Trifluoperazin schützt humane dopaminerge Zellen durch Aktivierung der Autophagie vor Wildtyp-Alpha-Synuklein-induzierter Toxizität

Die Parkinson-Erkrankung ist die zweithäufigste neurodegenerative Erkrankung nach der Alzheimer-Demenz. In industrialisierten Ländern liegt die Prävalenz der Erkrankung bei knapp zwei Prozent für alle Personen über 65 Jahren. Klinisch ist die Erkrankung charakterisiert durch Bradykinese, Rigor, Ruh...

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Bibliographic Details
Main Author: Goebel, Joachim
Contributors: Höglinger, Günter (Professor Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2018
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Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease and effects nearly two percent of all people aged 65 or older in industrialized countries. Clinically it is characterized by bradykinesia, rigidity, tremor, and postural instability. These motor symptoms are mainly caused by the demise of dopaminergic neurons in the substantia nigra pars compacta. The histopathological hallmark of Parkinson’s disease is the presence of intracellular protein inclusions, called Lewy bodies, in cells of the substantia nigra pars compacta. The main component of Lewy bodies is alpha-synuclein, a 140 amino acids long protein. Missense mutations, duplications or triplications of the alpha-synuclein gene (SNCA) lead to rare familial forms of Parkinson’s disease. Moreover, genome-wide-association studies identified variants of SNCA as risk factors for sporadic Parkinson’s disease. For these reasons it is undisputed, that alpha-synuclein plays a major role in the development of Parkinson’s disease. However, so far the precise alpha-synuclein species responsible for neuronal death is controversially debated. To date, pharmacological therapies of Parkinson’s disease are only symptomatic and there is no known therapy that halts or slows the progression of the disease. In our research group a new cell culture model of Parkinson’s disease was developed, in which adenoviral overexpression of wild-type alpha-synuclein leads to cell death in human postmitotic dopaminergic neurons (LUHMES cells). One cellular mechanism to dispose unwanted protein inclusions is macroautophagy. Therefore, the goal of this dissertation was to investigate if trifluoperazine, a known enhancer of macroautophagy in neurons, protects these dopaminergic cells from alpha-synuclein-mediated toxicity. First of all, Western blot analysis was used to show that trifluoperazine activated macroautophagy in LUHMES neurons. Then it could be proven that treatment with this compound protected LUHMES cells from alpha-synuclein-induced cell death in a concentration-dependent manner. The readout methods to measure cell death or survival comprised release of LDH into the cell culture medium as well as cell count after nuclear staining with DAPI. Besides toxicity, overexpression of alpha-synuclein in this cell model led to the appearance of a small oligomeric band of alpha-synuclein with a molecular weight of 37 kDa, which was not present in untransduced cells. Trifluoperazine reduced specifically this 37 kDa alpha-synuclein Western blot band in a dose-dependent manner without altering other alpha-synuclein bands. Inversely, inhibition of autophagy by chloroquine, a known inhibitor of autophagy, increased the toxicity of alpha-synuclein and this 37 kDa band, too. There was a significant positive correlation between toxicity and the strength of this band. These data strongly suggest that this 37 kDa band is a key form implicated in the toxicity of alpha-synuclein at least in our cell model of Parkinson’s disease. Additionally, from the experiments in this dissertation it can be concluded that overexpression of alpha-synuclein inhibits macroautophagy – the impact of alpha-synuclein itself on autophagy is so far a heavily debated question, too. In summary, it was shown that enhancement of autophagy saved human dopaminergic cells from alpha-synuclein-induced toxicity by degradation of a specific alpha-synuclein species only seen under pathological conditions. This suggests that activation of autophagy might be a possible target for a disease modifying pharmacotherapy for Parkinson’s disease in the future. Furthermore, a 37 kDa alpha-synuclein band was seen, which might be a key toxic species in Parkinson’s disease.