Piscopo, Valerio Emilio Crescenzo (2014) Overexpression of Mash1 and Nurr1 synergize to induce the Dopaminergic Neuron Phenotype during in vitro differentiation of Neural Stem Cells. [Tesi di dottorato]

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Abstract

Midbrain dopaminergic neurons (mDA) are essential for the control of voluntary movements and the regulation of emotions. They play a pivotal role in severe neurodegeneration, such as in Parkinson's disease. The development of dopaminergic neurons is a complex multi-step process in which cell-intrinsic factors as well as environmental cues are integrated in an accurate space-time sequence to define their phenotypic and functional specificity. A crucial task is to unveil the network of interactions and the hierarchy of molecular events underlying the acquisition of their function and circuitry. This thesis work was aimed at unveil some of these patterns and, more specifically, to validate my hypothesis that Mash1 and Nurr1 may cooperate in mDA lineage commitment, during the switch from the specification phase to the post-mitotic differentiation phase. Their interaction triggers a number of molecular events leading to the correct development of fully differentiated mDA neurons. Using as model system the differentiation of Neural Stem Cells towards DA phenotype I have been able to demonstrate the existence of a cooperative effect of Mash1 and Nurr1 in this process. The combined action of the two transcription factors drives the cells in the post-mitotic phase and leads to an increase in the expression of the catecholamine rate-limiting enzyme TH in cortex- and midbrain-derived NSCs. Although at lower levels, they positively influence also the expression of more mature DA markers as DAT, Vmat2 and Pitx3. In particular here I demonstrate that Mash1 enhances Nurr1-induced activation of TH transcription by binding to two distal enhancers sites on the TH promoter rather than by a physical interaction with Nurr1. This is consistent with a permissive rather than instructive role of Mash1 in the development of DA neurons. It is worth noticing that the cooperation of these two TFs is able to drive DA differentiation also in neurospheres derived by brain areas that normally do not give rise to DA neurons, as the cortex. Altogether these data shed light on the molecular and cellular events underlying the differentiation of DA neurons to enhance their generation in vitro and in vivo for possible future therapeutic purposes.

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