© 2014. Published by The Company of Biologists Ltd.

Neuronal activity alters BDNF-TrkB signaling kinetics and downstream functions

Wei Guo1,2, Yuanyuan Ji3, Shudan Wang1, Yun Sun4,5, Bai Lu1*

Journal of Cell Science

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1

: School of Medicine, Tsinghua University, 1 Qinghuayuan Road, Beijing, 100086,

China 2

: School of Life Sciences, Tsinghua University, 1 Qinghuayuan Road, Beijing, 100086,

China 3

: GlaxoSmithKline, R&D China,Building 3, 898 Halei Road,Zhangjiang Hi-tech Park,

Pudong, Shanghai, 201203,China 4

:National Institute of Biological Sciences, Beijing, 102206, China

5

: School of Life Sciences, Peking University, Beijing, 100871, China.

*: To whom the correspondence should be addressed at: bai. [email protected].

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JCS Advance Online Article. Posted on 14 March 2014

Summary Differential kinetics of the same signaling pathway may elicit different cellular outcomes. Here we show that high-frequency neuronal activity converts BDNF-induced TrkB signaling from a transient to a sustained mode. A prior depolarization (15 mM KCl, 1 hour) resulted in a long-lasting (>24 hours) activation of TrkB receptor and its downstream signals which otherwise lasts less than an hour. The LTP-inducing theta-burst stimulation but not the LTD-inducing low-frequency stimulation also induced sustained activation of TrkB. This sustained signaling

Journal of Cell Science

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facilitated dendritic branching and rescued neuronal apoptosis induced by glutamate. The change in TrkB signaling kinetics is mediated by calcium elevation and CaMKII activation, leading to an increase in TrkB expression on the neuronal surface. Physical exercise also alters the kinetics of TrkB phosphorylation induced by exogenous BDNF. Sustained TrkB signaling may serve as a key mechanism underlying synergistic effects of neuronal activity and BDNF. Running title :Activity sustains BDNF-TrkB signaling

Keywords: Signal transduction, Signaling kinetics, Neurotrophin, Neuronal activity, Neuronal surface receptor

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Introduction Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has multiple functions including neuronal survival, neuronal development and synaptic plasticity in the central nervous system (Huang and Reichardt, 2001; Lewin and Barde, 1996; Lu and Figurov, 1997; McAllister et al., 1999). For example, BDNF promotes dendritic outgrowth and branching, increases the density of dendritic spines and modulates spine morphological specializations (McAllister et al., 1995; Shimada et al., 1998). Many studies also suggest that BDNF plays a critical role in hippocampal

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activation of BDNF signaling pathways has been considered as a disease-modifying

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long-term potentiation (LTP) and long-term memory (Lu et al., 2008). Moreover,

of BDNF to neonatal hippocampal slices facilitates LTP, and this is mediated by an

strategy for neurological and psychiatric diseases ( Martinowich et al., 2007; Zuccato and Cattaneo, 2009; Nagahara and Tuszynski, 2011; Lu et al., 2013). The interplay between neuronal activity and BDNF is a subject of broad interest. On one hand, the effects of BDNF on neuronal activity and synaptic transmission have been observed in cultured neurons and in slices by many laboratories. For example, application enhancement of synaptic response to tetanic stimulation (Figurov et al., 1996). On the other hand, the effects of BDNF are modulated by neuronal activity. For example, blockade of neuronal activity and synaptic transmission prevents the increase of dendritic arborization induced by BDNF (McAllister et al., 1996). Presynaptic depolarization greatly facilitates the BDNF-induced synaptic transmission at the neuromuscular junction of Xenopus (Boulanger and Poo, 1999). BDNF effectively regulates repetitive excitatory synaptic responses only when the synapses were stimulated at 100 Hz or higher, which induces severe synaptic fatigue (Gottschalk et al., 1998). It remains ambiguous how neuronal activity modulates BDNF-TrkB signaling kinetics and downstream functions. BDNF binds TrkB, its high affinity receptor (Kaplan and Stephens, 1994; Squinto et al., 1991), leading to the activation of the downstream MAPK, PI3K and PLCγ pathways. After its activation, the TrkB receptor undergoes endocytosis, recycling or degradation (Haapasalo et al., 2002; Ji et al., 2010; Sommerfeld et al., 2000). In cultured hippocampal neurons, TrkB receptor on neuronal surface 3

increases after 15-second BDNF treatment and then declines rapidly(Haapasalo et al., 2002), while the total TrkB expression remains unchanged in the first few hours, followed by a reduction after several days through transcriptional regulation (Frank et al., 1996). It has been shown that the insertion and endocytosis of cell surface TrkB were acutely regulated by neuronal activity (Meyer-Franke et al., 1998; Du et al., 2000; Du et al., 2003). In the present study, we asked whether and how neuronal activity alters the kinetics of BDNF signaling. Surprisingly, we found that neuronal stimulation converts a transient TrkB activation and its downstream signals to the sustained mode, leading to an enhancement of dendritic branching and attenuation of neuronal apoptosis induced by TrkB induced by neuronal activity. Physical exercise known to enhance neuronal activity can also induce sustained TrkB signaling in vivo. These findings may provide new insights into the BDNF signaling and function in vivo.

