German Title: Engrailed Gene werden zell-autonom für das Überleben der dopaminergischen Neuronen benötigt

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Abstract

The dopaminergic neurons of the substantia nigra and ventral tegmentum are the main source of dopamine in the central nervous system. The progressive loss of dopaminergic neurons in the substantia nigra compacta over several decades leads to one of the most prominent human neurodegenerative disorder, Parkinson´s Disease. Accumulating evidence suggest that these neurons die by apoptosis in afflicted patients. Still little is known about the molecular mechanisms that lead to full differentiation and maintenance of these neurons. This neuronal population expresses the homeobox transcription factors En-1 and En-2, briefly, after the cells become postmitotic onwards. In mutant mice deficient for both genes, the neurons are induced at the proper location, express TH, but at a time, when En expression sets in, these neurons rapidly undergo apoptosis, so that at E14 the whole population is lost. Another remarkable feature of those neurons is the lack of axonal projection towards the telencephalon. We first addressed the question whether these neurons, lacking En, were dying because of their inability to project and interact with trophic clues. In our study we report that En genes are not regulating axonal outgrowth of mDA neurons, and that these neurons if cultured onto permissive substrate elongate neurites of the same length in comparison to the wild-type. Secondarily we addressed the question whether the large deletion of midbrain tissue in En-1-/-, En-2-/- mutant embryos deprives these cells of an essential trophic support released by the surrounding neuroepithelium or whether the En genes are cell-autonomously required for the survival of mDA neurons. To address this, we performed cell-mixing experiments of En1/2 double mutant and wild type cells in primary cell culture of E12 ventral midbrain and we observed that En-1-/-, En-2-/- mDA neurons, both in homotypic and heterotypic cultures, are lost within 72 hours of culture. To complement these experiments and verify whether the absence of any rescue effect of the mDA neurons from the wild-type neuroepithelium was not a result of a pre-commitment to cell death, at the time of dissociation, we silenced En-1 by RNAi in En-2-/- ventral midbrain cultures. We observed, thereby, that in En-1 interfered cultures the number of mDA neurons 4 days after transfection was always diminished about 25% and that interfered En-1 mDA neurons rapidly undergo apoptosis. These results taken together demonstrate that En genes are cell-autonomously required for the survival and the maturation of the dopaminergic neurons in the substantia nigra compacta and the ventral tegmentum. Finally overexpression experiments in chick demonstrate that En-1 is not involved in the specification of the dopaminergic precursors, since midbrain tissue, transiently overexpressing En-1, did not show any increase in the number of mDA neurons. Our data suggest that genes downstream of En-1 and En-2 are essential for the maturation and maintenance of this neuronal population and that these transcription factors may be involved in Parkinson´s Disease.