Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide affecting 1 - 2 % of the population older than 65. Patients develop characteristic motoric dysfunctions alongside early-onset non-motor symptoms including sleeping disorders, anxiety or depression and late-stage cognitive deficits such as dementia. To date, dopamine-replacement therapies are the gold standard for treating PD patients, improving motoric disorders by compensating for the loss of dopaminergic neurons in the substantia nigra, however no curative therapies to prevent disease progression are yet available. The pathomechanism underlying PD is complex, and the interplay of factors causing the disease is not entirely understood. The formation of α-synuclein protein aggregates, being one of the hallmarks associated with PD, is regarded as a major contributor to neuronal death and the spreading of PD pathology throughout different brain regions as the disease progresses. In the past, deficits in cellular protein clearance machinery have been affiliated with the accumulation of α-synuclein aggregates in PD. In particular, impairements in the macroautophagy-lysosomal pathway (here referred to as autophagy), which is involved in the degradation of large cytosolic components, were found to promote α-synuclein aggregation. In contrast, autophagic stimulation has been shown to benefit α-synuclein degradation and rescue PD phenotypes in cell and rodent models. In this study, I examined the role of FYCO1 in modulating neuronal autophagic processes for α-synuclein aggregate clearance in hiPSC-derived neurons. FYCO1 is an interaction partner of the central autophagic regulator RAB7 but was mostly unnoticed since it was not found detrimental to cellular homeostasis under basal conditions. Still, previous work of our group has identified FYCO1 to rescue PD phenotypes in model systems such as HEK cells and Drosophila, due to improved α-synuclein clearance following FYCO1 overexpression. Mechanistically, FYCO1 is involved in autophagosome-lysosome fusion events by binding to autophagic vesicles, which is required for autophagosome maturation and final degradation. In addition, FYCO1 affiliates autophagic vesicles with the cellular transport machinery via kinesin motor proteins. While fusion promotion can be assigned to an enhancing effect on autophagic clearance, FYCO1-induced anterograde transport promotion is opposite to the retrograde trafficking route of autophagic vesicles for maturation, which is of special importance in neuronal axons. Here, I illuminated FYCO1 effects on both axonal vesicle transport processes and somal vesicle pools to evaluate its ability to promote autophagy-related degradation in neurons. To this end, I established a lentiviral transduction-based model in hiPSC-derived neurons to express FYCO1 in the presence of either a fluorescently labelled marker for autophagic vesicles (LC3-TFL) or in the presence of α-synuclein. In neuronal axons, FYCO1 overexpression impaired retrograde autophagic transport resulting in less movement, implying an inhibitory effect on axonal autophagy. In contrast, FYCO1 enhanced autophagic processes in neuronal somata by upregulating LC3 levels, promoting the collection of α-synuclein in autophagic vesicle clusters and increasing the colocalisation of autophagosomes with lysosomal markers, pointing to the advance in autophagosome maturation. I could not fully resolve, whether α-synuclein degradation was promoted by this induction, as α-synuclein clearance was not indicated yet in the time course of three weeks. Still, studying mutant forms of FYCO1 revealed deficits in autophagosome maturation, which were not represented with wild-type FYCO1. In particular, the autophagosome-interaction domain was essential for autophagosome-lysosome fusion and additionally seemed to be relevant for autophagosomes entering axonal transport, while mutations in the kinesin binding domain caused autophagosome acidification impairments. The most pronounced effect of FYCO1 overexpression in neurons was the modulation of lysosomal vesicles. Besides increasing lysosomal localisation to autophagic vesicles, FYCO1 promoted retrograde trafficking of axonal lysosomal vesicles, by a so far unresolved mechanism. As increasing transport of lysosomes toward the neuronal soma can be connected to the upregulation of autophagy, I hypothesise FYCO1 to be a mediator in autophagy induction signalling. Nevertheless, such an effect needs to be verified in future studies. Conclusively, with this work, I contributed to the understanding of FYCO1’s role in enhancing neuronal autophagic processes but further studies in more advanced PD models are required to evaluate whether this could contribute to an increased clearance of α-synuclein aggregates.

