Utilization of a Human Induced Pluripotent Stem Cell Model of Microglial Fractalkine Signaling Dysfunction in Alzheimer's Disease

Abstract

Dysfunctional microglial activity has been identified as a potential mechanism leading to accumulation of amyloid beta and pTau and subsequent neurodegeneration in Alzheimer's Disease (AD). The CX3CR1/fractalkine axis serves as a mechanism for bidirectional communication between microglia and neurons, respectively, to promote an anti-inflammatory microglial state. Previous studies have demonstrated that deficiency in CX3CR1 signaling leads microglia to develop a more pro-inflammatory phenotype, induces phagocytic deficits, and increases susceptibility of neurons to cell death. The CX3CR1-V249I polymorphism was recently identified as a potential risk allele for AD. However, the role offractalk:ine dysfunction in human cells and the mechanisms by which microglia with the CX3CR1-V249I SNP contribute to neurodegeneration remain unclear. To address this shortcoming, we utilized human induced pluripotent stem cells and CRISPR/Cas9 technology to elucidate the effects of the V249I polymorphism on human microglia-like cells (hMGLs). We demonstrate effective differentiation from paired isogenic control and CX3CR1-V249I backgrounds into hMGLs. Transcriptional profiling via RNA-seq analyses demonstrated alterations in pathways such as apoptosis, toll-like receptor signaling, and the inflammasome due to the CX3CR1-V249I SNP. Both heterozygous and homozygous microglia bearing the V249I allele demonstrated decreased phagocytosis of amyloid beta in vitro compared to controls, with this effect modulated by the presence of fractalkine in heterozygous but not homozygous V249I hMGLs. Both heterozygous and homozygous V249I microglia exhibited increased stress-induced cell death compared to controls, with homozygous hMGLs demonstrating increased cell death at earlier time points. These findings suggest that the CX3CR1-V249I polymorphism may confer a dysfunctional microglia phenotype, which may subsequently contribute to neuronal dysfunction. Further investigation of microglia in neuron co-culture models demonstrated that the CX3CR1-V249I variant conferred altered neuronal excitability. Collectively, the results of this study highlight the importance of understanding CX3CR1 function in AD pathology to identify targetable mechanisms for intervention.