Probing Heterogeneity of Alzheimer's Disease Using iPSCs

PROJECT SUMMARY There is an increasing focus on understanding the role of microglia in neurodegenerative disorders including late-onset Alzheimer’s disease (LOAD), a disease defined by the accumulation of amyloid beta rich plaques and neurofibrillary tangles containing tau. Microglia are proposed to play a variety of critical roles during disease progression including synaptic engulfment, cytokine release, and phagocytosis of amyloid beta (Aβ). Further, recent GWAS studies have implicated innate immune processes in LOAD, supporting the importance of understanding the neuroimmunological processes underlying the risk and progression of AD. All recent large- scale GWAS have identified SNPs at the INPP5D locus that are significantly associated with AD. INPP5D expression is largely restricted to microglial cells in the human adult brain and we have confirmed previous findings that RNA levels of INPP5D are elevated in AD brain. However, through quantitative western blotting, we show that protein levels of full length, water-soluble INPP5D are reduced in AD brain. To study the functional consequences of reduced INPP5D, we used both pharmacological inhibition and CRISPR-Cas9 genome engineering to lower INPP5D activity in human iPSC-derived microglia. Through unbiased RNA and proteomic profiling and a series of pharmacological manipulations, we demonstrated that reduction of INPP5D activity induces changes in immune signaling and, more specifically, the activation of the inflammasome. These studies have raised important questions that we aim to address in this proposal regarding the constitution of INPP5D in microglia in the Alzheimer’s brain (aim 1), the molecular mechanism(s) linking INPP5D and inflammasome activation (aim 2), and the functional consequences of loss of INPP5D activity and inflammasome activation in microglia on astrocytes and neurons (aim 3). In aim 1, we will utilize quantitative immunostaining, sequential extraction and western blotting, and ELISA to deeply interrogate INPP5D levels and markers of inflammasome activation across a large cohort of human brain samples and iPSC-derived microglia cultures. Aim 2 interrogates candidate pathways that may link INPP5D activity with inflammasome activation that arose from our preliminary analyses. The first of these involves upregulation of PLA2G7, second through dysregulation of scavenger receptors and the third through disruption of lysosome function. Finally, in aim 3 we utilize co-culture models of iPSC-derived neurons, astrocytes and microglia and a conditional INPP5D knock out mouse model to determine the consequences of reduction of INPP5D levels in microglia on neuron and astrocyte biology in the context of AD relevant environments. Together, these studies will provide important new insights into our understanding of the fundamental role of INPP5D in microglia in health and disease, regulation of inflammasome activation in human microglia, and the consequences of sub-lytic inflammasome activation in microglia on neuron and astrocyte biology.