Parkinson’s disease is a common neurological disorder with a complex etiology. The disease is characterized by a progressive loss of dopaminergic cells in the substantia nigra, which leads to motor function and sometimes cognitive function disabilities. One of the pathological hallmarks in Parkinson’s disease is the cytoplasmic inclusions called Lewy bodies found in the dopamine neurons. The aggregated protein α-synuclein is a main component of Lewy bodies. In view of severe symptoms and the upcoming of problematic side effects that are developed by the current most commonly used treatment in Parkinson’s disease, new treatment strategies need to be elucidated. One such strategy is replacing the lost dopamine neurons with new dopamine-rich tissue. To improve survival of the implanted neurons, neurotrophic factors have been used. Glial cell line-derived neurotrophic factor (GDNF), which was discovered in 1993, improves survival of ventral mesencephalic dopamine neurons and enhances dopamine nerve fiber formation according to several studies. Thus, GDNF can be used to improve dopamine-rich graft outgrowth into the host brain as well as inducing sprouting from endogenous remaining nerve fibers. This study was performed on Gdnf gene-deleted mice to investigate the role of GDNF on the nigrostriatal dopamine system. The transplantation technique was used to create a nigrostriatal microcircuit from ventral mesencephalon (VM) and the lateral ganglionic eminence (LGE) from different Gdnf gene-deleted mice. The tissue was grafted into the lateral ventricle of wildtype mice. The results revealed that reduced concentrations of GDNF, as a consequence from the Gdnf gene deletion, had effects on survival of dopamine neurons and the dopamine innervation of the nigrostriatal microcircuit. All transplants had survived at 3 months independently of Gdnf genotype, however, the grafts derived from Gdnf gene-deleted tissue had died at 6 months. Transplants with partial Gdnf gene deletion survived up to 12 months after transplantation. Moreover, the dopaminergic innervation of striatal co-grafts was impaired in Gdnf gene-deleted tissue. These results highlight the role of GDNF for long-term maintenance of the nigrostriatal dopamine system. To further investigate the role of GDNF expression on survival and organization of the nigrostriatal dopamine system, VM and LGE as single or combined to double co-grafts created from mismatches in Gdnf genotypes were transplanted into the lateral ventricle of wildtype mice. Survival of the single grafts was monitored over one year using a 9.4T MR scanner. The size of single LGE transplants was significantly reduced by the lack of GDNF already at 2 weeks postgrafting while the size of single VM was maintained over time, independently of GDNF expression. The double grafts were evaluated at 2 months, and the results revealed that lack of GDNF in LGE reduced the dopamine cell survival, while no loss of dopamine neurons was found in VM single grafts. The dopaminergic innervation of LGE was affected by absence of GDNF, which also caused a disorganization of the striatal portion of the co-grafts. Small, cytoplasmic inclusions were frequently found in the dopamine neurons in grafts lacking GDNF expression. These inclusions were not possible to classify as Lewy bodies by immunohistochemistry and the presence of phospho-α-synuclein and ubiquitin; however, mitochondrial dysfunction could not be excluded. To further study the death of the dopamine neurons by the deprivation of GDNF, the attention was turned to how Lewy bodies are developed. With respect to the high levels of α-synuclein that was found in the striatum, this area was selected as a target to inject the small molecule – FN075, which stimulates α-synuclein aggregation, to further investigate the role of α-synuclein in the formation of cytoplasmic inclusions. The results revealed that cytoplasmic inclusions, similar to those found in the grafts, was present at 1 month after the injection, while impairment in sensorimotor function was exhibited, the number of dopamine neurons was not changed at 6 months after the injection. Injecting the templator to the substantia nigra, however, significantly reduced the number of TH-positive neurons at 3 months after injection. In conclusion, these studies elucidate the role of GDNF for maintenance and survival of the nigrostriatal dopamine system and mechanisms of dopamine cell death using small molecules that template the α-synuclein aggregation.

