Article |
Address correspondence to Bai Lu, Section on Neural Development and Plasticity, Laboratory of Cellular and Synaptic Neurophysiology, NICHD, NIH Building 49, Rm. 6A80, 49 Convent Dr., Bethesda, MD 20892-4480. Tel.: (301) 435-2970. Fax: (301) 496-1777. email: bailu{at}mail.nih.gov
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Abstract |
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Key Words: BDNF; endocytosis; calcium influx; hippocampus; activity dependent
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Introduction |
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Neuroelectric activity, like neurotrophins, is known to regulate the structure and function of synapses during development and refinement of neuronal connectivity in the adult (Katz and Shatz, 1996). The relationship between activity and neurotrophins in neuronal modulation remains largely unknown. Interestingly, neuronal activity often influences the effectiveness of neurotrophins, particularly BDNF. For example, regulation of dendritic arborization by BDNF requires neuronal activity and Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors (McAllister et al., 1996). BDNF regulation of the survival of retinal ganglion neurons is also dependent on neuronal depolarization (Meyer-Franke et al., 1995). Presynaptic depolarization greatly facilitates BDNF modulation of synaptic transmission at the neuromuscular junction (Boulanger and Poo, 1999). In the hippocampus, the effect of BDNF on CA1 synapses appears to be restricted to highly active presynaptic neurons (Gottschalk et al., 1998). Thus, whether or how well a neuron can respond to BDNF may depend on its intrinsic neuronal activity. One strategy is to increase the number of BDNF receptors on the cell surface. Indeed, treatment with depolarizing agents results in an increase in the amount of the BDNF receptor TrkB on the plasma membranes of retinal ganglion cells and spinal neurons (Meyer-Franke et al., 1998). In the hippocampus, tetanic stimulation, but not simple depolarization or low frequency stimulation, has been shown to facilitate the insertion of TrkB into the cell surface (Du et al., 2000). This effect requires Ca2+ influx through NMDA receptors or voltage-gated Ca2+ channels, but appears to be independent of ligand binding (Du et al., 2000). Binding of ligands to Trk receptor induces their tyrosine kinase activity and internalization, both of which are important for neurotrophin signaling (Kaplan and Stephens, 1994; Bothwell, 1995; Riccio et al., 1997; Zhang et al., 2000). Thus, an alternative and physiologically relevant way to control BDNF responsiveness is to regulate the tyrosine kinase activity and/or the internalization of TrkB receptor.
Here, we aimed to investigate whether and how neuroelectric activity and consequent Ca2+ influx regulates the internalization of the TrkB receptor and its relationship with TrkB tyrosine kinase function. Field electric stimulation was applied to hippocampal neurons to elicit action potentials. Three independent approaches were used to measure receptor internalization induced by BDNF. We show that neuroelectric activity facilitates the internalization of TrkB, as well as its tyrosine kinase activity. We also demonstrate that the tyrosine kinase activity of TrkB is critical for the activity-dependent modulation of TrkB internalization. These results identified a novel mechanism by which biological responses to BDNF might be regulated, and they provided new insights into the cell biology of tyrosine kinase receptors.
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Results |
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Although the imaging assay allowed visualization of BDNF receptor internalization, it was not quantitative. We used a BDNF binding assay that simultaneously quantified both cell surface receptors and internalized receptors. Cultured hippocampal neurons were incubated at 37°C with radiolabeled BDNF (125I-BDNF, 50 pM) with or without cold BDNF (50 nM). At the end of the 30-min incubation, 125I-BDNF bound to the receptors on neuronal surfaces was washed off by mild acid, and the amount of acid-washable radioactivity was used to quantify the cell surface BDNF receptors. The radioactivity inside the cells after acid wash was used to quantify receptor internalization. Both surface binding and internalization were markedly reduced when excess amount of cold BDNF was included in the incubation, suggesting that the assay is specific for BDNF receptors (Fig. 2 A). Time course studies indicated that binding was saturable within 30 min, whereas internalization continued to increase over 2 h (Fig. 2 B). Incubation of hippocampal neurons in 125I-BDNF at 4°C for 4 h still yielded high levels of surface binding, but there was virtually no radioactivity inside cells after acid wash (unpublished data), suggesting that the assay measures the true BDNF receptor-mediated internalization.
