Modest changes in Spi1 dosage reveal the potential for altered microglial

Genetic association studies have identified multiple variants at the SPI1locus that modify risk and age of onset for Alzheimer’s Disease (AD). Reports linking risk variants to gene expression suggest that variants denoting higher SPI1 expression are likely to have an earlier AD onset, and several other AD risk genes contain PU.1 binding sites in the promoter region. Overall, this suggests the level of SPI1 may alter microglial phenotype potentially impacting AD. This study determined how the microglial transcriptome was altered following modest changes to Spi1expression in primary mouse microglia. RNA-sequencing was performed on microglia with reduced or increased Spi1/PU.1 expression to provide an unbiased approach to determine transcriptomic changes affected bySpi1. In summary, a reduction in microglial Spi1 resulted in the dysregulation of transcripts encoding proteins involved in DNA replication pathways while an increased Spi1 results in an upregulation of genes associated with immune response pathways.

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Fig: Results of RNA-Seq experiment in primary mixed-glial cultures (A) Volcano plots summarising the distribution of genes in the Spi1 knock-down (purple) and Spi1 over-expression (orange). In the Spi1 knock-down dataset a 1462 genes were down-regulated (grey background) and 1615 up-regulated with a pvalue of ≤ 0.05 (solid lines), as indicated by the numbers on the graph. In theSpi1 over-expression dataset the majority of the genes, 284, were up-regulated and 62 genes had down-regulated expression. (B) Genes that were significantly changed in both the Spi1 knock down (KD, purple) and the Spi1 over expression (OE, orange) using a p ≤ 0.05 threshold. A Plot of adjusted p values from all genes in both datasets. Genes that were below the p ≤ 0.05 threshold in the Spi1knock-down dataset are highlighted in purple, those that were below the p ≤ 0.05 cut-off in the Spi1 over-expression are within the orange bar. In the bottom left corner the red line surrounds the 196 genes that were significantly changed in both datasets. (C) Venn diagrams summarising the gene expression that were significantly changed (p ≤ 0.05) in either the Spi1 knock-down dataset (purple), the Spi1 over-expression dataset (orange) or changed in both datasets (red). Of interest were the genes with expression that appears to be sensitive to the Spi1dose in microglia, namely the 194 genes that appear to be expressed relative to the dose of Spi1. (D) The log10 number of Spi1 mRNA fragments per Kilobase of transcript per Million mapped reads (FPKM) shows that Spi1 was lower in Spi1shRNA infected microglia than in control shRNA infected microglia (One-tailed Unpaired T-test on the log10 transformed data, p = 0.0166, n = 4 for control shRNA and n = 3 for Spi1 shRNA). PU.1 protein expression was normalised to the non-infected (NI) samples in each experiment (as described in methods). Microglia infected with the Spi1 shRNA (solid purple) have reduced PU.1 expression compared to cells infected with a control shRNA (lined purple) (One-tailed Paired T-test, p = 0.0013, n = 3 per group). (E) Spi1 mRNA expression (FPKM) was increased in the samples infected with the Spi1 over-expression construct (filled orange) compared to empty vector control samples (outline orange), (One-tailed Paired T-test on the Log10 transformed data, p = 0.0013, n = 4 per group).The Spi1 over-expression virus (Spi1 pSIEW) increased PU.1 protein expression in microglia by roughly half compared to cells infected with an empty vector control (lined orange). One-tailed Paired T-test, p = 0.0131, n = 4 per group) (D–E) Each dot represents the value from one biological replicate, the means are indicated by the horizontal lines and the error bars display the standard deviation about the mean. All experiments were performed using mixed glia cultures from 8-week-old female C57BL/6J mice. Figures (A–C) were made in GraphPad PRISM 6 (version 3.07) and (D–E) in GraphPad PRISM 8 (version 8.4.3; both GraphPad Software, Inc.).

Additionally, a subset of 194 Spi1 dose-sensitive genes was identified and pathway analysis suggests that several innate immune and interferon response pathways are impacted by the concentration of Spi1. Together these results suggestSpi1 levels can alter the microglial transcriptome and suggests interferon pathways may be altered in individuals with AD related Spi1 risk SNPs.

Jones, R.E., Andrews, R., Holmans, P. et al. Modest changes in Spi1dosage reveal the potential for altered microglial function as seen in Alzheimer’s disease. Sci Rep 11, 14935 (2021). https://doi.org/10.1038/s41598-021-94324-z

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