The neurodegenerative disorders Alzheimer??s Disease (AD) and Parkinson??s Disease (PD) are characterised by the accumulation of misfolded amyloid beta 1-42 peptide (Aβ1-42) or α-synuclein, respectively. In both cases, there is extensive evidence to support a central role for these aggregation-prone molecules in the progression of disease pathology. However, the precise mechanisms through which Aβ1-42 and α-synuclein contribute to neurodegeneration remain unclear. Organismal, cellular and in vitro models are under development to allow elucidation of these mechanisms. A cellular system for the study of intracellular Aβ1-42 misfolding and localisation was developed, based on expression of an Aβ1-42-GFP fusion protein in the model eukaryote Saccharomyces cerevisiae. This system relies on the known inverse relationship between GFP fluorescence, and the propensity to misfold of an N-terminal fusion domain. To discover cellular processes that may affect the misfolding and localisation of intracellular Aβ1-42, the Aβ1-42-GFP reporter was transformed into the S. cerevisiae genome deletion mutant collection and screened for fluorescence. 94 deletion mutants exhibited increased Aβ1-42-GFP fluorescence, indicative of altered Aβ1-42 misfolding. These mutants were involved in a number of cellular processes with suspected relationships to AD, including the tricarboxylic acid cycle, chromatin remodelling and phospholipid metabolism. Detailed examination of mutants involved in phosphatidylcholine synthesis revealed the potential for phospholipid composition to influence the intracellular aggregation and localisation of Aβ1-42. In addition, an existing S. cerevisiae model of α-synuclein pathobiology was extended to study the effects of compounds that have been hypothesized to be environmental risk factors leading to increased risk of developing PD. Exposure of cells to aluminium, dieldrin and compounds generating reactive oxygen species enhanced the toxicity of α- synuclein expression, supporting suggested roles for these agents in the onset and development of PD. Expression of α-synuclein-GFP in phosphatidylcholine synthesis mutants identified in the Aβ1-42-GFP fluorescence screen resulted in dramatic alteration of α-synuclein localisation, indicating a common involvement of phospholipid metabolism and composition in modulating the behaviours of these two aggregation-prone proteins.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 105-113). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Parkinson's disease (PD) is characterised by the loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain and the presence of intracellular aggregates, known as Lewy bodies (LBs), in the surviving neurons. The aetiology of PD is unknown but a causative role for α-Synuclein (SNCA) has been proposed. Although the function of αSyn is not well understood, a number of pathological mechanisms associated with αSyn toxicity have been proposed. In this study, nine induced pluripotent stem cells (iPSCs) lines from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of SNCA were differentiated to dopaminergic neurons (iDAn). All iPSC lines differentiated with similar efficiency to iDAn, indicating that they could be used for phenotypic analysis. Quantification of αSyn expression showed increased αSyn intracellular staining and the novel detection of increased αSyn oligomerization in PD iDAn. Analysis of mitochondrial respiration found a decrease in basal respiration, maximal respiration, ATP production and spare capacity in PD iDAn, but not in undifferentiated iPSCs, indicating the cell-type specificity of these defects. Decreased phosphorylation of dynamin-1-like protein at Ser616 (DRP1^Ser616) and increased levels of Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) in A53T SNCA iDAn suggest a new pathological mechanism linking αSyn to the imbalance in mitochondria homeostasis. Markers of endoplasmic reticulum (ER) stress were found to be up-regulated, along with increased β- Glucocerebrosidase (GBA) activity, perturbation of autophagy and decreased expression of fatty acids binding protein 7 (FAPB7) in PD iDAn. Lastly, lentiviral vectors for RNAi-mediated knockdown of αSyn were developed and these reduced αSyn protein levels in iDAn, resulting in increased expression of FABP7. These results describe a novel functional link between αSyn and FABP7. This work demonstrates that iDAn are a promising and relevant in vitro cell model for studying cellular dysfunctions in PD pathology, and the phenotypic analysis of A53T SNCA and SNCA triplication iDAn enabled the detection of novel pathological mechanisms associated with PD.

The pathological hallmark of many age-related neurodegenerative diseases is the presence of proteinaceous inclusions in nerve cells and glial cells. Alpha-synuclein is the main component of the inclusions of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, as well as of rarer diseases, collectively called synucleinopathies. For a long time, it was widely believed that neurodegenerative diseases were cell-autonomous; however, a more recent hypothesis has suggested that some misfolded proteins resemble prions. Thus, aggregated alpha-synuclein shares features of PrPSc, the scrapie form of the prion protein. The aim of this thesis was to further characterize the prion-like properties of aggregated alpha-synuclein by studying the pathways of seeded aggregation, and to identify the species of alpha-synuclein responsible. I present evidence, using a HEK 293T cell model, that filamentous protein was the most seed-potent form of alpha-synuclein. Recombinant aggregated protein, aggregated alpha-synuclein from mice transgenic for A53T alpha-synuclein, as well as alpha-synuclein aggregates from Parkinson’s disease and multiple system atrophy brains, seeded aggregation. The mechanisms of alpha-synuclein internalization and intracellular trafficking, and how these processes affect seeded aggregation, are not fully understood. I showed that internalization of alpha-synuclein aggregates occurs through clathrin- and dynamin-independent, Cdc42-, actin- and PI3K-dependent endocytosis. Alpha-synuclein aggregates are trafficked to the endolysosomal pathway; a small fraction of lysosomes ruptures, which induces aggregation of expressed cytoplasmic alpha-synuclein, and disruption of autophagy, which in turn enhances seeded aggregation. These findings expand knowledge of the prion-like properties of assembled alpha-synuclein and identify novel mechanisms with therapeutic potential.

