Fig 1.
Ibuprofen increased neurite length of human model neurons.
(A) Treatment with 100 μM and 500 μM Ibuprofen increased neurite outgrowth of human model neurons as measured after 24 hours. At 10 μM Ibuprofen treatment no difference to control could be detected. In 4 independent experiments, 778 to 2361 neurites were measured in total. (B) Neurite elongation under Ibuprofen treatment was slightly higher than in neurons treated with the membrane permeable analogue 8-Br-cAMP (5 experiments, 1015 to 2030 neurites measured. (C) Immunofluorescence staining of human model neurons under control conditions. (D) Neurons treated with 1 mM 8-Br-cAMP. (E-G) Neurons treated with 10 μM (E), 100 μM (F) and 500 μM (G) Ibuprofen. Cells are stained against beta-III-tubulin and counterstained with DAPI. ***p<0.001 with control by Kruskal-Wallis one-way ANOVA. Scale bars are 50 μm.
Fig 2.
Rho kinase (ROCK) inhibitors promote neurite elongation of human model neurons.
(A) Treatment with the ROCK inhibitor Y-27632 for 24 h resulted in dose-dependent increase of neurite lengths over a range from 1 μM to 50 μM Y-27632. At 50 μM Y-27632, treatment resulted in a doubling of neurite length compared to control conditions (3 experiments, 732 to 973 neurites measured). (B) Treatment with the ROCK inhibiting agent Fasudil led to increased neurite lengths of 125% of control at 10 μM and 175% at 100 μM Fasudil (3 experiments, 1511 to 2434 neurites measured) (C-F) Immunofluorescence staining of neurons treated with 1 μM (C), 5 μM (D), 10 μM (E), and 50 μM (F) of the ROCK inhibitor Y-27632. Neurons are stained against beta-III-tubulin and counterstained with DAPI. ***p<0.001 with control by Kruskal-Wallis one-way ANOVA. Scale bars are 50 μm.
Fig 3.
RhoA and Rho kinase inhibitors promote initiation of neurites.
(A) The percentage of cells bearing a neurite was significantly increased at 500 μM of Ibuprofen whereas 10 μM and 100 μM led to no difference to control conditions (3–7 independent experiments, 744 to 2358 neurites measured). (B) Application of cAMP did not change the number of cells bearing a neurite, however neurite lengths were increased to a similar amount compared to Ibuprofen (5 independent experiments, 1151 to 1775 neurites measured). (C) Blocking Rho kinases with increasing levels of Y-27632 resulted in a trend to more cells bearing a neurite. A highly significant effect was observed at 50 μM (3 independent experiments, 732 to 973 neurites measured). (D) Blocking Rho kinases with elevated levels of Fasudil (100 μM) resulted in a highly significant increase in neurite bearing cells (3 independent experiments, 1857 to 3422 neurites measured). **p<0.01 and *p<0.05 against control by Kruskal-Wallis test.
Fig 4.
Rho activation resulted in a growth cone collapse, but not decreased neurite lengths.
(A) Measurements of neurite outgrowth after 24 h incubation with elevated levels of the Rho activator lysophosphatidic acid (LPA) showed no change in neurite lengths, whereas Ibuprofen increased neurite length (2 independent experiments, 470 to 947 neurites measured). (B) Within 45 min, neurons cultured under control conditions showed a slowly advancing growth cone. (C) Inhibition of RhoA using 500 μM Ibuprofen elicited a weak positive effect on filopodial formation and extension of growth cone. (D) RhoA activation using 100 μM LPA resulted in a growth cone collapse and retraction of the neurite. ***p<0.001 against control by Kruskal-Wallis. Scale bar is 25 μm.
Fig 5.
(A) A RhoA bead pull down assay revealed lowered levels of activated RhoA in Ibuprofen treated cells. Treatment with downstream Rho kinase inhibitor Y-27632 showed no difference in RhoA level compared to control. After treatment with lysophosphatidic acid, significantly more phosphorylated RhoA could be detected in the Western Blot. Treatment with downstream Rho kinase inhibitor Fasudil showed no difference in RhoA level compared to control. Cell lysates were blotted and stained with RhoA-specific monoclonal antibody. Staining is visualized with horseradish peroxidase and the 3, 3′-diaminobenzidine substrate. Molecular weight of stained protein bands corresponds to about 20 kDa as reported for RhoA. (B) Relative RhoA level was decreased to approximately 50% of control after Ibuprofen treatment whereas RhoA level after LPA treatment was increased to 200% of control. Data are normalized grey values ± SEM of 4 independent experiments.