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The BDNF-biotin and 125I-BDNF assays cannot distinguish whether the internalized receptors are TrkB receptors or p75 NGF receptors (p75NR). Our third approach to detect receptor internalization was a biotinylation assay using a TrkB antibody. Cultured hippocampal neurons were incubated with BDNF on ice for 30 min to achieve saturated BDNF binding. All proteins on the cell surface were labeled with NHS-SS-biotin. By placing the cultures in a 37°C incubator for 30 min, we initiated BDNF-induced receptor internalization (because BDNF was already bound to the receptors on the surface of these cells). The internalization was terminated by placing the cultures on ice, and remaining biotinylated surface proteins were debiotinylated by cleavage of the NHS-SS-biotin disulfide bond with glutathione. The cells were lysed, and all internalized biotinylated proteins were precipitated by streptavidin, and separated by gel electrophoresis. Immunoblotting was then performed using an antibody against the extracellular domain of TrkB. The total surface biotinylated TrkB was determined in cells held on ice without glutathione cleavage, and background internalization was measured in cells that were debiotinylated without initiation of internalization (Fig. 3 A). A substantial amount of TrkB was internalized when the cultures were warmed to 37°C in the presence of BDNF (Fig. 3 A, +BDNF). Spontaneous internalization of TrkB was also detected in cells not treated with BDNF, although the levels were lower (Fig. 3 A, -BDNF). The difference in these two signals was used to quantify ligand-induced TrkB internalization. Using this assay, we found that BDNF induced significantly more TrkB internalization in active neurons (stimulated with TBS) than in inactive neurons (Fig. 3 B; TBS + CNQX/MK801 or Kyn). Quantitative analysis of the blots showed that the activity blockers reduced TrkB internalization to levels below those by application of BDNF alone (nonstimulated cultures, as indicated by the dashed line in Fig. 3 C). This result again suggests that spontaneous synaptic activity in nonstimulated cultures may also influence BDNF-induced TrkB endocytosis. However, TBS stimulation did not affect spontaneous internalization of insulin-like growth factor-1 receptors (IGF1-R) because reprobing of the same blots with antiIGF1-R antibody did not detect any significant decrease in cells treated with the activity blockers (Fig. 3, B and D). Thus, neuronal activity does not have a general effect on membrane protein endocytosis. Finally, we found that in the absence of exogenous BDNF, TBS has no effect on TrkB internalization (Fig. 3 E, compare lane 2 with lane 4), suggesting that activity selectively enhances ligand-induced TrkB internalization.
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In the next series of experiments, we determined whether neuronal activity also regulates TrkB tyrosine kinase activity. This was achieved by measuring tyrosine phosphorylation of the TrkB receptor itself, using Western blots with an antibody that specifically recognizes TrkB phosphorylated on the tyrosine residue 490 (pTrkB; Segal et al., 1996). In nonstimulated cultures, there was virtually no detectable pTrkB. Application of 2 nM BDNF to the cultures elicited a rapid phosphorylation of TrkB (Fig. 5 A, group I). Stimulation of the hippocampal neurons with TBS significantly enhanced the auto-phosphorylation of TrkB (Fig. 5 A, compare groups I and III; #, P < 0.05, t test). Moreover, blockade of synaptic transmission by a cocktail of CNQX/MK801 reduced pTrkB in stimulated cultures (Fig. 5 A, compare groups III and IV; *, P < 0.01, t test). Thus, the activity-dependent modulation TrkB tyrosine phosphorylation also involves action potentials coupled to excitatory synaptic transmission. In nonstimulated cultures, inhibition of excitatory transmission inhibited TrkB phosphorylation, suggesting that spontaneous firing and/or synaptic transmission in the hippocampal cultures potentiate the TrkB tyrosine kinase activity (Fig. 5 A, compare groups I and II; *, P < 0.01). Total TrkB served as an internal loading control, and did not show any significant change in any of these conditions (Fig. 5 B).
