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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 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.
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pone.0118536.g004: 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.

Mentions: Inhibition of RhoA and Rho kinases resulted in enhanced neurite outgrowth compared to control conditions. We tested whether a potent RhoA activator, such as lysophosphatidic acid (LPA) had the opposite effect leading to decreased neurite lengths. After 24 hours incubation with LPA, neurite lengths did not differ from control (Fig. 4). Neurite lengths ranged from 103.5±4.5 percent of control at 1 μM LPA to 104.3±4.9 percent of control at 10 μM LPA. Neurons cultured with 500 μM Ibuprofen on the same cell culture plate showed a mean neurite length of 138.3±4.9 percent of control (Fig. 4).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118536.g004: 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.
Mentions: Inhibition of RhoA and Rho kinases resulted in enhanced neurite outgrowth compared to control conditions. We tested whether a potent RhoA activator, such as lysophosphatidic acid (LPA) had the opposite effect leading to decreased neurite lengths. After 24 hours incubation with LPA, neurite lengths did not differ from control (Fig. 4). Neurite lengths ranged from 103.5±4.5 percent of control at 1 μM LPA to 104.3±4.9 percent of control at 10 μM LPA. Neurons cultured with 500 μM Ibuprofen on the same cell culture plate showed a mean neurite length of 138.3±4.9 percent of control (Fig. 4).

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