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First published online 13 June 2006
doi: 10.1242/jcs.03016

Journal of Cell Science 119, 2787-2796 (2006)
Published by The Company of Biologists 2006
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Neurotrophins support regenerative axon assembly over CSPGs by an ECM-integrin-independent mechanism

Feng-Quan Zhou1,2, Mark Walzer1, Yao-Hong Wu1, Jiang Zhou1, Shoukat Dedhar3 and William D. Snider1,*

1 Neuroscience Center, 8109 Neuroscience Research Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2 Departments of Orthopedic Surgery and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
3 BC Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada


Figure 1
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  		Fig. 1. Aggrecan and Nogo affect axon assembly from adult DRG neurons differently in the presence of extracellular axon promoting factors. (A) When adult DRG neurons (both naive and PCL neurons) were cultured on laminin (LM), treatment with the CNS myelin inhibitor Nogo had no effect on axon assembly. (B) NGF-induced axon assembly from adult naive DRG neurons plated on laminin was markedly inhibited by aggrecan. (C,D) Quantification of the inhibitory effect of aggrecan (Agg.) on axon assembly from adult naive neurons (C) and PCL neurons (D). Values are means ± s.e.m. *P<0.0001 compared with levels in the control. Magnification, 4x.

 

Figure 2
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  		Fig. 2. Neither acute elevation of cAMP nor inhibiting Rho kinase activity is sufficient to overcome the inhibitory effect of aggrecan on axon assembly from adult DRG neurons. (A) Neither addition of dbcAMP (1 mM) nor addition of Y27632 (25 µM) induced axon growth from naive neurons cultured on aggrecan and laminin in the presence of NGF. (B) Addition of the Rho kinase inhibitor Y27632 had little effect on axon assembly from the PCL neurons cultured on aggrecan and laminin. *P<0.01 and #P<0.05 compared with levels in the control group.

 

Figure 3
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  		Fig. 3. Activation of neurotrophin signaling is able to overcome the inhibitory effect of aggrecan on axon assembly from PCL neurons. (A,B) Laminin-induced axon assembly from PCL neurons (A) was markedly inhibited by aggrecan (B). (C) Addition of NGF overcame the inhibitory effect of aggrecan on axon assembly from PCL neurons. Magnification, 4x. (D,E) Quantification of the stimulatory effect of NGF on axon assembly from PCL neurons cultured on aggrecan. Note that the rescue effect of NGF depends on the activities of TrkA and PI3K, mediators of the NGF signaling, but is independent of integrin signaling and transcription (E). *P<0.0001 vs control; **P<0.0001 between indicated groups in D, and vs LM+NGF+Agg. in E. #P<0.01 vs LM+NGF+Agg. in D and E. DRB, transcription inhibitor; K252a, TrkA inhibitor; LY, LY 294002 PI 3-kinase inhibitor.

 

Figure 4
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  		Fig. 4. Either laminin or NGF alone is sufficient to mediate axon assembly from PCL neurons. (A) PCL neurons were unable to extend axons when cultured on poly-D-lysine (PDL) in the absence of extracellular cues. Neurons plated on laminin-coated coverslips extended axons robustly. (B) Neurons did not extend axons when cultured on poly-D-lysine-coated coverslips in serum-free medium. Addition of NGF induced robust axon assembly from the PCL neurons on poly-D-lysine. Magnification, 4x. (C) Quantification of laminin-induced axon assembly from PCL neurons. Please note that the addition of laminin (S, 20 µg/ml) directly to the culture medium induced a similar extent of axon assembly to that of coated laminin (C). Laminin-induced axon growth was independent of the neurotrophin receptor Trk activity. *P<0.0001 vs control. (D) Laminin-induced axon growth was significantly blocked by an integrin function-blocking antibody (20 µg/ml), whereas control IgG protein had no effect. *P<0.0001 vs control. (E,F) Quantification of NGF-induced axon growth from the PCL neurons. Note that other growth factors, such as EGF and IGF, failed to induce axon growth. In addition, inactivation of Rho kinase with inhibitor Y27632 (25 µM) had minimal effect on inducing axon assembly from the PCL neurons, only slightly enhancing basal axon growth. *P<0.005 and #P<0.05 compared with levels in the controls.

