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First published online July 23, 2007
doi: 10.1242/10.1242/jcs.003590

Journal of Cell Science 120, 2507-2516 (2007)
Published by The Company of Biologists 2007
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Neurotrophic factors switch between two signaling pathways that trigger axonal growth

Mikhail Paveliev1,*, Maria Lume1, Agne Velthut1, Matthew Phillips1,2, Urmas Arumäe1 and Mart Saarma1,*

1 Institute of Biotechnology, University of Helsinki, Helsinki FIN-00014, Finland
2 Division of Signal Transduction, Harvard Medical School, Beth Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, 10th Floor, Boston, MA 02115, USA


Figure 1
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  		Fig. 1. Discrimination between neurotrophic factors (NTF)-dependent and NTF-independent types of axonal growth in DRG neurons. (A) After 12 hours in culture DRG neurons exhibit substantial axonal growth in presence of NGF or NRTN or GDNF (b) but not in control culture (no neurotrophic factors) (a). For quantification see Fig. 2A. NTF in all figures stands for treatment with NGF or NRTN or GDNF. After 40 hours in culture, axonal growth occurs equally in the presence (d) or in the absence (c) of exogenous neurotrophic factors. For quantification see Fig. 2B and Fig. 7A. All cultures were maintained on coverslips precoated with laminin. No outgrowth was observed in the absence of laminin precoating both at 12 hours and 40 hours in culture. Bars, 20 µm. Control, absence of treatments that are specified otherwise. (B) The `truth table' summarizing the axonal growth dependence on laminin and NTF. Binary mode (`+' or `–') was used to describe output data of the assay (presence or absence of axonal growth, respectively). For input conditions: +, treatment with laminin or NTF; –, absence of treatment. (C) At 12 hours in culture, axonal growth takes place only if laminin and NTF are present together. This kind of input-output relation is described by the conjunctive (AND) operation. (D) At 40 hours in culture, laminin is able to trigger axonal growth in the absence of NTFs, meaning that a separate, NTF-independent pathway exists. NTF-dependent and NTF-independent pathways converge on the axonal growth machinery and each pathway alone is sufficient to cause outgrowth. This kind of input-output relationship is described by the disjunctive operation (OR). NTF-dependent pathway is shown in red, NTF-independent in blue, and the common part of the two pathways in green.

 

Figure 2
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  		Fig. 2. NTF-dependent and NTF-independent types of axonal growth differ in their sensitivity to signal transduction inhibitors. The SFK inhibitor SU6656 (SU) inhibits NTF-dependent axonal growth after 12 hours in culture (A,G,H). SU6656 (2 µM) does not affect axonal growth in the absence of exogenous NTFs after 40 hours in culture (B,J,K). (C) Axonal growth induced by NGF and NRTN is blocked by Src dominant negative protein transduced in an adenoviral vector (SrcDN AV). GFP-expressing adenoviral vector (GFP AV) was used as a control. (D) SrcDN AV does not inhibit the NTF-independent type of axonal growth. In C and D low laminin concentrations (20 and 50 ng/cm2, respectively) were used for precoating. Cultures were maintained for 90 hours. The Cdk5 inhibitor roscovitine (Rosc) does not affect NRTN-, GDNF- or NGF-dependent outgrowth at 12 hours in culture (E,I). Roscovitine inhibits axonal growth after 40 hours in culture (F,L) (no NTFs added). In A,C,E,G,H,I, NTFs were applied where indicated at 100 ng/ml. In A,B,E,F,H,I,K,L, all NTFs and inhibitors were applied when plating neurons. In A, a significant difference between the samples treated with NTFs versus NTFs+SU is indicated with asterisks. In E,F,I,L, roscovitine was applied at 50 µM. Cultures were maintained for the indicated time periods, then fixed and stained for PGP 9.5. All values are means ± s.e.m. of three experiments. Bars, 20 µm.

 

Figure 3
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  		Fig. 3. A Boolean model of the signaling network. (A) Signaling events induced by laminin and NTFs can be presented as a Boolean network based on axonal growth assay. (B) The generic equation for the network in (A). {cap}, AND; {cup}, OR; upper line, NOT. Boolean operator NOT means that the output is positive only if the input to the operator is negative. (C) Electronic scheme analog for the network in A. The color code in A and C is the same as in Fig. 1D.

