A common haplotype lowers PU.1 expression in myeloid cells and delays onset of Alzheimer's disease

Abstract

A genome-wide survival analysis of 14,406 Alzheimer's disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and 14 novel loci associated with age at onset. Linkage disequilibrium score regression of 220 cell types implicated the regulation of myeloid gene expression in AD risk. The minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability was enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affected the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.

Figure 1. Genetic and eQTL fine-mapping of AD

(a) The AD-survival association landscape at the CELF1/SPI1 locus resembles that of SPI1 eQTL association in monocytes and macrophages. (b) The AD-survival association landscape resembles that of MS4A4A/MS4A6A eQTL association in monocytes and macrophages.

Figure 2. SPI1 (PU.1) expression and ChIP-Seq analysis

(a) Rs1057233^G is associated with reduced SPI1 expression in a dosage-dependent manner. (b) The mouse homolog of SPI1, Sfpi1 or Spi1, is selectively expressed in microglia and macrophages in mouse brains based on the brain RNA-Seq database^–. OPCs contain 5% microglial contamination. (c) SPI1 (PU.1) binds to the promoter and regulatory regions of CD33, MS4A4A, MS4A6A, TREM2, and TREML2 in human CD14+ monocytes based on ChIP-Seq data.

Figure 3. PU.1 modulates the phagocytic activity of BV2 microglial cells

(a) Phagocytosis of zymosan labeled with red pHrodo fluorescent dye in BV2 cells with transient overexpression and knock-down of PU.1 was measured by flow cytometry. Cytochalasin D treatment was used as a negative control. Mean phagocytic index ± SD is shown: pcDNA 0.7373 ± 0.1772, pcDNA + 1 μM Cyt 0.0236 ± 0.0242, FLAG-PU.1 1.2630 ± 0.2503, shSCR 1.014 ± 0.3656, shA 0.4854 ± 0.1209, shB 0.2579 ± 0.06967, shD 0.2002 ± 0.05168. F(6,13) = 14.82, pcDNA vs pcDNA + 1 μM Cyt P=0.0078, pcDNA vs FLAG-PU.1 P=0.0295, shSCR vs shA P=0.0283, shSCR vs shB P=0.0020, shSCR vs shD P=0.0010, n = 3. (b) BV2 cells were transiently transfected with pcDNA3 (pcDNA) or pcDNA3-FLAG-PU.1 (FLAG-PU.1) and pCMV-GFP as described for phagocytosis assay. Note a shift in mobility of the band for exogenous FLAG-PU.1 in overexpression condition compared to endogenous PU.1 in control. (c) BV2 cells were transiently transfected with shRNA targeting PU.1 (shA, shB and shD) or non-targeting control (shSCR) in pGFP-V-RS vector. GFP⁺ cells were sorted with flow cytometer and analyzed for levels of PU.1 in western blotting in two independent experiments (b, c). (d) Quantification of PU.1 levels in c normalized to β-Actin as a loading control. Values are presented as mean ± SD: shSCR 100 ± 2.10, shA 50.34 ± 9.52, shB 16.03 ± 14.72, shD 12.13 ± 10.03. F(3,6) = 70.55, shSCR vs shA P=0.0014, shSCR vs shB P < 0.0001, shSCR vs shD P < 0.0001, n = 2. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Sidak’s post hoc multiple comparisons test between selected groups.

Figure 4. Genes regulated with differential expression of Spi1 in BV2 microglial cells

qPCR analysis in transiently transfected and sorted GFP⁺ BV2 cells with overexpression (FLAG-PU.1) and knock-down (shB) of Spi1. Changes in expression levels are grouped for genes with altered levels after overexpression and knock-down of Spi1 in (a) and genes with variable expression in BV2 cells either with overexpression (b) or knock-down (c) of Spi1. Values are presented as mean ± SD, n = 4 samples collected independently. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Dunnett’s post hoc multiple comparisons test between experimental and control groups, detailed statistical analysis is reported in Supplementary Table 11.