Most stem cells in the adult mouse brain exist in a state of reversible cell cycle arrest where they are no longer actively dividing or growing. These cells are known as ‘quiescent’ and exist on a continuum from deep to shallow levels, meaning some can be awoken more easily than others. For example, injury can bring some deeply quiescent cells into a shallower state, ready to be activated to replace damaged cells.

Research led by Piero Rigo, Sara Ahmed de Prado and Francois Guillemot and published recently in Science Advances showed which transcription factors awaken these sleeping cells. 

Waking up cells in a deep quiescence state

The researchers first focused on a gene called Ascl1 as it is known to play a role in actively proliferating neural stem cells. They deleted the Ascl1 gene in cells from the mouse hippocampus and compared them with wild-type cells, finding an almost complete absence of cell division in mice without the ASCL1 protein (0.55% of cells were actively dividing compared to 20% of control cells). 

By focusing on just the neural stem cells in the wild-type and knock-out mice, the team confirmed the lack of proliferating cells and also reported an increase in quiescent cells. And when focusing on the quiescent neural stem cells, they saw that those without ASCL1 were in a much deeper state of inactivity. 

The researchers then boosted ASCL1 stability by taking away a protein that marks it for removal. This led to more proliferating stem cells, an overall decrease in quiescent cells, and more quiescent cells existing in a shallower state. 

Waking up cells in a shallow quiescence state

Stem cells in a shallow quiescent state are primed to wake up if tissues need repairing. Previous research has shown that genes involved in mRNA transport and protein translation are switched on, so the team next looked at the role of Myc transcription factors, which regulate these pathways. 

They found that removing a Myc family member called Mycn led to fewer actively dividing neural stem cells and more quiescent cells. And these quiescent cells had a distinct phenotype, sitting halfway through the trajectory from deep to shallow states. These findings suggest that Mycn pushes neural stem cells through shallow states of quiescence to activate them. 

A defined order of events

Their findings so far led the team to consider that ASCL1 might induce the expression of MYCN, and this was confirmed in Ascl1 knock-out mice, where Mycn was the second most suppressed gene after Ascl1. 

Finally, the researchers analysed genetic pathways switched on by Ascl1 and Mycn in turn, finding that ASCL1 promoted activation from deep states by switching on pathways relating to cell adhesion and metabolism, whereas MYCN promoted activation from shallow states by increasing gene transcription and translation. 

Piero Rigo, PhD student in the Neural Stem Cell Biology Laboratory and first author, said:

“By analysing the transcriptome in single cells, we were able to characterise the genes controlling neural stem cells sitting on a continuum of deep to shallow quiescence. This explains how the mouse hippocampus maintains populations of stem cells in various states  and how some cells can be activated to repair damaged tissues. We know that ageing can involve slower repair and regeneration, suggesting that more stem cells might exist in deeper quiescent states and are harder to ‘wake up’.”

Francois Guillemot, Group Leader of the Neural Stem Cell Biology Laboratory and senior author, said:

“Although more evidence is needed to show if these processes exist in humans, researchers are starting to explore how to activate quiescent stem cells to generate new neurons. This could one day help to treat neurodegenerative disorders like Parkinson’s disease or dementia. Our collaborator on the paper is also looking at how to activate quiescent brain tumour cells to expose them to treatments that only work on proliferating cells. So there’s a lot to be excited about in the area, but we first need to explore how quiescence works in human brains.”

Francois and Piero also worked with Lachlan Harris’s team at the University of Queensland in Brisbane and the BRF and Genomics, Flow Cytometry and Light Microscopy STPs at the Crick.