The future utilization of stem cell therapy for neurodegenerative disease has been impeded by the limited numbers of stem cells that can be generated in vitro for transplantation, the potential immune response to the transplanted cells, and the sparse population of endogenous progenitor cells that exist in vivo. Thus, in addition to mastering the isolation and molecular guidance of stem cells, research is focusing on understanding mechanisms that increase stem cell numbers and survival, and minimize host rejection.
Notch encodes a transmembrane receptor with signaling integral to development and cancer. Notch signaling occurs through ligand-induced cleavage that frees the Notch intracellular domain (Nicd) to enter the nucleus and alter gene expression via tissue-specific transcription factors.1 Among numerous roles in nervous system development, Notch is imperative in stem cell biology because its signaling maintains an undifferentiated progenitor population.2, 3 However, the exact mechanism by which Notch prevents differentiation to prolong self-renewal and pluripotency, and its manipulation for therapeutic applications, has only recently been proposed (Figure 1).
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|Figure 1. Hypothesized mechanism of Notch-mediated neural stem cell expansion in vivo, based on infusion of soluble Notch ligand in the rat model. In vitro studies in fetal neural stem cells show that Notch activation by DLL4 induces a cytoplasmic signaling cascade that preserves fetal neural stem cell pluripotency and encourages cell expansion through STAT3-dependent Sonic hedgehog (Shh) release. (adapted from reference 4)|
Androutsellis-Theotokis and colleagues outlined a signaling cascade initiated by Notch ligands, Delta-like 4 (DLL4) and Jagged 1 (Jag1), that increased fetal neural stem cell (NSC) survival without inducing differentiation in vitro.4 This pathway includes the obligate cleavage of Nicd. However, the rapid timeline of cell expansion indicates that Nicd influences cytoplasmic effectors rather than, or in addition to, the commonly-associated yet delayed transcriptional response. Examination of kinase activation and administration of JAK and p38 kinase inhibitors identified that DLL4 and Jag1 led to activation of Akt kinase and mammalian target of rapamycin (mTOR). Both are serine/threonine kinases related to cell proliferation and differentiation, with downstream phosphorylation of transcription factor STAT3.
Other research has associated Notch signaling and STAT3 activation with differentiation to a glial cell fate.5, 6, 7 This is in apparent contrast to a Notch-dependent preservation of pluripotency. The discrepancy may be explained by different phosphorlyation sites. Rather than the previously reported phosphorlyation of STAT3 (Tyr705) that drives glial cell fate,7 Androutsellis-Theotokis et al. discovered that phosphorylation of STAT3 on Ser727 is integral to Notch-dependent cell expansion through its upregulation of both the Hes3 transcriptional repressor and expression of Sonic hedgehog (Shh).4 To further support the critical contribution of Notch to NSC, Basak and Taylor generated transgenic mice expressing Hes5-GFP under a Notch1 reporter and correlated the degree of self-renewal and pluripotency in vitro to the level of Notch1 activity in the developing nervous system.8 In combination, these results unveil the multiple paths of Notch influence on stem cell integrity and fate, thus providing molecular targets to manipulate stem cell populations.
To support their in vitro findings, Androutsellis-Theotokis et al. infused DLL4 and/or FGF basic into the normal adult rat brain. While FGF basic had little effect, DLL4 stimulated the proliferation of a subset of cells that expressed early neuronal markers, but lacked markers of mature glial cells and neurons.4 Supplementing previous studies that examined Notch signaling in experimental brain injury,9, 10 rats subjected to ischemic injury followed by DLL4 and FGF basic treatment exhibited improved motor recovery at 45 days post-injury.4 These results suggest that exogenous stimulation of Notch signaling enhances the endogenous progenitor cell population. This highlights a potential therapeutic approach to maximize an innate capacity for self-repair.
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