Role of VPS35 in the pathogenesis of Parkinson's disease
Author(s)
Primary Supervisor
Mellick, George
Other Supervisors
Wood, Stephen
Year published
2017-04
Metadata
Show full item recordAbstract
Parkinson’s disease (PD) is a heterogeneous and complex neurodegenerative disorder whose
aetiology is not fully understood. Studying familial Parkinson’s disease has identified several
mutations that cause the disease and investigating the functions of these genes has given us
clues to the cellular mechanisms that underlie the neurodegenerative phenotype in the
patients. A clear understanding of these cellular mechanisms is necessary to halt disease
progression. Next generation sequencing of a Swiss kindred with autosomal dominant
Parkinson’s disease has identified a causative D620N mutation in the VPS35 (vacuolar
sorting ...
View more >Parkinson’s disease (PD) is a heterogeneous and complex neurodegenerative disorder whose aetiology is not fully understood. Studying familial Parkinson’s disease has identified several mutations that cause the disease and investigating the functions of these genes has given us clues to the cellular mechanisms that underlie the neurodegenerative phenotype in the patients. A clear understanding of these cellular mechanisms is necessary to halt disease progression. Next generation sequencing of a Swiss kindred with autosomal dominant Parkinson’s disease has identified a causative D620N mutation in the VPS35 (vacuolar sorting protein 35) gene. The VPS35 protein is a subunit of a retromer complex, which is a key element of the endosomal sorting process. Little is known about the effects of this mutation and this thesis presents our efforts to gain a better understanding of the mechanism by which mutations in the VPS35 gene influences the pathogenesis of PD. This project also investigates disease specific differences in organelles along the retromer pathway in sporadic PD cases. The first cell model used in the study is a fibroblast model derived from a PD patient carrying the D620N mutation and healthy “wild-type” controls. It was found that the D620N mutation did not present any loss of endogenous VPS35 protein levels in the fibroblasts. However, in the presence of the mutation a redistribution of the endosomes was observed. This was indicative of altered endolysosomal trafficking within the cell. Further, defective trafficking of CIMPR, a well-known cargo molecule of the retromer complex was observed in the presence of the mutation. This trafficking defect resulted in compromised lysosomal function mainly through the impaired processing of its ligand cathepsin D, a lysosomal enzyme involved in the degradation of alpha synuclein. In addition to this, high content image analysis of the cells revealed a disease specific difference in features of organelles involved in the retromer trafficking pathway. The fibroblasts were also treated with rotenone to analyse the effects of mitochondrial stress in the presence of the mutation. Treatment with this pesticide which is also a known complex 1 inhibitor, that causes PD like pathology in animals, showed disease specific changes in cells with the mutation. The D620N mutation did not confer increased susceptibility to cell death, but caused disease specific changes in properties of the early endosome, lysosomal and retromer subunit of the fibroblasts. On treatment with rotenone, cathepsin D processing defects were also exacerbated in the patient cells with the mutation. Further, the effect of the D620N mutation on alpha synuclein processing was also investigated. Uptake of monomeric and fibrillar alpha synuclein forms was shown in the fibroblasts. Reduced clearance of the alpha synuclein fibrils was observed in the presence of the D620N mutation. In addition to this, the D620N mutation conferred an increased sensitivity to alpha synuclein toxicity within these cells. Although increased alpha synuclein aggregation in the presence of the D620N mutation was hypothesised, the mutation did not show increased aggregation under the experimental conditions described here. In order to investigate disease specific differences in sporadic patients, a second cell model was used in this study, namely the human olfactory neurosphere derived (hONS) model. Investigation surrounding the retromer functions in hONS cells derived from sporadic PD did not reveal any alterations in the retromer levels and cathepsin D processing defects relative to cells derived from healthy donors. However, a nonsignificant trend towards decreased cathepsin D levels was observed in the patient cells. Analysis of retromer related markers in these cells did not show any disease specific differences. In contrast to this, inducing mitochondrial stress using rotenone resulted in disease specific changes in several organelles within these cells. Treatment with rotenone brought about changes in cellular parameters of mitochondria, lysosomes and tubulin, indicating disease specific responses to mitochondrial stress that served as potential biomarkers for the disease. In conclusion, this study identified retromer trafficking defects and alpha synuclein processing defects in the presence of the D620N mutation. It is the first report that shows synuclein processing defects in fibroblasts from PD patients with the D620N mutation. This study also marks the identification of potential biomarkers for the disease by building on previously identified disease specific differences identified in hONS cells obtained from sporadic PD patients.
