Fig 2: TAM receptors expression and α-synuclein preformed fibril uptake by human microglia. All human primary microglia (hMGL) data are from in vitro hMGL, unless otherwise specified. (A and B) RNA-sequencing (RNAseq) assessment of TYRO3, AXL and MERTK expression in hMGL and induced pluripotent stem cell-derived microglia (iMGL). A one-way ANOVA with Sidak’s post hoc test was performed for pairwise comparisons between hMGL and iMGL in A. TPM = transcripts per million. Mean ± SEM of n = 4; ***P < 0.001. A two-way ANOVA with Dunnett’s post hoc test was performed to compare in vitro hMGL and iMGL to ex vivo hMGL in B. Mean ± SEM of n = 4; ***P < 0.001 versus ex vivo hMGL. (C) Western blot of MerTK, AXL and GAPDH. (D) Quantification of AXL and MerTK expression using GAPDH as a loading control. t-tests were performed. Mean ± SEM of n = 3; *P < 0.05. (E–I) hMGL and iMGL were challenged with pHrodoTM Green-labelled α-synuclein (α-syn) preformed fibrils (PFFs) for 2 h. (E) Merged phase contrast and green fluorescence images. Scale bar = 50 μm. (F) Quantification of mean green fluorescence intensity (MFI) in hMGL culture. A paired t-test was performed; n = 5; *P < 0.05; a.u. = arbitrary unit. (G) Representative fluorescence images of hMGL counterstained with Hoechst 33342 (blue). Scale bar = 500 μm. (H) Quantification of MFI in iMGL culture. A paired t-test was performed; n = 4; *P < 0.05; a.u. = arbitrary unit. (I) Representative fluorescence images of iMGL counterstained with Hoechst 33342 (blue). Scale bar = 300 μm.

Fig 3: Interaction between α-synuclein preformed fibrils and MerTK. Induced pluripotent stem cell-derived microglia (iMGL) were incubated with α-synuclein (α-syn) preformed fibrils (PFFs) or vehicle on ice for 30 min and a proximity ligation assay (PLA) was performed. (A) Representative flow cytometry plots. (B) Quantification of the PLA signals. A repeated measure one-way ANOVA was performed followed by Sidak’s post hoc test. Mean ± SEM of n = 3; **P < 0.01.

Fig 4: MerTK expression in Parkinson’s disease and Lewy body dementia. (A and B) Single-nuclei RNA-sequencing data from Kamath et al.47 were used to compare microglia from substantia nigra of seven Parkinson’s disease (PD) patients and three Lewy body dementia (LBD) patients versus eight control (CTL) donors. (A) Dot plot depicting examples of identified differentially expressed genes (DEGs) between PD + LBD and CTL. (B) Heatmap showing the average expression of genes encoding TAM receptors and their activating ligands. *P < 0.05 in DEG analysis. (C and D) Western blotting using human substantia nigra tissues of LBD and CTL donors. (C) Representative blots. Proteins were extracted in the presence of SDS and urea to solubilize α-synuclein (α-syn) aggregates. (D) Quantification of MerTK, CSF1R, phosphorylated α-syn (S129p) and α-syn expression. Mann–Whitney tests were performed; n = 9; ns = non-significant; *P < 0.05. (E and F) Western blotting using human cortex of LBD and CTL donors. (E) Representative blots. Proteins were extracted in the presence of SDS and urea to solubilize α-syn aggregates. (F) Quantification of MerTK, CSF1R, phosphorylated α-syn (S129p) and α-syn expression. Mann–Whitney tests were performed; n = 5; ns = non-significant; **P < 0.01.