Microglia and genetic risk in ALS and AD: insights from patient-derived models

In this thesis, we investigate the contribution of genetic risk factors in ALS and AD to disease pathology, with a particular focus on microglia biology and the use of patient-derived in vitro models. Additionally, we provide a comprehensive overview of neural organoid systems used for modeling neurodegenerative diseases and develop a novel organoid platform that incorporates microglia in a physiologically relevant environment. In Chapter 2, we review the general principles of neural organoid generation. We summarize the different existing protocols to obtain region-specific brain or spinal cord organoids and their application for studying neurodegenerative diseases. We next examine the limitations associated to using neural organoids for motor neuron disease modeling and discuss potential solutions. Lastly, we provide a framework to develop an organoid-on-a-chip platform that incorporates all the essential components of the neuromuscular system. In Chapter 3, we investigate the pathogenic effects of one of the strongest genetic risk factors for ALS – ATXN2 intermediate repeat expansions – by combining patient-derived and mouse models. First, we show that iPSC-derived motor neurons from ATXN2-ALS patients exhibit distinct ALS-relevant phenotypes, including impaired stress granule dynamics and altered electrophysiological properties. Next, we generate transgenic mice harboring ATXN2-Q33 repeats in a mutant TDP-43 ALS background, which reveals motor deficits, neuromuscular junction abnormalities, neuronal loss and altered stress granule dynamics in vitro. Transcriptomic analysis reveals mitochondrial dysfunction – which we confirm at a functional level – and altered inflammatory responses involving a microglial component, which we also study in a neural organoid model. In Chapter 4, we change focus to investigate how APOE-independent genetic risk for sAD influences microglia. For that, we generate neural organoids that innately develop microglia from healthy controls and sAD patients harboring APOE3 or APOE4 variants. We observe increased Aß accumulation and neuronal apoptosis in sAD organoids and a reduction in complexity in sAD organoid microglia. Next, we perform transcriptomic analysis of organoid microglia, which reveals distinct disease-associated transcriptional profiles. Furthermore, APOE3 sAD microglia exhibit gene expression changes related to metabolism, which we functionally validate as defects in glycolytic activity. In Chapters 3–4, we employ a neural organoid model that innately develops microglia, allowing for their co-development alongside other neural cell types. However, long-term culturing is impeded by limited oxygen and nutrient availability, and the study of microglia with a genetic background distinct from that of the neural environment is not feasible within this system. To circumvent these limitations, in Chapter 5 we develop a human iPSC-derived air-liquid-interface cortical organoid platform that contains exogenously added microglia. First, we provide a detailed stepwise protocol to generate this model. Next, we show the high physiological relevance of the system by providing examples of microglia morphology, density, even distribution and close interaction with neurons. Lastly, we outline several experimental analyses that can be performed to investigate human neuroimmune interactions in the 3D brain-like environment this newly developed in vitro system offers. Finally, the main findings presented in this thesis are summarized and discussed in the general discussion in Chapter 6.