Enhancement of dopaminergic properties and protection mediated by neuronal activation of Ras in mouse ventral mesencephalic neurones.
The poor differentiation and survival of dopaminergic neurones are practical constraints in their therapeutic applications. Here we explored the role of neuronally activated Ras in ventral mesencephalon-derived neurospheres generated from synRas mouse embryos. The expression of Val12 Ha-Ras transgene and enhanced Ras activity was evident after differentiation of the neurospheres with a corresponding activating phosphorylation of mitogen-activated protein kinase. Phosphorylation of Akt/PKB, the target kinase of phosphoinositide 3-kinase, along with phosphorylation of Bad and CREB were enhanced in synRas-derived differentiated neurosphere cultures. Furthermore, increased Nurr1 expression was associated with elevated numbers of dopaminergic neurones in synRas-derived cultures compared with the wild-type. Correspondingly, tyrosine hydroxylase promoter assays revealed enhanced transcriptional activation of the promoter in synRas-derived cultures. synRas-derived dopaminergic neurones were greatly resistant to degeneration induced by various noxious stimuli. Consistently, the transgenic expression of activated Ras attenuated the adverse 6-hydroxydopamine effects on dopaminergic neurones. Dopaminergic neurones derived from both wild-type and synRas cultures expressed voltage-gated potassium and sodium currents, fired action potentials and exhibited electrical network activity. Thus, expression of the transgene promotes survival and enhances differentiation towards a dopaminergic cell fate without altering their basic electrical properties. Our results suggest that intracellular cell therapy mimicking trophic signalling may offer potential benefit in models of human disease associated with dopamine neurone dysfunction.
Chakrabarty
Koushik
K
Department of Molecular Neurobiochemistry, NC7/174, Ruhr University, 44780 Bochum, Germany.
Serchov
Tsvetan
T
Mann
Stefan A
SA
Dietzel
Irmgard D
ID
Heumann
Rolf
R
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002454:Cell Differentiation; D049109:Cell Proliferation; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D060850:LIM-Homeodomain Proteins; D008636:Mesencephalon / Q000166:cytology*; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D018696:Neuroprotective Agents / Q000378:metabolism*; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D016627:Oxidopamine / Q000378:metabolism; D051762:PAX2 Transcription Factor / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011247:Pregnancy; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; C080330:En1 protein, mouse; D018398:Homeodomain Proteins; C112149:LIM homeobox transcription factor 1 beta; D060850:LIM-Homeodomain Proteins; C539882:NR4A2 protein, human; D018696:Neuroprotective Agents; C539883:Nr4a2 protein, mouse; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D051762:PAX2 Transcription Factor; C497319:Pax2 protein, mouse; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051025:bcl-Associated Death Protein; C433629:homeobox protein PITX3; D016627:Oxidopamine; D014446:Tyrosine 3-Monooxygenase; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins; D004298:Dopamine