Dopamine promotes the neurodegenerative potential of ß-synuclein.

A contribution of α-Synuclein (α-Syn) to etiology of Parkinson´s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related ß-Synuclein (ß-Syn) on these disorders remains enigmatic. ß-Syn has long been considered to be an attenuator of the neurotoxic effects of α-Syn, but in a rodent model of PD ß-Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of ß-Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced ß-Syn-induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with ß-Syn, thereby enabling structural and functional modifications. Interaction of DCH with ß-Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric ß-Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and ß-Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α-Syn. In conclusion, our results suggest that ß-Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons.

Neuronal Trans-differentiation by Transcription Factors Ascl1 and Nurr1: Induction of a Dopaminergic Neurotransmitter Phenotype in Cortical GABAergic Neurons.

Neurons with a desired neurotransmitter phenotype can be differentiated from induced pluripotent stem cells or from somatic cells only through tedious protocols with relatively low yield. Readily available cortical neurons isolated from embryonic rat brain, which have already undergone a complete neuronal differentiation process, might serve as alternative template source. These cultures consist of 85% glutamatergic and 15% GABAergic neurons, and we attempted to trans-differentiate them into dopaminergic neurons. Transcription factors Nurr1, Lmx1A and Pitx3, essential determinants of a dopaminergic cell fate during CNS development, were not sufficient to induce tyrosine hydroxylase expression in a significant number of cells. Combining Nurr1 with the generic neuronal differentiator and re-programming factor Ascl1, however, resulted in generation of neurons which express dopaminergic markers TH, AADC, VMAT2 and DAT. Only neurons of GABAergic phenotype could be trans-differentiated towards a dopaminergic neurotransmitter phenotype, while for glutamatergic neurons, this process proved to be neurotoxic. Intriguingly, GABAergic neurons isolated from embryonal midbrain could not be trans-differentiated into dopaminergic neurons by Ascl1 and Nurr1. Thus, in principle, post-mitotic embryonal neurons can serve as templates for neurons with a desired neurotransmitter phenotype. However, neurotransmitter phenotype plasticity critically depends on the differentiation history of the template neurons, which can result in relatively low yields of dopaminergic neurons.

Homogenous generation of dopaminergic neurons from multiple hiPSC lines by transient expression of transcription factors.

A major hallmark of Parkinson's disease is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The pathophysiological mechanisms causing this relatively selective neurodegeneration are poorly understood, and thus experimental systems allowing to study dopaminergic neuron dysfunction are needed. Induced pluripotent stem cells (iPSCs) differentiated toward a dopaminergic neuronal phenotype offer a valuable source to generate human dopaminergic neurons. However, currently available protocols result in a highly variable yield of dopaminergic neurons depending on the source of hiPSCs. We have now developed a protocol based on HBA promoter-driven transient expression of transcription factors by means of adeno-associated viral (AAV) vectors, that allowed to generate very consistent numbers of dopaminergic neurons from four different human iPSC lines. We also demonstrate that AAV vectors expressing reporter genes from a neuron-specific hSyn1 promoter can serve as surrogate markers for maturation of hiPSC-derived dopaminergic neurons. Dopaminergic neurons differentiated by transcription factor expression showed aggravated neurodegeneration through α-synuclein overexpression, but were not sensitive to γ-synuclein overexpression, suggesting that these neurons are well suited to study neurodegeneration in the context of Parkinson's disease.

Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free "GeneSwitch" vector.

Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3-4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - "Gene Switch" vector.