Define the RelationSHIP: INPP5D/SHIP1 regulates inflammasome activation in human microglia

Abstract

Microglia and neuroinflammation are implicated in the development and progression of Alzheimer’s disease (AD). However, there are limitations to studying human microglia and utilizing mouse models to study microglia function. Various iPSC-derived microglia-like cell (iMGLs) differentiation protocols have been published within the last few years. Here, one protocol was adapted with modifications to optimize for consistency and yield across genetic backgrounds. With the iMGLs, mix culture models were generated containing iPSC-derived neurons, astrocytes, and microglia. These mixed culture models were then characterized using single cell and single nucleus sequencing to understand then transcriptional changes between a monoculture and mixed culture. To better understand microglia-mediated processes in AD, the function of INPP5D/SHIP1, a gene linked to AD through GWAS was investigated in human brain tissue and iMGL cell cultures. Immunostaining and single nucleus RNA sequencing confirmed that INPP5D expression in the adult human brain is largely restricted to microglia. Examination of prefrontal cortex across a large cohort revealed reduced full length INPP5D protein levels in AD patient brains compared to cognitively normal controls. The functional consequences of reduced INPP5D activity were evaluated in human iMGLs using both pharmacological inhibition of the phosphatase activity of INPP5D and using CRISPR-Cas9 to reduce the number of functional INPP5D copy numbers in half. Unbiased transcriptional and proteomic profiling of these iMGLs suggested an upregulation of innate immune signaling pathways, lower levels of scavenger receptors, and altered inflammasome signaling with INPP5D reduction. INPP5D inhibition induced the secretion of IL-1ß and IL-18, further implicating inflammasome activation. Inflammasome activation was confirmed through visualization of inflammasome formation through ASC immunostaining in INPP5D-inhibited iMGLs, increased cleaved caspase-1, and through rescue of elevated IL-1ß and IL-18 with caspase-1 and NLRP3 inhibitors. In the INPP5D het iMGLs, an increased secretion of both IL-1ß and IL-18 were observed, which was rescued with NLRP3 inhibitor treatment. This work implicates INPP5D as a regulator of inflammasome signaling in human microglia and provides further insight as to how INPP5D function could be linked to the development of AD.