Investigating the effects of APOE and APOE-related AD risk genes on human microglia activity and lipid metabolism in aging and disease

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SUMMARY Understanding the molecular and cellular mechanisms linking Alzheimer’s disease (AD) and aging-associated genetic variants to reduced risk and increased longevity is a critical bottleneck for the translation of genetic findings into effective therapeutics. Our proposal aims to fill this gap by focusing on biological processes (cholesterol/lipid clearance), cell types (microglia), and genes implicated by human genetics. The brain is the most cholesterol/lipid-rich organ in the body. Hence, tissue damage in the aged/diseased brain produces large amounts of cholesterol/lipid-rich cellular debris. Macrophages (MΦ, like microglia in the brain) are cell types specialized in maintenance and restoration of tissue homeostasis, mainly via their ability to 1) phagocytose and clear apoptotic cells and other cellular debris (efferocytosis) and 2) orchestrate an innate immune response. When challenged with cholesterol/lipid-rich cellular debris, MΦ upregulate the expression of several genes involved in cholesterol/lipid metabolism, efferocytosis and AD. This gene expression profile is often referred to as DAM/LAM (for disease-associated microglia/ lipid-associated macrophages). The most upregulated DAM/LAM gene is Apolipoprotein E (APOE), a major gene for cholesterol metabolism and AD, and one of very few genes associated with longevity. We and others showed that common non-coding AD risk alleles identified in genome-wide association studies (GWAS) are enriched in MΦ-specific enhancers, strongly implicating these cell types and MΦ-specific gene expression regulation (and transcription factors like SPI1/PU.1) in the etiology of AD. Pathway analysis of these alleles also implicated cholesterol metabolism, phagocytosis, and innate immunity. Similarly, rare coding AD risk alleles impact the structure/function of genes highly or specifically expressed in MΦ and with critical roles in efferocytosis (e.g., TREM2, ABCA7, ABI3). Our main hypothesis is that AD/aging-associated genetic variants modulate risk/longevity by affecting expression or structure and thus activity of genes involved in phagocytic clearance of cholesterol/lipid-rich cellular debris, thereby impairing microglia ability to maintain brain tissue homeostasis during aging and disease. Our approach is to use isogenic human iPSC-derived microglia (iMGL) carrying AD/aging-associated genotypes and gene modifications to assess their impact on microglial lipid metabolism, gene expression, and functional roles, at baseline and in aging/disease-relevant contexts, both in vitro (2D and 3D cultures) and in vivo (mouse brain xenografts43). In particular, we will focus on APOE genotypes (Aim 1), BHLHE40/41 and NR1H2/3 transcription factors (Aim 2), and their therapeutic interactions (Aim 3).