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First published online 26 October 2004
doi: 10.1242/jcs.01511

Journal of Cell Science 117, 5803-5814 (2004)
Published by The Company of Biologists 2004
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Truncated TrkB receptor-induced outgrowth of dendritic filopodia involves the p75 neurotrophin receptor

Matthias Hartmann1,2, Tanja Brigadski1, Kai S. Erdmann2, Bettina Holtmann3, Michael Sendtner3, Frank Narz2 and Volkmar Leßmann1,*

1 Institute of Physiology and Pathophysiology, Johannes Gutenberg-University Mainz, Duesbergweg 6, 55128 Mainz, Germany
2 Department of Molecular Neurobiochemistry, Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany
3 Institute for Clinical Neurobiology, Julius-Maximilians University Würzburg, Sanderring 2 - 97070 Würzburg, Germany



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  		Fig. 1. Topology of cloned TrkB and p75 fusion proteins. Green fluorescent protein (GFP) and red fluorescent DsRed was fused C-terminally to full length rat TrkB (TrkB.FL), yielding TrkB.FL-GFP and TrkB.FL-DsRed. Similarly, the truncated TrkB isoforms were constructed to yield TrkB.T1-GFP and TrkB.T2-GFP. As a control, unfused TrkB.T1 was cloned into the same vector. The intracellular domain of TrkB.T1 (black bar) was deleted in the construct T1{Delta}ICD-GFP. C-terminal fusion of GFP to rat p75NTR yielded p75-GFP. A dominant negative p75NTR (lowermost construct) was generated by fusing DsRed to the transmembrane domain of p75NTR. The grey vertical bar shows the location of the plasma membrane; the extracellular space is to the left. The p indicates the pre sequence directing the receptor mRNAs to the rough ER.

 


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  		Fig. 2. TrkB.FL-GFP and TrkB.T1-GFP are localized in the plasma membrane of hippocampal neurons and are targeted to dendrites and axons. (A) Hippocampal neurons, transfected with TrkB.FL-GFP (left) and TrkB.T1-GFP (right) expression plasmids at 8 days in vitro (DIV), showed a clear accumulation of GFP fluorescence at the plasma membrane (2 days after transfection). (B) Immunocytochemistry of transfected neurons with an antibody against the dendritic marker MAP2 (at 16 DIV, 8 days after transfection). TrkB.FL-GFP and TrkB.T1-GFP in hippocampal neurons (green), were targeted to both, dendrites (MAP2-positive, red) and axons (MAP2-negative, arrows). Filopodia (small protrusions) were restricted to dendrites, whereas the appearance of axons was generally smooth. Scale bars: 5 µm (A); 20 µm (B).

 


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  		Fig. 3. TrkB.T1 expression in hippocampal neurons increases the number of dendritic filopodia. (A) Typical examples of the morphology of TrkB.T1-GFP-, TrkB.FL-GFP- and GFP-expressing hippocampal neurons (at 11 DIV, 3 days after transfection). The increased number of filopodia in TrkB.T1-GFP expressing neurons lead to a ruffled appearance (left) compared to the much smoother TrkB.FL-GFP and GFP controls. (B) The increase in density of filopodia was highly significant (n=26-91 cells, 5-13 independent experiments, P<10–5). (C) Sholl analysis of all neurons from B. The expression of TrkB.T1-GFP, had no significant effect on the complexity of the dendritic tree. (D) Typical dendrite of a TrkB.T1-GFP expressing hippocampal neuron (green, at 10 DIV). Phalloidin-TRITC staining (red) of TrkB.T1-GFP-induced filopodia revealed their filamentous actin content, which is characteristic for filopodia. Scale bars: 10 µm (A); 5 µm (D).

 


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  		Fig. 4. The induction of filopodia by TrkB.T1 is independent from ligand binding, TrkB kinase activity, and from the intracellular domain of TrkB.T1. (A) Hippocampal neurons were transfected with TrkB.T1-GFP, TrkB.FL-GFP or GFP expression plasmids. On the day of transfection, either TrkB ligand (BDNF or NT-4/5) was added (100 ng/ml), or endogenously released ligand was scavenged with TrkB-Fc receptor bodies (0.4 µg/ml). The density of filopodia was determined 2 days later. Neither exogenous application of ligand nor scavenging of endogenously released ligands with TrkB-Fc receptor bodies influenced the density of dendritic filopodia (n=10-50 cells; two to five independent experiments, *P<10–4, compared with TrkB.T1-GFP positive control). (B) K252a (200 nM), did not affect the growth of filopodia, indicating the lack of an effect of Trk kinase signalling. *Significantly different from TrkB.T1-GFP with P<0.05. (C) The induction of filopodia by a deletion mutant of TrkB.T1 without intracellular domain (T1{Delta}ICD-GFP) was not significantly different (P>0.14) from the TrkB.T1-GFP-induced effect. The increase in filopodia caused by T1{Delta}ICD-GFP is statistically significant compared to TrkB.FL-GFP transfected controls (*P<0.05, n=8-26 cells).

