Microglia are resident innate immune cells of the central nervous system with potent phagocytic and inflammatory capabilities. These cells are crucial in both health and disease, including neurodegenerative diseases. Genetics studies have linked Alzheimer’s disease and other diseases to genes that affect microglial functions, and evidence indicates that microglial phagocytosis and inflammation control neuronal function and survival. Some microglial activity may be beneficial during neurodegeneration, but excessive release of pro-inflammatory cytokines and reactive oxygen/nitrogen species is a hallmark of neurodegenerative disease and can promote neurodegeneration. Additionally, microglial phagocytosis of the protein aggregates driving proteinopathic diseases may be beneficial, but excessive microglial phagocytosis of synapses or neurons may contribute to neurodegeneration. Overall, study of microglia-mediated neurodegeneration is essential when working towards microglia-targeted therapies for dementias, such as Alzheimer’s disease, and other conditions.

In order to study these interactions between microglia and neurons, in vitro model systems are required that include both cell types. Use of these models has often been limited to low-throughput experiments due to practical challenges and the need for careful manual analysis of individual cell types. In this work, I used a neuron-glia coculture model in which inflammatory activation of microglia with lipopolysaccharide (LPS) or other stimuli results in neuronal loss, addressing the above limitations by building an imaging and analysis workflow using recent methods and machine learning tools. This enabled accurate, automated analysis of images from primary cocultures.

Early tests of these higher-throughput assays identified potential roles for urokinase (uPA) and spleen tyrosine kinase (SYK) in microglia-mediated neurodegeneration. uPA is an extracellular protease that may also regulate migration, inflammation and proliferation in association with its receptor uPAR. SYK signals downstream of other microglial cell surface receptors that have been linked to brain diseases, including Alzheimer’s, such as TREM2, CR3 and CSF1R. In this work, uPA and SYK were investigated further using assays for microglial survival, inflammation, and phagocytosis.

Here, I found that uPA may influence inflammatory neurodegeneration, as well as microglial proliferation and phagocytosis, but it remains unclear which of uPA’s many signalling mechanisms drive this. A broad uPA inhibitor affecting both proteolysis and receptor binding prevented LPS-induced, microglia-dependent neuronal loss in cocultures, potentially by depleting microglia and affecting their morphological and phagocytic response to LPS. However, more specific inhibitors of either proteolysis or receptor binding produced only weak effects, if any. Interestingly, exogenous uPA caused proliferation of microglia, suggesting a further role for uPA signalling in these cells. Meanwhile, inhibitor studies found that SYK also regulates neurodegeneration while affecting microglial survival, inflammation, and phagocytosis, which fits with existing knowledge on SYK and its upstream receptors. Finally, a high-content screen for drugs and targets that control microglia-mediated neurodegeneration was developed, using the primary neuron-glia cocultures and new image analysis methods. This novel proof-of-concept validated the use of neuron-glia cocultures in high-content assays when combined with the image analysis developed here. The data identified contributions from steroid hormones, adrenergic receptors, and MAPK signalling (amongst other pathways).

Overall, this work has used updated image analysis methods to investigate the roles of uPA and SYK in microglial biology and microglia-mediated neurodegeneration, as well as showing proof-of-concept for using neuron-glia cocultures in screens for drugs and targets influencing neurodegenerative disease. This adds to the increasing literature on targeting microglia for therapies against neurodegeneration, while validating new assays to study neuron-glia interactions for both target discovery and investigation of the complex mechanisms controlling microglial function.