UC Irvine

Alzheimer’s disease (AD) is a progressive neurodegenerative disease for which there is no cure. Worldwide, AD is estimated to effect 50 million people with 10 million new cases each year. Developing therapeutics for Alzheimer’s disease has been particularly difficult given the complex etiology of this disease. Alzheimer’s disease is characterized by an accumulation of parenchymal beta-amyloid protein plaques, tau neurofibrillary tangles, and neuroinflammation. For sporadic AD, which accounts for around 95 % of AD cases, the direct trigger of neurodegeneration remains unclear. Understanding the mechanistic pathophysiology of this disease will allow for the development of more effective targeted therapeutics and biomarker studies to help patients. In the last decade, genome-wide association studies (GWAS) have renewed interest in neuroinflammation as a potential disease-modifying mechanism. These large-scale genetic studies have uncovered a striking enrichment of immune-specific genes as risk-factors for Alzheimer’s disease. However, the function of many of these GWAS loci are not well understood. Additionally, many of these genes have poor homology between human cells and traditional murine disease-models. Thus, there is a critical need to develop a model of human microglia in order to understand how these immune risk factors influence human microglial function. The focus of this dissertation is to develop and characterize a model of human microglia that can be readily studied without the need for isolation of human brain tissue surgically or post-mortem. Building on previous research in the lab, we have developed a model of human microglia differentiated from induced pluripotent stem cells (iPS-Microglia). This model follows developmental ontogeny with a primary differentiation into CD43+ hematopoietic progenitor cells before transition into a microglial differentiation medium containing neuron and astrocyte derived cytokines to educate our microglia in a homeostatic brain-like environment. The result is a highly pure population of iPS-Microglia which perform key microglial functions and cluster alongside human microglial transcriptomes (Chapter 1). Because this microglial model is fully defined beginning from iPS cells, it is possible to combine this approach with modern molecular biological manipulation such as CRISPR gene editing. This dissertation focuses on studies surrounding the AD-risk loci Triggering Receptor Expressed on Myeloid Cells II (TREM2). Predicted loss of function mutations in TREM2 increase Alzheimer’s disease risk up to 2-3 fold making it the highest microglial-specific risk factor for AD. By performing CRISPR-mediated knockout of TREM2 in human iPSCs and differentiating into microglia, we find that TREM2-knockout locks microglia in a homeostatic state. Specifically, microglia lacking TREM2 are deficient in SYK-mediated phagocytosis, CXCR4-mediated migration, and are more sensitive to MCSF-mediated cell death. In vivo, this translates to an inability to perform chemotaxis towards and compaction of beta-amyloid plaques leading to a build-up of pathology in the brain. We also characterize a distinct lack of transcriptional activation in TREM2 knockout cells suggesting that induction of the Disease Associated Microglia (DAM) profile may be an essential immune response in shielding the brain from dementia (Chapter 2). Because TREM2-knockout cells are locked in a homeostatic state, they express high levels of homeostatic microglia markers P2RY12 and P2RY13. These purinergic receptors are critical for microglial communication with neurons and clearance of dead cells. We show that purinergic signaling has a profound effect on microglial motility and process extension and that hyper-expression and response of purinergic signaling in TREM2-knockout microglia may render these cells unable to sense gradients and activate against disease pathology. Furthermore, we highlight that partial inhibition of purinergic receptors will rescue migratory deficits characterized in TREM2-knockout microglia suggesting a therapeutic mechanism (Chapter 3). Taken as a whole, the development of the iPS-Microglia model has yielded critical insight into the interaction of microglial function and the development of Alzheimer’s disease. This model has been readily adapted by many academic labs as well as pharmaceutical companies with the aim of studying human microglial function or other disease risk loci. By further studying AD risk genes in this fashion, we may be able to converge on several mechanisms by which microglial functions are able to attenuate or drive neurodegeneration. Focusing therapeutic efforts on these pathways could lead to the development of immunotherapies for Alzheimer’s disease.