Regulation Of Midbrain Dopaminergic Neuron Development By Wnts, Sfrps And bHLH Proteins

Sammanfattning: Parkinson´s disease (PD) is a chronic neurodegenerative disorder. The main pathology is characterized by progressive degeneration of the dopaminergic (DA) neurons of the substantia nigra leading to loss of striatal dopamine innervation. The knowledge about the fundamental mechanisms behind the degenerative process has been limited. During recent years a promising approach is evolving based on DA cell replacement therapy. However, in order to find a cure for PD an increased understanding is required about the intrinsic and extrinsic signals involved in the development of DA progenitor cells during embryogenesis. This thesis identifies novel players in midbrain development and investigates the mechanisms by which three major signaling modulators, Wnts, soluble Frizzled Related Proteins (sFRPs) and basic-Helix-Loop-Helix (bHLH) family of proteins, regulate DA neuron development. Proneural genes belong to the bHLH family of transcription factors and are crucial regulators of neurogenesis and subtype specification in many areas of the nervous system. Their function in DA neuron development has been unknown. In this thesis it is reported that the proneural genes Neurogenin2 (Ngn2) and Mash1 have an intricate pattern of expression in the ventricular zone (VZ) of the ventral midbrain (vMB), where DA neurons are generated. To examine the function of these genes, mice were analyzed in which one or two of these genes were deleted (Ngn1,Ngn2 and Mash1) or substituted (Mash1 in the Ngn2 locus). Our results demonstrate that Ngn2 is required for the differentiation of Sox2+ VZ progenitors into Nurr1+ postmitotic DA neuron precursors in the intermediate zone (ImZ), and that it is also required for their subsequent differentiation into DA neurons in the marginal zone (MZ). Although Mash1 normally has no detectable function in DA neuron development, it could partially rescue the generation of DA neuron precursors in the absence of Ngn2. These results demonstrate that Ngn2 is uniquely required for the development of vMB DA neurons. The Wnt signaling pathway regulates several developmental processes in the mammalian CNS; neural patterning, cell fate determination, proliferation, differentiation, neuronal maturation, cell migration and axon guidance. Our results present evidence that Wnt-1, -3a, and -5a expression is differentially regulated during vMB development. Wnt-3a promoted the proliferation of precursor cells expressing the orphan nuclear receptor-related factor 1 (Nurr1) but did not increase the number of DA neurons. Conversely, Wnt-1 and -5a increased the number of rat vMB DA neurons in rat embryonic day 14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of Nurr1-precursors. In contrast, Wnt-5a primarily increased the proportion of Nurr1 precursors that acquired a neuronal DA phenotype. These findings indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during vMB neurogenesis and that different Wnts have specific and unique activity profiles. Furthermore temporal expression profiles of Wnt components during critical phases of MB development revealed Frizzled (Fz) 9, a Wnt receptor, to be highly expressed in DA progenitors but not in newborn DA neurons. A possible function of Fz9 during early MB development might be to regulate proliferation of DA progenitors and inhibit differentiation, since Fz9 reduced Wnt5a signaling in DA cells in vitro. Finally, we set to examine the function of sFRPs in the developing ventral midbrain. sFRPs are a secreted family of factors that sterically hinder the Wnt ligand-Fz receptor complex to form and thereby block the Wnt signling pathway. sFRP1-3, but not 4 were expressed in the developing vMB, during DA neurogenesis. We therefore examined whether sFRP1-3 could work as Wnt antagonists in a dopaminergic cell line. We found that high doses of sFRP1 and sFRP2, but not sFRP3, acted as competitive antagonists of Wnt signaling. Treatment of vMB precursor cultures with sFRP1 resulted in DA neuron cell death, an effect that is compatible with Wnt1 blocking. However, treatment with sFRP2 lead to increased proliferation of progenitors and increased number of DA neurons, an effect incompatible with a Wnt blocking activity. Analysis of the sFRP2-/- mice also suggested that sFRP2 does not block Wnt function. On the contrary, we found that sFRP2 is partially required for several sequential steps in DA neuron development, including DA neurogenesis, differentiation of DA precursors into neurons and neuritogenesis. Thus, our results unravel several novel functions of sFRPs and identify sFRP2 as a novel player in DA neuron development. In summary, these results presented identify several novel players in midbrain DA neuron development and reveal new functions of proneural bHLHs, Wnts and sFRPs, thereby extending our knowledge and identifying factors that may be used to develop novel DA cell replacement therapies for the treatment of PD.