(645e) Experimental and Computational Analysis of Notch1 Receptor Signaling in Adult Neural Stem Cell Differentiation

The discovery of active neurogenesis (the generation of new neurons) in the adult human brain, and the isolation and culture of the neural stem cells that underlie this process, has created a host of possibilities for the therapeutic treatment of a number of neurodegenerative diseases. Strategies including direct modulation of endogenous stem cells pools, or harvest, ex vivo expansion, and implantation of these stem cells, can be pursued for central nervous system cell replacement therapy. However, before this scientific promise can translate into clinical reality, significant progress must be made in understanding the basic mechanisms that govern the behavior of these stem cells. In particular, an improved understanding of how these cells respond to various extracellular microenvironmental cues by either proliferating in their undifferentiated state or differentiating into a specific cell fate is required. We have analyzed how the Notch1 receptor and signaling cascade controls the differentiation of adult rat neural stem cells. Notch1 along with its downstream signal transducers is an evolutionarily conserved signaling network known to be involved in various cell fate decisions during development and in the adult. Here we employ a quantitative and molecular approach to analyze how the Notch1 system controls differentiation between astrocytes vs. neurons. Constitutive activation of Notch1 receptor signaling induces the selective differentiation of the adult neural stem cells into astrocytes rather than neurons or oligodendrocytes, the two other major cell types of the adult nervous system, as assessed by quantitative RT-PCR and immunostaining. Furthermore, overexpressing a dominant negative variant of Hes1, a transcription factor whose expression is activated by Notch1 signaling, at various times after constitutive Notch1 activation revealed a critical temporal duration of Notch1 activation for this fate switch to occur. Transcriptional profiling of Notch1 activated cells has enabled us to identify several potential downstream effecter pathways, including the TGF-β receptor pathway. A detailed time course study following Notch1 receptor activation will further help to elucidate the signaling cascade involved in the full differentiation of the cells 6 days post Notch1 activation.Furthermore, we have developed mathematical and computational models incorporating the key components of this signaling network, which along with our experimental data will allow us to understand how the Notch1 signaling circuit induces this cell fate switch in our cells. We believe a quantitative understanding of the dynamics of this pathway could be a key step towards engineering neural stem cells to differentiate into a desired cell type ? by either stimulating or inhibiting Notch1 signaling to generate astrocytes or neurons, respectively.