Carol Hicks

Transient notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells

The genesis of vertebrate peripheral ganglia poses the problem of how multipotent neural crest stem cells (NCSCs) can sequentially generate neurons and then glia in a local environment containing strong instructive neurogenic factors, such as BMP2. Here we show that Notch ligands, which are normally expressed on differentiating neuroblasts, can inhibit neurogenesis in NCSCs in a manner that is completely dominant to BMP2. Contrary to expectation, Notch activation did not maintain these stem cells in an uncommitted state or promote their self-renewal. Rather, even a transient activation of Notch was sufficient to cause a rapid and irreversible loss of neurogenic capacity accompanied by accelerated glial differentiation. These data suggest that Notch ligands expressed by neuroblasts may act positively to instruct a cell-heritable switch to gliogenesis in neighboring stem cells.

Notch Receptor Activation Inhibits Oligodendrocyte Differentiation

In this study, we show that oligodendrocyte differentiation is powerfully inhibited by activation of the Notch pathway. Oligodendrocytes and their precursors in the developing rat optic nerve express Notch1 receptors and, at the same time, retinal ganglion cells express Jagged1, a ligand of the Notch1 receptor, along their axons. Jagged1 expression is developmentally regulated, decreasing with a time course that parallels myelination in the optic nerve. These results suggest that the timing of oligodendrocyte differentiation and myelination is controlled by the Notch pathway and raise the question of whether localization of myelination is controlled by this pathway.

Fringe differentially modulates Jagged1 and Delta1 signalling throughNotch1 and Notch2

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.

Nuclear Notch1 signaling and the regulation of dendritic development

To understand the function of Notch in the mammalian brain, we examined Notch1 signaling and its cellular consequences in developing cortical neurons. We found that the cytoplasmic domain of endogenous Notch1 translocated to the nucleus during neuronal differentiation. Notch1 cytoplasmic-domain constructs transfected into cortical neurons were present in multiple phosphorylated forms, localized to the nucleus and could induce CBF1-mediated transactivation. Molecular perturbation experiments suggested that Notch1 signaling in cortical neurons promoted dendritic branching and inhibited dendritic growth. These observations show that Notch1 signaling to the nucleus exerts an important regulatory influence on the specification of dendritic morphology in neurons.

A secreted Delta1‐Fc fusion protein functions both as an activator and inhibitor of Notch1 signaling

Signaling induced through interactions between DSL (Delta, serrate, LAG‐2) ligand‐signaling cells and Notch‐responding cells influences the developmental fate of a wide variety of invertebrate and vertebrate cell types. Consistently with a requirement for direct cell–cell interactions, secreted DSL ligands expressed in flies do not appear to activate Notch signaling but rather produce phenotypes reminiscent of losses in Notch signaling. In contrast, secreted DSL ligands expressed in Caenorhabditis elegans or supplied to mammalian cells in culture produce effects indicative of Notch activation. In fact, engineered secreted DSL ligands have been used to study Notch signaling in neurogenesis, gliogenesis, hematopoeisis, neurite morphogenesis and ligand‐induced nuclear translocation of the Notch intracellular domain. Using a recombinant, secreted form of the DSL ligand Delta1, we found that antibody‐induced oligomerization (termed “clustering”) was required for this soluble ligand to bind specifically to Notch1‐expressing cells, undergo internalization, and activate downstream signaling. Interestingly, clustering with either limiting or excess antibody led to ligand binding in the absence of Notch signaling, indicating that ligand binding is necessary but not sufficient for activation of Notch signaling. Moreover, such antibody clustering conditions blocked Notch1 signaling induced by membrane‐bound DSL ligands. We propose that multimerization influences whether ligand binding to Notch results in activation or inhibition of downstream signaling and suggest that differences in ligand presentation might account for why secreted forms of DSL ligands have been reported to function as agonists and antagonists of Notch signal transduction.

