MerTK is a mediator of alpha-synuclein fibril uptake by human microglia

Abstract

Mer tyrosine kinase (MerTK) is a receptor tyrosine kinase that mediates non-inflammatory, homeostatic phagocytosis of diverse types of cellular debris. Highly expressed on the surface of microglial cells, MerTK is of importance in brain development, homeostasis, plasticity and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with ageing and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK-dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway in synucleinopathies was assessed through burden analysis of MERTK variants and analysis of MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the non-inflammatory nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization was not observed to induce secretion of pro-inflammatory cytokines such as IL-6 or TNF, and downmodulated IL-1β secretion from microglia. Burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinson's disease in one of the cohorts (P = 0.002). Despite a small upregulation in MERTK mRNA expression in nigral microglia from Parkinson's disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (P = 5.08 × 10-21), no significant upregulation in MerTK protein expression was observed in human cortex and substantia nigra lysates from Lewy body dementia patients compared to controls. Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein fibrils by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinson's disease. Upregulation of this pathway in synucleinopathies could have therapeutic values in enhancing alpha-synuclein fibril clearance in the brain.

Keywords: MerTK; Parkinson’s disease; alpha-synuclein; microglia; phagocytosis.

Figure 1

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 pHrodo^TM 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.

Figure 2

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.

Figure 3

Effect of UNC2025 on microglial uptake of α-synuclein preformed fibrils. Microglia were pretreated with vehicle, UNC2025 (3 μm) or cytochalasin D (1 μm) for 1 h, then challenged with pHrodo^TM Green-labelled α-synuclein (α-syn) preformed fibrils (PFFs), myelin debris or IgG-opsonized red blood cells (IgG-RBC) for 2 h. (A and B) Quantification of mean green fluorescence intensity (MFI) in induced pluripotent stem cell-derived microglia (iMGL) culture. Paired t-tests were performed; n = 4; *P < 0.05, **P < 0.01. (C) Representative images of iMGL counterstained with Hoechst 33342 (blue). Scale bar = 150 μm. (D) Quantification of mean green fluorescence intensity (MFI) in human primary microglia (hMGL) culture. A paired t-test was performed; n = 5; **P < 0.01. (E) Representative images of hMGL counterstained with Hoechst 33342 (blue). Scale bar = 200 μm.

Figure 4

Effect of AXL/MERTK knockdown on microglial uptake of α-synuclein preformed fibrils. Microglia were transfected with siCON, siAXL and/or siMERTK. (A) Cell surface expression of AXL and MerTK assessed by flow cytometry on live induced pluripotent stem cell-derived microglia (iMGL) 48 h following transfection. Repeated measure one-way ANOVA were performed followed by Dunnett’s post hoc test. Mean ± SEM of n = 5; *P < 0.05, **P < 0.01; MFI = median fluorescence intensity. (B) Quantification of mean green fluorescence intensity (MFI) in induced pluripotent stem cell-derived microglia (iMGL) culture challenged with pHrodo^TM Green-labelled α-synuclein (α-syn) preformed fibrils (PFFs) for 2 h. A repeated measure one-way ANOVA was performed followed by Dunnett’s post hoc test. Mean ± SEM of n = 5; **P < 0.01. (C) Representative images of iMGL challenged with pHrodo^TM Green-labelled α-syn PFFs for 2 h and counterstained with Hoechst 33342 (blue). Scale bar = 150 μm. (D) qRT-PCR assessment of AXL and MERTK expression in fetal microglia (fMGL) 48 h following transfection. Repeated measure one-way ANOVA were performed followed by Dunnett’s post hoc test. Mean ± SEM of n = 3; *P < 0.05, ***P < 0.001. (E) Quantification of mean green fluorescence intensity (MFI) in fMGL culture challenged with pHrodo^TM Green-labelled α-syn PFFs for 2 h. A repeated measure one-way ANOVA was performed followed by Dunnett’s post hoc test. Mean ± SEM of n = 4; *P < 0.05, **P < 0.01. (F) Representative fluorescence images of fMGL challenged with pHrodo^TM Green-labelled α-syn PFFs for 2 h and counterstained with Hoechst 33342 (blue). Scale bar = 200 μm. (G) Viability of fMGL 48 h following transfection. A repeated measure one-way ANOVA was performed followed by Dunnett’s post hoc test. Mean ± SEM of n = 4.

Figure 5

Inflammatory response of human microglia to α-synuclein preformed fibrils. Microglia were treated for 24 h with vehicle, Pam₃CSK₄ (100 ng/ml), lipopolysaccharide (LPS; 100 ng/ml) and/or α-synuclein (α-syn) preformed fibrils (PFFs) (1 μm). (A) Cytokine secretion from induced pluripotent stem cell-derived microglia (iMGL). Repeated measure one-way ANOVA were performed followed by Dunnett’s post hoc test. Mean ± SEM of n = 6; *P < 0.05, **P < 0.01. (B) Cytokine secretion from iMGL pretreated or not with UNC2025 (3 μm) or cytochalasin D (cyt D; 1 μm) for 1 h prior to vehicle/α-syn PFF treatment. Repeated measure one-way ANOVA were performed followed by Sidak’s post hoc test. Mean ± SEM of n = 4; **P < 0.01, ***P < 0.001. (C) Cytokine secretion from hMGL. Paired t-tests were performed. Mean ± SEM of n = 6; *P < 0.05. (D) Cytokine secretion from human primary microglia (hMGL). Repeated measure one-way ANOVA were performed followed by Sidak’s post hoc test. Mean ± SEM of n = 3; **P < 0.01. IL-1β = interleukin-1β; IL-6 = interleukin-6; IL-10 = interleukin-10; TNF = tumor necrosis factor.

Figure 6

MerTK expression in Parkinson’s disease and Lewy body dementia. (A and B) Single-nuclei RNA-sequencing data from Kamath et al. 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.