Pitx3 and Nurr1 in control of terminal differentiation of meso-diencephalic dopamine neurons

Abstract

Slow progressive degeneration of meso-diencephalic dopaminergic (mdDA) neurons is the hallmark of Parkinson’s disease (PD). We believe that studying the molecular cascades involved in the establishment of the mdDA neuronal field during embryonic development may reveal the key processes underlying the dramatic neuronal loss of dopamine (DA) neurons in the substantia nigra (SNc) later in life. One of the key proteins involved during development is the transcription factor Pitx3. For reasons unknown, Pitx3-deficiency in mice leads to a morphological phenotype remarkably similar to the pathological endstate of PD, as the majority of SNc DA neurons are lost. The overall aim of this thesis is to unravel the downstream cascade of Pitx3 during terminal differentiation, in order to ultimately define a molecular basis for its critical role in development of SNc DA neurons. The first downstream target of Pitx3 we identified was the Ahd2-gene, being physically bound and regulated by Pitx3 through a highly conserved region within its promoter. In the absence of Pitx3, Ahd2 fails to be expressed in mdDA neurons in vivo, pointing to a strict dependence of the Ahd2 gene for regulation by Pitx3. Interestingly, the expression of Ahd2 in DA neurons also requires the orphan nuclear receptor Nurr1, a well-described essential transcription factor for mdDA development. Thusfar, Nurr1 and Pitx3 had been assumed to be part of two independent developmental pathways. In this thesis, we establish Pitx3 as essential potentiator of Nurr1’s transcriptional acitivity and show that recruitment of Pitx3 physically alters the Nurr1 transcriptional complex with dramatic consequences for the expression of previously described Nurr1 target genes. This positions Pitx3 next to Nurr1 as crucial factor for the DA phenotype, being involved in the expression of a multitude of DA-related genes. In line with these findings, further investigation into the Nurr1 downstream cascade identified three novel downstream targets for Nurr1 in vivo, which are also affected in Pitx3 deficient embryos. In order to further investigate the functional relevance of the Pitx3 and Nurr1 transcriptional cascades we focussed on the gene function of Ahd2. The Ahd2-gene encodes an enzyme involved in the generation of retinoic acid (RA), a crucial vitamine A-derivate for neuronal differentiation during embryonic development. We showed that supplementation of RA to Pitx3-deficient embryos bypasses the requirement for Pitx3 and Ahd2 and efficiently counteracts the developmental defects caused by Pitx3 deficiency. For the first time, this has put forward an important role for Ahd2 and RA with respect to mdDA neuron development and survival. Based on these observations, we could subdivide the Pitx3-downstream cascade in two pathways. Pitx3, in cooperation with Nurr1, is directly involved in the regulation of Ahd2, Ptpru, Vmat2 and Dat, and through the Pitx3-Ahd2-RA pathway, Pitx3 indirectly affects the expression of the RA-sensitive genes Th, Dlk1 and D2R in the SNc. Overall, this thesis establishes Pitx3 next to Nurr1 as crucial factor for multiple aspects of mdDA neuron differentiation and implicates novel players and pathways in the Pitx3 and Nurr1 driven gene cascades, essential for development of a healthy mdDA neuronal population. The findings lead to a re-evaluation of their roles in relation to each other and with regards to their functionality in distinctive mdDA neuronal subsets. Finally, the implication of Ahd2-mediated RA-signaling in differentiation and maintenance of SNc neurons has provided a molecular basis for the selective requirement of SNc for Pitx3, and has opened up new avenues to explore in relation to mdDA neuronal pathology as observed in PD.