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. 2017 Jul;18(7):1186-1198.
doi: 10.15252/embr.201743922. Epub 2017 May 8.

TREM2 Deficiency Impairs Chemotaxis and Microglial Responses to Neuronal Injury

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Free PMC article

TREM2 Deficiency Impairs Chemotaxis and Microglial Responses to Neuronal Injury

Fargol Mazaheri et al. EMBO Rep. .
Free PMC article

Abstract

Sequence variations in the triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to an increased risk for neurodegenerative disorders such as Alzheimer's disease and frontotemporal lobar degeneration. In the brain, TREM2 is predominantly expressed in microglia. Several disease-associated TREM2 variants result in a loss of function by reducing microglial phagocytosis, impairing lipid sensing, preventing binding of lipoproteins and affecting shielding of amyloid plaques. We here investigate the consequences of TREM2 loss of function on the microglia transcriptome. Among the differentially expressed messenger RNAs in wild-type and Trem2-/- microglia, gene clusters are identified which represent gene functions in chemotaxis, migration and mobility. Functional analyses confirm that loss of TREM2 impairs appropriate microglial responses to injury and signals that normally evoke chemotaxis on multiple levels. In an ex vivo organotypic brain slice assay, absence of TREM2 reduces the distance migrated by microglia. Moreover, migration towards defined chemo-attractants is reduced upon ablation of TREM2 and can be rescued by TREM2 re-expression. In vivo, microglia lacking TREM2 migrate less towards injected apoptotic neurons, and outgrowth of microglial processes towards sites of laser-induced focal CNS damage in the somatosensory cortex is slowed. The apparent lack of chemotactic stimulation upon depletion of TREM2 is consistent with a stable expression profile of genes characterizing the homoeostatic signature of microglia.

Keywords: Alzheimer's disease; TREM2; chemotaxis; microglia; neurodegeneration.

Figures

Figure 1
Figure 1. Disturbed expression of gene clusters involved in chemotaxis in the absence of Trem2

Scatter plot of the normalized and averaged log2 gene expression level of Trem2−/− mice (KO) against wild‐type (WT) samples. The upper red line corresponds to a fold change of 2 while the lower red line indicates a fold change of 0.5.

Gene expression profile of the normalized log2 transformed levels (for genes with P < 0.05 after Student's t‐test and FDR < 0.158) from the NanoString‐based chips represented by a heat map. For generation of the heat map, the heatmap.2 function within the gplots package of R statistical software was used. Hierarchical clustering by the hclust function (method = “complete”) was applied to group experimental conditions (columns) as well as intensities of genes (rows) for the heat map. Rows are scaled and represented as z‐score. A dendrogram is shown only for the columns. For the assignment of genes to functional categories at the right site of the heat map, the enrichment analysis of the GeneRanker was used but also a pathway analysis with help of the Pathway Studio software (Copyright 2014, Elsevier) as well as manual literature searches. WT N = 5, Trem2−/− N = 7.

Source data are available online for this figure.
Figure EV1
Figure EV1. Heat map of significantly expressed genes
The heat map is based on normalized log2 expression levels of 57 significantly changed genes (FDR < 0.05) from the NanoString‐based chips. For generation of the heat map, the heatmap.2 function within the gplots package of R statistical software was used. Hierarchical clustering by the hclust function (method = “complete”) was applied to group experimental conditions (columns) as well as intensities of genes (rows). Rows were scaled and represented as z‐score. A dendrogram is shown for the samples (columns) as well as for gene expression levels (rows). Genes which show decreased expression levels in Trem2−/− mice (KO) compared to wild‐type mice (WT) and known to be involved in the functional categories chemotaxis and migration are highlighted by an asterisk.
Figure 2
Figure 2. Migration deficits of Trem2−/− microglia in an ex vivo assay

Cx3cr1‐GFP‐ (green) and CD68 (red)‐positive young microglial cells migrate towards old tissue in co‐cultures of old APP/PS1 and young Cx3cr1+/GFP brain slices (7 DIV). DAPI (blue) was used to counterstain the nuclei.

CD68‐positive cells migrate out of WT and Trem2−/− brain slices cultured alone (7 DIV). Trem2−/− microglia migrate much shorter distances compared to WT.

Enhanced migration of CD68‐positive microglia in co‐cultures of old APP/PS1 and young WT brain slices is abolished in the absence of Trem2.

Quantitative analysis of the distance migrated by WT or Trem2−/− CD68‐positive cells in (B) and (C). N = 4 WT and 4 Trem2−/− mice, from each mouse, two slices were prepared, and on average, 230–550 cells per genotype were analysed. Data are presented as mean ± s.e.m., ***P < 0.001 by using a one‐way analysis of variance with Tukey post hoc comparison. P‐values: WT young only versus Trem2−/− young only: ***P = 0.001; WT young only versus WT co‐culture: ***P = 0.001; Trem2−/− young only versus Trem2−/− co‐culture: P = 0.8999; WT co‐culture versus Trem2−/− co‐culture: ***P = 0.001.

