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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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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.
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pone.0118536.g001: 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.

Mentions: Culturing human NT2 2wkRA neurons with Ibuprofen resulted in an increased length of the longest neurite to 114.0% of control at 100 μM (Fig. 1A and F) and 132.2% of control at 500 μM (Fig. 1A and G). Lower concentrations (10 μM) failed to increase neurite length in a significant way (Fig. 1A and E). Moreover, we used a membrane permeable cAMP analogue to elevate intracellular levels of cAMP. Incubation with 1 mM 8-Br-cAMP resulted in a less, but still significant elongation of neurites to 121% of control (Fig. 1B and D).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118536.g001: 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.
Mentions: Culturing human NT2 2wkRA neurons with Ibuprofen resulted in an increased length of the longest neurite to 114.0% of control at 100 μM (Fig. 1A and F) and 132.2% of control at 500 μM (Fig. 1A and G). Lower concentrations (10 μM) failed to increase neurite length in a significant way (Fig. 1A and E). Moreover, we used a membrane permeable cAMP analogue to elevate intracellular levels of cAMP. Incubation with 1 mM 8-Br-cAMP resulted in a less, but still significant elongation of neurites to 121% of control (Fig. 1B and D).

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