networks in the archaic brain organoid less adaptable than in modern human organoids? We can do this by stimulating these organoids using a machine interface. We will challenge these networks to solve simple problems, revealing another layer of complexity that we could not yet investigate where these experiments are ongoing," Muotri said.

Highlighting limitations to the research, Muotri noted that CRISPR can cause unintentional alterations in a cell’s genome, and it is important to pay attention to this possibility. Furthermore, the limitations of brain organoids mean that comparisons with adult human brains are extrapolations.

A new Archealization Center at UCSD will further this work.

Alysson Muotri was speaking to Molly Campbell, Science Writer for Technology Networks.

Reference: Trujillo CA, Rice ES, Schaefer NK, et al. Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment. Science. 2021;371(6530). doi:10.1126/science.aax2537.