Identification of novel pathways in the differentiation of mesencephalic dopaminergic neurons

The research project of this PhD thesis is focused on the study of molecular mechanisms involved in the acquisition and maturation of midbrain dopaminergic neurons and on the study of transcription factors essential for mesDA differentiation. As previously described, in the mammalian brain the circuits formed by DA neurons are the nigrostriatal and meso-cortico-limbic pathways. These pathways are essential for homeostasis, motor control, reward mechanisms and modulation of affective and emotional states. Dysfunctions of these circuits are involved in serious neurological and psychiatric diseases (Parkinson’s disease, schizophrenia, ADHD, DOPA-responsive dystonia, etc.). The mesDA neurons are relatively few, therefore it is useful to generate in vitro model systems enriched in DA neurons to dissect the molecular events involved in mesDA differentiation and to possibly use these mesDA neurons in disease animal models and in regenerative medicine. Thus, the first aim of my PhD thesis was to generate in vitro a large amount of DA neurons from different cell types and dissect the overlapping and distinct roles of the transcription factors Nurr1 and Lmx1a. As described in the Introduction, Nurr1 is an orphan nuclear receptor with no known ligands. It is involved in the mesDA specification by direct regulation of many DA genes (such as Th, Vmat2 and Dat) and its loss determines agenesis of mesDA neurons (Zetterström et al. 1997, Saucedo-Cardenas et al. 1998). Lmx1a has been recently described as positive regulator of Th, Vmat2 and Dat (Chung et al. 2012). Considering the importance and the role of these two transcription factors in vivo, my first step was to analyze whether Nurr1 and Lmx1a could have a role during in vitro DA differentiation and consequently to set-up a new protocol for mesDA differentiation useful to apply on different cell lines. After the generation of this in vitro model enriched in DA neurons, the second step was to verify if it could exist a possible interaction between Nurr1 and Lmx1a during mesDA differentiation and maturation. As afore mentioned, in the last few years several experimental results have shown the importance of miRNAs in brain development and function. miRNAs dysregulation has also been suggested to be involved in specific dopaminergic (DA) neurons-affecting pathologies, such as Parkinson’s disease (PD) or schizophrenia. However miRNAs exclusively related to DA neurons development and function, have not yet been identified. Thus, the second aim of my PhD thesis was to identify miRNAs involved in DA development and differentiation and if there was a synergy between miRNAs and the transcription factors Nurr1 and Lmx1a to promote DA differentiation. This work contributes to add new and interesting information on the molecular mechanisms underlying the mesDA differentiation and maintenance, leading to the identification of new molecular targets for potential therapeutic strategies to cure pathological disorders affecting the DA system