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glutamate. This sustained TrkB activity is mediated by prolonged expression of surface

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Results Priming by depolarization converts TrkB signaling from transient to sustained mode A number of methods, including high K+ and electrical stimulation, have been used to enhance neuronal activity (Boulanger and Poo, 1999; Du et al., 2000; Meyer-Franke et al., 1998). We used 15 mM KCl to depolarize cultured hippocampal neurons. Similar to previous reports (Grubb and Burrone, 2010; Tongiorgi et al., 1997), our calcium imaging experiments showed that 15 mM KCl induced a sustained neuronal activation (supplementary material Fig. S1). Primary cultured hippocampal neurons (DIV 12-14)

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hour, followed by application of 1 nM BDNF. Proteins were collected at different time

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were pretreated with 15 mM KCl or 15 mM NaCl (as a control for ionic strength) for 1

and 4-hour time points after BDNF application remained about 80% of the maximum for

points for quantitative Western blotting. Acute application of BDNF (1 nM, 25ng/ml) triggered a robust but transient increase in TrkB phosphorylation: the level of phospho-TrkB (pTrkB) reached its peak at about 15 min but declined rapidly and approached baseline 4 h after BDNF application (Ji et al., 2010). Interestingly, same concentration of BDNF (1 nM) elicited a sustained TrkB phosphorylation when neurons were pretreated with 15 mM KCl for 1 hrs (Fig. 1A, left). The levels of pTrkB at 2-hour the 15 mM KCl group, but declined to baseline for the 15 mM NaCl group (Fig. 1B). In absence of BDNF, TrkB phosphorylation was undetectable with15 mM KCl alone (supplementary material Fig. S2A).Prior exposure of neurons to 15 mM NaCl (osmotic and Cl- control) resulted in a similar transient pattern of BDNF-induced TrkB phosphorylation (Fig. 1A, right). Depolarization-induced sustained TrkB signaling lasted as long as 24 hours, the longest time point examined. The levels of pTrkB at 8-hour and 24-hour time points after BDNF application remained about 100% and half of those at the 15-min time point respectively for the 15 mM KCl group, but only about 20% at the same time point for the 15 mM NaCl group (Fig. 1 C, D). The results suggest that priming with neuronal depolarization could convert BDNF-induced TrkB activation from a transient to a sustained mode. In subsequent experiments, we focused on kinetic changes in signaling within the first 4 hours, which exhibit a clear difference between these two modes. Next, we examined whether the high K+-induced sustained TrkB activation could lead to the same sustained activation of the three major signaling pathways downstream 5

of TrkB: MAPK, PI3K, and PLCγ. In hippocampal neurons, BDNF-induced activation of MAPK pathway was detectable by an antibody against phosphorylated Erk (pErk) on Western blots. A sustained pattern of Erk phosphorylation was evident in cells pretreated with 15 mM KCl followed by BDNF application (Fig. 2A, B). For comparison, BDNF induced a marked increase in pErk, which peaked around 15 min, followed by a rapid decline to basal level within the next 4 hours in the presence of 15 mM NaCl (Fig. 2A, B). For PI3K and PLC pathways, we examined the phosphorylation of Akt and PLC-1 (pAkt and pPLC-1). Similarly, sustained increases in pAkt and pPLC-1were induced by BDNF in the presence of high K+, while transient ones were induced in the presence

