Chorea Acanthocytosis (ChAc) is an autosomal recessive inherited disease caused by loss- of-function mutation in the VPS13A gene which encodes CHOREIN protein. This study used induced pluripotent stem cells (iPSCs) as well as neural progenitor cells (NPCs) to generate medium spiny neurons (MSN) as well as midbrain dopaminergic neurons (mDAN). The first objective of this thesis was to generate and characterize a stem cell based disease model of ChAc. The second objective was to establish two different differentiation protocols that yield different neuronal sub types that are affected in ChAc, and compare whether they harbor similar phenotypes and whether the faster protocol can be used to model the disease accurately. The generated iPSCs were characterized using AP staining as an early marker for reprogramming, qPCR for analysis of residual expression of exogenous transcription factors, immunocytochemistry (ICC) for staining of pluripotency markers as well as markers for mesoderm, ectoderm and endoderm formation upon three germ layer formation. Karyotyping was conducted to exclude aberrant clones. Western blot using CHOREIN antibody revealed that the cell lines retained their disease identity. There were no differences observed between wild type and ChAc lines in stem cell and neuron populations in either protocol. qPCR analysis, investigating the expression of previously described markers for characterization, revealed no significant clustering between wild type and ChAc lines in either protocol. A disturbed ratio of globular and filamentous actin is causative for the aberrant shape of ChAc erythrocytes. Investigation of the ratio in mature neurons revealed a significant reduction of this ratio in MSN but no difference in mDAN cultures. When the ratio of cytosolic and filamentous tubulin and the acetylation of tubulin were investigated, no differences were found between wild type and ChAc lines. Mature neurons of both differentiation protocols were subjected to treatment with the proteotoxic stress inducer L-canavanine and the unfolded protein response (UPR) inducer tunicamycin. Survival was analyzed with the PrestoBlue assay as well as lactate dehydroxylase (LDH) release assay. Both cultures of mature neurons showed an increased susceptibility to the respective drugs. Furthermore the data suggests that MSN cultures are more vulnerable against proteotoxic stress (L-canavanine). Kinetics of tunicamycin poisoning were not different within MSN cultures but indicated a late cell death of ChAc lines under mDAN differentiation conditions. DNA damage plays a major role in the progression of neurodegenerative diseases. The amount of double strand breaks (DSB) was assessed in mature cultures of MSN and mDAN differentiations. There was no difference in basal level of DSB. When etoposide was applied to induce DNA damage, increased susceptibility of ChAc lines was observed. Albeit significant, the effect size was very small. Seahorse was used to characterize energy metabolism. Glycolysis was not impaired in ChAc lines in either protocol. Furthermore, MSN differentiation showed no difference in any parameter related to oxidative phosphorylation, while under mDAN conditions, coupling efficiency and spare respiratory capacity was increased for ChAc lines. The non-respiratory oxygen consumption was increased in ChAc lines in MSN cultures but decreased in mDAN cultures. The yeast homolog of VPS13A interacts with vesicle and mitochondrial membranes. Therefore, this study focuses on vesicle and mitochondria homeostasis. Live cell imaging of mature neurons of MSN differentiations revealed a decreased amount and reduced motility of mitochondria. Even though mitochondria were normally shaped their size was reduced. mDAN differentiations harbored a reduced amount and shortened mitochondria. These mitochondria, however, showed an increased motility. When analyzing aligned mature neurons in microfluidic chambers (MFCs), a strong phenotype was already observed in proximal regions, which resembled the distal parts of the channels. Hence, the dysregulation, that occurs distal in healthy controls, happens closer to the soma in diseased cells. The mitochondria potential marker JC-1 showed a hyperpolarization of mitochondria in MSN culture and a depolarization in mDAN cultures. When investigated in MFCs of mDAN cultures, there was a significant increase in potential observed at the distal position of ChAc lines, while wild type cultures showed no difference. Experiments conducted on the lysosomal compartments showed a decrease in proximal parts of ChAc MSN cultures when compared to wild type. Their shape was altered as well. mDAN cultures featured no significant morphological changes. Trafficking analysis revealed an increase in motility in MSN cultures but a decrease in mDAN cultures. When lysosomes were analyzed in MFCs only mDAN cultures showed an increase in retrograde transport. In order to investigate whether the in vitro phenotypes of Huntington (Htt) and ChAc are similar, some of the previous experiments were conducted in MSN differentiations of one Htt line. Cells from Htt behaved similar to ChAc lines when DNA damage response was investigated. Analysis of mitochondrial parameters showed no difference as well. However, the non-respiratory oxygen consumption was not increased and resembled wild type. When Htt neurons were investigated during live cell imaging, shortened mitochondria were found. Their number was not reduced significantly. However, a trend for reduction was observed. Mitochondria of Htt cells were more motile than ChAc or wild type lines. Mitochondrial potential was increased in Htt and comparable to ChAc. Lysosomal count showed a reduction and the area of Htt lysosomes was significantly smaller than wild type or ChAc. Lysosomes of Htt cells were more motile than their wild type or ChAc counterparts.

