Evidence that α-syn mediates microglial directional migration. (A) Quantitative analysis of microglial passage across the insert filters and migration to the bottom wells of 24-well Boyden chambers. The closed red bar indicates total input (1.0 × 10⁵). Transmigrated microglia (isolectin-positive cells) were counted. n = 4. (B) Representative images showing microglial migration toward neurons. Rat neuron-enriched cultures in the 24-well plates were infected with the AAV2-blank, AAV2-α-syn, lentiviral scrambled (Scram), or rat α-syn–specific shRNA-carrying vectors. Then 3.0 × 10⁵ microglia were directly added to the wells and allowed to migrate toward neurons overnight before staining with Alexa Fluor 594-conjugated isolectin, calcein-AM, and Hoechst. (C) Quantitative analysis of microglial migration toward neurons. Images, as represented in B, were captured, and the microglia that overlapped neurons were counted. n = 5. (D) Chemotaxis assays of microglial migration toward rH α-syn aggregates or fMLP in the 96-well Boyden chambers. After overnight incubation, the transmigrated microglia were measured using a CytoQuant assay kit. n = 4. Group comparisons were tested by ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. (Scale bar: 100 µm.)

CD11b is involved in the regulation of α-syn–mediated microglial migration. (A) Effect of fMLP receptor antagonist cyclosporin H and Ab against either α-syn or CD11b on migration of rat microglia toward purified rH α-syn aggregates or fMLP. Here 1.0 × 10⁵ microglia were loaded onto each insert. (B) Effect of anti–α-syn or anti-CD11b Ab on migration of WT mouse microglia toward purified rH α-syn aggregates or fMLP. (C) Migration of CD11b^−/− mouse microglia toward purified rH α-syn aggregates or fMLP. In A, B, and C, the transmigrated microglia were measured in the 96-well Boyden chambers using a CytoQuant assay kit. (D) Migration of rat primary microglia toward rat neuron-enriched cultures in which α-syn expression was intact, enhanced or knocked down. “Input” control reflects addition of 1.0 × 10⁵ microglia directly to the well. (E) Migration of mouse WT or CD11b^−/− microglia toward mouse or rat neuron-enriched cultures with or without an anti-CD11b Ab. Microglial migration in D and E was measured in the 24-well Boyden chambers. In A–E, n = 4. The Student t test was performed in E, whereas ANOVA followed by the Newman–Keuls multiple-comparisons test were performed in A–D. (F) Representative images showing rat primary microglial migration toward rat neurons with or without an anti-CD11b blocking Ab. Here 3.0 × 10⁵ microglia were loaded onto each insert. (G) Quantitative analysis of microglia that overlapped neurons, counted in images as represented in F. n = 5, Student’s t test. In A–E and G, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding BSA or IgG controls; ^##P < 0.01 and ^###P < 0.001 compared as indicated. (Scale bar: 100 µm.)

α-Syn aggregates directly bind to CD11b, which activates Nox2 to induce a migratory conformation of microglia. (A) In vitro binding assays. Myc-DDK–fused human CD11b was incubated with rH α-syn aggregates, and binding was detected via immunoblot for DDK and α-syn. Incubation of CD11b or α-syn alone served as controls. n = 3. (B) In vivo binding assays. Purified rH α-syn aggregates were mixed with WT or CD11b^−/− microglial lysates to allow α-syn to react with CD11b, and the mixtures were further incubated in the IgG- or Ab-conjugated magnetic beads. Binding was detected via immunoblot for CD11b and α-syn. n = 3. (C) O₂⁻ production in mouse microglial cultures on stimulation using PMA (positive control) or α-syn. (D) Representative immunoblots showing plasma membrane (PM) translocation of p47^phox from cytosol (Cyt) in PMA (positive controls)- or α-syn–stimulated HAPI cells. (E) Quantitative analysis of p47^phox membrane translocation based on the immunoblot data. ANOVA was performed. n = 3. ***P < 0.001 compared with the BSA control. (F) Membrane translocation of p47^phox and polarization of the cellular morphology in BSA- or α-syn–stimulated HAPI cells. (G) Rearrangement of the actin cytoskeleton in α-syn–stimulated HAPI cells in the presence or absence of an anti–α-syn or anti-CD11b Ab. The samples were immunostained for p47^phox, F-actin (by phalloidin), and nuclei (by Hoechst). The arrows indicate the polarized distribution of p47^phox in the lamellipodia. (Scale bar: 10 μm.)

Activation of Nox2 is essential to α-syn–induced microglia directional migration. (A) Migration of WT or gp91^phox−/− mouse microglia toward fMLP or rH α-syn aggregates. n = 5. The Student t test was performed. (B) Effect of the Nox2 inhibitor Apo on mouse microglial migration toward fMLP or α-syn. One-way ANOVA followed by the Newman–Keuls multiple-comparisons test were performed. n = 5. The transmigrated cells in A and B were detected in the 96-well Boyden chambers using a CytoQuant kit; 1.0 × 10⁵ microglia were loaded. (C) Effect of the Nox2 inhibitor Apo on rat microglial migration toward neuron-enriched cultures. ANOVA and the Newman–Keuls multiple-comparisons test were applied. n = 5. The transmigration assays were performed in the 24-well Boyden chambers. (D) Representative images showing rat microglial migration toward neurons with or without Apo. Here 3.0 × 10⁵ microglia were loaded. (E) Quantitative analysis of the microglia that overlapped neurons after the direct addition to neuron-enriched cultures overnight. Images, as represented in D, were captured, and the number of microglia that overlapped neurons was compared between DMSO treatment and the corresponding Apo treatment. The Student t test was performed. n = 5. *P < 0.05; **P < 0.01; ***P < 0.001. (Scale bar: 100 µm.)