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Next, we determined whether the activity-dependent enhancement of TrkB tyrosine phosphorylation also requires Ca2+ influx. The effect of electric stimulation was significantly attenuated when normal culture medium was replaced by Ca2+-free medium (Fig. 6 A). TrkB phosphorylation was also severely reduced when hippocampal neurons were stimulated with TBS in the presence of the general Ca2+ channel blockers Cd2+ (0.2 mM) or Co2+ (3 mM), or the NMDA receptor blocker MK801 (80 µM; Fig. 6 A). Thus, Ca2+ influx through voltage-gated Ca2+ channels and/or NMDA receptors play an important role in activity-induced potentiation of TrkB tyrosine kinase activity in hippocampal neurons. Specific types of Ca2+ channels involved in modulating TrkB tyrosine phosphorylation remain unknown. To determine whether neuronal activity/Ca2+ influx enhances the TrkB tyrosine kinase by facilitating TrkB internalization, we measured the effects of electric stimulation on TrkB phosphorylation in the presence of monodansylcadavenrine (MDC), a widely used agent known to block clathrin-mediated receptor internalization for a variety of cell types (Schutze et al., 1999). Application of BDNF to cultured hippocampal neurons induced a significant TrkB internalization over the spontaneous or ligand-independent internalization of TrkB (Fig. 6 B, right, lanes 1 and 2; and Fig. 3 A, lanes 3 and 4). Pretreatment with 10 µM MDC for 15 min reversed BDNF-induced TrkB internalization (Fig. 6 B). The residual bands are likely to represent spontaneous TrkB internalization. To determine the specificity of MDC in hippocampal neurons, we compared its effect with that of a dynamin proline-rich domain peptide, which is known to block clathrin-mediated endocytosis (Wang and Linden, 2000). Treatment of the hippocampal cultures with this peptide, but not the control, scrambled peptide, inhibited BDNF-induced endocytosis to the same extent as MDC (Fig. 6 B). These results suggest that MDC could effectively block BDNF-induced TrkB endocytosis in hippocampal neurons. In MDC-treated cultures, there was no significant change in the TBS-induced increase in TrkB tyrosine phosphorylation (Fig. 6 C), suggesting that the enhancement of TrkB tyrosine kinase by electric stimulation does not require TrkB internalization. Thus, activity-dependent modulation of TrkB tyrosine phosphorylation occurs upstream of TrkB internalization.
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Discussion |
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Unlike other tyrosine kinase receptors, internalization of Trk receptors often serves as an important step that mediates some biological functions of neurotrophins, rather than as a process that inactivates neurotrophin signaling. For example, substantial evidence suggests that TrkA internalization is required to initiate cell body responses to target-derived NGF (Grimes et al., 1996; Bhattacharyya et al., 1997; Riccio et al., 1997). Blockade of TrkA internalization by dominant-negative dynamin prevents NGF-induced neurite outgrowth in PC12 cells (Zhang et al., 2000). At the neuromuscular synapses, acute application of NT3 rapidly potentiates transmitter release (Lohof et al., 1993), whereas long-term treatment with NT3 induces both structural and functional changes at the neuromuscular synapses (Wang et al., 1995). Using dominant-negative dynamin and bead-conjugated NT3, we recently found that the long-term, but not acute, synaptic modulation by NT3 requires dynamin-dependent internalization of the NT3 receptor TrkC (unpublished data). Thus, the internalization of Trk receptors is critical for many neurotrophin actions, and modulation of this process by neuronal activity has a profound physiological relevance.
The internalization process could be measured semi-quantitatively by 125I-BDNF binding or the TrkB biotinylation. However, the latter seemed to detect bigger effects of neuronal activity (Fig. 3 C; TBS-stimulated neurons exhibited twice as much internalization compared with neurons stimulated by TBS in the presence of activity blockers) than the former (Fig. 2 D; internalization was 3355% more in active neurons than in inactive neurons). A possible explanation is that 125I-BDNF assay may reflect the internalization of both TrkB and p75NR. The presence of p75NR, which is expressed at a quite low level in these hippocampal neurons (Frade et al., 1996), could interfere with the measurement of the TrkB signals that are specifically regulated by neuroelectric activity. Thus, although less efficient, the TrkB-biotinylation assay was more reliable in quantifying TrkB internalization. Using this assay, we often observed that manipulation of neuroelectric activity affected the internalization of both full-length and truncated TrkB receptors, which lack the kinase domain. In contrast, several lines of experiments clearly indicated that TrkB tyrosine kinase activity is important for the activity-dependent modulation of internalization. Therefore, it is puzzling why the internalization of the kinase-deficient truncated TrkB was still regulated by neuronal activity. One possibility is that the full-length and the truncated TrkB receptors are located very close to each other on the cell surface. When exposed to BDNF, the full-length receptor is internalized, carrying the truncated TrkB in the same endocytotic vesicles into the hippocampal neuron.
