Mechanisms controlling midbrain dopaminergic neuron development

Sammanfattning: The degeneration of midbrain dopaminergic (mDA) neurons accounts for some of the main motor symptoms of Parkinson’s disease (PD). Efforts during the last decades have focused on understanding how mDA neurons are generated and maintained with the hope of developing novel stem cell-based replacement therapies. In this thesis, I address some of these questions in four papers. Paper I: Wnt signaling controls multiple developmental processes in the embryo. Here we report that Wnt1 deletion causes the loss ofLmx1a and Ngn2 expression in the midbrain floorplate resulting in the loss of mDA progenitor specification and neurogenesis in this region. Only a few ectopic LMX1A+, NURR1 + and TH+ cells were transiently found in the basal plate. This phenotype and the morphogenesis defect found in Wnt5a−/− mice were worsened in Wnt1−/−;Wnt5a−/− mice, indicating the existence of a previously unsuspected cooperation between Wnt1 and Wnt5a in mDA neuron development in vivo. Based on these results, we developed a combined Wnt protocol to promote the generation of mDA neurons from neural and embryonic stem cells in vitro. We conclude that coordinated Wnt actions promote mDA neuron development in vivo and in stem cells. Paper II: In this study we report that the chemokine Cxcl12 is expressed in the meninges, surrounding the ventral midbrain (VM) and that its cognate receptor CXCR4, is present and activated in NURR1 + mDA precursors and neurons. We found that VM meninges or CXCL12 promoted migration and neuritogenesis of TH+ cells in a CXCR4-dependent manner in vitro. Consistently, pharmacological blockade of CXCR4 or genetic deletion of Cxcr4 resulted in an accumulation of TH+ cells in the lateral aspect of the intermediate zone in the VM. Moreover, the processes of TH+ cells in Cxcr4−/− mice were no longer radially distributed but disoriented. Thus our results indicate that CXCL12/CXCR4 regulate the radial migration of mDA neurons. Paper III: We report that the homeodomain transcription factor ZEB2, is present at a high level in progenitor cells of the ventricular zone in the midbrain floor plate and that its expression diminishes in NURR1 + post-mitotic precursors. We found that ZEB2 upregulated miR200c, which in turn repressed Zeb2, to form a negative feedback loop. Overexpression of Zeb2 reduced the levels of CXCR4 and NR4A2 in the developing VM in vivo, resulting in migration and mDA differentiation defects. This phenotype was phenocopied by mir200c knockdown, indicating that the Zeb2-miR200c loop prevents the premature differentiation of mDA progenitors into postmitotic cells and their migration. Paper IV: We demonstrate that the extracellular matrix protein, laminin 511 (LM511), promotes midbrain dopaminergic neuron survival and differentiation via binding to integrin α3β1 and activation of the Yes-Associated protein, YAP. We found that LM511-YAP enhances mDA neuron survival by inducing the expression of miR-130a, which reduces the levels of PTEN, a negative regulator of the Akt/PKB pro-survival pathway, both in vitro and in vivo. Additionally, YAP up-regulates the expression of mDA differentiation genes such as LMX1A, LMX1B and PITX3, and prevents the loss ofmDA neurons by oxidative stress. Thus our results suggest the LM511-YAP pathway as a possible target for the development of novel therapies for PD. In sum, the data presented in this thesis provide evidence that multiple factors control common aspects of mDA development such as neurogenesis, positively controlled by WNT1, WNT5A and miR200c and negatively by ZEB2. Similarly, the migration of mDA neurons is controlled by WNT5A, miR200c and CXCL12/CXCR4. Lastly, we found that the survival and differentiation of mDA neurons is not only controlled by neurotrophic factors, but also by the extracellular molecule, LM511, via YAP activation.