Id proteins facilitate self-renewal and proliferation of neural stem cells.

Members of helix-loop-helix (HLH) protein family of Id (inhibitor of differentiation) dimerize with bHLH transcription factors and function as negative regulators of differentiation during development. Most of inhibitory roles of Id proteins have been demonstrated in non-neural tissues, and their roles in the developing nervous system are not clearly demonstrated. In this study, we show that Id1, Id2, and Id3 increase self-renewing and proliferation potential of cortical neural stem cells (NSCs) while inhibiting neuronal differentiation. In electrophoretic mobility gel shift and luciferase assays, Id proteins interfered with binding of NeuroD/E47 complexes to the E-box sequences and inhibited E-box-mediated gene expression. Overexpression of Id proteins in NSCs increased both the number and the size of neurospheres in colony-forming assays. Expression of Hes1 and Hes5 was not increased by overexpression of Id proteins under the condition in which Nestin expression was increased. In utero electroporation of Id yielded higher numbers of Ki67-positive and Sox2-positive cells in the mouse embryonic brain. The study suggests Id proteins play independent roles in the maintenance of neural stem properties.

Mash1 and neurogenin 2 enhance survival and differentiation of neural precursor cells after transplantation to rat brains via distinct modes of action.

Neural precursor cells (NPCs) are regarded as a promising source of donor cells in transplantation-based therapies for neurodegenerative disorders. However, poor survival and limited neuronal differentiation of the transplanted NPCs remain critical limitations for developing therapeutic strategies. In this study, we investigated the effects of the proneural basic helix-loop-helix (bHLH) transcription factors Mash1 and Neurogenin 2 (Ngn2) in neuronal differentiation and survival of NPCs. Induction of Mash1 or Ngn2 expression strikingly enhanced neuronal differentiation of cultured NPCs in vitro. Ngn2-transduced NPCs underwent a rapid cell cycle arrest, which often accompanies differentiation. In contrast, cells continuously expanded upon Mash1 expression during NPC differentiation. Notably, sonic hedgehog (SHH) was upregulated by Mash1 and mediated the proliferative and survival effects of Mash1 on NPCs. Upon transplantation into adult rat brains, Mash1-expressing NPCs yielded large grafts enriched with neurons compared to control LacZ-transduced NPCs. Interestingly, enhancements in neuronal yield, as well as in donor cell survival, were also achieved by transplanting Ngn2-transduced NPCs. We show that a differentiation stage- and cell density-dependent survival effect of Ngn2 involves neurotrophin3 (NT3)/TrkC-mediated signaling. Together, these findings suggest potential benefits of bHLH gene manipulation to develop successful transplantation strategies for brain disorders.

Differential regulation of tyrosine hydroxylase expression by sonic hedgehog.

Sonic hedgehog functions to induce floor plate in early stages, and spinal motor neurons and midbrain dopaminergic neurons in later stages of development. Here, we investigated the effects of sonic hedgehog on tyrosine hydroxylase expression in three cell lines that correspond to different stages of neural development. Sonic hedgehog increased the tyrosine hydroxylase gene expression in pluripotent P19 cells but repressed it in tyrosine hydroxylase-producing PC12 cells. Promoter analysis in mouse neural stem cells indicated that the N-terminal of sonic hedgehog repressed both the basal and cAMP-dependent protein kinase A-mediated tyrosine hydroxylase activity. These results suggest that the N-terminal of sonic hedgehog increases tyrosine hydroxylase gene expression in cells to acquire dopaminergic phenotypes, but decreases expression in late born neurons by antagonizing the protein kinase A cAMP-responsive element binding protein pathway.