Fig 1: Loss of Mertk rescues microglia signature in Grn knock-out mice, whereas loss of Axl worsens it.We sequenced 166,224 single-nuclei across all mice. A UMAP plot of all single nuclei and their annotated cell types with their top marker genes. OPC = oligodendrocyte precursor cells. B Dot plot showing expression of cell marker genes for each cell type. C UMAP plots by genotype. Note cluster 3 neurons are lost in Axl−/− and Grn−/−;Axl−/−. D, E UMAP plot of all 4668 microglial cells analyzed and reclustered into homeostatic (cluster 1, salmon) and disease-associated (cluster 2, blue) cells. E UMAP of microglia by genotype. F Proportion of cells in clusters 1 and 2 by genotype. ****p < 0.0001, two-sided Fisher’s exact test. ns = not significant. G Volcano plot of DEGs defining cluster 2 compared to cluster 1. Cutoffs for inclusion: |Log2FC| > 0.1 and FDR < 0.05. H Feature plots of transcript expression overlaid onto UMAP of all microglial cells, for example homeostatic markers (P2ry12, Cx3cr1) and disease-associated markers (Apoe, Gpnmb) markers. Colored scale bar denotes normalized expression level. I Scatterplot showing the correlation between our comparison of DEGs in the microglial cluster 2 versus cluster 1 (x axis) and a previously published44 comparison of disease-associated microglia (DAM/MGnD) versus homeostatic microglia (y axis). R = 0.5979, ****p < 2.2 × 10−16, Pearson’s correlation. J Dot plot showing that disease-associated genes upregulated in Grn−/− relative to WT microglia are further upregulated in Grn−/−;Axl−/−. This effect is driven by interaction between loss of Grn and loss of Axl as Axl−/− does not show this profile. By contrast, Grn−/−;Mertk−/− rescues the Grn−/− induced profile. Expression average denoted by color, percent of microglia expressing this marker denoted by circle size.
Fig 2: Progranulin regulates MERTK and AXL RNA expression but binds neither MERTK nor AXL proteins.A The upregulation of MERTK RNA in GRN−/− relative to control human induced microglia (Sidak’s multiple comparison test p < 0.0001) was normalized by exogenous administration of 100 nM progranulin (Sidak’s multiple comparison test p < 0.98). B While there is no baseline difference in AXL mRNA expression in untreated WT or GRN−/− microglia (Sidak’s multiple comparison test p = 0.91), 100 nM exogenous progranulin significantly decreased AXL RNA expression in GRN−/− but not WT human induced microglia (Sidak’s multiple comparison test p < 0.05). C, D We found no interaction of PGRN over a wide range of concentrations with either MERTK or AXL by surface plasmon resonance (SPR). E, F As controls, we show that Gas6, a known binder of MerTK and Axl, binds these receptors with 27.7 nM and 193 pM affinities respectively and theoretical Rmax of 135 RU. Data representative of three experiments (red) were fit to a 1:1 kinetic model (black) to obtain binding parameters. We also find no binding of PGRN to GAS6 after immobilizing GAS6 with an anti-GAS6 antibody (G) or H6-tagged-GAS6 antibody (H), which anchor Gas6 at different sites. The inverted sensograms in (H) suggests nonspecific binding between PGRN and the Anti-H6 reference surface. Error bars = SEM. *p < 0.05, ***p < 0.001.
Fig 3: Dysregulated phagocytic pathways in post-mortem GRN+/− FTD microglia.A Sex and genotypes of human donors used for snRNA-seq. 108,227 nuclei were isolated from the superior frontal cortex of patients with GRN+/− FTD (n = 8) and GRN+/+ controls without neurologic disease (Ctrl; n = 7). B Uniform Manifold Approximation and Projection (UMAP) plot of all single nuclei and their annotated cell types. OPC = oligodendrocyte precursor cells. C Proportion of cell types by genotype. D Dot plot showing expression of cell marker genes for each cell type. E Number of up-regulated (red) and down-regulated (blue) differentially expressed genes (DEGs) between GRN-FTD versus non-FTD control samples for each cell type. FDR < 0.05, log2FC cutoff 0.1. F Volcano plot of significant DEGs (FDR < 0.05) between GRN-FTD and non-FTD control samples in microglia. G Bar plots of Ingenuity Canonical pathways enriched in DEGs identified in (F). P-values were calculated using right-tailed Fisher’s exact test with threshold of significant enrichment as p-value ≤ 0.05 (indicated by a dashed line of −log (p-value) = 1.3). H Violin plot showing expression of MERTK and AXL in all cell types.
Fig 4: CSF MERTK but not AXL is associated with decreased CSF PGRN and is decreased in symptomatic GRN-FTD patients.A, B Pearson correlations of CSF PGRN with CSF MERTK (A; n = 116) and CSF AXL (B; n = 70) shows that CSF MERTK but not AXL is associated with lower CSF PGRN. Data points are symbol and color-coded by GRN mutation carriers (purple triangle) and controls (CTL; blue circle). C CSF MERTK is significantly lower in symptomatic (Sx) but not presymptomatic (PreSx) GRN, C9orf72 and MAPT mutation carriers compared to controls (CTL) (Kruskal-Wallis p < 0.0001; Dunn’s multiple comparisons test vs CTL **p < 0.01, ***p < 0.001). D CSF AXL is not significantly different between groups (Kruskal-Wallis p = 0.66). Horizontal dotted line represents median CSF MERTK or AXL levels in CTL. Units for all aptamers are expressed as log2 RFU (relative fluorescence unit). Box plots represent the median and 25th and 75th percentiles, and box hinges represent the interquartile range of the two middle quartiles within a group. Min and max data points define the extent of whiskers (error bars).
Fig 5: Ablation of Mertk, but not Axl, rescues thalamic microgliosis in Grn−/− mouse.A- F Representative hemibrain images strained with anti-Iba1 microglia marker from the 6 genotypes with thalamic regions outlined. Inset (blue box) is depicted to illustrate differences in microglia morphology. G Grn−/− increased Iba1+ microglia count compared to WT (Tukey’s multiple comparison test p < 0.001) whereas Grn−/−;Mertk−/− restored microglia counts to WT baseline (Tukey’s multiple comparison test, p = 0.14). Grn−/−;Axl−/− exacerbated microgliosis compared Grn−/− (one-way ANOVA F(5,18) = 24, p < 0.001, Tukey’s multiple comparison test, Grn−/−; Axl−/− vs WT p < 0.0001, Grn−/−; Axl−/− vs Grn−/− p < 0.05). ns not significant. Error bars = SEM.
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