 

Figure 4
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  		Fig. 4. NTF-dependent and NTF-independent types of axonal growth do not differ in axonal morphology. Neurons cultured for 12 hours in the presence, or for 40 hours in the absence, of NGF exhibit similar mean values of total axonal length (A) and number of branches (B). (C) Distribution of total length and number of branches of the longest axon of each neuron at 12 hours in the presence of NGF (squares) and at 40 hours in the absence of NGF (triangles). The data are presented for 40 neurons from four independent experiments; the data in A and B are means ± s.e.m.

 

Figure 5
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  		Fig. 5. NTF-dependent and NTF-independent types of axonal growth differ in their response to signal transduction inhibitors in terms of axonal morphology. (A-C) The SFK inhibitor SU6656 reduces dramatically axonal length and branching during the NGF-dependent axonal growth at 12 hours in culture. The number of branches (E,F) but not axonal length (D,F) was significantly reduced by SU6656 during axonal growth in the absence of NTFs at 40 hours in culture. The Cdk5 inhibitor roscovitine (Rosc) does not affect NGF-dependent axonal length (G) and number of branches (H) at 12 hours in culture. In contrast, roscovitine strongly suppresses axonal length (J) and branching (K) during axonal growth in the absence of NTFs at 40 hours in culture. (C,F,I,L) Distribution of the length and the number of branches of the longest axon of individual neurons. {blacktriangleup}, SU6656 treatment (C,F) and roscovitine treatment (I,L). For each condition, data are shown for 30 neurons from three independent experiments; the data in A,B,D,E,G,H,J,K are means ± s.e.m. Where indicated, NGF was applied at 100 ng/ml, SU6656 at 2 µM, roscovitine at 50 µM.

 

Figure 6
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  		Fig. 6. The roscovitine-sensitive outgrowth at 40 hours is not induced by endogenous NTFs. (A) Axonal outgrowth at 40 hours is not inhibited by TrkB-FC chimera (200 ng/ml), anti-NGF (250 ng/ml) and anti-GDNF (183 ng/ml) blocking antibodies, and by the glial inhibitor cytosine 1-beta-D-arabinofuranoside (1 µM) (AraC). (B) Blocking antibodies to GDNF and NGF block outgrowth in GDNF- and NGF-treated cultures, respectively. Anti-GDNF and anti-NGF antibodies were applied at the same concentration as in A. (C) Conditioned medium collected from DRG cultures at 40 hours does not activate axonal growth in newly plated neurons. All values are means ± s.e.m. of three experiments

 

Figure 7
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  		Fig. 7. NTF-dependent axonal growth at 40 hours in culture. (A) Treatment with NRTN and NGF (100 ng/ml each) does not increase significantly axonal growth at 40 hours in culture compared with the control. Roscovitine (Rosc) blocks axonal growth at 40 hours in the absence of NTFs (A,B,D) but fails to affect outgrowth in sister cultures treated with NTFs (A,C,E). Simultaneous treatment with roscovitine and SU6656 (SU) inhibits axonal growth (A). Roscovitine was applied at 50 µM, SU6656 at 2 µM. (B,C) The schemes show which elements of the network are active (bright colors) according to the model and which elements are inactive (faint colors), under particular conditions. Bars, 20 µm.

 

Figure 8
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  		Fig. 8. The two-pathway model correctly predicts axonal growth response in separate DRG subpopulations. (A) NRTN and NGF induce outgrowth in responsive subpopulations under roscovitine (Rosc) treatment (50 µM). This NTF-dependent, roscovitine-insensitive outgrowth is inhibited by SU6656 (SU) (2 µM). The schemes below the histogram show which elements of the network are active (bright colors) according to the model and which elements are inactive (faint colors) under particular conditions. (B) Two large and different subpopulations of mature mouse DRG neurons express TrkA and Ret receptor tyrosine kinases. These two receptors perceive NGF and NRTN ligand inputs, respectively. (C) The values of axonal growth activated by NRTN and NGF (100 ng/ml each) were roughly additive. All values are means ± s.e.m. of three experiments. *P<0.05; ***P<0.005.

 

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© The Company of Biologists Ltd 2007