Towards Uncovering the Role of Pre-fibrillar Oligomers of á-Synuclein in the Pathogenesis of Parkinson's Disease
by Madhu Babu Gajula Balija
Date of Examination:2010-07-02
Date of issue:2010-08-06
Advisor:Prof. Dr. Herbert Jäckle
Referee:Prof. Dr. Herbert Jäckle
Referee:Prof. Dr. Christian Griesinger
Referee:Dr. Stefan Eimer
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Description:Dissertation
Abstract
English
Parkinson s disease (PD) is a progressive neuropathological disorder that is characterized by the presence of intra-cytoplasmic inclusions called Lewy Bodies (LBs). LBs represent insoluble protein aggregates with á-Synuclein (áS) as a major component. LBs also mark lesions found in a range of related disorders, collectively referred to as synucleinopathies. Recent studies suggest that pathogenesis could result from small pre-fibrillar intermediates of the aggregation pathway, i.e. soluble oligomers of misfolded á-Synuclein. So, it is not decided yet whether the soluble oligomers or the final products of the áS aggregation (i.e. the áS fibrils) are cause of the disease, meaning that the nature of pathogenic áS species is still unresolved. To understand the nature of pathogenic áS species and to investigate whether accumulation of soluble pre-fibrillar oligomers of áS is responsible for the increased neurotoxicity in PD, I have used rationally designed structural variants of áS with different aggregation properties and tested their biological properties in Drosophila melanogaster, which lacks an áS gene in the wild type genome. Two of the designed variants, TP-áS and A56P-áS, have a remarkably decreased propensity to aggregate, and they form more pre-fibrillar soluble oligomeric species in aggregation assays in vitro. In order to examine their biological effects in vivo, I have employed Drosophila as a tool to explore possible PD-like effects of the áS mutants. The áS mutant genes were inserted into the Drosophila genome. To avoid possible position effects affecting the expression of the individual transgenes, I used the phiC31 site-specific integration system. The site specific genomic integration of the áS gene and the different áS mutants permit to directly compare the effects of the different áS variants on various biological parameters such as longevity, climbing (negative geotactic and phototactic responses), sleep behavior and circadian rhythmicity that were used as sensitive readouts for neurotoxicity in response to áS activity expressed specifically in the Dopaminergic (DA) neurons. The results show that the TP-áS and A56P-áS mutants caused increased neurotoxicity as compared to wild type áS or expression of the bacterial lacZ gene serving as controls. Immunohistochemistry on adult fly brains confirm that neurotoxicity as revealed by the behavior tests correlates well with the degeneration of DA neurons in response to the expression of the áS variants. The data show that the pre-fibrillar oligomer-forming TP-áS mutant is most toxic among the áS variants. The results are consistent with the conclusion that pre-fibrillar oligomers of áS are high ly toxic pathogenic species in the neurodegeneration process associated with PD. Aside from supporting the hypothesis that the pre-fibrillar status of áS is already toxic for neurons, the Drosophila system presented here delivers a novel experimental system in which the non-motor aspects of PD, the mechanism of cellular action and likely even the efficacy of lead compounds leading to therapeutic drugs can be addressed.
Keywords: á-Synuclein; Parkinson s Disease; Neurodegeneration; Dopaminergic System; Motor Symptoms and Non-motor Symptoms; Circadian Rhythms and Sleep; Drosophila melanogaster.; á-Synuclein; Parkinson s Disease; Neurodegeneration; Dopaminergic System; Motor Symptoms and Non-motor Symptoms; Circadian Rhythms and Sleep; Drosophila melanogaster.