Understanding the functional evolution of human genes through Neanderthal and Denisovan genes

In early 2021, an interesting paper attracted attention and praise from the scientific community. Clever A. Trujillo and his colleagues from California, the UK and Brazil worked together to generate brain organoids expressing an allelic variant of the gene neuro-oncological ventral antigen 1 (NOVA1) as it was expressed in Neanderthal and Denisovan genomes. To do so, they used CRISPR-Cas9 technology to introduce the gene variant in human induced pluripotent stem cells, thus expressing the archaic gene in a modern human genetic background. Their work led to the above mentioned paper in Science (Trujillo et al., Science 371, eaax2537, 2021). Let’s take a look at why this peculiar work is of interest for the scientific community.

Why express a Neanderthal gene in human brain organoids?
Researchers made a genomic comparison between the modern (human) genome and the archaic (Neanderthal and Denisovan) one. They looked for autosomal genomic regions in which not a single human genome analysed carries alleles that are present also in archaic genomes (i.e. regions carrying autosomal fixed derived mutations) and they found 61 of such regions. They then looked at the size of these genomic regions containing exclusively human allelic variants and no archaic mutation at all. NOVA1 resulted to be the third largest human-specific haplotype, right after OR8B2 and NCOA6. Due to its role in neurological disorders, NOVA1 became the candidate for the study in brain organoids. NOVA1 is in fact an RNA-binding protein that regulates alternative splicing in the central and peripheral nervous system (Jensen et al., Neuron 2000). In particular, it regulates splicing of genes responsible for synapse formation (Ule et al., Nat Gen 2005) and is required for neuronal survival.

Besides expressing the archaic gene variant, they also generated brain organoids expressing the modern human variant of NOVA1. This allowed them to study the role of NOVA1 by investigating the effects of the archaic form in a human genetic background on early embryonic brain development, and comparing it with the early development of brain organoids expressing the human variant of NOVA1.

Effects of the archaic NOVA1 gene variant on the development of brain organoids
Trujillo and colleagues performed several analyses on the brain organoids they generated, thus identifying some effects of the archaic variant. Despite the fact that the early stages in the protocol looked very similar, once they reached the proliferative phase of organoids generation researchers noticed the first differences between those expressing the archaic NOVA1 (NOVA1Ar/Ar) and those expressing the human variant (NOVA1Hu/Hu). NOVA1Ar/Ar led to smaller organoids, with a more irregular surface. This is probably the result of a higher rate of apoptosis (cell death), a smaller pool of neural progenitors and their slower proliferation rate - all effects observed by the researchers. Additionally, neurons with the archaic variant express different genes than neurons with the human variant.

But NOVA1 is a regulator of gene splicing, so: is the splicing of genes changing? As it turns out, more than 100 genes expressed in the NOVA1Ar/Ar organoids are spliced differently than they are in NOVA1Hu/Hu organoids. The process of splicing removes non-coding regions of a pre-mRNA to join together different coding regions and obtain the final mature messenger RNA, which will serve as a template for the creation of the protein. Thanks to this process, a certain gene can be processed into different mRNAs depending on the cell type or the developmental stage. Thus, over 100 human genes are not producing the correct mature form of the protein in the presence of the archaic NOVA1 gene. The majority of these genes are involved in the generation of synapses and of neural connections. Another consequence of the different splicing pattern is that synaptic proteins in NOVA1Ar/Ar organoids bind to a network of proteins different from NOVA1Hu/Hu organoids. An interesting example of this identified by the researchers is the upregulation of the binding of SHANK, HOMER, GluR1, and mGluR5, a protein cluster that is commonly dysregulated in some human neurodevelopmental disorders.

Given the effects of NOVA1Ar/Ar on neuronal proteins, what are the consequences on neurons and their functions? Trujillo and the other researchers involved in the study report that, while they still fire action potentials, neurons in NOVA1Ar/Ar organoids show an increased number of bursts, a firing pattern that can be associated with some neuropsychiatric disorders such as depression. There is also reduced neuronal synchrony, a phenomenon of synchronisation of the patterns of action potentials leading to neuronal oscillations that can be measured, e.g via electroencephalogram recordings.

As interesting as this work is, one thing is clear. Conversely to what inferred from some titles reporting the news of this paper, Trujillo et al. does not report the generation of Neanderthal brain organoids! What the authors did instead was simply to express a specific allele of the gene NOVA1 that was present in Neanderthal and Denisovan genomes (but is not in human genomes) within the genetic background of humans. To have a real archaic mini brain in a dish recapitulating neurodevelopment in a species of archaic humans sharing a common ancestor with us, one would first need to generate a line of induced pluripotent stem cells expressing the genes from the Neanderthal or Denisovan genomes, or at least what has been isolated so far. Perhaps in the years to come, this will be possible, and then we could start studying archaic neural development.

The work of this paper helps to show how an archaic gene behaves in a modern human genetic environment. What is the advantage of this? Firstly, it helps to better understand the role of the human gene. Also, it provides insights on the function of the mutation that has occurred in humans. By comparing NOVA1Ar/Ar and NOVA1Hu/Hu organoids, it becomes in fact evident the importance that the human fixed mutation has on neural development, regulating physiological functions essential to the viability and proliferation of neural progenitors, to the viability of neurons, the generation of synapses and of synaptic networks. Ultimately, this experimental approach can shed light on the functions of human genes, while also providing some understanding of how human neural development diverged from the one of archaic species of humans or hominids.

 

Written by Chiara Galante; Edited by Gabrielle Sant. Featured Image: NGC/Design.

 

Resources:

Trujillo et al., Science 371, eaax2537, 2021 (Link)

Jensen et al., Neuron 2000 (Link)

Ule et al., Nat Gen 2005 (Link)

Cohen, Science 360:1284, 2021 (Link)

Nature 590:376-377, 2021 (Link)