 

Figure 5
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  		Fig. 5. ECM-integrin and neurotrophin signaling mediate axon assembly from PCL neurons by different signal pathways. (A) NGF-induced axon assembly from the PCL neurons was significantly inhibited by specific inhibitors of PI3K (LY294002, 20 µM), classical PKC (Go6976, 200 nM), general PKC (BIS, 10 µM), Src (PP1, 10 µM) and ILK (KP74728, ILKi, 20 µM). *P<0.0001 vs control. (B) In contrast to NGF-induced axon assembly, neither the specific PI3K inhibitor wortmannin (200 nM) nor the classical PKC inhibitor Go6976 (200 nM) inhibited laminin-induced axon assembly of the PCL neurons. Similarly to NGF-induced axon assembly, laminin-induced axon assembly of the PCL neurons was significantly inhibited by the PKC inhibitor BIS (10 µM), the specific Src inhibitor PP1 (10 µM), and the specific ILK inhibitor KP74728 (20 µM). *P<0.0001 vs control. (C) Expression of a dominant-negative (DN) PI3K construct had no effect on laminin-induced axon extension from PCL neurons. (D) Expression of a dominant-negative C-Raf construct had no effect on laminin-induced axon extension from PCL neurons. (E) Expression of a dominant-negative Src construct significantly inhibited laminin-induced axon extension from PCL neurons. *P<0.005 vs control.

 

Figure 6
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  		Fig. 6. The GSK-3ß–APC pathway is required for integrin-induced regenerative axon assembly from PCL neurons. (A) Overexpression of EGFP had no effect on integrin-mediated axon assembly. (B) Expression of the constitutively activated GSK-3ß mutant (yellow) significantly blocked axon assembly. (C) Axon assembly was abolished in neurons expressing C-EB1 (yellow), a mutant protein that interferes interactions between APC and microtubule plus ends. (D) Axon assembly in neurons expressing C-EB1{Delta}APC was not affected. Arrows indicate transfected cells that express GFP. Axons of transfected neurons are yellow. Neurofilament staining (red) reveals axons of untransfected cells. Magnification, 4x. (E,F) Quantification of axon assembly defect in neurons expressing the active GSK-3ß mutant or the EB1 mutant (*P<0.001 vs control). (G) Laminin stimulation alone was sufficient to phosphorylate GSK-3ß in adult PCL neurons, but had no effect on phospho-ERK and phospho-Akt levels. By contrast, laminin stimulation was unable to phosphorylate GSK-3ß in embryonic day 14 (E-14) DRG neurons in the absence of NGF.

 

Figure 7
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  		Fig. 7. A proposed model for regulation of axon assembly of adult DRG neurons. To promote efficient axon growth, extracellular axon growth promoting factors are required. Adult naive neurons require the activation of both integrin and neurotrophin signaling pathways simultaneously for efficient axon growth. Both the CNS myelin-based inhibitor Nogo and glial scar-based inhibitor CSPGs can inhibit basal axon assembly by activation of Rho. However, CSPGs and myelin-based inhibitors act differently to affect axon growth when the extracellular axon growth promoting factors are present. CSPGs can interfere with ECM-integrin signaling, thus preventing extracellular factors from stimulating axon growth. On the contrary, activation of ECM-integrin pathway is able to antagonize the inhibitory effect of the myelin-based inhibitors. Peripheral nerve-injury-mediated conditioning lesion separates the neurotrophin and ECM-integrin pathways, thus allowing either ECM-integrin signaling or neurotrophin signaling to mediate axon assembly independently. As a result, neurotrophins can promote robust axon assembly of PCL neurons over CSPGs by bypassing the requirement of integrin activation for axon assembly.

 

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© The Company of Biologists Ltd 2006