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Enhanced neurite outgrowth of human model (NT2) neurons by small-molecule inhibitors of Rho/ROCK signaling.

Roloff F, Scheiblich H, Dewitz C, Dempewolf S, Stern M, Bicker G - PLoS ONE (2015)

Bottom Line: Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth.Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction.Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15/102, 30173, Hannover, Germany.

ABSTRACT
Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.

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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.
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pone.0118536.g002: 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.

Mentions: Culturing the 2wkRA neurons with an inhibitor for the downstream effector of RhoA, the Rho Kinase resulted also in significantly longer neurites. Even low concentrations of 1 μM of the ROCK inhibitor Y-27632 caused an elongation to 129% of control (Fig. 2A), the level reached by application of 500 μM Ibuprofen (Fig. 1A and C). The dose dependent effect of Y-27632 showed an increased elongation to 150% of control at 5 μM (Fig. 2A and D), 170% of control at 10 μM (Fig. 2A and E), and a doubling to 202% of control at the highest Y-27632 concentration used (50 μM, Y-27632, Fig. 2A and F). To obtain independent support for the strategy of neurite growth enhancement by RhoA/ROCK inhibition, we used a second ROCK blocker. Treatment with the ROCK inhibitor Fasudil led to a similar increase in neurite lengths as seen for Y-27632 (Fig. 2B). The increase in neurite lengths was highly significant for Fasudil levels of 10 μM and 100 μM.


Enhanced neurite outgrowth of human model (NT2) neurons by small-molecule inhibitors of Rho/ROCK signaling.

Roloff F, Scheiblich H, Dewitz C, Dempewolf S, Stern M, Bicker G - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4340918&req=5

pone.0118536.g002: 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.
Mentions: Culturing the 2wkRA neurons with an inhibitor for the downstream effector of RhoA, the Rho Kinase resulted also in significantly longer neurites. Even low concentrations of 1 μM of the ROCK inhibitor Y-27632 caused an elongation to 129% of control (Fig. 2A), the level reached by application of 500 μM Ibuprofen (Fig. 1A and C). The dose dependent effect of Y-27632 showed an increased elongation to 150% of control at 5 μM (Fig. 2A and D), 170% of control at 10 μM (Fig. 2A and E), and a doubling to 202% of control at the highest Y-27632 concentration used (50 μM, Y-27632, Fig. 2A and F). To obtain independent support for the strategy of neurite growth enhancement by RhoA/ROCK inhibition, we used a second ROCK blocker. Treatment with the ROCK inhibitor Fasudil led to a similar increase in neurite lengths as seen for Y-27632 (Fig. 2B). The increase in neurite lengths was highly significant for Fasudil levels of 10 μM and 100 μM.

Bottom Line: Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth.Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction.Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15/102, 30173, Hannover, Germany.

ABSTRACT
Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.

Show MeSH
Related in: MedlinePlus