MSEL-1L: L'OROLOGIO MOLECOLARE DEL DIFFERENZIAMENTO NEURALE

The gene SEL-1L codifies for a protein involved in the retrotranslocation of misfolded peptides from the lumen on the endoplasmic reticulum to the cytoplasm, where they are degraded by the ubiquitin-proteasome mechanism in the ERAD (Endoplasmic Reticulum Associated Degradation) pathway. Recently, it was found that the murine protein mSEL-1L (murine SEL-1L) plays an essential role during embryonic development, since its homozygous deletion is lethal during mid-gestation, altering the correct organogenesis. This transgenic model allowed us to investigate mSEL-1L contribution in differentiation and brain development in vivo and to study its role in neural stem cell (NSC) biology in vitro, focusing on its relationship with Notch signaling. We have shown that during embryogenesis, mSEL-1L expression is ubiquitous in the developing brain with high levels detected in the ventricular regions, densely populated by neural progenitors. As development proceeds, mSEL-1L protein levels significantly decrease, allowing the detection of rare cells still expressing the protein only in the ventricular zones and in the dentate gyrus, the only adult neurogenic ¡§niches¡¨ in which a population of undifferentiated and quiescent NSCs is retained. It has been subsequently proved that mSEL-1L expression plays as essential role in directing and ensuring a harmonious NSC differentiation, as the protein deletion is associated with an early cellular differentiation that determines the progenitor pull depletion, both in vivo and in vitro. In particular, mSEL-1L absence in vivo compromises the corticogenesis, because it affects the correct sequence of neurogenic and astrogliogenic phases, altering both the residual stem cell population and its differentiated progeny. The similarity of this transgenic model with Notch mutants led us to investigate the possible involvement of mSEL-1L with this pathway. Co-immunoprecipitation analysis has revealed an interaction between these two proteins, while expression studies have shown a specific inhibition of Notch-1 signaling associated to mSEL-1L down-regulation. The expression of the nuclear activated form of the receptor, as well as that of its main effectors HES-1 and HES-5, are significantly inhibited when mSEL-1L is totally or partially depleted, promoting an erroneous up-regulation of the transcriptional factor Neurogenin-2 (NGN-2) and the following increase of the neuronal marker ƒÒIII¡VTubulin expression. The main NSC properties primarily governed by Notch pathway, such as self-renewal, differentiation, proliferation and cell survival, are drastically affected by mSEL-1L misregulation. Moreover, mSEL-1L deficient mouse models show significant vasculogenic and angiogenic defects during embryonic development, probably due to an alteration of Notch signaling. The proper control of mSEL-1L expression is therefore of paramount importance to enable the correct embryonic development, as well as its regulation during differentiation by specific and sophisticated mechanisms. In fact, it has emerged that mSEL-1L is abundantly expressed in mouse embryonic stem cells (ESCs) and is maintained stable through their in vitro differentiation into neural progenitors (NEPs) first, and then into radial glia-like NSCs, while its expression is inhibited during their final maturation into neurons, astrocytes and oligodendrocytes. So, the protein is not affected by passing from a state of pluripotency (ESCs), to multipotency (NEPs), up to tripotency (NSCs), but it must be silenced to ensure IV NSC terminal differentiation. In fact, it has been demonstrated that mSEL-1L expression is regulated in a post-transcriptional way by mmu-miR-183. This microRNA, whose expression is induced during differentiation both in vitro and in vivo, is able to negatively regulate mSEL-1L, as well as other proteins with an important function in stemness, such as ƒÒ-Integrin and Bmi-1. Differently, mmu-miR-183 down-modulation can promote an increase in the protein levels in NSCs derived from the telencephalic cortex of embryos with only one mSEL-1L functional allele (mSEL-1L HET), ensuring a high protein expression, although in presence of a reduced quantity of its messenger. In conclusion, this research has highlighted an ERAD-independent role of mSEL-1L concerning NSC biology, a role that is essential to guarantee the proper embryonic development and the homeostasis of the whole organism. Therefore, the protein appears as a sort of ¡§molecular clock¡¨ that can drive the transition from a stemness state to a differentiate one, only when it is appropriate. The data here presented may provide a good starting point for the development of specific therapeutic strategies for degenerative pathologies and for regenerative medicine applications.