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of NaCl (Fig. 2A, B). We then examined the phosphorylation of CREB, the transcription factor downstream of MAPK known to mediate BDNF-induced gene expression and synaptic modulation (Finkbeiner et al., 1997). As expected, BDNF triggered a sustained phosphorylation of CREB with 15 mM KCl pretreatment (supplementary material Fig. S3). It should be noted that treatment with 15 mM KCl alone (but not NaCl alone) could induce CREB activation, which was shown at “0” time point in KCl + BDNF group. This is because high K+ can also increase phosphorylation of CREB through calcium influx (Bito et al., 1996). However, high K+ alone induced only transient activation of CREB (Sala et al., 2000). Taken together, these results suggest that neuronal activity (depolarization) could modulate the kinetics of BDNF-induced TrkB activation and its downstream signaling. High K+ facilitates BDNF regulation of survival and neurite branching Previously we reported that neurite branching is regulated by sustained activation of TrkB induced by gradual stimulation with BDNF (Ji et al., 2010). Here we report that sustained TrkB activation by depolarization coupled with BDNF stimulation also facilitated neurite branching (Fig. 3A). Hippocampal neurons (DIV3) treated with BDNF either in the presence of 15 mM NaCl or 15 mM KCl for 3 days were analyzed for dendritic complexity after immunostaining with anti-MAP2 antibody, and dendritic morphology was analyzed. Treatment with BDNF alone increased both the number of primary neurites and number of branch points (Fig. 3A, p < 0.01, ANOVA). Treatment with high K+ alone had no effect on either parameter. However, high K+ coupled with BDNF selectively facilitated dendritic branching, without affecting the number of 6

primary dendrites (Fig. 3A). The number of branch points (3.35 ± 0.17 per cell) in neurons treated with BDNF + 15 mM KCl was significantly higher than that treated only with either 15 mM KCl (2.05 ±0.14) or BDNF + 15 mM NaCl (2.46 ±0.16) (p < 0.001, ANOVA). Neuronal depolarization therefore augmented the effect of BDNF on dendritic branching. BDNF has been shown to promote neuronal survival in vitro (Arancibia et al., 2008; Mattson et al., 1995). Here we examined whether depolarization alters the neuronal survival function of BDNF in a glutamate-induced neuronal excitotoxicity (Almeida et al., 2005; Mattson et al., 1995). Hippocampal neurons were treated with 15 mM NaCl, 15

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mM KCl or control medium, followed by incubation with BDNF (1 nM) for 6 hours. Subsequently, the cultures were treated with vehicle or glutamate (125 µM) for 15 min and then returned to original medium. Seven hours later, neuronal apoptosis, as reflected by intracellular ATP level, was measured (Fig. 3B). Application of glutamate generally reduced cell survival by around 50%. In either control cultures or cultures pretreated with NaCl, BDNF failed to reverse glutamate-induced cell death. In contrast, prior neuronal depolarization by high K+ significantly attenuated the toxic effect of glutamate (p120, three independent experiments, N=3). Data are presented as mean  s.e.m., and analyzed by ANOVA. **: p < 0.01, ***: p < 0.001. A complete 26

table of ANOVA analysis is shown in supplementary material Table S1. (B) Attenuation of glutamate-induced toxicity by BDNF is facilitated by neuronal depolarization. Cultured hippocampal neurons were pretreated with either 15 mM NaCl or 15 mM KCl, and then BDNF (25 ng/ml) for 6 hours. The cultures were exposed to vehicle or glutamate (125 µM) for 15 min and then replaced with neurobasal medium with or without BDNF. Neuronal survival was determined later by measuring intracellular ATP seven hours later. N=3 three independent experiments, and in each experiment, same treatment was repeated 6 times (n=6). *: p < 0.05; ANOVA. BDNF significantly

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Fig. 4. TBS or tetanic stimulation sustains BDNF-induced TrkB phosphorylation.

Journal of Cell Science

attenuated the toxicity in high K+ group but not in NaCl group.

Western blots (E, G) and quantitative plots (F, H) are presented. Data from 4 independent

(A-D) Cultured hippocampal neurons were stimulated by two different stimulation patterns: TBS or LIS for one hour. The neurons were then treated with 1 nM BDNF and proteins were collected at different time points. Representative Western blots (A, C) and quantitative plots (B, D) are presented. (E-H) Cultured hippocampal neurons were stimulated by tetanic stimulation or LFS. Then neurons were treated with 1 nM BDNF and proteins were collected at different time points (from 0 to 4 h). Representative experiments (N = 4) were averaged and plotted. Dotted line indicates pTrkB level before BDNF application. Data are presented as mean  s.e.m. and compared by paired t-test. *: p < 0.05, **: p < 0.01. Fig. 5. Role of glutamate receptors, CaMKII and calcium channels in activity-dependent BDNF-induced sustained TrkB activation. (A-C) Hippocampal neurons were pre-treated with 100 nM K252a, 50 M MK801, 20 M KN-93 or 20 M U0126 for 30 min before incubation with KCl (15 mM, one hour) and then 1 nM BDNF. Representative Western blots (A) and quantitative plots of 2-hour time point (B) and 4-hour time point (C) are presented. Note: at 2 or 4-hour time point, pTrkB level in each condition was compared to 15 mM KCl + 1 nM BDNF group, respectively. (D-F) Hippocampal neurons were pre-treated with 100 nM K252a, 50 M MK801, 20 M KN-93 or 20 M U0126 for 30 min before TBS stimulation and then 1 nM BDNF treatment. Representative Western blots (D) and quantitative plots of 2-hour time point (E) and 4-hour time point (F) are presented. (G, H) Hippocampal neurons 27

