Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Aging-related transcriptional factor Dbx2 is a critical regulator of the properties of neural stem cells and their progeny

Neural stem cells (NSCs) persist into adulthood within specialized brain regions called neurogenic niches, where they continually generate neurons throughout life. In the adult mammalian brain, these neurogenic areas are limited to the subventricular zone (SVZ), adjacent to the lateral ventricles, and the subgranular zone (SGZ) within the hippocampal dentate gyrus. Aging leads to intrinsic changes in NSCs and alterations in the niche microenvironment, resulting in a progressive reduction in adult-born neurons generation and contributing to cognitive decline in rodents. The impact of age-related changes on neurogenic decline remains largely unexplored. This project aims to investigate the role of the Developing brain homeobox type 2 (Dbx2), recently identified as an age-related gene in rodent NSCs. Our previous studies indicate that Dbx2 levels increase in aged NSCs, and when overexpressed in young NSCs impairs their growth, suggesting a role in regulating NSC proliferation. Additionally, RNA-bulk analysis suggests Dbx2 may be involved in regulating fate commitment and cell metabolism, though this has not been experimentally confirmed. The effect of Dbx2 on NSC metabolism was studied through lipidomic analysis. We found that Dbx2 overexpression alters lipid class distribution compared to control cultures (GFP-NSC), increasing lipid accumulation as lipid droplets, and reducing cholesterol production. Our lipidomic data well match with previous transcriptomic analyses suggesting that Dbx2 might control different pathways involved in NSC quiescence. Consistently, we found that Dbx2 overexpressing cells are more prone to reactivate upon BMP4/bFGF-driven cell cycle exit. Moreover, in vitro differentiation analysis revealed that increased Dbx2 expression affects neuronal and glial differentiation. These findings support the idea that Dbx2 is critical controller of NSC properties. Interestingly, recent studies have shown that Dbx2 is also expressed in astrocytes, and its levels are higher in astrocytes isolated from aged mice compared to young astrocytes. This prompts us to investigate whether high Dbx2 expression in astrocytes can influence NSC properties through non-cell-autonomous mechanisms, along with cell-autonomous mechanisms. To tackle this question, we assessed the role of Dbx2 on astrocyte molecular properties (i.e. astrocytes reactivity) and function (astrocyte-NSC communication). We generated astrocytes from NSCs overexpressing Dbx2 and evaluated astrocyte reactivity following their activation with pro-inflammatory cytokines. We found that both in unstimulated Dbx2 astrocyte cultures and following stimulation, the expression of several inflammatory cytokines was downregulated, indicating that Dbx2 mitigates the effect of cytokines. Preliminary results showed that astrocytes constitutively expressing Dbx2 had no effect on inducing neuronal fate in NSCs, but it may affect their proliferation, as suggested by Ki-67 immunostaining. This was observed under both conditions examined: astrocytes and reactive astrocytes. To address whether Dbx2-overesspressing astrocytes mediates non-cell-autonomous mechanisms we also established NSC inducible lines allowing the doxycycline-regulated expression of Dbx2 by a piggyBac-transposase mediated integration. These experiments yielded surprising results. We found that NSC properties were strongly affected by the integration into their genome of the exogenous DNA sequences driving inducible transgene expression. NSCs fault to differentiate into astrocytes, regardless of the type of transgene used. We speculate that PiggyBac transposase-mediated integration might preferentially occurs next to certain genes which become activated when doxycycline is administered. These findings open new avenues for the identification of unknown genes involved in NSC maintenance and differentiation.