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Fig. 3 | Molecular Neurodegeneration

Fig. 3

From: Pathological α-synuclein recruits LRRK2 expressing pro-inflammatory monocytes to the brain

Fig. 3

Microglia-like polarization suppresses α-synuclein fibril induction of LRRK2 expression and activity. A Flow chart of PBMC differentiation into three monocyte lineages, with validation (mRNA-seq and fluorescent imaging of canonical markers), and treatment timeline. The JAK inhibitor (AZD1480, 100 nM) was applied 1 h before human ɑ-synuclein fibrils (1 μg・mL− 1), and cell lysates were collected 48 h later. B Representative phase-contrast images of human monocytes differentiated to dendritic-like cells (GMCSF), macrophage-like cells (MCSF), and microglia-like cells (MDMi). C Principal component analysis of the resultant transcriptomes (circle: female, triangle: male, orange: GMCSF, cyan: MCSF, pink: MDMi). D Representative immunostaining of canonical markers for the different lineages (CD163 for macrophages MCSF; CCR2 for dendritic cells GMCSF; and P2RY12 for microglia cells MDMi). Quantification presented in Supplemental Fig. 4. Scale bars are 10 μm. E Representative immunoblots, and F quantification for HLA-DRα upregulation, 24 h after human α-synuclein fibril treatment in both macrophages and microglia-like (MDMi) cells. G Representative immunoblots of lysates from cells treated with or without human ɑ-synuclein fibrils (1 μg・mL− 1, 48 h), with or without the JAK2 inhibitor AZD1480 (100 nM). IFNγ (20 ng・mL− 1) treated macrophages (MCSF) were used as a positive control for LRRK2 upregulation and functional activation in the different cell lineages. H Quantification of total LRRK2 and the ratio of pT73-Rab10 to total Rab10 in dendritic (GMCSF) and microglia-like cells (MDMi). I From cell culture media, human IL-6 levels were measured by ELISA in untreated cells (control), cells treated with LPS (100 ng・mL− 1, 24 h), or human ɑ-synuclein fibrils (1 μg・mL− 1, 24 h). All data show mean ± SEM for n = 3 independent experiments, except for transcriptional analysis in panel C that includes a male and female pair (healthy controls, age range 24 to 34 years old). Significance is assessed by one-way ANOVA with Tukey’s post hoc test where *p < 0.01, **p < 0.01, ***p < 0.001

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