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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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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.
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pone.0118536.g003: 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.

Mentions: In an attempt to test whether RhoA/ROCK inhibition directly effects elongation of existing neurites or whether the initiation of new neurites is altered, we categorized ß-tubulin III-positive cells into neurons with and without neurites. Under control conditions, approximately 60% of the neurons had grown at least one neurite (Fig. 3). Culturing neurons for 24 hours under Ibuprofen treatment resulted in a dose dependent increase from 57.52% at 10 μM, 67.85% at 100 μM, and to 75.62% at 500 μM Ibuprofen (Fig. 3A). However, only at the highest applied Ibuprofen concentration of 500 μM we obtained statistical significance. Culturing neurons under elevated levels of cAMP resulted in a weak but not significant increase of neurite bearing cells to 65.77% of ß-tubulin III-positive cells (Fig. 1B). Application of the ROCK inhibitor Y-27632 led to increased neurite formation shown as a dose dependent increase of neurite bearing cells (Fig. 3C). Under control conditions approximately 67% of all cells generated a neurite. After application of Y-27632, the percentage increased in a dose dependent manner to 86% at 50 μM Y-27632, which was significantly different from control. Application of the ROCK inhibitor Fasudil led to an increase in neurite bearing cells to approximately 75% at 100 μM (Fig. 3D).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118536.g003: 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.
Mentions: In an attempt to test whether RhoA/ROCK inhibition directly effects elongation of existing neurites or whether the initiation of new neurites is altered, we categorized ß-tubulin III-positive cells into neurons with and without neurites. Under control conditions, approximately 60% of the neurons had grown at least one neurite (Fig. 3). Culturing neurons for 24 hours under Ibuprofen treatment resulted in a dose dependent increase from 57.52% at 10 μM, 67.85% at 100 μM, and to 75.62% at 500 μM Ibuprofen (Fig. 3A). However, only at the highest applied Ibuprofen concentration of 500 μM we obtained statistical significance. Culturing neurons under elevated levels of cAMP resulted in a weak but not significant increase of neurite bearing cells to 65.77% of ß-tubulin III-positive cells (Fig. 1B). Application of the ROCK inhibitor Y-27632 led to increased neurite formation shown as a dose dependent increase of neurite bearing cells (Fig. 3C). Under control conditions approximately 67% of all cells generated a neurite. After application of Y-27632, the percentage increased in a dose dependent manner to 86% at 50 μM Y-27632, which was significantly different from control. Application of the ROCK inhibitor Fasudil led to an increase in neurite bearing cells to approximately 75% at 100 μM (Fig. 3D).

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