Engineering of Dominant Active Basic Helix-Loop-Helix Proteins That Are Resistant to Negative Regulation by Postnatal Central Nervous System Antineurogenic Cues

Neural precursor cells (NPCs) are present in most regions of the adult central nervous system (CNS). Using NPCs in a therapeutical perspective, that is, to regenerate CNS tissue after injury or in neurodegenerative diseases, will require the efficient manipulation of their fate. Proneural gene overexpression in NPCs represents a promising strategy to promote neuronal differentiation. The activity of the proneural proteins is, however, context-dependent and can be inhibited/modulated by binding with other bHLH (basic helix-loop-helix) or HLH transcription factors. In this study, we show that the two proneural proteins, Ngn2 and Mash1, are differentially sensitive to negative regulation by gliogenic factors or a gliogenic substrate (i.e., postnatal spinal cord slices). Coexpressing E-proteins with proneural proteins was efficient to rescue proneural proteins neurogenic activity, suggesting a central role for E-protein sequestration in mediating postnatal CNS gliogenic inhibition. Tethering of proneural proteins with E47 further insulated Mash1 from negative environmental influences whereas this strategy was not successful with Ngn2, suggesting that mechanisms of inhibition differ in between these two proneural proteins. Our results demonstrate that a better understanding of proneural protein modulation by environmental cues is a prerequisite to develop innovative approaches that will permit the manipulation of the fate of NPCs in the adult CNS after trauma or disease. STEM CELLS 2009;27:847-856