Chorea Acanthocytosis (ChAc) is an autosomal recessive inherited disease caused by loss- of-function mutation in the VPS13A gene which encodes CHOREIN protein. This study used induced pluripotent stem cells (iPSCs) as well as neural progenitor cells (NPCs) to generate medium spiny neurons (MSN) as well as midbrain dopaminergic neurons (mDAN). The first objective of this thesis was to generate and characterize a stem cell based disease model of ChAc. The second objective was to establish two different differentiation protocols that yield different neuronal sub types that are affected in ChAc, and compare whether they harbor similar phenotypes and whether the faster protocol can be used to model the disease accurately. The generated iPSCs were characterized using AP staining as an early marker for reprogramming, qPCR for analysis of residual expression of exogenous transcription factors, immunocytochemistry (ICC) for staining of pluripotency markers as well as markers for mesoderm, ectoderm and endoderm formation upon three germ layer formation. Karyotyping was conducted to exclude aberrant clones. Western blot using CHOREIN antibody revealed that the cell lines retained their disease identity. There were no differences observed between wild type and ChAc lines in stem cell and neuron populations in either protocol. qPCR analysis, investigating the expression of previously described markers for characterization, revealed no significant clustering between wild type and ChAc lines in either protocol. A disturbed ratio of globular and filamentous actin is causative for the aberrant shape of ChAc erythrocytes. Investigation of the ratio in mature neurons revealed a significant reduction of this ratio in MSN but no difference in mDAN cultures. When the ratio of cytosolic and filamentous tubulin and the acetylation of tubulin were investigated, no differences were found between wild type and ChAc lines. Mature neurons of both differentiation protocols were subjected to treatment with the proteotoxic stress inducer L-canavanine and the unfolded protein response (UPR) inducer tunicamycin. Survival was analyzed with the PrestoBlue assay as well as lactate dehydroxylase (LDH) release assay. Both cultures of mature neurons showed an increased susceptibility to the respective drugs. Furthermore the data suggests that MSN cultures are more vulnerable against proteotoxic stress (L-canavanine). Kinetics of tunicamycin poisoning were not different within MSN cultures but indicated a late cell death of ChAc lines under mDAN differentiation conditions. DNA damage plays a major role in the progression of neurodegenerative diseases. The amount of double strand breaks (DSB) was assessed in mature cultures of MSN and mDAN differentiations. There was no difference in basal level of DSB. When etoposide was applied to induce DNA damage, increased susceptibility of ChAc lines was observed. Albeit significant, the effect size was very small. Seahorse was used to characterize energy metabolism. Glycolysis was not impaired in ChAc lines in either protocol. Furthermore, MSN differentiation showed no difference in any parameter related to oxidative phosphorylation, while under mDAN conditions, coupling efficiency and spare respiratory capacity was increased for ChAc lines. The non-respiratory oxygen consumption was increased in ChAc lines in MSN cultures but decreased in mDAN cultures. The yeast homolog of VPS13A interacts with vesicle and mitochondrial membranes. Therefore, this study focuses on vesicle and mitochondria homeostasis. Live cell imaging of mature neurons of MSN differentiations revealed a decreased amount and reduced motility of mitochondria. Even though mitochondria were normally shaped their size was reduced. mDAN differentiations harbored a reduced amount and shortened mitochondria. These mitochondria, however, showed an increased motility. When analyzing aligned mature neurons in microfluidic chambers (MFCs), a strong phenotype was already observed in proximal regions, which resembled the distal parts of the channels. Hence, the dysregulation, that occurs distal in healthy controls, happens closer to the soma in diseased cells. The mitochondria potential marker JC-1 showed a hyperpolarization of mitochondria in MSN culture and a depolarization in mDAN cultures. When investigated in MFCs of mDAN cultures, there was a significant increase in potential observed at the distal position of ChAc lines, while wild type cultures showed no difference. Experiments conducted on the lysosomal compartments showed a decrease in proximal parts of ChAc MSN cultures when compared to wild type. Their shape was altered as well. mDAN cultures featured no significant morphological changes. Trafficking