A striking feature of Parkinson's disease (PD) is that the distal axonal terminals of neurons degenerate prior to the soma, a process referred to as 'dying-back'. Another hallmark of the disease is the pathological accumulation of abnormal protein aggregates in soma and axons. Lysosomes, a critical component of the protein quality control machinery, have thus been thought to be altered in PD. LRRK2 G2019S, a gain-of-kinase-function mutation, is one of PD's most common known causative mutations, and LRRK2-specific small molecule inhibitors have been developed as possible therapeutics. However, LRRK2 G2019S is incompletely penetrant, and its role in axonal degeneration is unclear. LRRK2 phosphorylates a subset of Rab GTPases, including Rab10. Since Rab GTPases are mediators of organelle trafficking, we speculated that LRRK2 G2019S affects the transport of organelles, such as lysosomes, thereby contributing to early PD pathogenesis. Using neural progenitor cell-derived neurons from two LRRK2 G2019S-PD patients; we developed a model of axonal trafficking of lysosomes to characterize the impact of mutant LRRK2 on lysosomal trafficking. In comparison to their isogenic gene-corrected controls, we observed a subtle reduction in mutant axonal lysosomal speed, which could indicate that mutant LRRK2 mildly disrupts retrograde lysosomal transport. We also observed that this trafficking phenotype was only partially rescued by LRRK2 kinase inhibitors, which could indicate the importance of other factors regulating axonal transport. Consistent with this idea, we found that mutant LRRK2 was associated with increased co-localization of phosphorylated Rab10 on a small subset of distal axonal lysosomes. Furthermore, the over-expression of Rab10 only mildly affected lysosomal trafficking in axons. Interestingly, damaging the lysosomal membrane increased LRRK2-dependent Rab10 phosphorylation, leading us to speculate that membrane damage in the axon might induce LRRK2 activity. Since lysosomes have been shown to mediate plasma membrane repair, we speculated that membrane damage might exacerbate LRRK2-dependent phenotypes in distal axons. Axotomy was used to test this idea, and we observed an inconsistent delay in the regrowth of mutant axons after axotomy. Moreover, we identified an association between mutant LRRK2 and the transient increase in lysosomes at the injury site, indicating that LRRK2 G2019S might potentially affect damage-prone distal axons. Since the LRRK2 G2019S-associated phenotypes observed in our assays were relatively mild in one isogenic pair, we were curious about the clinical and genetic phenotypes of the patients from whom the somatic cells for neural progenitor cell generation were sourced. Interestingly, we observed that clinical features of PD, including age-of-onset, motor symptoms, cognitive impairment, and the level of cerebrospinal fluid biomarkers, were heterogeneous between the two patients. Additionally, genetic analysis of specific PD risk-associated loci in MAPT and SNCA revealed that one patient was more at risk of developing PD than the other, indicating influence from genetic factors in addition to LRRK2 G2019S. These factors might affect the axonal phenotypes observed in our assays. Overall, we have developed assays to investigate the effects of LRRK2 G2019S on axonal lysosomes. These assays can potentially be a useful tool to better understand early pathogenesis in heterogeneous PD patients and test targeted therapeutics that can be successful over an eclectic cohort of PD patients, all of whom are diagnosed based on deteriorating motor symptoms.:TABLE OF CONTENTS I LIST OF FIGURES IV LIST OF TABLES VI ABBREVIATIONS VII 1 INTRODUCTION 1 1.1 Neurodegenerative diseases 1 1.2 Parkinson’s disease 2 1.2.1 General Features 2 1.2.2 Phenomenon of “dying back” in PD 6 1.2.3 Contribution of axonal architecture and function to “dying back” 7 1.2.4 Etiology of PD 10 1.2.4.1 Environmental factors 10 1.2.4.2 Genetic factors linked to axonal function 11 1.3 Lysosomes 12 1.3.1 Composition and biogenesis of lysosomes 13 1.3.2 Lysosomes as digestive centers 15 1.3.3 Lysosomes as secretory organelles 18 1.3.4 Lysosomes in PD 20 1.3.4.1 Genetic PD factors linked to lysosomal function 21 1.4 Leucine-rich repeat kinase 2 (LRRK2) 22 1.4.1 LRRK2 domain organization and function 22 1.4.2 Clinical features of PD patients with LRRK2 mutations (LRRK2-PD) 24 1.4.3 LRRK2 animal models 24 1.4.4 LRRK2 induced pluripotent stem cell (iPSC)-based models 25 1.4.5 Animal and iPSC-based models demonstrate a role for LRRK2 in the endo-lysosomal system 27 1.4.6 LRRK2 kinase inhibitors 30 2 AIMS OF THE THESIS 32 3 MATERIALS AND METHODS 33 3.1 Materials 33 3.1.1 Chemicals 33 3.1.2 Purchased kits 34 3.1.3 Plasmids 34 3.1.4 Antibodies 35 3.1.5 Dyes 36 3.1.6 Primers and oligonucleotides 36 3.1.7 Cell culture media and reagents 37 3.1.8 Small molecules 38 3.1.9 Compounds 38 3.1.10 Cell culture media 39 3.1.11 Human Neural Progenitor Cell (NPC) lines 40 3.2 Methods 41 3.2.1 Ethics statement 41 3.2.2 Licenses 41 3.2.3 Information about iPSC and NPC line generation 41 3.2.4 Preparation of cell culture coated plates 41 3.2.5 Maintenance of NPCs 42 3.2.6 Differentiation of NPCs to neurons 42 3.2.7 Preparation of microfluidic chambers 43 3.2.8 Seeding neurons as single cells 44 3.2.9 HEK293T cell culture 45 3.2.10 Treatment of neurons with compounds 45 3.2.11 Genomic DNA isolation 46 3.2.12 Polymerase-Chain Reaction (PCR) 46 3.2.13 Agarose gel electrophoresis 46 3.2.14 Plasmid DNA isolation 46 3.2.15 Lentiviral vector production 47 3.2.16 Lentiviral infection of human neurons 48 3.2.17 Protein isolation and quantification 48 3.2.18 Capillary electrophoresis 49 3.2.19 Axotomy 49 3.2.20 Immunostaining 50 3.2.21 Live cell imaging 51 3.2.22 Quantification of axonal trafficking using kymographs 52 3.2.23 Quantification of axonal trafficking using an object based method 53 3.2.24 Apotome imaging and quantification 54 3.2.25 Confocal imaging and quantification 54 3.2.26 Clinical and biomarker data collection 55 4 RESULTS 57 4.1 Establishing an axonal lysosomal trafficking assay 57 4.1.1 NPCs from LRRK2 G2019S patients and their respective isogenic controls differentiate into neurons 57 4.1.2 Axons can be spatially separated from soma and dendrites 60 4.1.3 Setting up the axonal trafficking assay 62 4.2 Axonal lysosomal trafficking assay detects LRRK2 G2019S associated changes in lysosome movement 65 4.3 Axonal lysosomal trafficking assay detects partial rescue by a small molecule LRRK2 inhibitor 71 4.4 LRRK2 G2019S is associated with an increase in the proportion of lysosomes co-localizing with phosphorylated Rab10 76 4.5 Rab10 over-expression mildly affects lysosomal trafficking in axons 78 4.6 Lysosomal membrane damage increases LRRK2-mediated Rab10 phosphorylation 81 4.7 LRRK2 G2019S is not associated with consistent effects on long-term axonal regrowth after axotomy 82 4.8 LRRK2 G2019S is associated with transient accumulation of lysosomes at the injury site after axotomy 86 4.9 Assessment of clinical, biomarker and genetic data from the LRRK2 G2019S patient donors 88 5 DISCUSSION 92 6 APPENDIX 101 7 SUMMARY 104 8 ZUSSAMENFASSUNG 106 9 BIBLIOGRAPHY 108 10 ACKNOWLEDGEMENTS 136 11 DECLARATIONS 138

