Repeat expansions in ALS: Lessons from patient-derived models

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease that affects motor neurons in the cerebral cortex, brain stem and spinal cord. Several genetic mutations have been identified that cause or increase the risk of developing ALS. Among them, repeat expansions in intronic or exonic regions of several genes have been linked to ALS, including in C9ORF72 and Ataxin-2 (ATXN2) genes. Expanded repeats in either case lead to cytotoxicity by protein loss-of-function as well as toxic gain-of-function of the repeat. The GGGGCC hexanucleotide repeat expansion (HRE) in C9orf72 is the most common cause of ALS identified to date. The HRE results in C9orf72 haploinsufficiency, HRE RNA foci, and dipeptide repeats resulting from repeat-associated non-ATG translation of the HRE. In ATXN2 protein, intermediate expansion of the polyQ stretch is a risk factor for developing ALS. In this thesis, we approach different aspects of C9ORF72 and ATXN2 repeat expansions in ALS mainly using induced pluripotent stem cell (iPSC) -derived models. In chapter 2 we review the use of central nervous system organoid models in neurodegeneration. We discuss limitations and future perspectives and ultimately provide a framework for the development of organoid-based organ-on-a-chip models for motor neuron disease. In chapter 3 we describe a cerebral organoid model where microglia innately develop. Organoid-derived microglia are similar to adult post-mortem microglia in morphology, transcriptional profile, and inflammatory response. These microglia are competent in phagocytosis and overall represent a valuable model for studying the complex interplay of different brain cell populations in development and disease. In chapter 4 we use the model developed in chapter 3 to study C9orf72 hexanucleotide HRE-associated brain pathology. We show that C9ORF72 HRE makes embryoid bodies significantly smaller than controls. Additionally, we find that at a mature stage, C9orf72 ALS organoids have altered expression of neural progenitor and forebrain markers, hinting at abnormal organoid development. At a later timepoint, C9ALS organoids are smaller than controls which could be a reflection of ALS-associated neural atrophy. In chapter 5, we combine mouse and patient iPSC-derived models to study the contribution of the Ataxin-2 polyQ intermediate repeat expansion in ALS. In both models, transcriptomic signatures show altered expression of genes involved in lipid metabolism, known to be associated with ATXN2 polyQ. We show that ATXN2 intermediate repeat expansion is sufficient to induce both loss- and toxic gain-of-function phenotypes which impact lipid metabolism. Finally, in chapter 6 we discuss technical aspects of iPSC models and of the origin of microglia in cerebral organoids. We then discuss a broader role of lipid metabolism in ALS. Lastly, we highlight the contribution, synergy and overlap between Ataxin-2 and C9orf72. The findings described in this thesis will hopefully contribute to the understanding of repeat expansions in ALS

Repeat expansions in ALS: Lessons from patient-derived models

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease that affects motor neurons in the cerebral cortex, brain stem and spinal cord. Several genetic mutations have been identified that cause or increase the risk of developing ALS. Among them, repeat expansions in intronic or exonic regions of several genes have been linked to ALS, including in C9ORF72 and Ataxin-2 (ATXN2) genes. Expanded repeats in either case lead to cytotoxicity by protein loss-of-function as well as toxic gain-of-function of the repeat. The GGGGCC hexanucleotide repeat expansion (HRE) in C9orf72 is the most common cause of ALS identified to date. The HRE results in C9orf72 haploinsufficiency, HRE RNA foci, and dipeptide repeats resulting from repeat-associated non-ATG translation of the HRE. In ATXN2 protein, intermediate expansion of the polyQ stretch is a risk factor for developing ALS. In this thesis, we approach different aspects of C9ORF72 and ATXN2 repeat expansions in ALS mainly using induced pluripotent stem cell (iPSC) -derived models. In chapter 2 we review the use of central nervous system organoid models in neurodegeneration. We discuss limitations and future perspectives and ultimately provide a framework for the development of organoid-based organ-on-a-chip models for motor neuron disease. In chapter 3 we describe a cerebral organoid model where microglia innately develop. Organoid-derived microglia are similar to adult post-mortem microglia in morphology, transcriptional profile, and inflammatory response. These microglia are competent in phagocytosis and overall represent a valuable model for studying the complex interplay of different brain cell populations in development and disease. In chapter 4 we use the model developed in chapter 3 to study C9orf72 hexanucleotide HRE-associated brain pathology. We show that C9ORF72 HRE makes embryoid bodies significantly smaller than controls. Additionally, we find that at a mature stage, C9orf72 ALS organoids have altered expression of neural progenitor and forebrain markers, hinting at abnormal organoid development. At a later timepoint, C9ALS organoids are smaller than controls which could be a reflection of ALS-associated neural atrophy. In chapter 5, we combine mouse and patient iPSC-derived models to study the contribution of the Ataxin-2 polyQ intermediate repeat expansion in ALS. In both models, transcriptomic signatures show altered expression of genes involved in lipid metabolism, known to be associated with ATXN2 polyQ. We show that ATXN2 intermediate repeat expansion is sufficient to induce both loss- and toxic gain-of-function phenotypes which impact lipid metabolism. Finally, in chapter 6 we discuss technical aspects of iPSC models and of the origin of microglia in cerebral organoids. We then discuss a broader role of lipid metabolism in ALS. Lastly, we highlight the contribution, synergy and overlap between Ataxin-2 and C9orf72. The findings described in this thesis will hopefully contribute to the understanding of repeat expansions in ALS

C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits

Objective: How hexanucleotide (GGGGCC) repeat expansions in C9ORF72 cause amyotrophic lateral sclerosis (ALS) remains poorly understood. Both gain- and loss-of-function mechanisms have been proposed. Evidence supporting these mechanisms in vivo is, however, incomplete. Here we determined the effect of C9orf72 loss-of-function in mice. Methods: We generated and analyzed a conditional C9orf72 knockout mouse model. C9orf72fl/fl mice were crossed with Nestin-Cre mice to selectively remove C9orf72 from neurons and glial cells. Immunohistochemistry was performed to study motor neurons and neuromuscular integrity, as well as several pathological hallmarks of ALS, such as gliosis and TDP-43 mislocalization. In addition, motor function and survival were assessed. Results: Neural-specific ablation of C9orf72 in conditional C9orf72 knockout mice resulted in significantly reduced body weight but did not induce motor neuron degeneration, defects in motor function, or altered survival. Interpretation: Our data suggest that C9orf72 loss-of-function, by itself, is insufficient to cause motor neuron disease. These results may have important implications for the development of therapeutic strategies for C9orf72-associated ALS