In a recent study published in Scienceresearchers demonstrated how expression of the transketolase-like 1 (TKTL1) gene influences neocortical neurogenesis in modern humans.
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Intriguingly, a single amino acid substitution in TKTL1, from lysine in apes and archaic humans to arginine in modern humans, altered the function and organization of brain layers, such as the neocortex, especially in the frontal lobe. Evolutionary changes in the neocortex of the brain and the simultaneous increase in neuron production have enhanced the cognitive abilities seen in modern humans. Endocrane analyzes revealed that the endocranial volume of modern humans and Neanderthals was similar, suggesting a similar size of the neocortex. However, the fossils failed to assess whether similar size of the neocortex implied similar production of neocortical neurons.
A previous study by Pinson et al. analyzed the effect of a single amino acid change in the TKTL1 protein on basal radial glia (bRG) production, the cells that generate most parts of the neocortex. They observed that in organoids, the human variant of TKTL1 (hTKTL1) generated more neuroprogenitors than the archaic variant (aTKTL1) found in Neanderthals, Denisovans and other primates. This may be why modern humans have more neocortices in their brains than Neanderthals.
About the study
In this study, the researchers examined the role of TKTL1 in the development of the neocortex, which, in turn, affects the number of neuroprogenitors. Additionally, they determined whether aTKTL1 and hTKTL1 have similar effects on neuroprogenitors during neocortical development.
The study used overexpression of the gene in the developing neocortex of mice and ferrets. Likewise, they used fetal human neocortical tissue knockout technology and genome editing technology to study brain organoids.
Using clustered regularly interspaced short palindromic repeats (CRISPR) hTKTL1 knockout technology and CRISPR-associated protein 9 (Cas9), they showed that hTKTL1 knockout in fetal human neocortical tissue significantly reduced the number from brG. The team then used brain organoids to validate their results. These brain mini-structures made from human embryonic stem cells displayed aTKTL1 instead of hTKTL1.
Specifically, the team obtained total ribonucleic acid (RNA) from human organoids derived from simulated and genetically modified H9 cells. She used quantitative real-time PCR (qPCR) to confirm that these organoids expressed TKTL1 messenger RNA (mRNA), and then Sanger sequencing of the PCR products. They matched the simulated, genetically modified organoids to the human TKTL1 gene to confirm the expression of hTKTL1 mRNA in the simulated human organoids and archaic TKTL1 mRNA in the genetically modified human organoids.
Prior to immunofluorescence, they cut coronal cryosections 20-50 µm thick using a cryostat and stored these samples at -20˚C. The team first subjected a single-cell suspension of mouse embryonic neocortex to cell surface staining. Then they microdissected it under an epifluorescence stereomicroscope. In this way, they obtained the electroporated region of the neocortex. Finally, the team used mass spectrometry (MS) to determine levels of acetyl coenzyme A (acetyl-CoA) in bRG isolated by fluorescence-activated cell sorting (FACS).
Study results
Naturally, the mouse embryo neocortex lacks TKTL1 expression. Introduction of the hTKTL1 gene into the neocortex of mouse embryos increased the abundance of bRGs without affecting basal intermediate progenitors (bIPs) and the apical progenitor population, which, in turn, increased neuron production corticals over time, especially late-developing upper-layer neurons. Conversely, aTKTL1, which differs only by one amino acid, did not increase bRG abundance.
Additionally, the authors noted that hTKTL1 expression increased the abundance of bRGs and the proportion of bRGs with multiple processes, a feature of bRGs that can self-amplify, in the developing ferret neocortex. As a result, the size of the ferrets’ gyrus increased.
Furthermore, the researchers observed that cellular organoids expressing aTKTL1 contained fewer bRG and neuroprogenitor cells. This finding confirmed that the substitution of lysine for arginine in hTKTL1 was essential for maintaining the bRG population in this human brain model. Similarly, hTKTL1 expression in neuroprogenitors increased during neurogenesis in human fetal neocortex. Intriguingly, this expression was higher in the developing frontal lobe than in the developing occipital lobe.
Another interesting observation was that hTKTL1 increased the bRG population via two consecutive metabolic pathways, the pentose phosphate (PPP) pathway and fatty acid synthesis. Therefore, when the researchers suppressed the PPP or fatty acid synthesis pathways using a series of specific inhibitors, the hTKTL1-induced increase in bRG population ceased in the mouse embryonic neocortex. . Similarly, the number of bRGs was reduced in human fetal neocortical tissue.
Additionally, the authors noted that hTKTL1-stimulated bRGs, but not aTKTL1-induced bRGs, had a higher concentration of acetyl-co A, a crucial metabolite for fatty acid synthesis. Thus, only hTKTL1 promoted the synthesis of membrane lipids containing a type of fatty acid necessary for the growth of bRG processes, which increased their population.
Conclusions
The current study demonstrated what distinguished the neocortical neurogenesis of modern humans from that of Neanderthals. Although these archaic humans had similar brains (in size) to modern humans, a single lysine to arginine substitution in the hTKTL1 gene led to the abundance of bRGs but not that of bIPs, which, in turn , generated more neocortical neurons in modern humans. Furthermore, the researchers demonstrated that the hTKTL1 effect mediates PPP and fatty acid synthesis, and that inhibition of these pathways reduced the abundance of bRGs in fetal human neocortical tissue.
Journal reference:
- Anneline Pinson, Lei Xing, Takashi Namba, Nereo Kalebic, Jula Peters, Christina Eugster Oegema, Sofia Traikov, Katrin Reppe, Stephan Riesenberg, Tomislav Maricic, Razvan Derihaci, Pauline Wimberger, Svante Pääbo, Wieland B. Huttner. (2022). Human TKTL1 involves greater neurogenesis in the frontal neocortex of modern humans than Neanderthals. Science. doi : 10.1126/science.abl6422 https://www.science.org/doi/10.1126/science.abl6422