Cell culture models of Chorea Acanthocytosis and their evaluation

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 Appendi

The Erythrocyte Sedimentation Rate and Its Relation to Cell Shape and Rigidity of Red Blood Cells from Chorea-Acanthocytosis Patients in an Off-Label Treatment with Dasatinib

Background: Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease with deformed red blood cells (RBCs), so-called acanthocytes, as a typical marker of the disease. Erythrocyte sedimentation rate (ESR) was recently proposed as a diagnostic biomarker. To date, there is no treatment option for affected patients, but promising therapy candidates, such as dasatinib, a Lyn-kinase inhibitor, have been identified. Methods: RBCs of two ChAc patients during and after dasatinib treatment were characterized by the ESR, clinical hematology parameters and the 3D shape classification in stasis based on an artificial neural network. Furthermore, mathematical modeling was performed to understand the contribution of cell morphology and cell rigidity to the ESR. Microfluidic measurements were used to compare the RBC rigidity between ChAc patients and healthy controls. Results: The mechano-morphological characterization of RBCs from two ChAc patients in an off-label treatment with dasatinib revealed differences in the ESR and the acanthocyte count during and after the treatment period, which could not directly be related to each other. Clinical hematology parameters were in the normal range. Mathematical modeling indicated that RBC rigidity is more important for delayed ESR than cell shape. Microfluidic experiments confirmed a higher rigidity in the normocytes of ChAc patients compared to healthy controls. Conclusions: The results increase our understanding of the role of acanthocytes and their associated properties in the ESR, but the data are too sparse to answer the question of whether the ESR is a suitable biomarker for treatment success, whereas a correlation between hematological and neuronal phenotype is still subject to verification

Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation.

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy

Mitochondria-Endoplasmic Reticulum Contact Sites Dynamics and Calcium Homeostasis Are Differentially Disrupted in PINK1-PD or PRKN-PD Neurons

Background: It is generally believed that the pathogenesis of PINK1/parkin-related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria-endoplasmic reticulum contact sites (MERCSs). Objective: The aim of our study was to perform an in-depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin-linked PD.MethodsIn our study, we used induced pluripotent stem cell-derived dopaminergic neurons from patients with PD with loss-of-function mutations in PINK1 or PRKN. We employed a split-GFP-based contact site sensor in combination with the calcium-sensitive dye Rhod-2 AM and applied Airyscan live-cell super-resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis. Results: Our results showed that thapsigargin-induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild-type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1-PD or PRKN-PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin-induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted. Conclusions: Our results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild-type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Cell culture models of Chorea Acanthocytosis and their evaluation

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 Appendi

Cell culture models of Chorea Acanthocytosis and their evaluation

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 Appendi

Bayes for ALS/MND

These are the synthetic tofersen data described in the clinical trials section of the manuscript

Human Spinal Motor Neurons Are Particularly Vulnerable to Cerebrospinal Fluid of Amyotrophic Lateral Sclerosis Patients

Amyotrophic lateral sclerosis (ALS) is the most common and devastating motor neuron (MN) disease. Its pathophysiological cascade is still enigmatic. More than 90% of ALS patients suffer from sporadic ALS, which makes it specifically demanding to generate appropriate model systems. One interesting aspect considering the seeding, spreading and further disease development of ALS is the cerebrospinal fluid (CSF). We therefore asked whether CSF from sporadic ALS patients is capable of causing disease typical changes in human patient-derived spinal MN cultures and thus could represent a novel model system for sporadic ALS. By using induced pluripotent stem cell (iPSC)-derived MNs from healthy controls and monogenetic forms of ALS we could demonstrate a harmful effect of ALS-CSF on healthy donor-derived human MNs. Golgi fragmentation—a typical finding in lower organism models and human postmortem tissue—was induced solely by addition of ALS-CSF, but not control-CSF. No other neurodegenerative hallmarks—including pathological protein aggregation—were found, underpinning Golgi fragmentation as early event in the neurodegenerative cascade. Of note, these changes occurred predominantly in MNs, the cell type primarily affected in ALS. We thus present a novel way to model early features of sporadic ALS

Functional and Molecular Properties of DYT-SGCE Myoclonus-Dystonia Patient-Derived Striatal Medium Spiny Neurons.

Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca(2+) content and lower frequency of spontaneous Ca(2+) signals in SGCE MSNs. Blocking of voltage-gated Ca(2+) channels by verapamil was less efficient in suppressing KCl-induced Ca(2+) peaks of SGCE MSNs. Ca(2+) amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca(2+) channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia