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First published online 2 January 2007
doi: 10.1242/jcs.03323

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A_1-42 stimulates actin polymerization in hippocampal neurons through Rac1 and Cdc42 Rho GTPases

¹ Laboratory of Cellular, Molecular Biology and Neuroscience, Department of Biology, Faculty of Sciences and
² Department of Neurological Sciences, Universidad de Chile, Las Palmeras 3425, Nunoa, Santiago, Chile

View larger version (45K): [in this window] [in a new window]   Fig. 1. F-actin levels are increased in hippocampal neurons treated with A. (A) Control hippocampal neurons grown on poly-D-lysine for 4 days were stimulated with 10 µM of fibrillar A1-42 for 4 hours and double stained for Tub-Tyr (green) and F-actin filaments with Rhodamine-phalloidin. Overlay of Tub-Tyr with phalloidin is shown. Scale bar, 20 µm. (B) Hippocampal cells, untreated or stimulated for 4 hours with 10 µM A1-42 were immunostained with phalloidin-TRITC and the F-actin intensity was analyzed. The values are expressed as the main ± s.e.m. Data are representative for three different experiments, with at least 12 determinations per experiment (*P<0.05). (C) F-actin levels in control and 4-hour and 24-hour A-stimulated neurons were measured with Actin Polymerization Assay kit. Phalloidin (P) was used as a positive control. The ratio of F-actin/total actin was determined from the blots by densitometric measurements. Significant differences are indicated by asterisks (*P<0.05, ***P<0.005; Student's t-test).

View larger version (33K): [in this window] [in a new window]   Fig. 2. Increase in F-actin levels correlates with enhancement in growth cone area and filopodia number. Hippocampal neurons control and stimulated with A1-42 were immunostained with phalloidin-TRITC (top panels; higher magnification of the boxed regions is shown in the insets) and growth cone area and filopodia number were analyzed (bottom panels). Scale bar, 20 µm. Values are expressed as mean ± s.d.

View larger version (40K): [in this window] [in a new window]   Fig. 3. GTPase activities of Rac1 and Cdc42-GTPase are increased in neurons stimulated with A1-42. Hippocampal neurons cultured for 4 days were treated with (A) 0.1, 1 and 10 µM A1-42 for 24 hours and (B) with 10 µM of peptide for 30 min, 2, 4 and 24 hours. Active GTP-Rac1 was pulled down using the PAK-PBD conjugated with agarose and then tested by immunoblotting with anti-Rac1 monoclonal antibody. Values were normalized against total Rac1. Graphs show data from four independent experiments; *P<0.05, **P<0.01. (C,D) Hippocampal neurons stimulated 4 hours with 10 µM fibrilar A1-42 were pre-treated with pertussis toxin and roscovitin. The GTP bound to Rac1 (C) and to Cdc42 (D) was determined using the Rac/Cdc42 Activation Assay Kit, according to manufacturer's recommendations. Values were normalized, with respect to total Rac1 and Cdc42; *P<0.05.

