Abstract
Reduced levels of cellular ubiquitin (Ub) pools due to disruption of the polyubiquitin gene Ubb lead to dysregulation of neural stem cell (NSC) differentiation and impaired neuronal maturation in cells isolated from Ubb−/− mouse embryonic brains. However, it is currently unknown whether Ub is required for the specific stage of neuronal development or whether it plays a pleiotropic role throughout the process. To answer this question, we aimed to downregulate Ubb expression temporally during neuronal development, which could not be achieved in Ubb−/− cells. Therefore, we exploited lentivirus-mediated knockdown (KD) of Ubb at different stages of neuronal development, and investigated their phenotypes. Here, we report the outcome of Ubb KD on two independent culture days in vitro (DIV): DIV1 and DIV7. We observed that NSCs did not differentiate properly via Ubb KD on DIV1, but the maturation of already differentiated neurons was intact via Ubb KD on DIV7. Intriguingly, Ubb KD activated Notch signaling when it had been suppressed, but exerted no effect when it had already been activated. Therefore, our study suggests that Ub plays a pivotal role in NSC differentiation to suppress Notch signaling, but not in the subsequent maturation stages of neurons that had already been differentiated.
Highlights
Ubiquitin (Ub) is one of the most abundant eukaryotic proteins involved in post-translational modifications[1,2,3]
On days in vitro 1 (DIV1), cells were infected with the lentivirus harboring shUbb to investigate how reduced levels of cellular Ub pools via Ubb KD affect the differentiation of neural stem cell (NSC) into neurons
NSCs are differentiated into neurons and astrocytes, in a process where neurogenesis precedes gliogenesis[25,26]
Summary
Ubiquitin (Ub) is one of the most abundant eukaryotic proteins involved in post-translational modifications[1,2,3]. Reduced levels of cellular Ub via the disruption of the polyubiquitin gene Ubb compromised the degradation of substrates and resulted in the dysregulation of NSC differentiation with inhibition of neurogenesis and impaired neuronal maturation[12,13,14]. In Ubb−/− cells, increased steady-state levels or delayed degradation of NICD resulted in the activation of Notch signaling even before the start of culture in vitro, which was sufficient to reverse the neurogenic and gliogenic potentials of NSCs12. Our data suggest that maintenance of Ub levels are important to suppress Notch signaling during early stage of neurogenesis or generation of neurons from NSCs, but not for neuronal maturation, if the neurons were generated under sufficient supply of cellular Ub levels
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