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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:90019 |
| Date | 20 February 2024 |
| Creators | Bhatia, Priyanka |
| Contributors | Sterneckert, Jared Lynn, Falkenburger, Björn, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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