Microglia Purinergic Receptor-Mediated Neuroinflammation In Alzheimer’s Disease Neurodegeneration involves a complicated cascade of homeostatic dysfunction that converges on neuron loss and cognitive decline, involving complex immune, metabolic, and cell cell crosstalk pathways. The complicated interplay and heterogeneous nature of these factors in the brain make therapeutic development challenging. Recent advances have placed the immune system as an important driver of neurodegeneration both mechanistically and genetically. Microglia are the professional phagocytes that inhabit the brain and direct these inflammatory pathways, which can have reparative or destructive outcomes on the brain parenchyma. While various genetic risk factors for neurodegeneration reside in microglia, how these trigger and facilitate disease requires further investigation.
In the present dissertation, I investigate inflammatory activation in microglia upon various damage or pathology-associated stimuli by utilizing a primary human monocyte-derived microglia-like cell (MDMi) model from a diverse donor cohort, which allows for the examination of genetically driven differences. I find that MDMi stimulated through ATP-mediated P2RX7 activation display reduced phagocytic function for amyloid beta uptake, and this pathway is also influenced by individual donors’ SPI1 genotype which has been associated with Alzheimer’s disease in previous computational studies. These experiments demonstrate functional outcomes related to AD genetics in immune cells.
Previous computational studies have identified cognitive-decline associated gene modules expressed in human brain tissues from late-stage AD. I conducted in vitro follow up experiments to interrogate these genetic findings which is crucial for validating RNA sequencing data in a biological model. To interrogate differential MDMi inflammatory pathways, I treated cells with the toxic immunostimulatory molecule lipopolysaccharide (LPS), or its non-toxic derivative monophosphoryl lipid A (MPLA) which has positive immune properties currently utilized in vaccine adjuvants. My results indicated that individual gene expression in this module does not shift in a uniform manner upon LPS or MPLA challenge, suggesting more nuanced in vitro interrogation is required to identify conditions propagating this end stage disease phenotype. Microglia serve as the primary immune cells of the brain but also interact closely with astrocytes, large glial cells that facilitate neuronal homeostasis and are central players in AD due to their high apolipoprotein (APOE) production. Given the newly appreciated role of cellular crosstalk in neurological disease pathogenesis, I sought to optimize a protocol for isolation of primary mouse astrocytes for coculture with MDMi and investigation of non-direct cell contact interactions through astrocyte supernatants. Described in this dissertation is my optimized protocol for purified mouse astrocyte isolation from mice expressing humanized APOE2, APOE3, or APOE4.
By developing this model, I was able to discern differential changes to MDMi gene expression in the presence of APOE2, 3, or 4 astrocyte supernatants. Verification of these tools allows further exploration of APOE genotype on glial crosstalk and downstream AD pathology. Overall, this work uncovers important mechanisms of human microglia activation through AD genetics and extracellular P2RX7 receptor behavior. By interrogating these scientific questions in a human microglia model derived from donors of various genetic and age backgrounds, we can assess how real biological variation modulates canonical inflammatory pathways. This adds powerful clinical relevance as AD and other neurodegenerative conditions can present a very heterogenous phenotype pathologically and therefore may require the nuance of more personalized medicine therapeutically.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/z9yj-bj44 |
| Date | January 2024 |
| Creators | Heavener, Kelsey Sarah |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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