Fig. 6
RhoA/ROCK pathway contributed to regulation of Nogo/NgR pathway on microglia spread and membrane protrusion formation. To explore the effect of Nogo-P4 on cytoskeleton reorganization of microglia and the associated mechanism, F-actin staining and the mean spread area of the microglia were examined. a, b Nogo-P4 inhibited microglia spread and membrane protrusion formation induced by Aβ fibrils. Microglia from 3-month-old mice were plated in 96-well plates pre-coated with BSA (0.01% in PBS), fAβ1–42 (10 μM), Nogo-P4 (100 μg/ml), or Nogo-P4 (100 μg/ml) + fAβ1–42 (10 μM) for 8 h. a Photomicrographs of microglia stained with rhodamine-conjugated phalloidin. c–e The effects of Nogo-P4 on spread and membrane protrusion formation induced by Aβ fibrils were mediated by NgR. Before adding to the wells pre-coated with BSA (0.01% in PBS), fAβ1–42 (10 μM) or Nogo-P4 (100 μg/ml) + fAβ1–42 (10 μM), microglia from 3-month-old mice were pre-treated with NEP1-40 (10 μM) or PI-PLC (0.3 U/ml) for 30 min to interrupt the function of NgR. c Photomicrographs of microglia stained with rhodamine-conjugated phalloidin. d, e Quantitative assessment of cell spreading by determining the spread area of the microglia. f–h RhoA/ROCK pathway contributed to the regulation of Nogo-P4 on spread and membrane protrusion formation induced by Aβ fibrils. After pre-treatment with PBS or Y27632 (50 μM) for 30 min, microglia from 3-month-old mice were plated in 96-well plates pre-coated with BSA (0.01% in PBS), fAβ1–42 (10 μM), or Nogo-P4 (100 μg/ml) + fAβ1–42 (10 μM) for 8 h. f Photomicrographs of microglia stained with rhodamine-conjugated phalloidin. Values were reported as the mean ± SD, as a percentage of values determined in BSA group (control, 100%). *p < 0.05; **p < 0.01; ***p < 0.01, when compared with the BSA group, n = 3. ###p < 0.001, when compared with the Nogo-P4 + fAβ1–42 group, n = 3