View less >
View more >Parkinson’s disease (PD) is a heterogeneous and complex neurodegenerative disorder whose aetiology is not fully understood. Studying familial Parkinson’s disease has identified several mutations that cause the disease and investigating the functions of these genes has given us clues to the cellular mechanisms that underlie the neurodegenerative phenotype in the patients. A clear understanding of these cellular mechanisms is necessary to halt disease progression. Next generation sequencing of a Swiss kindred with autosomal dominant Parkinson’s disease has identified a causative D620N mutation in the VPS35 (vacuolar sorting protein 35) gene. The VPS35 protein is a subunit of a retromer complex, which is a key element of the endosomal sorting process. Little is known about the effects of this mutation and this thesis presents our efforts to gain a better understanding of the mechanism by which mutations in the VPS35 gene influences the pathogenesis of PD. This project also investigates disease specific differences in organelles along the retromer pathway in sporadic PD cases. The first cell model used in the study is a fibroblast model derived from a PD patient carrying the D620N mutation and healthy “wild-type” controls. It was found that the D620N mutation did not present any loss of endogenous VPS35 protein levels in the fibroblasts. However, in the presence of the mutation a redistribution of the endosomes was observed. This was indicative of altered endolysosomal trafficking within the cell. Further, defective trafficking of CIMPR, a well-known cargo molecule of the retromer complex was observed in the presence of the mutation. This trafficking defect resulted in compromised lysosomal function mainly through the impaired processing of its ligand cathepsin D, a lysosomal enzyme involved in the degradation of alpha synuclein. In addition to this, high content image analysis of the cells revealed a disease specific difference in features of organelles involved in the retromer trafficking pathway. The fibroblasts were also treated with rotenone to analyse the effects of mitochondrial stress in the presence of the mutation. Treatment with this pesticide which is also a known complex 1 inhibitor, that causes PD like pathology in animals, showed disease specific changes in cells with the mutation. The D620N mutation did not confer increased susceptibility to cell death, but caused disease specific changes in properties of the early endosome, lysosomal and retromer subunit of the fibroblasts. On treatment with rotenone, cathepsin D processing defects were also exacerbated in the patient cells with the mutation. Further, the effect of the D620N mutation on alpha synuclein processing was also investigated. Uptake of monomeric and fibrillar alpha synuclein forms was shown in the fibroblasts. Reduced clearance of the alpha synuclein fibrils was observed in the presence of the D620N mutation. In addition to this, the D620N mutation conferred an increased sensitivity to alpha synuclein toxicity within these cells. Although increased alpha synuclein aggregation in the presence of the D620N mutation was hypothesised, the mutation did not show increased aggregation under the experimental conditions described here. In order to investigate disease specific differences in sporadic patients, a second cell model was used in this study, namely the human olfactory neurosphere derived (hONS) model. Investigation surrounding the retromer functions in hONS cells derived from sporadic PD did not reveal any alterations in the retromer levels and cathepsin D processing defects relative to cells derived from healthy donors. However, a nonsignificant trend towards decreased cathepsin D levels was observed in the patient cells. Analysis of retromer related markers in these cells did not show any disease specific differences. In contrast to this, inducing mitochondrial stress using rotenone resulted in disease specific changes in several organelles within these cells. Treatment with rotenone brought about changes in cellular parameters of mitochondria, lysosomes and tubulin, indicating disease specific responses to mitochondrial stress that served as potential biomarkers for the disease. In conclusion, this study identified retromer trafficking defects and alpha synuclein processing defects in the presence of the D620N mutation. It is the first report that shows synuclein processing defects in fibroblasts from PD patients with the D620N mutation. This study also marks the identification of potential biomarkers for the disease by building on previously identified disease specific differences identified in hONS cells obtained from sporadic PD patients.
View less >
Thesis Type
Thesis (PhD Doctorate)
Degree Program
Doctor of Philosophy (PhD)
School
School of Natural Sciences
Copyright Statement
The author owns the copyright in this thesis, unless stated otherwise.
Subject
Parkinson's disease
Pathogenes
VPS35 vacuolar sorting protein
Cellular dysfunctions
Retromer complex
Alpha synuclein