View larger version (71K): [in this window] [in a new window]   Fig. 4. Rac1 and Cdc42 display enhanced F-actin colocalization in hippocampal cells treated with A. (A) Four-day hippocampal neurons, untreated (control) and exposed to A1-42 fibrils for 4 hours were double immunostained with Rac1 or Cdc42 and Rhodamine-phalloidin and analyzed by confocal laser-scanning microscopy. Images in the upper panel are overlays of Rac1 or Cdc42 vs. Rhodamine-phalloidin staining. Arrows indicate increased actin protusions in amyloid-stimulated neurons. Lower panels are higher magnification of the boxed regions showing Rac1 and Cdc42 overlaying with F-actin in control conditions, as well as the augmented colocalization of these proteins with F-actin (arrowhead) after A stimulation. Scale bar, 20 µm. (B) Respective scatter plots of fluorescence intensity distribution for Rac1/F-actin and Cdc42/F-actin from control and A-stimulated neurons. (C) Colocalization coefficients between Rac1/F-actin and Cdc42/F-actin were evaluated using Zeiss colocalization coefficient function software. Data are expressed as mean ± s.e.m.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Rac1 and Cdc42 increased in the plasma membrane after A1-42 stimulation. (A) Four-day hippocampal neurons, untreated (control) and exposed to A1-42 fibrils for 4 hours, were fixed after detergent extraction performed under cytoskeleton-stabilizing conditions and double immunostained with Rac1 or Cdc42 and Rhodamine-phalloidin. Both GTPases were recruited to the plasma membrane and were frequently found to colocalize with F-actin after amyloid stimulation (arrowheads). Bar, 20 µm. (B) Embryonic hippocampal cells, untreated and treated for 4 and 24 hours with -amyloid, were lysed and cytoplasmic and membrane fractions obtained. Rho GTPases recruitment to the membrane fraction was analyzed using Rac1 and Cdc42 antibodies and the values were normalized against flotillin as a membrane protein control. Samples from the cytoplasmic fraction were analyzed by immunoblotting using Rac1 and Cdc42, along with actin antibody as a loading control. Significant differences are indicated by asterisks (*P<0.05, **P<0.01; Student's t-test).

View larger version (57K): [in this window] [in a new window]   Fig. 6. Rac1 and Cdc42 are essential for the increased F-actin polymerization induced by A1-42 treatment. Primary hippocampal cells treated with A1-42 were transfected with the dominant negative forms of RacT17N-GFP and Cdc42T17N-GFP (arrows), and then stained for F-actin using Rhodamine-phalloidin. The untransfected neurons are indicated by arrowheads. Scale bar, 20 µm. (B) Changes in F-actin levels were determined and differences between transfected (t) vs. untransfected (ut) control and A-stimulated neurons were determined using Student's t-test. The values were expressed as mean ± s.e.m. for three different experiments, with at least 12 determinations per experiment; *P<0.05.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Tiam1 is activated in A-stimulated hippocampal cells. (A) Membrane fractions from hippocampal cells, either untreated (control) and stimulated for 4 and 24 hours with 10 µM of fibrillar A1-42, were obtained. Samples were analyzed by immunoblotting using Tiam1 antibody and the values were normalized against flotillin. Tiam1 expression in the cytoplasmic fraction was analyzed by immunoblotting using Tiam1 antibody and actin as a loading control. (B) Tiam1 was immunoprecipitated from control hippocampal neurons and neurons treated for 4 hours with A1-42. Phosphorylation levels were tested by immunoblotting using P-Thr monoclonal antibody. *P<0.05.

View larger version (35K): [in this window] [in a new window]   Fig. 8. A-mediated Tiam1 activation is affected by Ca2+ signaling. (A) Time lapse experiments were performed to analyze the Ca2+ increase in hippocampal cells after A1-42 addition, using the fluorescent tracer Fluo3-AM. (B) Phospho-Thr levels from total immunoprecipitated Tiam1 were tested by immunoblot, in neurons treated with A1-42 in the presence of BAPTA-AM; *P<0.05. (C) Hippocampal neurons stimulated with A1-42 were analyzed for Tiam1 association to active Rac1 after BAPTA-AM or Gö6976 treatments. Rac1 was pulled down using PAK-PBD agarose and the co-immunoprecipitated Tiam1 was tested by immunoblotting using anti-Tiam1 antibody. Values were normalized against total Tiam1 in the crude homogenized. *P<0.05.

View larger version (26K): [in this window] [in a new window]   Fig. 9. Tiam1 mediates Rac1 activation through Ca2+ signaling in A-stimulated hippocampal cells. Hippocampal neurons were incubated for 4 hours with A1-42 in the presence of BAPTA-AM (A) or Gö6976 (B) and then the active Rac1 fraction was pulled-down using the PAK-PBD binding domain. The active form of Rac1 was revealed using Rac1 monoclonal antibody. **P<0.01.