Data information: Scale bars, 100 μm. For (A–C), white arrows indicate migration of microglia cells.
Figure 3
Figure 3. Migration of N9 microglia cells towards chemotactic stimuli in a transwell assay

N9 cell migration in the presence and absence of the general tyrosine kinase inhibitor genistein and the Syk‐selective inhibitor piceatannol. P = 0.00001, N = 12 for each condition.

Migration capacity of wild‐type N9 microglia (WT) or Trem2‐deficient N9 microglia (N9 mu) towards 100 ng/ml recombinant mouse Ccl2. P = 0.00041, N = 18 for WT and N = 18 for Trem2−/−.

Migration capacity of wild‐type N9 microglia (WT) or Trem2‐deficient N9 microglia (N9 mu) towards 25 ng/ml recombinant mouse C5a. P = 0.00001, N = 19 for WT and N = 17 for Trem2−/−.

The migration deficit of Trem2‐deficient N9 cells is fully restored upon expression of mouse Trem2. P = 0.00001, N = 6.

Western blot analysis of lysates (upper panel) and media (lower panel) from WT, mutant and N9 cells re‐expressing TREM2 using the anti‐murine TREM2 antibody 5F4 raised against the murine TREM2 extracellular domain. sTREM2, soluble TREM2. Calnexin (middle panel), loading control.

Data information: In panels (A–D), DAPI‐stained cells are shown on the left and quantitation is shown on the right side. Values are normalized to WT N9 cells and represent mean ± s.e.m., normalized to the WT N9 mean. P‐values were determined according to a two‐tailed Student's t‐test. Scale bars, 200 μm. Source data are available online for this figure.
Figure 4
Figure 4. Reduced migration of Trem2‐deficient microglia towards apoptotic neurons in vivo

Migration of microglia towards injected Alexa 488‐labelled apoptotic neurons (green) in WT (A) and Trem2−/− (B) mouse brains. Microglial cells are visualized with the anti‐P2ry12 antibody (red) and nuclei with DAPI (blue). Dashed line indicates injection channel. Scale bar, 25 μm.

Quantification of the number of microglia (labelled with white dots) clustered around the injection site. N = 6 individual mice per genotype. **P = 0.004 according to a two‐tailed Student's t‐test. The scatter plot shows data points from individual experiments. Horizontal lines represent mean values, error bars indicate standard deviations.

Source data are available online for this figure.
Figure 5
Figure 5. Impaired wound response in Trem2‐deficient microglia in vivo

Early response of Iba1‐GFP WT or Trem2−/− microglia towards a laser lesion (indicated in magenta). White arrows point to the microglial processes. Time between each frame is 278 seconds. Scale bar, 25 μm.

Areas used to quantitate process outgrowth speeds.

Quantification of the speed of process outgrowth. Data are presented as mean ± s.e.m., **P = 0.0016 according to Mann–Whitney U‐test (N = number of injuries: WT = 13 injuries in five mice; Trem2−/− = 8 injuries in three mice).

Source data are available online for this figure.
Figure 6
Figure 6. Sustained expression of microglial homoeostatic genes in the absence of Trem2

Heat map demonstrating altered expression levels of microglial genes in FACS‐sorted microglia from WT and Trem2−/− mice as determined by NanoString analysis. Heat map and hierarchical clustering of microglia were analysed with the MG400 chip. Results were log‐transformed, normalized and centred, and populations and genes were clustered by Pearson correlation using MeV v4.6.

Expression levels of homoeostatic microglial genes in FACS‐sorted microglia derived from WT and Trem2−/− mice as determined by NanoString analysis. Bars show levels of mRNA transcripts in the respective models normalized to six independent housekeeping genes. Values indicate mean ± s.e.m. per 100 ng of total RNA. *P < 0.05, **P < 0.01, ***P < 0.001 by two‐tailed Student's t‐test. P‐values: Trem2−/− versus WT, Bin1 *P = 0.012, Cx3cr1 *P = 0.012, Il10ra ***P = 0.001, Mertk ***P = 0.002, Olfml3 **P = 0.005, P2ry12 *P = 0.011, Sall1 **P = 0.005, Tgfbr1 **P = 0.006, Tmem119 *P = 0.02, Csf1r P = 0.081, Gpr34 P = 0.505, Rhob P = 0.164, Siglech P = 0.062. Data were generated from the same samples as in Fig 1; WT N = 5, Trem2−/− N = 7.

Source data are available online for this figure.

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