Neuronal activity in the brain is tightly regulated. During operation in real time, for instance, feedback and feedforward loops limit excessive excitation. In addition, cell autonomous processes ensure that neurons’ average activity is restored to a setpoint in response to chronic perturbations. These processes are summarized as homeostatic plasticity (Turrigiano in Cold Spring Harb Perspect Biol 4:a005736–a005736, 2012). In the basal ganglia, information is mainly transmitted through disinhibition, which already constraints the possible range of neuronal activity. When this tightly adjusted system is challenged by the chronic decline in dopaminergic neurotransmission in Parkinson’s disease (PD), homeostatic plasticity aims to compensate for this perturbation. We here summarize recent experimental work from animals demonstrating that striatal projection neurons adapt excitability and morphology in response to chronic dopamine depletion and substitution. We relate these cellular processes to clinical observations in patients with PD that cannot be explained by the classical model of basal ganglia function. These include the long duration response to dopaminergic medication that takes weeks to develop and days to wear off. Moreover, dyskinesias are considered signs of excessive dopaminergic neurotransmission in Parkinson’s disease, but they are typically more severe on the body side that is more strongly affected by dopamine depletion. We hypothesize that these clinical observations can be explained by homeostatic plasticity in the basal ganglia, suggesting that plastic changes in response to chronic dopamine depletion and substitution need to be incorporated into models of basal ganglia function. In addition, better understanding the molecular mechanism of homeostatic plasticity might offer new treatment options to avoid motor complications in patients with PD.