Aspartate Mutations in Presenilin and γ‐Secretase Inhibitors Both Impair Notch1 Proteolysis and Nuclear Translocation with Relative Preservation of Notch1 Signaling

It has been hypothesized that a presenilin 1 (PS1)‐related enzymatic activity is responsible for proteolytic cleavage of the C‐terminal intracellular protein of Notch1, in addition to its role in β‐amyloid protein (Aβ) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand‐induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full‐length Notch1‐enhanced green fluorescent protein‐transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C‐promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notchs physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP γ‐secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP γ‐secretase inhibitors block Notch1 nuclear translocation with an IC50 similar to that reported for APP γ‐secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or γ‐secretase inhibitors. Our results support the hypotheses that (a) PS1‐dependent APP γ‐secretase‐like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimers disease by targeting γ‐secretase processing of APP to reduce Aβ production.

CADASIL Notch3 Mutant Proteins Localize to the Cell Surface and Bind Ligand

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementia arising from abnormal arteriolar vascular smooth muscle cells. CADASIL results from mutations in Notch3 that alter the number of cysteine residues in the extracellular epidermal growth factor–like repeats, important for ligand binding. It is not known whether CADASIL mutations lead to loss or gain of Notch3 receptor function. To examine the functional consequences of CADASIL mutations, we engineered 4 CADASIL-like mutations into rat Notch3 and have shown that the presence of an unpaired cysteine does not impair cell-surface expression or ligand binding.

Extrinsic and Intrinsic Factors Governing Cell Fate in Cortical Progenitor Cultures

Central nervous system germinal zones contain stem cells that generate both neurons and glia. In the recent past, these cells have been isolated, maintained in a variety of culture systems and used in vitro for subsequent characterization of molecular mechanisms underlying brain development. Factors that govern cell fate choices of these neural stem cells have not been fully elucidated, but recent studies suggest that age at the time of culture is an important intrinsic mechanism. Stem cell mitogens and Notch-DSL signaling are significant extrinsic factors. In the current study, we compare neurosphere cultures propagated from animals on embryonic day 12, embryonic day 18 and the day of birth and stimulated to divide by either basic fibroblast growth factor (bFGF) or transforming growth factor-α (TGF-α). As described for other systems, when bFGF was used, clonal neurospheres derived from the youngest age gave rise to a greater percentage of neurons. When TGF-α, acting via the epidermal growth factor receptor, was used, this effect was not observed, with neurospheres from younger animals giving rise to a similar percentage of neurons as those derived from older animals suggesting that this growth factor was either stimulating a different population of stem cells to proliferate, or that it was capable of overriding intrinsic mechanisms. Other differences were also observed when the two growth factors were compared, including age-dependent differences in the numbers of putative astrocytes and oligodendrocytes formed. We further assessed age-dependent influences on cell fate by assessing the effects of a lentivirally transduced constitutively activated Notch receptor on cell fate. At all ages studied, Notch activation resulted in a significantly greater number of GFAP-positive cells, seemingly overriding the greater neurogenic potential of younger stem cells. These data suggest that both extrinsic and intrinsic factors differentially regulate cell fate choices of progenitors during cortical development.

Rapid Notch1 Nuclear Translocation after Ligand Binding Depends on Presenilin‐associated γ‐Secretase Activity

Abstract: Recent data suggest an intimate relationship between the familial Alzheimer disease gene presenilin 1 (PS1) and proteolytic processing of both the amyloid precursor protein (APP) and the important cell signaling molecule, Notch1. We now show, using mammalian cells transfected with full‐length Notch1, that the C terminal domain of Notch1 rapidly translocates to the nucleus upon stimulation with the physiologic ligand Delta and initiates a CBF1‐dependent signal transduction cascade. Using this assay, we demonstrate that the same aspartate mutations in PS1 that block APP processing also prevent Notch1 cleavage and translocation to the nucleus. Moreover, we show that two APP γ‐secretase inhibitors also diminish Notch1 nuclear translocation in a dose‐dependent fashion. However, Notch1 signaling, assessed by measuring the activity of CBF1, a downstream gene, was reduced but not completely abolished in the presence of either aspartate mutations or γ‐secretase inhibitors. Our results support the hypothesis that similar PS1‐related enzymatic activity is necessary for both APP and Notch1 processing, yet suggest that Notch signaling may remain relatively preserved with moderate levels of γ‐secretase inhibition.