Generation of midbrain dopaminergic neurons in vivo and in vitro: the role of Neurogenin2

Sammanfattning: Parkinsons disease (PD) is a neurodegenerative disorder where dopaminergic neurons of the substantia nigra (SNc) in the mesencephalon are progressively eliminated. The ensuing loss of dopaminergic innervation of the basal ganglia manifests itself as severe motor deficits in PD patients. Clinical trials have shown that cell replacement therapy, where dopaminergic neuroblasts derived from fetal ventral mesencephalon (VM) are transplanted to the striatum, may be an alternative to pharmacological treatment of PD patients. The limited access and ethical concerns with using fetal tissue have prompted the use of stem cells as a renewable and limitless source of dopaminergic neurons. However, the mechanisms of specification of mesDA neurons in vivo need to be elucidated for identification and generation of mesencephalic dopaminergic (mesDA) neurons from stem cells in vitro.

In this thesis I have identified expression of the proneural gene Neurogenin2 (Ngn2) in a restricted pattern in the embryonic VM during mesDA neurogenesis. The protein was expressed in the progenitor population in the ventricular zone but not in mature neurons in the mantle zone. When isolating the Ngn2-expressing cells and their direct descendants by FACS from an Ngn2-GFP-KI mouse, I found that the Ngn2-GFP-positive cell fraction contained dopaminergic neurons, in contrast to Ngn2-GFP-negative cells. This shows that Ngn2 label early mesDA neuron precursors. Furthermore, when I analysed the Ngn2 knockout mutants, I found that they displayed an early loss of mesDA neurons that was partially maintained at postnatal stages, showing that Ngn2 has a role in the generation of the mesDA neurons. No other neuronal subtype in the VM was affected suggesting that this role for Ngn2 is specific for the mesDA neurons.

Using embryonic mouse tissue obtained at the stage of mesDA genesis, I was able to generate cultures of neural stem and progenitor cells, so called neurosphere cultures, that were neurogenic and maintained a ventral midbrain character over several passages. Although the neurospheres did not spontaneously give rise to dopaminergic neurons when differentiated, TH-positive cells were detected when Nurr1 was over-expressed in the cultures. The frequency with which this occurred, and the morphology of the TH-positive cells, differed from the results obtained when over-expressing Nurr1 in forebrain-derived expanded cells. This suggests that neurosphere expanded cells derived from VM specifically contain progenitors that can generate dopaminergic neurons under certain conditions. When over- expressing Ngn2 together with Nurr1 TH-positive cells were generated that displayed a mature neuronal morphology. Furthermore, I found that they expressed other dopaminergic markers which were not seen when either Nurr1 or Ngn2 were over-expressed alone. This suggests that Nurr1 and Ngn2 interact to specify a more mature dopaminergic phenotype.

The results in this thesis have identified a new cellular marker of mesDA progenitors in the developing embryo and also provided new insight into the development of mesDA neurons.