Abstract: Background: In Parkinson’s disease, postural instability and falls are of particular socioeconomic relevance. Although effective fall prevention and the prophylaxis of fall-related injuries depend on low-threshold symptom monitoring, validated instruments are lacking. Objectives: To develop a self-report questionnaire for the assessment of falls, near falls, fear of falling, fallrelated injuries, and causes of falls for patients with Parkinson’s disease (PwPD). - Methods: A pool of potential items was generated from a literature review and by discussion in an expert panel. The first version of the Dresden Fall Questionnaire (DREFAQ) was tested in a group of German-speaking movement disorder specialists as well as PwPD. The resulting 5-item questionnaire was assessed in a validation cohort of 36 PwPD who documented fall events and near-fall events in a calendar for 3 months and completed the DREFAQ at the end of the study. The questionnaire was subsequently used in a separate cohort of 46 PwPD to determine test–retest reliability and confirm the factor structure. - Results: The DREFAQ showed good internal consistency (Cronbach’s α = 0.84) and good test–retest reliability (intraclass correlation coefficient, 0.76; 95% confidence interval, 0.60–0.86). The total DREFAQ score showed good concurrent validity with fall events (Spearman’s ρ = 0.82) and near-fall events (Spearman’s ρ = 0.78) as determined by fall and near-fall diaries. Factor analysis revealed a 2-factor structure composed of near falls with fear of falling and severe falls with injuries. - Conclusions: The DREFAQ is a reliable and valid 5-item questionnaire for determining the incidence of falls, near falls, fear of falling, fall-related injuries, and causes of falls in PwPD.

Introduction: The OPTIPARK study confirmed the effectiveness and safety of opicapone as adjunct therapy to levodopa in patients with Parkinson’s disease (PD) and motor fluctuations under real-world conditions. The aim of this sub-analysis was to evaluate opicapone in the German patient cohort of OPTIPARK in order to provide country-specific data. Methods: OPTIPARK was an open-label, single-arm study conducted in routine clinical practice across Germany and the UK. Patients with PD and motor fluctuations received once-daily opicapone 50 mg for 3 months in addition to levodopa. The primary endpoint was Clinicians’ Global Impression of Change (CGI-C). Secondary assessments included Patients’ Global Impressions of Change (PGI-C), Unified Parkinson’s Disease Rating Scale (UPDRS) I–IV, Parkinson’s Disease Questionnaire (PDQ-8), and Non-Motor Symptoms Scale (NMSS). This sub-analysis reports outcomes from the German patients only. Results: Overall, 363 (97.6%) of the 372 patients included in the German cohort received ≥1 dose of opicapone and 291 (80.2%) completed the study. Improvements on CGI-C and PGI-C were reported by 70.8% and 76.3% of patients, respectively. UPDRS scores improved for activities of daily living during OFF time by −3.3 ± 4.5 points and motor scores during ON time by −5.3 ± 7.9 points. PDQ-8 and NMSS scores also demonstrated improvements. Treatment emergent adverse events considered at least possibly related to opicapone occurred in 37.7% of patients, with most being of mild or moderate intensity. Conclusion: Opicapone added to levodopa in patients with PD and motor fluctuations was effective and generally well tolerated in routine clinical practice across Germany.

