In vitro and in vivo CRISPR-Cas9 screens reveal drivers of aging in neural stem cells of the brain

Abstract

Aging impairs the ability of neural stem cells to transition from quiescence to activation (proliferation) in the adult mammalian brain. Neural stem cell (NSC) functional decline results in decreased production of new neurons and defective regeneration upon injury during aging1–9, and this is exacerbated in Alzheimer’s disease10. Many genes are upregulated with age in NSCs3, 11–13, and the knockout of some of these boosts old NSC activation and rejuvenates aspects of old brain function14–18. But systematic functional testing of genes in old NSCs – and more generally in old cells – has not been done. This has been a major limiting factor in identifying the most promising rejuvenation interventions. Here we develop in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screening pipeline in primary cultures of young and old NSCs identifies over 300 gene knockouts that specifically restore old NSC activation. Interestingly, the top gene knockouts are involved in glucose import, cilium organization and ribonucleoprotein structures. To determine which gene knockouts have a rejuvenating effect for the aging brain, we establish a scalable CRISPR-Cas9 screening platform in vivo in old mice. Of the 50 gene knockouts we tested in vivo, 23 boost old NSC activation and production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes for the GLUT4 glucose transporter, is a top rejuvenating intervention for old NSCs. GLUT4 protein expression increases in the stem cell niche during aging, and we show that old NSCs indeed uptake ∼2-fold more glucose than their young counterparts. Transient glucose starvation increases the ability of old NSCs to activate, which is not further improved by knockout of Slc2a4/GLUT4. Together, these results indicate that a shift in glucose uptake contributes to the decline in NSC activation with age, but that it can be reversed by genetic or external interventions. Importantly, our work provides scalable platforms to systematically identify genetic interventions that boost old NSC function, including in vivo in old brains, with important implications for regenerative and cognitive decline during aging.

Tracking cell-type-specific temporal dynamics in human and mouse brains

“…For validation, we utilized a recently published dataset 54 ( Figure 5G , left) that employed a genome-wide CRISPR screen to systematically dissect the roles of various genes in neurogenesis by quantifying the enrichment of gene-specific single-guide RNAs (sgRNAs) within proliferation-active (Ki67+) primary neural stem cells in vitro . We examined the downregulated genes in aged NPCs ( Figure 5G , middle) and detected a significant reduction in their sgRNA enrichment compared to a set of randomly chosen genes ( Figure 5G , right).…”

CellBiAge: Improved single-cell age classification using data binarization

“…Although the functional implications of these two genes in the context of aging remain unexplored, a recent study showed that knockout of genes encoding glucose import, such as Slc2a4 (encodes the GLUT4 glucose transporter), rejuvenated old NSCs. 53 Another top gene, Cd34 , which encodes endothelial cell glycoprotein, has been implicated in degeneration. 54 Thus, interpretation of the ELN model identifies potential genes related to the aging process.…”

Understanding neural development and diseases using CRISPR screens in human pluripotent stem cell-derived cultures

“…While hPSC-derived 2D and 3D neural systems are well suited to recapitulate features of the developing fetal brain, it could be challenging to use them to model the aging brain and its related disorders. In this regard in vivo CRISPR screens are gaining traction ( Wertz et al, 2020 ; Ruetz et al, 2021 ). A recent study utilized both in vitro and in vivo CRISPR screening platforms in old mice and uncovered over 300 factors that boosted the activation of aged neural stem cells (NSCs) ( Ruetz et al, 2021 ).…”

“…In this regard in vivo CRISPR screens are gaining traction ( Wertz et al, 2020 ; Ruetz et al, 2021 ). A recent study utilized both in vitro and in vivo CRISPR screening platforms in old mice and uncovered over 300 factors that boosted the activation of aged neural stem cells (NSCs) ( Ruetz et al, 2021 ). The in vivo screen was useful to validate top hits from the in vitro screen performed in primary NSCs from old mice.…”