were pre-treated with the calcium channel blockers CdCl2, Nimodipine or the AMPA receptor antagonist CNQX for 30 min before stimulation by 15 mM KCl (one hour). Then 1 nM BDNF was added on the cultured neurons which were collected 4h later for pTrkB measurement. Representative Western blots (G) and quantitative plots (H) are presented. Data from 4 independent experiments (N = 4) were averaged and plotted. Note: at 2 or 4-hour time point, pTrkB level in each condition was compared to TBS + 1 nM BDNF group, respectively. *: p < 0.05, **: p < 0.01, ***: p < 0.001; ANOVA. Fig. 6. Neuronal activity attenuates BDNF-induced down-regulation of TrkB

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Hippocampal neurons were treated with 15 mM KCl, 15 mM NaCl, TBS, or LIS for one

Journal of Cell Science

expression on neuronal surface.

BDNF treatment). *: p < 0.05, **: p < 0.01; ANOVA.

hour before 1 nM BDNF application. Cell surface proteins were biotinylated at different time points after BDNF application, and collected using immobilized Streptavidin. Equal amounts of biotinylated proteins were loaded to analyze surface TrkB expression. Representative Western blots (A, C) and quantitative plots (B, D) are presented. Data from 4 independent experiments (N = 4) were averaged and plotted. Note: surface TrkB expression level in each condition was compared to the control (“0” time point of 1 nM

Fig. 7. Prolonged surface TrkB expression induced by neuronal activity depends on NMDA receptor and CaMKII. (A-D) Hippocampal neurons were pre-treated with 50 M MK801, 20 M KN-93 or 20 M U0126 for 30 min before incubation with 15 mM KCl, 15 mM NaCl or stimulation with TBS for one hour, and then 1 nM BDNF for 4 hours. Cell surface proteins were biotinylated and collected with Immobilized Streptavidin. Equal amounts of biotinylated proteins were loaded to analyze surface TrkB expression. Representative Western blots (A, C) and quantitative plots (B, D) are presented. Data from 4 independent experiments (N = 4) were averaged and plotted. *: p < 0.05, **: p < 0.01; ANOVA. Fig. 8. Running on the treadmill prolonged TrkB activation induced by exogenous BDNF in vivo. Rats were forced to run on the treadmill for half an hour and rest for another half an hour. BDNF was injected into the right but not left hippocampal CA1. About 1mg tissue 28

around the injection site was dissected out at 1h and 4h time points and processed for TrkB phosphorylation by Western blot. (A) Sample Western blot showing the effect of running on TrkB phosphorylation induced by BDNF injection to the right hippocampus. As controls, the left hippocampus (PBS injection) was processed the same way (the right 4 lanes). (B) Quantification of the effect of running on TrkB phosphorylation kinetics (N=4). The values were derived from the formula shown on the right. Data are presented

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as mean  s.e.m. and compared by paired t-test. *: p < 0.05.

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Fig. 1 A

15 mM KCl + 1 nM BDNF 15 mM NaCl + 1 nM BDNF Time (h) Ctrl 0 0.25 0.5 1

0 0.25 0.5 1

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D pTrkB (normalized to total TrkB)

8 pT r k B ( n o r m a liz e d to to ta l Tr k B )

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pTrkB

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15 mM KCl + 1 nM BDNF

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0 0.25 8

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15 mM KCl + 1 nM BDNF Time (h)

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pErk Erk pAkt Akt pPLCγ PLCγ

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15 mM KCl + 1 nM BDNF 15 mM NaCl + 1 nM BDNF

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pX (normalized to total X)

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2.5 4.0

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Fig. 3 A

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6.47 6.47±0.22 Number of primary dendrites 1.64 1.64±0.14

1 nM BDNF

15 mM KCl

15 mM NaCl

15 mM KCl + BDNF

15 mM NaCL 15 mM KCl + 1 nM BDNF

7.72 7.72±0.26** 6.74 6.74±0.21

7.43 7.43±0.27

2.41 2.41±0.17 ** 1.92 1.92±0.12

2.46 2.46±0.16

6.6 6.60±0.24

2.05 2.05±0.14

15 mM KCl + 1 nM BDNF

6.85 6.85±0.23 3.35 3.35±0.17***

** significant between Ctrl and BDNF; p

Neuronal activity alters BDNF-TrkB signaling kinetics and downstream functions.

Differential kinetics of the same signaling pathway might elicit different cellular outcomes. Here, we show that high-frequency neuronal activity conv...
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