Fig. 1. Stable expression of the wild-type TrkA and receptor mutants in nnr5 cells. (A) Schematic representation of the cytosolic domain of the wild-type TrkA receptor and selected receptor mutants. Y794F represents a TrkA receptor in which the PLC-γ-binding site was inactivated by substituting the tyrosine residue Y794 with a phenyl alanine, while in Re794Y all tyrosine residues have been replaced by phenylalanines, with the exception of Y794. Tyrosine residues Y679, Y683 and Y684 are essential for receptor autophosphorylation and were maintained in all mutants. (B) Western blot of wild-type (nnr5-TrkA) and mutated TrkA receptors (nnr5-Y794F and nnr5-Re794Y) stably expressed in nnr5 cells. Wild-type receptor and mutants were assayed for their levels of expression. We selected clones that, by probing with an anti-pan-Trk antibody, showed comparable levels of receptor protein.

Fig. 3. High potassium- and NGF-mediated BDNF secretion from nnr5-TrkA, nnr5-Y794F and nnr5-Re794Y. Cells were perfused at a flow rate of 0.1 ml/min. Fractions were collected every 5 min. After collecting two fractions, a first stimulation by 50 mM KCl was initiated and four additional fractions collected. After a recovery of 30 min, cells were stimulated by 100 ng/ml NGF. BDNF concentrations were measured by two-site ELISA. Increased BDNF concentrations (expressed in pg/ml) occurred immediately after stimulation. The peak concentration occurred in the following fraction. The 5 min delay between the onset of stimulation and the peak of BDNF secretion is due to the dead volume of the perfusion system. NGF induced BDNF secretion in nnr5-TrkA and nnr5-Re794Y cells, but not in nnr5 or nnr5-Y794F cells. The values given represent the mean ± SE (n = 6).

Fig. 5. BDNF secretion from cultured hippocampal neurons expressing TrkA, Y794F and Re794Y receptors. (A) Hippocampal neurons transduced with AdCMV-TrkA, AdCMV-Y794F and AdCMV-Re794Y were stimulated with 100 ng/ml NGF for 10 min under ‘static’ conditions. NGF-induced BDNF secretion occurred only in neurons expressing wild-type TrkA and the Re794Y receptors, but not Y794F or the green fluorescence protein (GFP) taken as a control of virus infection. The values given are the mean ± SE (n = 10). (B) Time course of BDNF secretion induced by NT4/5 via endogenous TrkB or by NGF via transuced Re794Y. Cells were perfused and fractions were collected before, during and after stimulation with 100 ng/ml NT4/5. A second stimulation was initiated by the administration of 100 ng/ml NGF. NT4/5 activated endogenous TrkB receptors, and NGF the TrkA receptor mutant Re794Y. The values given are the mean ± SE (n = 6).

Fig. 7. Dependence on intact Ca²⁺ stores of NT-mediated NT secretion in hippocampal neurons. (A) BDNF secretion from hippocampal neurons in the absence of extracellular Ca²⁺ (BAPTA), chelation of intracellular calcium by BAPTA-AM and after depletion of the intracellular Ca²⁺ stores by caffeine and thapsigargine. Cultured hippocampal neurons co-infected with AdCMV-BDNF and AdCMV-Re794Y were perfused as described in Figure 3. After stimulation with 100 ng/ml NT4/5 for 5 min in normal Ca²⁺, a second stimulation with 100 ng/ml NGF was performed in Ca²⁺-free medium or medium containing the Ca²⁺ chelator BAPTA-AM (10 µM). Removal of extracellular Ca²⁺ (BAPTA) did not affect the ability of NGF to induce BDNF secretion. However, the effect of NGF on BDNF secretion was abolished by pre-treating hippocampal neurons for 30 min with the cell-permeable BAPTA-AM. Similarly, the depletion of intracellular Ca²⁺ stores with a combination of caffeine (3 mM) and thapsigargine (10 µM) abolished the NGF-mediated BDNF secretion. The values given represent the mean ± SE (n = 6). (B) Role of Ca²⁺ in BDNF secretion from nnr5-TrkA or nnr5-Re794Y clones. Similarly to the effects shown in hippocampal neurons, NGF induced BDNF secretion from nnr5 clones in the absence of extracellular Ca²⁺ (BAPTA). In contrast, chelating the intracellular Ca²⁺ with BAPTA-AM or depleting intracellular Ca²⁺ stores with caffeine/thapsigargine prevented NGF-mediated secretion of BDNF. The values given represent the mean ± SE (n = 7).

Fig. 9. Analysis of the signal transduction pathway(s) leading to BDNF secretion by glutamate. (A) BDNF secretion initiated by glutamate receptor agonists in cultured hippocampal neurons. Primary cultures of hippocampal neurons were infected with AdCMV-BDNF for 36–48 h. After an equilibration time of 60 min, basal levels of secreted BDNF were determined in the medium collected over a 10 min period under ‘static’ conditions. Stimulation of neurons for 10 min with glutamate (50 µM), AMPA (100 µM) or t-ACPD (100 µM) resulted in increased concentrations of BDNF in the incubation medium. NMDA had no effect. (B) Hippocampal neurons, pre-treated with the specific antagonists of AMPA and mGluRI receptors, CNQX (50 µM) and AIDA (500 µM), respectively, were tested for the effects of AMPA and t-ACPD. (C) Influence of Ca²⁺ stores on AMPA- and t-ACPD-mediated BDNF secretion. In hippocampal neurons, treatment with thapsigargine (10 µM) and caffeine (3 mM) initiated BDNF secretion but abolished the subsequent secretion of BDNF induced by AMPA and t-ACPD. The values given represent the mean ± SE (n = 6).

