|dc.description.abstracteng||Parkinson’s disease (PD) is the second most common neurodegenerative disease in industrialized countries. However, the molecular mechanisms leading to neuronal death in PD still remain unclear. About 5-10% of PD patients suffer from familial forms of the disease and so far, 16 chromosomal regions have been associated with familial PD. As disease progression and outcome of familial and sporadic forms of the disease are very similar, studying the cellular function of genes associated with familial PD might hopefully yield in a better under-standing of the molecular pathways that are involved in the disease pathogenesis. Two major pathways have been proposed to be causing neuronal cell death in PD: dysfunction of the cellular protein degradation machineries and accumulation of misfolded proteins as well as mitochondrial dysfunction. The aim of the present study was to investigate the cellular function and toxicity of two PD-associated genes, α-synuclein (αS)/PARK1 and ATP13A2/PARK9, in the nematode C. elegans.
Protein aggregations in the brain of PD patients, the so-called “Lewy bodies”, are one of the main pathological hallmarks in PD. The main component of Lewy bodies is the protein αS. Mutations, as well as increased expression levels of αS, have been shown to cause familial PD. In both cases increased aggregation rates of αS have been demonstrated. Therefore, until now it was believed that fibrillar αS aggregates cause neurodegeneration in PD. It re-mained unclear however, whether insoluble fibrillar aggregates of αS or rather smaller oligo-meric precursors are causing toxicity. To address this question we decided to compare the aggregation properties and toxicity of different variants of αS including synthetic variants that do not form fibrillar aggregates but stop aggregation at the stage of small soluble oligomers. This analysis revealed that the oligomer-promoting variants exhibit higher toxicity than wild type (wt) αS or the PD mutants. This suggests that not the fibrils but rather the soluble oligo-mers are the toxic species of αS. However, it remained unresolved which molecular mecha-nisms are involved in mediating αS toxicity. To shed light on this aspect, the effects of αS on mitochondrial morphology were investigated. It was previously reported that αS can loca¬lize to mitochondria and that overexpression can cause mitochondrial pathology. Electron mi-croscopy (EM) and spinning disk confocal microscopy analysis demonstrated that mitochon-drial morphology is severely affected in both C. elegans muscle cells and neurons expressing αS. While expression of wt human αS in muscle cells induces both mitochondrial frag-mentation and the occurrence of long and thin mitochondrial tubules, expression of the oli-gomer-promoting variant of αS, for which increased toxicity was demonstrated, pre¬domi¬nant-ly leads to long, thin and interconnected mitochondria. Based on these observations we pro-pose a model in which the effects of αS expression on mitochondrial morphology are de-pendent on the relative abundance of different species of αS (monomeric vs. different oligo-meric forms and fibrils) and their relative amounts. The finding that mitochondrial frag¬men¬ta-tion is also occurring in aged worms without expression of αS suggests that expression of αS accelerates the physiological aging process.
The second part of my PhD thesis is concerned with the cellular function and toxicity of an-other PD-related gene, the lysosomal P-type ATPase catp 6, which is the C. elegans ortho¬log of human ATP13A2 (PARK9). Phenotypic analysis of deletion mutants of this gene revealed severely impaired egg laying which causes a strong reduction in brood size. The mutants also exhibit a markedly delayed postembryonic development. Thus, asynchronous development of different tissues might be causing the defects in the egg laying apparatus as coordinated differentiation is crucial for establishing the connection of the different tissues involved in egg laying. Furthermore, a locomotion defect, which is already apparent at larval stages and can be rescued by restoring CATP 6 function specifically in muscle cells, was detected in catp 6 mutants. This raised the question what causes these defects on a cellular level. As a functional interaction of CATP 6 and αS was reported previously and given that αS causes drastic reorganization of the mitochondrial network, it was decided to investigate mitochondrial morphology and function in deletion mutants of catp 6. Loss of function of CATP 6 leads to severe changes in mitochondrial morphology with formation of networks of long and very thin mitochondrial tubules and clustering of mitochondria around the nucleus. The observations that the mitochondrial fission protein DRP-1 forms large clusters in catp 6 mutants and that RNAi against drp 1 did not change mitochondrial morphology in catp 6 mu-tants suggest that loss of function of DRP 1 might be the cause for reorganization of the mi-tochondrial network in catp 6 mutants. Besides morphological defects, also functional im-pairments of the mitochondria were observed upon loss of function of CATP 6, with deletion mutants exhibiting a reduced membrane potential, changes in the composition of complex IV as well as increased oxygen consumption. A possible explanation for the latter might be an up-regulation of mitochondrial mass. catp 6 mutants more¬over exhibited increased sensitivity to oxidative stress and a reduced lifespan. In agreement with this, the mitochondrial stress response was found to be strongly up-regulated in catp 6 mutants. Future investigations are needed to determine whether these effects could be due to excess reactive oxygen species (ROS) production by the mutants caused by defects in the respiratory chain. In line with mi-tochondrial function being compromised in the mutants, we see an increased activation of the cellular energy sensor AMPK, which, in reaction to a rise in the cellular AMP/ATP ratio, acts to inhibit anabolic process and stimulate catabolic processes in the cell in order to maintain the cellular energy balance. In future studies it will be important to determine the exact molecular and cellular mechanisms how loss of function of the lysosomal P-type ATPase CATP 6 can cause such drastic effects on mitochondrial morphology.||de