 


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  		Fig. 5. Expression of TrkB.FL inhibits the TrkB.T1-induced growth of filopodia in a dominant negative fashion. Hippocampal microcultures were cotransfected with TrkB.T1-GFP and TrkB.FL-DsRed (relative amounts of DNA: 1:3). (A) A typical neuron, expressing both TrkB.T1-GFP and TrkB.FL-DsRed. Note the smooth dendritic surface, compared to B, a neuron expressing TrkB.T1-GFP only. (Insets in A an B show boxed areas at higher magnification.) (C) The reduction in the density of filopodia by coexpression of TrkB.FL is highly significant compared to controls expressing TrkB.T1 only (P<10–5 n=16-22 cells; three independent experiments). Both groups of neurons from a given experiment were located on the same glass cover slip. All experiments were performed in the presence of 200 nM K252a. Therefore, TrkB receptor tyrosine kinase activity is not required for the dominant negative action of TrkB.FL. Scale bars: 20 µm (A); 5 µm (inset).

 


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  		Fig. 6. TrkB.T1-induced formation of filopodia is dependent on p75NTR but can not be mimicked by application of NGF. Application of 8 µg/ml MC192 (a monoclonal antibody that binds to an extracellular epitope of p75NTR) to TrkB.T1-overexpressing neurons reduced the outgrowth of filopodia to control levels (**significantly different from TrkB.T1-GFP; P<0.01). Basal levels of filopodia remained unaffected (see GFP controls). Application of 100 ng/ml NGF to GFP transfected controls does not mimic the effect of TrkB.T1 expression (n>30cells; five experiments).

 


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  		Fig. 7. Coexpression of a dominant negative p75NTR variant (p75{Delta}ICD-DsRed) inhibits the TrkB.T1-induced growth of filopodia. Neurons were cotransfected with TrkB.T1-GFP and p75{Delta}ICD-DsRed (relative amounts of DNA: 3:1). (A) Typical neuron, expressing both, TrkB.T1-GFP and p75{Delta}ICD-DsRed. Note the lack of filopodia compared with the neuron expressing TrkB.T1-GFP only (on the same coverslip) shown in B. Insets in A and B show boxed areas at higher magnification. (C) The reduction in the density of filopodia by coexpression of p75{Delta}ICD-DsRed is highly significant (P<0.0001). (D) Neurons were cotransfected with GFP and p75{Delta}ICD-DsRed (relative amounts of DNA: 3:1). Cells coexpressing p75{Delta}ICD-DsRed showed a significant (P<0.0001) reduction of filopodia compared with neurons expressing GFP only, on the identical coverslips, highlighting the physiological significance of TrkB.T1-induced filopodial growth (n=13-30 cells from three independent experiments). Scale bars: 20 µm (A); 5 µm (inset).

 


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  		Fig. 8. TrkB.T1 is endogenously expressed in hippocampal neurons and glia. (A) untransfected rat hippocampal microcultures were stained at 10 DIV with an antibody that selectively reacts with truncated TrkB receptors (TrkB[TK–]). (B) As a positive control, rat hippocampal neurons were transfected at 9 DIV with wt TrkB.T1 receptor and also processed for TrkB [TK–] immunocytochemistry. Neurons were identified by co-staining with a MAP2 antibody. Truncated TrkB receptors can be immunostained in untransfected neurons (arrows) and glia (asterisk). Specificity of the TrkB[TK–] antibody was evident from the intense signal in TrkB.T1-overexpressing neurons in B. Owing to the tenfold shorter exposure time for the overexpressed truncated receptor staining in the glia is not visible in B (lower panel). Scale bar: 10 µm.

 


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  		Fig. 9. Reduction of TrkB.T1-induced filopodia formation in hippocampal neurons from p75 knockout mice. Neurons from wt or p75NTR knockout mice were transfected with either TrkB.T1-GFP or GFP. The reduction in the density of TrkB.T1-induced filopodia in p75NTR knockout compared with wild-type mice is highly significant (P<0.0005). Both TrkB.T1-transfected groups were also significantly different from the respective GFP-transfected negative controls (*P<0.0001). The number of filopodia in GFP-expressing cells from p75NTR knockout mice was not significantly different from GFP-expressing wild-type cultures (P>0.15) (n=28-52 cells from two to three independent experiments).

 


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  		Fig. 10. Proposed model of TrkB.T1 action in the induction of filopodia. Interaction of TrkB.T1 with the p75NTR, leads to the formation of filopodia (grey arrow). This effect can be blocked by either the MC192 antibody or the dominant negative p75{Delta}ICD (inhibitory agents in red). The dominant negative action of the TrkB.FL receptor can be explained by its competition with TrkB.T1 for binding to either p75NTR (right margin) or by scavenging TrkB.T1 (left margin). NGF, BDNF, TrkB-IgG or K252a were without effect on this signalling mechanism. The short p75NTR (in blue) indicates a possible (albeit weaker) such signalling via a p75NTR lacking an intact extracellular domain (see p75NTR knockout mice experiments)

 

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© The Company of Biologists Ltd 2004