Kinase Inhibitors as Mediators of Neuronal Differentiation.

Abstract

Neuronal differentiation is an elaborate developmental program that relies on both intrinsic and extrinsic signaling. Previous research has demonstrated that basic helix-loop-helix (bHLH) transcription factors are critical regulators of both the initiation of neuronal differentiation and the specification of distinct neuronal phenotypes. However, the precise molecular mechanisms underlying proneural bHLH action are not fully understood. In this dissertation, a potential effector of the proneural bHLH protein Ascl1 (PKIβ) as well as a novel transcriptional target (Gadd45γ) were identified in microarray hybridization studies. Both of these targets have established roles as inhibitors of kinase activity. In response to Ascl1 overexpression, P19 cells undergo a transient increase in mRNA and protein expression of the endogenous PKA inhibitor PKIβ. shRNA constructs targeting PKIβ reduce levels of PKIβ mRNA and protein, and also prevent the neuronal differentiation of P19 cells. This obstruction of differentiation is partially rescued by overexpressing PKIβ protein, and this rescue is dependent on the binding of PKIβ to PKA. These findings define a requirement for PKIβ and its association with PKA during Ascl1-mediated neuronal differentiation of P19 cells. A novel model system of P19 cells with doxycycline-inducible expression of Ascl1 was developed to study earlier events mediating neuronal differentiation. The gene encoding the cell cycle regulator Gadd45γ was increased earliest and to the greatest extent following Ascl1 induction. Additional studies provided the first evidence identifying Gadd45γ as a direct transcriptional target of Ascl1. Furthermore, overexpression of Gadd45γ itself was sufficient to initiate some aspects of neuronal differentiation independent of Ascl1. This thesis describes the development and characterization of a novel model system consisting of P19 embryonic carcinoma cells with doxycycline-inducible expression of the proneural bHLH factor Ascl1 and use of this system to elucidate the Ascl1 regulatory network. The studies detailed in this dissertation provide a foundation for future studies of the transcriptional programs initiated by bHLH proteins. The continued elucidation of the molecular mechanisms that drive neurogenesis will be important for neuronal replacement therapies, which require the directed differentiation of cells to specific neural subtype identities.