H₂O₂, a product of α-syn–activated Nox2, serves as a direct signal to regulate microglial directional migration on the interaction between α-syn and CD11b. (A) Microglial chemotaxis toward H₂O₂ with or without catalase (Cat) based on an under-agarose gel migration assay. n = 4. ANOVA, followed by the Newman–Keuls multiple-comparisons test. (B) Polarized microglial morphology and F-actin distribution after direct exposure of cells to 10 µM H₂O₂ for 30 min. (Scale bar: 10 μm.) (C) Effect of α-syn stimulation on extracellular H₂O₂ in WT mouse microglia with or without catalase, as measured by an Abcam kit. BSA served as a control (Ctr). (D) Extracellular H₂O₂ in stimulated CD11b^−/− mouse microglial cultures. (E) Extracellular H₂O₂ in stimulated gp91^phox−/− mouse microglial cultures. (F) Quantitative analysis of the changes in intracellular H₂O₂ concentration in WT mouse microglia. (G) Quantitative analysis of the changes in intracellular H₂O₂ concentration in WT mouse microglia pretreated with catalase overnight. In C, D, E, F, and G, ANOVA was performed comparing with the BSA-treated control. n = 4. (H) BSA- or catalase-preincubated microglia migrated toward BSA, fMLP, or α-syn. Assays were performed in the 96-well Boyden chambers using a CytoQuant kit, with 1.0 × 10⁵ microglia loaded onto each insert. n = 4. ANOVA followed by the Newman–Keuls multiple-comparisons test were performed. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding BSA-treated control. ^###P < 0.001 as indicated.

Lyn acts as an H₂O₂ sensor to regulate the phosphorylation of cortactin. (A) Exposure to either α-syn or H₂O₂ induced the phosphorylation of SFKs (pSFK) in WT primary mouse microglia. Tubulin (Tub) blots served as protein loading controls. (B) Lyn, an SFK family member, is phosphorylated in WT mouse microglia stimulated using either α-syn or H₂O₂. (C) Effect of DMSO (DM) or catalase (Cat) on α-syn– or H₂O₂-induced Lyn phosphorylation in WT mouse microglia. (D) Effect of DMSO (DM) or PP2 on α-syn– or H₂O₂-induced Lyn phosphorylation in WT mouse microglia. (E) Lyn phosphorylation (pLyn) in CD11b^−/− mouse microglia stimulated by α-syn or H₂O₂. (F) Lyn phosphorylation (pLyn) in gp91^phox−/− microglia stimulated by α-syn or H₂O₂. (G) Effect of DMSO (DM) or PP2 on α-syn–induced cortactin (Cort) phosphorylation (pCort) in WT mouse primary microglia. (H) Effect of DMSO (DM) or PP2 on H₂O₂-induced cortactin phosphorylation (pCort) in WT mouse primary microglia. (I) Effect of scrambled (Scram) or Lyn siRNA treatment on α-syn– or H₂O₂-induced cortactin phosphorylation in rat microglia-derived HAPI cells. In A–H, ANOVA and the Newman–Keuls multiple-comparisons test were performed. In I, the Student t test was performed. ***P < 0.001 comparing Lyn shRNA- and its corresponding scrambled shRNA-treated HAPI cells, stimulated by either α-syn or H₂O₂. In A–I, representative Western blots are shown on the top, and a corresponding summary of three to five independent experiments is shown on the bottom.

Activation of Lyn and cortactin facilitates microglial migration toward the source of H₂O₂, fMLP or α-syn. (A) Effect of the Src-inhibitor PP2 on the α-syn–induced distribution of F-actin and phosphorylated cortactin (pCortactin) in WT mouse primary microglia. (B) Effect of the Lyn siRNA on the α-syn–induced distribution of F-actin and phosphorylated cortactin in HAPI cells. Arrows are indicating the polarized localization of F-actin and pCortactin. (Scale bar: 10 μm.) (C) Effect of PP2 or Lyn siRNA on rat primary microglial or HAPI cell chemotaxis toward the source of H₂O₂ by under-agarose migration assays. (D) Effect of PP2 or Lyn siRNA on rat primary microglial or HAPI cell chemotaxis toward the source of fMLP or α-syn in the 96-well Boyden chambers. In C and D, 1.0 × 10⁵ microglia were loaded onto each well or insert. Cell migration with DMSO (DM) or PP2 is shown on the left, and migration of cells transfected with scrambled (Scram) or Lyn siRNA for 3 or 10 d is shown on the right. n = 4–5. The Student t test was performed. *P < 0.05 and **P < 0.01 compared with the corresponding DMSO- or scrambled siRNA-treated controls.