High throughput identification of genetic regulators of microglial inflammatory processes in Alzheimer's disease.

UNLABELLED: Greater than a hundred genetic risk factors for Alzheimer’s disease (AD) have been identified by genome-wide association studies (GWAS), many of which are primarily expressed in microglia. However, the pathogenic role for most of them remains unclear. We sought to assess at scale how AD GWAS hits influence human microglial inflammatory responses. Thus, we conducted CRISPR inhibition (CRISPRi) screens of 119 AD GWAS hits in hiPSC-derived microglia (iMGLs), with reactive oxygen species (ROS) produced in response to the viral mimic poly(I:C) as a readout. Top hits that either decreased or increased ROS in response to poly(I:C) when knocked down were then interrogated in CROP-seq experiments, where CRISPRi was combined with single cell RNA-sequencing (scRNA-seq). These analyses identified 9 unique microglial clusters, including a poly(I:C)-driven inflammatory cluster (2). Emerging evidence supports a pathogenic role of viral infections in AD and our scRNA-seq showed significant overlap with AD-relevant microglial clusters. Knockdown (KD) of two hit genes, MS4A6A and EED, which lead to high levels of ROS in the presence of poly(I:C), increased the proportion of inflammatory cluster 2 cells and led to functionally related changes in gene expression. In addition, KD of MS4A6A reduced the proportion of iMGLs in the disease-associated microglia (DAM) cluster under all conditions, suggesting that MS4A6A may modulate the DAM response. In contrast, KD of hit genes, INPP5D or RABEP1 which led to low levels of ROS in the presence of poly(I:C), did not significantly affect the proportion of cells in inflammatory cluster 2 but rather shaped the inflammatory response. This included upregulation of a poly(I:C)-independent HLA inflammatory cluster (6) by INPP5D KD under all conditions. Despite INPP5D or RABEP1 being involved in disparate biological processes, their perturbation led to similar changes in gene expression, which included changes in genes related to metabolism such as oxidative phosphorylation, suggesting a shared conversion point by which AD GWAS hits affect cell state. Overall, our data begin to elucidate the functional roles played by various AD GWAS hits, including how they regulate and shape inflammatory responses such as those observed in AD. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12974-025-03562-9.