First, the researchers isolated and cultured qNSCs from young (3–4 months old) or old mice (18–21 months old) that constitutively expressed Cas9, before exposing cells to a genome-wide single-guide RNA library. As described by the study’s corresponding author, Anne Brunet, this approach enabled the researchers “to test all genes in the genome (23,000) for their ability, when knocked out, to improve the activation of old NSCs,” on the basis of the proportion of proliferative cells observed in culture. Ultimately, the authors identified 301 gene knockouts that selectively improved activation of qNSCs from old (versus young) mice, including genes linked to cilium organization and glucose transport.
Next, the authors were interested in whether they could translate the results of this screen to improve qNSC activation in old mice. Brunet explained that “Tyson [Ruetz] had the idea of leveraging the spatial nature of the neurogenic niche (with NSCs being at a separate location from the new neurons they generate)”. For example, qNSC activation in the subventricular zone niche leads to migration of these cells to the olfactory bulb. After individually knocking out 30 of the 301 genes identified from their in vitro screen, the authors reported 7 gene knockouts that reproducibly improved qNSC activation in older mice; these included Slc2a4, which encodes the GLUT4 insulin-dependent glucose transporter. The authors observed that GLUT4 levels and glucose uptake were higher in qNSCs from old versus young mice. Additionally, they reported that knocking out Slc2a4 or temporarily starving qNSCs of glucose improved the activation rate of old, but not young, qNSCs. These results suggest that older NSCs may be in a state of high glucose uptake that potentially stems from increased GLUT4 expression during aging. In the words of Brunet, Slc2a4 could be “one of these genes that when knocked out boosts old NSC activation and improves neurogenesis in old mice”.
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