miR-155 expression modulates microglia functions in vitro and in the APP/PS1 mouse model of Alzheimer’s disease

Thesis (Ph.D.)--University of Washington, 2019Alzheimer’s Disease (AD) is characterized by the accumulation of extracellular Amyloid-β (Aβ) as well as both CNS and systemic inflammation. Microglia, myeloid cells resident to the CNS, use microRNAs to rapidly respond to inflammatory signals. MicroRNA (miRNA) profiles are altered in the tissue, circulating monocytes, and serum of AD patients. MiR-155 is a specific miRNA that modulates the phasic inflammatory responses of innate immune cells, however its precise role in AD pathogenesis remains unknown. We hypothesized that miR-155 participates in AD pathophysiology by regulating microglia responses to Aβ in vitro and in vivo. In cultured neonatal microglia, we observed that modulation of miR-155 expression impacts the internalization of fibrillar Aβ at the plasma membrane and to low-pH compartments. In mouse models of AD, microglia specific knock-out of miR-155 decreased accumulation of Aβ. In addition, we also observed that microglia specific deletion of miR-155 acutely increased seizures and seizure-related mortality in two mouse models of AD. Reduced Aβ plaques after miR-155 deletion in microglia suggests increased clearance and the hypothesis that in AD models, microglia facilitate epileptogenesis by increased internalization of synaptic material along with removal of Aβ. Together, these findings identify miR-155 expression in microglia as a potential regulator of synaptic homeostasis and microglia responses to Aβ in mouse models of AD

Modulation of Hematopoietic Lineage Specification Impacts TREM2 Expression in Microglia-Like Cells Derived From Human Stem Cells

Microglia are the primary innate immune cell type in the brain, and their dysfunction has been linked to a variety of central nervous system disorders. Human microglia are extraordinarily difficult to obtain for experimental investigation, limiting our ability to study the impact of human genetic variants on microglia functions. Previous studies have reported that microglia-like cells can be derived from human monocytes or pluripotent stem cells. Here, we describe a reproducible relatively simple method for generating microglia-like cells by first deriving embryoid body mesoderm followed by exposure to microglia relevant cytokines. Our approach is based on recent studies demonstrating that microglia originate from primitive yolk sac mesoderm distinct from peripheral macrophages that arise during definitive hematopoiesis. We hypothesized that functional microglia could be derived from human stem cells by employing BMP-4 mesodermal specification followed by exposure to microglia-relevant cytokines, M-CSF, GM-CSF, IL-34, and TGF-β. Using immunofluorescence microscopy, flow cytometry, and reverse transcription polymerase chain reaction, we observed cells with microglia morphology expressing a repertoire of markers associated with microglia: Iba1, CX3CR1, CD11b, TREM2, HexB, and P2RY12. These microglia-like cells maintain myeloid functional phenotypes including Aβ peptide phagocytosis and induction of pro-inflammatory gene expression in response to lipopolysaccharide stimulation. Addition of small molecules BIO and SB431542, previously demonstrated to drive definitive hematopoiesis, resulted in decreased surface expression of TREM2. Together, these data suggest that mesodermal lineage specification followed by cytokine exposure produces microglia-like cells in vitro from human pluripotent stem cells and that this phenotype can be modulated by factors influencing hematopoietic lineage in vitro