Neuronal activity has recently been shown to rapidly activate TrkB, but this effect has been interpreted as a consequence of activity-dependent secretion of BDNF (Aloyz et al., 1999; Patterson et al., 2001). Two pieces of evidence suggest that the activity-dependent enhancement of TrkB tyrosine kinase in our cultured hippocampal neurons is not due to an elevated BDNF secretion: (1) in the absence of BDNF, electric stimulation did not activate TrkB receptor; and (2) electric stimulation still increases TrkB tyrosine phosphorylation induced by BDNF at a saturated concentration. Thus, we have observed a direct effect of neuroelectric activity on TrkB tyrosine kinase. The fact that electric stimulation potentiates TrkB tyrosine phosphorylation when TrkB internalization is blocked by MDC, and that inhibition of TrkB tyrosine kinase attenuates TrkB internalization, suggests that activation of TrkB tyrosine kinase is upstream of its internalization. Neuroelectric activity and Ca2+ influx potentiate both the insertion and the internalization of TrkB, but these two effects differ in several ways. The internalization of TrkB is triggered by the ligand BDNF, and is regulated by its tyrosine kinase. In contrast, TrkB insertion is ligand independent, and is not influenced by TrkB tyrosine kinase activity. Moreover, high frequency stimulation is required for TrkB insertion, whereas low frequency neuronal firing seems to be sufficient to enhance Trk internalization. Thus, the molecular mechanisms underlying the insertion and internalization of TrkB receptor may be quite different.
The results in this work may have a number of implications in the cell biology of tyrosine kinase receptors. First, we report the potentiation of TrkB tyrosine kinase by neuronal activity and Ca2+ influx. To our knowledge, this is the first demonstration for Ca2+-dependent modulation of Trk kinases, and perhaps receptor tyrosine kinases in general. Thus, our results suggest a cross-talk between Ca2+ and tyrosine kinase signaling pathways. The molecular mechanisms underlying such a cross-talk remain to be investigated. Second, this work reveals an important regulatory effect of neuronal activity and Ca2+ influx on the internalization/endocytosis of the TrkB, a tyrosine kinase receptor. This process resembles in many ways the endocytosis of AMPA-type glutamate receptors, which is implicated in the mechanism for long-term depression in the hippocampus (Carroll et al., 2001). It will be interesting to determine whether the trafficking of AMPA receptors and TrkB receptors share similar underlying mechanisms. Finally, we show that inhibition of TrkB tyrosine kinase dramatically attenuates the activity and Ca2+ regulation of TrkB internalization, suggesting a key role of tyrosine phosphorylation in TrkB endocytosis. Ligand-induced endocytosis of EGF receptor has been shown to require the tyrosine kinase activity of the receptor (Lamaze and Schmid, 1995). This is achieved by EGF receptor-dependent activation of Src tyrosine kinase, leading to phosphorylation and redistribution of clathrin, a major player in ligand-induced endocytosis (Wilde et al., 1999). It is tempting to speculate that TrkB tyrosine kinase also phosphorylates and modulates some common molecules involved in endocytosis. Together, these results suggest a general role of tyrosine kinase in the endocytosis of growth factor receptors.
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Materials and methods |
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Biotinylation of BDNF and imaging assay
100 µg BDNF (provided by Regeneron Pharmaceuticals, Inc.) was incubated with 2 mg NHS-LC-Biotin (Pierce Chemical Co.) in 100 µl PBS with Ca2+ and Mg2+ for 2 h at 4°C. Biotinylated BDNF and unbound biotin were separated with a desalting gel column (D-SaltTM polyacrylamide 1800; Pierce Chemical Co.). 20 nM biotinylated BDNF (BDNF-biotin; determined by Bio-Rad Laboratories protein assay) with or without cold BDNF (200-fold excess) was applied to cultured neurons in DME containing 0.5% protamine and 10 mM Hepes for 30 min on ice. Unbound BDNF-biotin was washed out with culture medium. Internalization was initiated by applying warm media (37°C) to the cultures with or without various inhibitors. After 30 min of incubation, the cultures were washed with ice-cold acid for 20 min to remove the surface-bound BDNF. The cultures were fixed in 4% PFA in PBS and permeabilized; and the internalized BDNF-biotin was visualized by Cy3-conjugated avidin (1:500; Jackson ImmunoResearch Laboratories) in 0.4% Triton X-100, 5% goat serum in PBS. The cells were mounted by mounting media and visualized by a 510-meta confocal microscope. Three-dimensional images were reconstructed using the Z stack function (20 sections from top to bottom, 1 µm/section). The images in the middle range of the Z stack were used to examine the internalization particles.