analysis revealed an increase in motility in MSN cultures but a decrease in mDAN cultures. When lysosomes were analyzed in MFCs only mDAN cultures showed an increase in retrograde transport. In order to investigate whether the in vitro phenotypes of Huntington (Htt) and ChAc are similar, some of the previous experiments were conducted in MSN differentiations of one Htt line. Cells from Htt behaved similar to ChAc lines when DNA damage response was investigated. Analysis of mitochondrial parameters showed no difference as well. However, the non-respiratory oxygen consumption was not increased and resembled wild type. When Htt neurons were investigated during live cell imaging, shortened mitochondria were found. Their number was not reduced significantly. However, a trend for reduction was observed. Mitochondria of Htt cells were more motile than ChAc or wild type lines. Mitochondrial potential was increased in Htt and comparable to ChAc. Lysosomal count showed a reduction and the area of Htt lysosomes was significantly smaller than wild type or ChAc. Lysosomes of Htt cells were more motile than their wild type or ChAc counterparts.:List of abbreviations Introduction 1. Neurodegenerative diseases 1.1. Chorea-acanthocytosis – a clinical overview 1.2. Chorea-Acanthocytosis – genetic considerations 2. Disease modelling 2.1. Human disease models 2.2. Induced pluripotent stem cells 2.3. Multipotent neuronal progenitor cells 3. Objectives of this thesis Materials & Methods 1. Cell culture procedures 1.1. Coating 1.2. Matrigel 1.3. PLO/laminin 1.4. Gelatin coating 1.5. Mouse embryonic fibroblast isolation 1.6. Generation of feeder cells 1.7. Human fibroblast culture 1.8. Reprogramming 1.9. iPSC culture 1.10. Culture of small molecule neuronal precursor cells (smNPC) 1.11. MSN differentiation 1.12. mDAN differentiation 2. Nucleic acid biochemistry 2.1. mRNA isolation 2.2. cDNA generation 2.3. Polymerase chain reaction (PCR) 2.4. Agarose gel electrophoresis 3. Cell survival analysis 3.1. PrestoBlue cell viability assay 3.2. Cytotoxicity detection kit: 3.3. DNA damage analysis 4. Metabolic characterization 5. Protein biochemistry 5.1. Alkaline phosphatase staining 5.2. Preparation of immunocytochemistry samples 5.3. Isolation of globular and filamentous actin 5.4. Whole cell protein Isolation 5.5. Cytosolic protein isolation 5.6. Protein concentration measurement 5.7. Western blot 6. Live cell imaging 7. Statistics Results 1. Generation of induced pluripotent stem cells 1.1. Silencing of exogenous transcription factors 1.2. Karyotyping of iPSC clones 1.3. Evaluation of pluripotency 1.4. Alkaline phosphatase staining 1.5. Staining of pluripotency markers 1.6. Three germ layer formation 1.7. Confirmation of ChAc phenotype by CHOREIN western blot 2. Characterization of differentiation potential 2.1. Differentiation efficiency 2.2. Characterization by qPCR 2.3. Ratio of polymerized and unpolymerized cytoskeleton proteins 2.4. Cell survival upon stress induction 2.5. DNA damage in mature neurons 2.6. Characterization of metabolism 3. Live cell imaging 3.1. Mitochondrial dynamics 3.1.1. Morphological analysis 3.1.1.1. Undirected neurons (96 well plate format) 3.1.1.2. Microfluidic chambers 3.1.2. Trafficking analysis 3.1.2.1. 96 well 3.1.2.2. Microfluidic chambers 3.1.3. JC-1 3.1.3.1. 96 well 3.1.3.2. Microfluidic chambers 3.2. Lysosomal dynamics 3.2.1. Morphological analysis 3.2.1.1. 96 well 3.2.1.2. Microfluidic chambers 3.2.2. Trafficking 3.2.2.1. 96 well 3.2.2.2. Microfluidic chambers 4. Comparison with Huntington’s disease 4.1. DNA damage 4.2. Characterization of metabolism 4.3. Live cell imaging 4.3.1. Mitochondria 4.3.1.1. Morphological analysis 4.3.1.2. Trafficking 4.3.1.3. JC-1 4.3.2. Lysosomes 4.3.2.1. Morphological analysis 4.3.2.2. Trafficking Discussion 1. Characterization of ChAc lines 1.1. ChAc stem cell lines show no impaired differentiation potential 1.2. Neurons from MSN differentiation have an altered G/F actin ratio 1.3. Mature neurons from ChAc lines are susceptible to UPR, proteotoxicity and DNA damage 1.4. ChAc neurons are not susceptible to DNA damage 1.5. Energy dynamics in ChAc and Huntington lines feature a shift to glycolysis 2. Live cell imaging of ChAc lines 2.1. Video analysis is reproducible and sensitive 2.2. ChAc lines have altered mitochondria shape and trafficking 2.3. Treatments are not selective on ChAc lines mitochondria 2.4. Mitochondrial potential is altered in ChAc lines 2.5. ChAc lysosomes feature normal morphology but altered trafficking 2.6. Lysosomes of MSN cultures respond poorly to treatments 3. MSN and mDAN differentiation highlight different aspects of the disease References List of figures List of tables Acknowledgments Appendix