iPSC-derived microglia carrying the TREM2 R47H/+ mutation are proinflammatory and promote synapse loss

Abstract

Genetic findings have highlighted key roles for microglia in the pathology of neurodegenerative conditions such as Alzheimer's disease (AD). A number of mutations in the microglial protein triggering receptor expressed on myeloid cells 2 (TREM2) have been associated with increased risk for developing AD, most notably the R47H/+ substitution. We employed gene editing and stem cell models to gain insight into the effects of the TREM2 R47H/+ mutation on human-induced pluripotent stem cell-derived microglia. We found transcriptional changes affecting numerous cellular processes, with R47H/+ cells exhibiting a proinflammatory gene expression signature. TREM2 R47H/+ also caused impairments in microglial movement and the uptake of multiple substrates, as well as rendering microglia hyperresponsive to inflammatory stimuli. We developed an in vitro laser-induced injury model in neuron-microglia cocultures, finding an impaired injury response by TREM2 R47H/+ microglia. Furthermore, mouse brains transplanted with TREM2 R47H/+ microglia exhibited reduced synaptic density, with upregulation of multiple complement cascade components in TREM2 R47H/+ microglia suggesting inappropriate synaptic pruning as one potential mechanism. These findings identify a number of potentially detrimental effects of the TREM2 R47H/+ mutation on microglial gene expression and function likely to underlie its association with AD.

Keywords: Alzheimer's disease; induced pluripotent stem cells; inflammation; microglia; neurodegeneration; triggering receptor expressed on myeloid cells 2 (TREM2).

Figure 1.. The TREM2 R47H/+ mutation alters microglial gene expression and induces a pro-inflammatory signature.

(A) Schematic of CRISPR mutagenesis and generation of TREM2 R47H/+ mutant microglia. (B) Diagram of TREM2 protein and downstream effectors. Locations of the R47H/+ mutation is indicated. Chromatogram traces showing successful editing of the R47H sequence in TREM2. (C) Quantification of TREM2 mRNA level in TREM2 R47H/+ and isogenic control microglia. n=6 for each of CTRL-A, CTRL-B, R47H-A and R47H-B. (D) Images and (E) quantification of western blots for different forms of TREM2. TREM2 null iPSC-microglia (see Methods) were included to control for antibody specificity. Actin serves as loading control. n=6 for each of CTRL-A, CTRL-B, R47H-A and R47H-B. (F) Quantification of ELISA for soluble TREM2 levels in culture media from control and TREM2 R47H/+ microglia. n=3 for each of CTRL-A, CTRL-B, R47H-A, R47H-B and null. ***p<0.001. Student’s t-test. (G) PCA plot for control and TREM2 R47H/+ biological and technical replicates. (H) Volcano plot and (I) enriched GO terms for genes differentially expressed between control and TREM2 R47H/+ microglia. (J) Heatmap of the gene expression Z-Score for genes in the ‘Inflammatory response’ GO term in control and TREM2 mutant microglia that showed significantly increased expression in one or both of TREM2 R47H/+ and T66M microglia.

Figure 2.. TREM2 R47H/+ microglia show exaggerated responses to inflammatory stimuli.

(A) Quantification of IL6 ELISAs from culture media following treatment with 100 ng/mL LPS (left) or 50 ng/mL IFNγ (right). n=8 for CTRL-A, CTRL-B, R47H-A and R47H-B for all baseline conditions; n=9 for CTRL-A, CTRL-B, R47H-A and R47H-B for all LPS and IFNγ treated conditions. ***p<0.001. 1-way ANOVA with Sidak’s test. (B) Quantification of TNFα ELISAs from culture media following treatment with 100 ng/mL LPS (left) or 50 ng/mL IFNγ (right). n=8 for CTRL-A, CTRL-B, R47H-A and R47H-B for all baseline conditions; n=9 for CTRL-A, CTRL-B, R47H-A and R47H-B for all LPS and IFNγ treated conditions. *p<0.05, ***p<0.001. 1-way ANOVA with Sidak’s test. (C) Volcano plots for genes differentially expressed by LPS treatment in control and TREM2 R47H/+ microglia. (D) The number of differentially expressed genes and enrichment p-value (inset) for LPS-differentially expressed genes from the ‘Response to LPS’ and ‘Cell activation’ GO terms. (E) Volcano plots for genes differentially expressed by IFNγ treatment in control and TREM2 R47H/+ microglia. (F) The number of differentially expressed genes and enrichment p-value (inset) for IFNγ-differentially expressed genes from the ‘Type II interferon’ and ‘Cell activation’ GO terms. (G) Heatmap of the gene expression Z-Score for selected cytokine and chemokine genes induced by LPS and/or IFNγ in control and TREM2 R47H/+ microglia. Note that one CTRL-A LPS-treated sample (and thus also a corresponding untreated control) was removed from analysis due to poor read quality.

Figure 3.. The TREM2 R47H/+ mutation impairs microglial uptake of brain-relevant substrates.