Die posturale Instabilität zählt zu den am meisten beeinträchtigenden Symptomen der Parkinson-Krankheit. Die Störung der motorischen Gleichgewichtskontrolle ist progressiv im Verlauf und weder durch medikamentöse noch durch operative Methoden zufriedenstellend einzudämmen. In der Bewegungstherapie werden häufig Gleichgewichtsübungen empfohlen, um ein Fortschreiten der körperlichen Einschränkungen zu verringern. Der aktuelle wissenschaftliche Stand lässt allerdings eine Einschätzung zur Effektivität von Gleichgewichtstraining bei Parkinsonpatienten kaum zu. Dies ist u. a. auf einen Mangel an geeigneten Testverfahren zur Beurteilung der posturalen Instabilität zurückzuführen. In der vorliegenden Untersuchung wurden die Auswirkungen eines 12-wöchigen Gleichgewichtstrainings bei Parkinsonpatienten auf die posturale Stabilität in einem umfassenden Testdesign, bestehend aus alltagsmotorischen, biomechanischen und subjektiven Testverfahren evaluiert. In nahezu allen eingesetzten Testverfahren zeigte sich ein mehr oder weniger deutlicher Trend zu einer Verbesserung der posturalen Stabilität. Durch den Einsatz einer Testbatterie konnte somit insgesamt von einem positiven Einfluss eines Gleichgewichtstrainings auf die posturale Stabilität von Parkinsonpatienten ausgegangen werden. Weiterhin werden zahlreiche positive Nebeneffekte bzgl. der allgemeinen Fitness, der psychischen und sozialen Situation durch ein zielgerichtetes Gruppentraining unter geschulter Anleitung vermutet.

Bis heute ist die Ätiopathogenese der Parkinson Krankheit noch nicht geklärt. Verschiedene Abweichungen im Stoffwechsel von Betroffenen konnten zwar detektiert werden (z.B. Komplex I-Mangel, erhöhte Eisen- und 8-OHdG Werte im Gehirn), aber bis heute gibt es keine eindeutigen Hinweise, wodurch es zur Entstehung der Krankheit kommt. Da es am wahrscheinlichsten ist, dass die Krankheit multifaktoriell bedingt ist, könnten auch Mutationen der mitochondrialen DNA eine wichtige Rolle spielen. Entscheidende Hinweise darauf lieferten Experimente mit Cybrid–Zellen. Bisherige Screeninguntersuchungen des mitochondrialen Genoms konnten allerdings noch keine eindeutigen krankheitsspezifischen Mutationen nachweisen. Die Theorie, dass oxidativer Stress in Verbindung mit der Parkinsonschen Krankheit stehen könnte, fand Unterstützung, als signifikant erhöhte Produkte der Lipidperoxidation (Malondialdehyd, Lipidhydroperoide) in der Substantia nigra (Dexter et al., 1989 b, 1994) und ein abnormaler Eisenstoffwechsel in den Basalganglien des Gehirns (Dexter et al., 1987; Dexter et al., 1989a; Cadet, 2001; Hirsch et al., 1991) einiger Patienten nachgewiesen worden. Erhöhte Eisenwerte in Neuromelaninaggregationen, sowie verringerte Ferritinspiegel unterstützen diese Untersuchungen (Cadet, 2001; Dexter et al., 1987, 1989b; Riederer et al., 1989; Sofic et al., 1988). Besonders anfällig für reaktive Sauerstoffverbindungen im Gehirn ist die Substantia nigra. Zum einen kommt es während des Dopaminstoffwechsels zur Freisetzung von Wasserstoffperoxid, des weiteren enthält sie Neuromelanin, welches selektiv Metalle (z.B. Eisen) bindet. Reduziertes Eisen kann mit Wasserstoffperoxid via Fentonreaktion reagieren und das äußerst schädliche Hydroxylradikal bilden (Klein & Ackerman, 2003). Die Menge der in den Mitochondrien frei werdenden Radikale ist von einer Reihe von verschiedenen Faktoren abhängig. Umwelteinflüsse und Ernährungsfaktoren spielen dabei eine ebenso wichtige Rolle, wie der mitochondriale Stoffwechsel selbst (Adachi et al., 1993; Simic, 1991; Menegon et al., 1997). Als ein Biomarker für den oxidativen Stress hat sich in den letzten Jahren 8-Hydroxy-2’-deoxyguanosin (8-OHdG) etabliert, welches als Folge von Angriffen des Hydroxyl-Radikals auf die Doppelbindungen der DNA-Basen am häufigsten gebildet wird (Simic, 1991; Dizdaroglu et al., 1991, Kasai, 1997). 8-OHdG ist in der Lage sich mit Adenin zu paaren (ca. 1% der Fälle), was wiederum bei der nächsten Replikation zu einer Transversion von Guanin zu Thymin führt (Richter, 1992; Croteau & Bohr, 1997).

No description available.

In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.

In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.