125I-BDNF receptor binding and internalization assays
The 125I-BDNF binding and internalization assays were performed as described previously (Du et al., 2000). BDNF surface binding was obtained by acid wash to remove the 125I-BDNF bound to cell surface (0.2 M acetic acid and 0.5 M NaCl). BDNF receptor internalization was obtained by measuring the remaining radioactivity inside the cells. To determine the total BDNF surface binding without internalization, the binding assay was performed at 4°C for 4 h.
Biotinylation assay of TrkB internalization
Hippocampal neurons were treated with 8 nM BDNF for 30 min on ice. Unbound BDNF was washed off three times with cold PBS. The cell surface proteins were labeled with 0.5 mg/ml NHS-SS-biotin (Pierce Chemical Co.) in PBS with Ca2+ and Mg2+ for 2.5 min at 37°C, and then washed extensively with ice-cold PBS. Internalization was initiated by switching to warm media (37°C) for 30 min. The remaining, biotinylated surface proteins were debiotinylated by washing with glutathione buffer (50 mM reduced glutathione, 100 mM NaCl, 1 mg/ml BSA, 1 mg/ml glucose, and 50 mM Tris, pH 8.6) for 3x 30 min at 4°C. The cells were washed an additional two times with PBS and harvested with the lysis buffer (the same as that for Western blot). The internalized, biotinylated proteins were precipitated by immobilized streptavidin, separated by SDS-PAGE, and subjected to Western blot using a monoclonal anti-TrkB antibody (1:200; Transduction Labs), or a polyclonal anti-IGF1 receptor antibody (1:100; Sigma-Aldrich).
BDNF-induced TrkB internalization was effectively inhibited by either MDC or dynamin proline-rich peptide (dyn-pep; Wang and Linden, 2000). We made the dyn-pep with a leading Tat sequence from HIV known to facilitate penetration of the peptide into the cells (RKKRRQRRRQVPSRPNRAP). A scrambled peptide, RKKRRQRRRQPPASNPRVR, was used as a control. Hippocampal neurons (12 d in culture) were incubated with the peptides (80 µM) for 1 h before the TrkB biotinylation experiments.
Western blot
Western blots were performed as described previously (Du et al., 2000). TrkB phosphorylation was detected by an antibody specifically recognizing the phosphorylated tyrosine residue 490 (1:500; New England Biolabs, Inc.) in 0.5% BSA in TBST (1:500), followed by ECL detection (Pierce Chemical Co.). Because these neurons do not express TrkA, and TrkC runs at a lower molecular mass, the 145-kD band seen on the gel represents phospho-TrkB.
Data analysis
The blots after ECL reactions were exposed to film (Kodak) at different exposure times. The films were scanned, and the intensities of the bands, as well as that of the background near the bands, were measured by Kodak 3.0 software. The specific signals were obtained by subtracting the background values from the total intensities. To ensure that the densitometric values faithfully reflected the relative levels of TrkB phosphorylation or internalization, the following measures were taken. First, only the films in which immunoreactive bands fell within the linear range (not saturated) were used for quantification. Second, multiple lanes of the same samples were often included in the same blot to obtain average values of a specific condition. Finally, the same experiments were repeated at least three to five times, using independent samples. For 125I-BDNF assay, raw data (quadruplicates) from a specific experimental condition were normalized to the mean in TBS-stimulated condition. The results in 832 experiments were pooled and averaged, and presented as mean ± SEM.
To facilitate cross-experiment comparison, we generally set the average value for internalization in TBS-stimulated cells as 100%, and normalize data on all other conditions to that in "stimulation alone" condition in both 125I-BDNF binding assay and TrkB biotinylation assay. To distinguish the "phosphorylation" data from the "internalization" data, we normalized all the TrkB phosphorylation data to "nonstimulated" condition.
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Acknowledgments |
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This work was supported by funds from NICHD Intramural program (to B. Lu).
Submitted: 28 May 2003
Accepted: 3 September 2003
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References |
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