| dc.description.abstracteng | Objective: α-synuclein and degeneration of dopaminergic neurons have been closely associated to Parkinson´s disease. The interaction of α-synuclein with the neurotransmitter dopamine has been connected to neurodegeneration, and suggested to be causatively involved in Parkinson´s disease (PD). Like α-synuclein, ß-synuclein is extensively expressed in the central nervous system. Recent findings in rodent models of PD suggest that ß-synuclein can aggregate and induce degeneration of dopaminergic neurons (Taschenberger et al. 2013). This suggests a link between ß-synuclein and the dopaminergic neurotransmitter phenotype that has not been investigated so far. Therefore, the objective of this thesis is to generate a robust cell-based model with dopaminergic neurotransmitter phenotype, and to study the neurodegeneration induced by ß-synuclein in the context of dopamine.
Methods: Primary cortical neurons, isolated from rat pups during embryonic development day 18, were used to develop the cell-based model with dopaminergic neurotransmitter phenotype. Two approaches were employed. In a transdifferentiation approach, transcription factors known to induce the dopaminergic neurotransmitter phenotype were expressed in cortical neurons. In the second approach, enzymes, substrates, and transporter proteins necessary to mimic the dopaminergic neurotransmitter phenotype, were introduced into cortical neurons. Neurotoxicity of α-synuclein, ß-synuclein, and γ-synuclein were comparatively elucidated in the developed cell-model.
Results: In the first approach to develop the cell-model by transdifferentiation, transcription factors Ascl1, Nurr1, and Lmx1a (“ANL”) induced the expression of classical dopaminergic neurotransmitter markers tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), vesicular monoamine transferase 2 (VMAT2), and dopamine transporter (DAT) in 15-22% of total cortical neurons. Results further revealed that “ANL” induced the dopaminergic neurotransmitter phenotype only in cortical GABAergic neurons. GABAergic neurons lost their GABA neurotransmitter-determining marker GAD65 by DIV 21, suggesting a true neuronal phenotype switch. However, “ANL” caused degeneration of glutamatergic neurons. Moreover, “ANL” was unable to transdifferentiate midbrain GABAergic neurons in culture. In conclusion, neuronal transdifferentiation was achieved in principle. However, due to low yield of desired neurons, and due to the profound loss of cells, the second approach was employed.
In the second approach to develop the cell-based model, AADC, VMAT2 and multiple doses of extracellular L-3,4-dihydroxyphenylalanine (L-DOPA) were introduced into the cortical neurons. This resulted in robust dopamine production. Results suggested that most of the dopamine was incorporated into and presumably released by glutamatergic vesicles in the cell culture supernatant due to endogenous electrical activity of cortical neurons. Similarly, the introduction of DAT, VMAT2, and multiple doses of extracellular dopamine dramatically enhanced the intracellular dopamine levels. In conclusion, a cell-model based on AADC-VMAT2-L-DOPA, and another cell-model based on DAT-VMAT2-dopamine were established.
Using the developed cell-models, it was found for the first time that dopamine aggravates neurotoxic properties of ß-synuclein, and to a similar extent, of α-synuclein. It has been reported that α-synuclein impairs endogenous network activity by decreasing the action potential frequency (Tolo et al. 2018). However, in this study, it was found that dopamine production in α-synuclein and ß-synuclein overexpressing cells does not impair the endogenous network activity in vitro. Furthermore, NMR studies revealed that the binding affinity of dopamine with ß-synuclein is 10-100 fold less in comparison to the binding affinity of dopamine with α-synuclein, suggesting that ß-synuclein-dopamine binding might not be directly responsible for aggravated neurodegeneration.
Conclusion: The results in this study provide new perspectives on the neurodegeneration induced by ß-synuclein in the context of the neurotransmitter dopamine. | de |