The aggregation of alpha-synuclein (αS) protein from soluble monomer into solid amyloid fibrils in the brain is associated with a range of devastating neurodegenerative disorders such as Parkinson’s disease. Soluble oligomers formed during the aggregation process are highly neurotoxic and are thought to play a key role in the onset and spreading of disease. Despite their importance, these species are difficult to study by conventional experimental approaches owing to their transient nature, heterogeneity, low abundance and a remarkable sensitivity of the oligomerisation process to the chosen experimental conditions. In this thesis, well-established single-molecule techniques have been utilised to study the aggregation and oligomerisation of αS in solution.

Misfolding and accumulation of insoluble protein aggregates in the form of amyloid fibrils is associated with a number of prevalent and debilitating mammalian disorders. In addition, amyloid-like nanostructures exhibit robust material properties, biological compatibility and replicative properties, making them of particular interest in the development of novel nanomaterials. Understanding fibril formation is essential to the development of strategies to control, manipulate or prevent fibril growth. The amyloid hypothesis is that since amyloid-like fibrils share a common core structure, they also share common formation mechanisms. Utilising a combination of turbidity and extrinsic fluorescence techniques this thesis provides insight into the diagnostic strength of simple, inexpensive kinetic measurements of aggregate growth. These simple techniques are found to be capable of delivering a substantial amount of information about the growth mechanisms controlling aggregation, and the effect of solution and environmental conditions, forming a solid basis for further investigation. Two competing fibrillar pathways are observed for hen egg white lysozyme at low pH in the presence of salt. These two pathways, leading to the formation of either curvilinear, worm-like fibrils or to the more widely recognised rigid, straight fibrils are not particular to hen egg white lysozyme, and similar competition may affect growth curve analysis in many other protein assays, including a-synuclein. Many proteins aggregate in the presence of membranes and detergents, and the kinetics of a-synuclein aggregation in the presence of SDS are strongly influenced by SDS concentration. Most descriptions of amyloid fibril growth currently lack heterogeneous nucleation events, and these may be important for predicting aggregation of membrane-active species in vivo. It is clear that simple analytical solutions to growth models are unable in many cases to capture the complexities of filament growth. Even in relatively simple in vitro experiments different growth processes can dominate growth rate over time, competing fibrillar species can result in composite kinetic growth signals and some growth mechanisms have not yet been sufficiently incorporated into an overall description of fibril growth.

Accumulating evidence links oligomeric species of the protein alpha-synuclein to the neuronal death associated with Parkinson's disease. However, the direct detection of alpha-synuclein oligomers in post-mortem brain has been challenging and this has limited our understanding of their structure, distribution and effects in Parkinson's disease. The work presented in this thesis addresses two aspects of the role of alpha-synuclein oligomers in Parkinson's disease. Firstly, I describe the development of a novel technique, the alpha-synuclein proximity ligation assay (AS-PLA), which specifically detected alpha-synuclein oligomers in vitro and in post-mortem brain tissue. In a blinded study with post-mortem brain tissue from eight Parkinson's disease patients and eight controls, AS-PLA revealed widespread, previously unrecognised pathology in the form of extensive diffuse deposition of alpha-synuclein oligomers. Furthermore, AS-PLA preferentially detected early-stage, loosely compacted Parkinson's disease lesions such as pale bodies, whereas Lewy bodies, considered heavily compacted late lesions were only very exceptionally stained. The oligomeric species detected by AS-PLA displayed a unique, intermediate proteinase K resistance profile, suggesting the detection of a conformer that is different from both physiological pre-synaptic alpha-synuclein (proteinase K sensitive) and highly aggregated alpha-synuclein within Lewy bodies (proteinase K resistant). In addition, AS-PLA revealed the age-dependent accumulation of alpha-synuclein oligomers in the substantia nigra of a BAC transgenic mouse model of Parkinson's disease that overexpresses human wild-type alpha-synuclein, SNCA-OVX. Secondly, the detection of early pathology in Parkinson's disease brain tissue using AS-PLA suggests that oligomeric species of alpha-synuclein could represent a potential target for therapeutic intervention. Therefore, I undertook a screen to identify compounds that can prevent the formation of alpha-synuclein oligomers in vitro. Using bimolecular fluorescence complementation constructs, I identified nine compounds capable of reducing the fluorescence indicative of the formation of alpha-synuclein oligomers. Two of these compounds showed dose-dependent activity. Future work will confirm the hits in vitro before studying whether Parkinson's-like phenotypes in the SNCA-OVX mice can be ameliorated or reversed by treatment with the compounds.

