The Roles of Nucleoporins in Mouse Neural Stem Cell Quiescence

Abstract

Neural stem cells (NSCs) are multipotent, self-renewing cells in the central nervous system (CNS), which exist in either a proliferative or quiescent state. Quiescence con- sists of reversible cell cycle arrest accompanied by low biosynthetic activity. This state preserves stem cells from replicative exhaustion and protects them from environmental insults, thus ensuring tissue homeostasis during development and into adulthood.

Though only few NSCs persist post-natally, they become increasingly quiescent as a function of age, and by adulthood most of them are in this state. Due to the lack of specific markers, quiescence is difficult to study, leaving many unanswered questions surrounding it. Importantly, when acquired by cancer stem cells, quiescence makes them refractory to therapy, highlighting the clinical imperative to understand this state.

Work in Drosophila showed that downregulation of nucleoporins (Nups), the structural components of the nuclear pore complex (NPC), in NSCs induced quiescence. More- over, nucleocytoplasmic partitioning of polyadenylated RNA was altered in this state in both Drosophila and mammalian NSCs. The work presented here aimed to test whether the causal link between Nup levels and quiescence induction was conserved in mammalian NSCs, and to further probe the function of Nups in NSC quiescence regulation by using a mammalian in vitro model for this state. Furthermore, I investigated whether Nups fulfilled a transport-independent role in gene expression regulation via binding to chromatin in NSCs.

Longitudinal proteome profiling showed that most Nups were downregulated in mouse NSC cultures as they transitioned from proliferation into increasingly deeper quiescence, without affecting the abundance and density of NPCs. The knockdown of Nup96-98 and Nup93, but not of Nup50 nor Nup210, resulted in features of NSC quiescence, such as nuclear accumulation of polyadenylated RNA and mild but significant decrease in proliferation, which appeared to be reversible.

Hundreds of genes changed their expression significantly in response to both Nup knockdowns, but only the downregulation of Nup98-96, though mild, resulted in the small but significant repression of a handful of cell-cycle genes. Both knockdowns resulted in the downregulation of genes associated with transcription and translation, but concomitantly upregulated ones involved in synapse development and glial differentiation. However, no cells expressing markers of either lineage could be detected using an independent approach. Strikingly, there were no transcripts which significantly changed their nucleocytoplasmic bias following either knockdown, despite the nuclear accumulation of polyadenylated RNA seen by microscopy. Regardless of abundant changes in transcript expression, the levels of only a few proteins, mostly ribosomal, changed significantly in response to either knockdown, confirming a likely decrease in translation in these populations.

A transport-independent role for NUP98 was also considered, and its interactions with chromatin in NSCs were profiled by CUT&RUN. NUP98 bound to 412 genes with high confidence in active NSCs, mostly at their promoter, but no binding was detected in quiescent NSCs in vitro. NUP98-bound genes were enriched in motifs for transcriptional repressors that are known regulators of stemness, hinting at the possible involvement of NUP98 in its regulation. Two-thirds of NUP98-bound genes were differentially ex- pressed in quiescent NSCs (compared to active ones), and NUP98 bound to down- regulated genes involved in transcription, development and Notch signalling, which supports its contribution to the transcriptional regulation of quiescence.

Together these results suggest that the Nup96-98 and Nup93 knockdowns alone are not sufficient to induce quiescence in NSCs, but they might promote a more subtle state transition towards either the quiescent or differentiated state. This hypothesis will be probed further with functional experiments taking advantage of the amenable in vitro NSC system and with stronger reversible knockdowns which will better elucidate the role of Nups in NSCs.

Date of Award	1 Mar 2025
Original language	English
Awarding Institution	King's College London
Supervisor	Rita Sousa-Nunes (Supervisor) & Francois Guillemot (Supervisor)