(A) Images of TREM2 R47H/+ and isogenic control microglia stained for IBA1 (red) and SVP38 (green) following 3 hours uptake of mouse synaptosomes. A no-synaptosome condition using control cells is included to verify SVP38 specificity. Scale bar = 10 μm. (B) Images of TREM2 R47H/+ and control microglia stained for IBA1 (red) and MBP (green) following 3 hours uptake of mouse myelin. A no myelin condition using control cells is included to verify MBP specificity. Scale bar = 10 μm. (C) Images of TREM2 R47H/+ and control microglia stained for IBA1 (red) following 3 hours uptake of Aβ₄₂-488 (green). A no-Aβ₄₂-488 condition using control cells is included to verify signal specificity. Scale bar = 10 μm. (D) Quantification of pHrodo-synaptosome signal in flow cytometry experiments using TREM2 R47H/+ and control microglia. n=6 for CTRL-A, CTRL-B, R47H-A and R47H-B. *p<0.05. Student’s t-test. (E) Quantification of pHrodo-myelin signal in flow cytometry experiments using TREM2 R47H/+ and control microglia. n=7 for CTRL-A, CTRL-B, R47H-A and R47H-B. ***p<0.001. Student’s t-test. (F) Quantification of Aβ₄₂-488 signal in flow cytometry experiments using TREM2 R47H/+ and control microglia. n=6 for CTRL-A, CTRL-B, R47H-A and R47H-B. ***p<0.001. Student’s t-test. (G) Live imaging of microglial Aβ₄₂-488 (green) uptake. Microglia are labeled with Vybrant-DiD (red). Scale bar = 10 μm. (H) Quantification of Aβ₄₂-488 uptake over the 2-hour time series. n=>18 for CTRL-B and R47H-B. Representative of 2 independent experiments, one using each clone ***p<0.001. Student’s t-test.

Figure 4.. TREM2 R47H/+ microglia show an impaired response following injury in an in vitro model.

(A) Schematic of laser injury experiment. (B) Images of NGN2 induced-neuron co-cultures with control and TREM2 R47H/+ microglia stained with β-Tubulin 3 (TUJ1) (red) and IBA1 (green). Scale bar = 50 μm. (C) Images of live NGN2 induced-neuron co-cultures with control and TREM2 R47H/+ microglia. Microglia are labeled with Vybrant-DiO dye (white). Imaged before neuronal injury (left), immediately following neuron injury (middle) and 90 minutes after injury induction (right). Scale bar = 50 μm. (D) Vectors showing movement of tracked cells corresponding to the time courses in (C). Arrows represent direction and net distance traveled by each tracked cell. Scale bar = 50 μm. (E) Quantification of directed movement for each cell toward the center of the injury site over the 90-minute session. >1000 cells tracked for both genotypes. n=7 time series for CTRL-A, 4 for CTRL-B, 3 for R47H-A, 6 for R47H-B. **p<0.01. Student’s t-test. (F) Quantification of total movement (in any direction) for each cell between time points. >25000 cell movement events tracked for both genotypes, from the same injury response time series as in (E). ***p<0.001. Student’s t-test. (G) Quantification of P2RY12 mRNA levels in TREM2 R47H/+ and isogenic control microglia. n=6 for each of CTRL-A, CTRL-B, R47H-A and R47H-B. ***p<0.001. Student’s t-test. (H) Images and (I) quantification of western blots for P2RY12 protein from control and TREM2 R47H/+ microglia. Actin serves as loading control. n=4 for each of CTRL-A, CTRL-B, R47H-A and R47H-B. *p<0.05. Student’s t-test.

Figure 5.. Transplanted TREM2 R47H/+ microglia reduce synapse density in the mouse brain.

(A) Tile-scan image of representative hippocampus following xenotransplant using control iPSC-microglia, stained with DAPI (blue), the neuronal marker β-Tubulin 3 (TUJ1, red), the microglia marker IBA1 (green) and the human nuclei-specific marker STEM101 (white). Scale bar = 100 μm, inset scale bar = 10 μm. (B) Images of transplanted control and R47H/+ microglia in hippocampal area CA1, stained for DAPI (blue), IBA1 (green) STEM101 (red). Scale bar = 50 μm. (C) Quantification of the number of STEM101 positive cells in brain sections from mice transplanted with control and TREM2 R47H/+ microglia. n=6 for CTRL-A, 5 for CTRL-B, 5 for R47H-A, 4 for R47H-B. (D) Images of DAPI (blue) and MBP (red) in hippocampi from mice transplanted with PBS or control or TREM2 R47H/+ microglia. Scale bar = 50 μm. (E) Images of DAPI (blue) and SVP38 (green) in hippocampi from mice transplanted with PBS or control or TREM2 R47H/+ microglia. Scale bar = 50 μm. (F) Quantification of MBP signal in hippocampi of transplanted mice. n=12 from 6 mice for CTRL-A, 10 from 5 mice for CTRL-B, 10 from 5 mice for R47H-A and 8 from 4 mice for R47H-B. (G) Quantification of SVP signal in hippocampi of transplanted mice. n=12 from 6 mice for CTRL-A, 10 from 5 mice for CTRL-B, 10 from 5 mice for R47H-A and 8 from 4 mice for R47H-B. *p<0.05, **p<0.01. 1-way ANOVA with Tukey’s test. (H) High magnification images of PSD95 (green) and VGlut1 (white) in hippocampi of transplanted mice. Scale bar = 5 μm. (I) Processed images of synaptic puncta as defined by co-localization of the pre- and post-synaptic markers VGlut1 and PSD95 corresponding to the images in (H) (see Methods). (J) Quantification of the number of synaptic puncta per field of view from mice injected with PBS, control or TREM2 R47H/+ microglia. n=6 for CTRL-A, 5 for CTRL-B, 5 for R47H-A, 4 for R47H-B. *p<0.05, **p<0.01. 1-way ANOVA with Tukey’s test. (K) Heatmap of the gene expression Z-Score in control and TREM2 mutant microglia for complement system genes from our original RNA-seq experiments. Only complement system genes within the top 10,000 highest expressed genes in our iPSC microglia were included.