Abstract
The known genetic network of the redox-sensitive transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) is expanding and with it, the discovery of novel roles. Of these, a critical contribution to stem cell maintenance and fate has been revealed through its direct upregulation of stemness transcription factor Notch1. In spite of this knowledge, the Nrf2-Notch1 axis has yet to be studied in relation to the growing literature implicating Nrf2 in the regulation of neural stem cell behaviour and neurogenesis in both development and in adulthood. Using the human SH-SY5Y cell line as an in vitro model of neurogenesis, this mechanistic link was explored to provide a framework for how Nrf2 influences the neurogenic process.As known inducers of endogenous Nrf2 transcriptional activity, hypoxia (1 kPa O2) and diethylmaleate (DEM), were investigated to do as such in SH-SY5Y cells. While hypoxia was found to be ineffective, DEM successfully upregulated expression of key Nrf2 target genes in an Nrf2-dependent manner, without effecting cell viability. Demonstrating for the first-time existence of an Nrf2-Notch1 axis in cells of a neural lineage, reporter gene assay showed overexpression of Nrf2 or its endogenous activation with DEM increased transactivation of the Notch1 promoter in undifferentiated SH-SY5Y cells. Moreover, Nrf2 enhanced Notch1 signalling events at CBF-1 response element sites with a corresponding rise in transactivation of Notch1 target genes: Hes1 and Hey1. When an enzyme essential to the canonical pathway of Notch signalling was inhibited, Nrf2 could no longer increase notch signalling. This regulation of Notch1 by Nrf2 was found to promote SH-SY5Y proliferation. An increase in the proportion of Ki67-positive cells was found following Nrf2 overexpression, which could be attenuated using a Notch signalling inhibitor. Taken together, these data showed the importance of the Nrf2-Notch1 axis to the undifferentiated state of neural SH-SY5Y cells.
Upon SH-SY5Y neuronal differentiation with retinoic acid (RA), the Nrf2-Notch1 axis was disrupted. While Nrf2 overexpression still led to Notch1 promoter transactivation it no longer resulted in signalling events at CBF-sites. Upon closer inspection, the programme of differentiation disrupted an event post S3 cleavage in the canonical Notch pathway, thereby preventing Nrf2 from enhancing Notch signalling.
These findings in SH-SY5Y cells suggest the presence of the Nrf2-Notch1 axis in neural cells is state-dependent, occurring in undifferentiated but not differentiating neurons. Given Notch1 is famously tasked with inhibition of the differentiated state, this finding could indicate the Nrf2-Notch1 axis is required to maintain the undifferentiated state while needing to be suppressed for neuronal differentiation to transpire. Considering that pathologies characterised by a dysregulation of Nrf2 signalling have been linked with abnormalities in neurogenesis, whether a disturbance in the Nrf2-Notch1 axis underlies these abnormalities could present a promising line of investigation for future research.
| Date of Award | 1 Jan 2022 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Giovanni Mann (Supervisor), Patricia Zunszain (Supervisor) & Richard Killick (Supervisor) |