Fig. 2. Comparison between receptor tyrosine and PLC-γ phosphoryl ation in wild-type and mutated TrkA receptors in stably transfected nnr5 cells and adenovirally transduced hippocampal neurons. (A) NGF-mediated tyrosine phosphorylation in nnr5-TrkA, nnr5-Y794F and nnr5-Re794Y clones. Each clone was stimulated for 0, 5 or 10 min with 100 ng/ml NGF and assayed for receptor tyrosine phosphorylation. Wild-type and mutated receptors were immunoprecipitated from cell lysates by an anti-pan-Trk antibody and the samples separated on an SDS–polyacrylamide gel. Western blots were probed with an anti-phosphotyrosine antibody. NGF administered for 5 or 10 min produced a marked tyrosine phosphorylation in wild-type and receptor mutants. (B) NGF-mediated PLC-γ tyrosine phosphorylation in nnr5 clones. The individual clones were stimulated with 100 ng/ml NGF for 0 or 5 min. The cell lysates were immunoprecipitated by agarose-linked anti-phosphotyrosine antibodies and subjected to western blot analysis with a specific anti-PLC-γ antibody. NGF treatment for 5 min was sufficient to induce PLC-γ phosphorylation in nnr5-TrkA and nnr5-Re794Y clones, whereas nnr5-Y794F clones did not show a PLC-γ phosphorylation signal in either the presence or absence of NGF. (C) NGF-mediated tyrosine phosphorylation in cultured hippocampal neurons transduced with TrkA and Re794Y receptors. Transduced neurons were stimulated for 0 and 5 min with 100 ng/ml NGF and assayed for receptor tyrosine phosphorylation. The western blot was performed as in (A). NGF produced a marked tyrosine phosphorylation in the wild-type receptor and mutant. (D) NGF- and BDNF-mediated PLC-γ tyrosine phosphorylation in cultured hippocampal neurons transduced with Re794Y. Cultured hippocampal neurons infected with the adenovirus coding for the Re794Y were stimulated for 0 or 5 min with 100 ng/ml either NGF or BDNF. Lysates of hippocampal neurons were immunoprecipitated by an agarose-conjugated anti-phosphotyrosine antibody. PLC-γ phosphorylation was identified by western blotting as described in (A). Both NGF and BDNF induced PLC-γ tyrosine phosphorylation to a similar extent.

Fig. 4. Adenovirally transduced nnr5 cells. (A) Time course of BDNF secretion from nnr5 cells double transduced with AdCMV-Re794Y and AdCMV-BDNF. Perfusion was performed as described in Figure 3. Two subsequent stimulations by 50 mM KCl and 100 ng/ml NGF were separated by a recovery period of 30 min. Fractions were collected every 5 min and the BDNF concentrations were determined by ELISA. The values presented are the mean ± SE (n = 4). (B) Expression of BDNF and Re794Y mutant in nnr5 cells co-transduced with AdCMV-BDNF and AdCMV-Re794Y. The expression of both BDNF and Re794Y was detected by probing with the hybridoma supernatant (9E10), which recognizes the myc epitope added to the C-terminal domain of BDNF, and with the anti-pan-Trk antibody, which recognizes the C-terminus of Re794Y. Cells were analysed by confocal microscopy. The pictures represent a single confocal scan along a given x–y plane. The BDNFmyc-infected nnr5 cells showed a discontinuous staining pattern characteristic for the localization of BDNF in ER and Golgi compartments (green). The staining obtained with an anti-pan-Trk antibody was localized predominantly at the plasma membrane (red).

Fig. 6. Changes of intracellular Ca²⁺ concentrations in hippocampal neurons expressing the Re794Y receptors. (A) Non-transduced hippocampal neurons (controls) and those transduced by Re794Y were loaded with Fura-2/AM. Changes in intracellular Ca²⁺ concentrations were determined by the ratio of the fluorescence at excitation wavelengths of 340 and 380 nm. In the absence of extracellular Ca²⁺ (BAPTA), hippocampal neurons expressing the TrkA receptor mutant showed an NGF-mediated increase in [Ca²⁺]_i that could not be distinguished from that obtained by BDNF acting via endogenous TrkB receptors. This is a representative example of eight independent experiments. (B) Effect of the depletion of the intracellular Ca²⁺ stores by thapsigargine and caffeine on the subsequent Ca²⁺ signalling by BDNF or NGF. In the absence of extracellular Ca²⁺ (BAPTA), caffeine/thapsigargine initiated a strong, prolonged Ca²⁺ signal resulting from the depletion of the Ca²⁺ stores. Subsequent administration of BDNF and NGF did not elicit a detectable Ca²⁺ signal, in distinct contrast to the hippocampal neurons not treated with caffeine and thapsigargine. This is a representative example of three independent experiments.

Fig. 8. Time course of BDNF secretion in native, non-transduced hippocampal slices. Acute hippocampal slices were perfused and fractions were collected over consecutive 5 min periods. BDNF concentrations were determined by ELISA. After a recovery period of 30 min, an initial stimulation by 50 µM glutamate was followed by a second stimulation with 100 µM t-ACPD, AMPA or NMDA. The values given are the mean ± SE (n = 4).