Fig. 4
From: Pathological α-synuclein recruits LRRK2 expressing pro-inflammatory monocytes to the brain
α-Synuclein fibrils induce Lrrk2 expression in classical and non-classical monocytes recruited to the mouse brain. A Analysis of median-fluorescence intensity (MFI) in spleen cells (live CD45+/CD11b+) isolated from naive Lrrk2 knockout (KO) and WT-Lrrk2 BAC mice with monoclonal antibody c41–2. B Sagittal aspect of a mouse brain showing the site of human α-synuclein fibril (10 μg, bilateral) injection (red arrow). After 72 h, the indicated midbrain region (boxed in red lines) was isolated and triturated for flow cytometry analysis. Scale bars shown for 5 mm. C Lrrk2 expression in immune cells (live CD45+/CD11b+) isolated from the midbrain. Representative cytographs with representative gates (dashed boxes) depicting CD45Lo, CD45hi, and lymphocyte populations. Extended gating strategy is detailed in Supplemental Fig. 6. D Ly6C+ (enriched in classical monocytes) and Ly6C- (enriched in non-classical monocytes) populations are shown from the CD45hi gate. E Representative Lrrk2 MFI (relative to Lrrk2 KO) in different immune cell populations calculated (by subtracting MFI signals from Lrrk2 KO mice from each cell population) of brain cell suspensions from WT-Lrrk2 BAC mice as well as Lrrk2 KO mice. Data are plotted as the mean MFI (red bars) ± SEM for n = 3 mice (2 males and 1 female for WT-Lrrk2 BAC and 2 males for Lrrk2 KO), aged 2–3 months. F Lrrk2 MFI in CD45hi/CD11b+/Ly6C+ and CD45hi/CD11b+/Ly6C- cell populations were evaluated from midbrain cell suspensions and matched whole blood samples 72 h after human α-synuclein fibril (10 μg, bilateral midbrain) injection. The ratio of brain/blood Lrrk2 MFI, as measured via flow cytometry, is depicted. Data are plotted as the mean MFI (red bars) ± SEM for n = 5 mice in each group (3 males and 2 females for WT-Lrrk2 BAC mice aged 4 months. Significance was assessed by 2-sided t-test or one-way ANOVA with Tukey’s post hoc test, **p < 0.01, ***p < 0.001