α-Synuclein (αSyn) is highly abundant cytosolic protein whose conversion into insoluble fibrils is a pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Despite decades of research, fundamental questions regarding αSyn biology are unresolved. Soluble, prefibrillar oligomers, not their fibrillar end products, are believed to be neurotoxic in humans and in disease models, but their mechanism of action remains unknown. Evidence from our lab and others increasingly suggests that, in healthy cells, αSyn does not exist purely as an unfolded monomer, as the field has long believed, but also as aggregation-resistant, α-helical oligomers; however, their physiological role remains controversial. Thus, my aim was twofold: to characterize toxic αSyn species in the context of mitochondrial dysfunction, a central phenotypic feature of PD; and to purify helical αSyn oligomers from human brain to enable further characterization of physiological αSyn.

Ziel der Studie war, phosphoryliertes und trunkiertes Alpha-Synuclein in Nervenfasern der Haut zu untersuchen und herauszufinden, ob die posttranslationalen Modifikationen Phosphorylierung und Trunkierung des Alpha-Synucleins als potenzielle Biomarker für eine Diagnosestellung des M. Parkinson geeignet sind. Die Besonderheit der vorliegenden Studie war zum einen, dass ausschließlich Patienten in frühen Erkrankungsstadien (Hoehn-und-Yahr-Stadien I und II) des idiopathischen M. Parkinson untersucht wurden und zum anderen der Versuch, die Detektionsrate anhand von Stufenschnitten zu erhöhen. Zusammenfassend kann man sagen, dass phosphoryliertes Alpha-Synuclein ein hohes Potenzial als Biomarker für die Diagnosestellung und zur Differenzialdiagnostik eines M. Parkinson hat und Stufenschnitte die Nachweisrate deutlich erhöhen können. In der Immunfluoreszenzdoppelfärbung mit dem Anti-Phospho-Alpha-Synuclein-Antikörper von BioLegend® (San Diego, USA) konnte bei beinahe 80% der Patienten das gesuchte Protein gefunden werden (Nachweisrate Hoehn-und-Yahr-Stadium I: 58,3%; Hoehn-und-Yahr-Stadium II: 93,8%), in der Immunfluoreszenzdoppelfärbung mit dem Anti-Phospho-Alpha-Synuclein-Antikörper von Prothena Biosciences Inc (San Francisco, USA) nur in etwas mehr als 46% der Patienten (Nachweisrate Hoehn-und-Yahr-Stadium I: 41,7%; Hoehn-und-Yahr-Stadium II: 50%). In Hoehn-und-Yahr-Stadium I ist die Sensitivität jedoch noch nicht ausreichend hoch. Da insbesondere in frühen Stadien der Erkrankung eine Differenzierung zwischen atypischen Parkinson-Syndromen und idiopathischem M. Parkinson klinisch sehr schwierig ist, ist jedoch vor allem das frühdiagnostische Potential eines Biomarkers entscheidend. In Hoehn-und-Yahr-Stadium I müsste die Detektionsrate noch erhöht werden, um einen sinnvollen Einsatz des Biomarkers Phospho-Alpha-Synuclein in der Klinik gewährleisten zu können. / The aim of the study was to investigate phosphorylated and truncated alpha-synuclein in dermal nerve fibres and to find out whether the posttranslational modifications phosphorylation and truncation of the alpha-synuclein are suitable as potential biomarkers for a diagnosis of Parkinson's disease. The peculiarity of the present study was, on the one hand, that only patients in early disease stages (Hoehn and Yahr stages I and II) of idiopathic Parkinson's disease were examined and, on the other hand, an attempt to increase the detection rate by serial sections. In summary, it can be said that phosphorylated alpha-synuclein has great potential as a biomarker for the diagnosis and differential diagnosis of Parkinson's disease and that step-cuts can significantly increase the detection rate. The protein searched for was found in nearly 80% of patients in the immunofluorescence double staining with the anti-phospho-alpha-synuclein antibody from BioLegend® (San Diego, USA) (detection rate Hoehn and Yahr stage I: 58.3% ; Hoehn-und-Yahr stage II: 93.8%), in the immunofluorescence double staining with the anti-phospho-alpha-synuclein antibody from Prothena Biosciences Inc (San Francisco, USA) only in a little more than 46% of the patients ( Detection rate of Hoehn-und-Yahr stage I: 41.7%; Hoehn-und-Yahr stage II: 50%). In Hoehn-und-Yahr stage I, however, the sensitivity is not yet sufficiently high. Since it is clinically very difficult to differentiate between atypical Parkinson's syndrome and idiopathic Parkinson's disease, especially in the early stages of the disease, the early diagnostic potential of a biomarker is crucial. In Hoehn-und-Yahr stage I, the detection rate would have to be increased in order to be able to ensure a sensible use of the biomarker phospho-alpha-synuclein in the clinic.