TrkB phosphorylation by Cdk5 is required for activity-dependent structural plasticity and spatial memory

Journal name:
Nature Neuroscience
Volume:
15,
Pages:
1506–1515
Year published:
DOI:
doi:10.1038/nn.3237
Received
Accepted
Published online

Abstract

The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB participate in diverse neuronal functions, including activity-dependent synaptic plasticity that is crucial for learning and memory. On binding to BDNF, TrkB is not only autophosphorylated at tyrosine residues but also undergoes serine phosphorylation at S478 by the serine/threonine kinase cyclin-dependent kinase 5 (Cdk5). However, the in vivo function of this serine phosphorylation remains unknown. We generated knock-in mice lacking this serine phosphorylation (TrkbS478A/S478A mice) and found that the TrkB phosphorylation–deficient mice displayed impaired spatial memory and compromised hippocampal long-term potentiation (LTP). S478 phosphorylation of TrkB regulates its interaction with the Rac1-specific guanine nucleotide exchange factor TIAM1, leading to activation of Rac1 and phosphorylation of S6 ribosomal protein during activity-dependent dendritic spine remodeling. These findings reveal the importance of Cdk5-mediated S478 phosphorylation of TrkB in activity-dependent structural plasticity, which is crucial for LTP and spatial memory formation.

At a glance

Figures

  1. TrkB is phosphorylated at S478 at neuronal synapse.
    Figure 1: TrkB is phosphorylated at S478 at neuronal synapse.

    (a) Western blotting confirmed that S478 phosphorylation was absent in the brains of TrkbS478A/S478A mice. TrkB phosphorylation at Y490 was similar between wild-type (Trkb+/+) and TrkbS478A/S478A mice, although TrkB expression was slightly reduced in TrkbS478A/S478A brains. Data are expressed as mean ± s.e.m. and were pooled from three experiments (three pairs of mice for each genotype; *P < 0.05, ***P < 0.001, Student's t-test). Full-length blots were presented in Supplementary Figure 11. (b) TrkB was phosphorylated at S478 in SPM and PSD fractions of rat brains. Enrichment of specific pre- and postsynaptic proteins in the SPM and PSD fractions was verified by antibodies to synaptophysin and PSD-95. (c) Colocalization of phospho-S478 TrkB immunoreactivity with the synaptic marker PSD-95 (arrows) in cultured hippocampal neurons. (d) TrkB was concentrated in the SPM fraction in both wild-type and TrkbS478A/S478A brains. Similar enrichment of TrkB was observed from two different wild-type and TrkbS478A/S478A brains. (e) Immunofluorescence staining of dissociated hippocampal neurons showed similar colocalization of total TrkB (red) with PSD-95 (green) in TrkbWT/WT and TrkbS478A/S478A neurons. (f) Biotinylation of cultured cortical neurons revealed similar levels of surface TrkB expression (normalized with total TrkB in whole cell lysate) in both TrkbWT/WT and TrkbS478A/S478A neurons (data are expressed as mean ± s.e.m. and were pooled from three experiments; P > 0.05, Student's t-test; difference in total TrkB level between TrkbWT/WT and TrkbS478A/S478A neurons was significant; **P <0.01, Student's t-test). (g) Surface staining with antibody to the extracellular domain of TrkB showed distinct puncta of surface TrkB (red) in the dendrites (blue) of TrkbWT/WT and TrkbS478A/S478A neurons (middle and right). The specificity of the surface staining was verified by the absence of red puncta without TrkB antibody (left). Scale bars represent 10 μm.

  2. S478 phosphorylation of TrkB is required for BDNF-induced spine morphogenesis and glutamate-induced spine enlargement.
    Figure 2: S478 phosphorylation of TrkB is required for BDNF-induced spine morphogenesis and glutamate-induced spine enlargement.

    (a) Hippocampal neurons were co-transfected with GFP and vector (mock), wild-type TrkB (WT), or TrkB S478A construct. Neurons were treated with BDNF for 24 h. Representative images are shown. BDNF increased the spine density (middle; ***P < 0.001, Student's t test) and spine area (bottom; ***P < 0.001, Kolmogorov-Smirnov test) in mock or wild-type TrkB–expressing neurons, but not in neurons that overexpressed the TrkB S478A mutant. We measured 36 dendrites from 12 neurons for each condition. Scale bars represent 10 μm. (b) GFP-transfected hippocampal neurons derived from control (TrkbWT/WT) or TrkbS478A/S478A mice were treated with BDNF for 24 h. Representative images are shown. Treatment with BDNF significantly increased the spine density of the control neurons, but not that of neurons lacking TrkB S478 phosphorylation (middle, 9–16 dendrites from 5–10 neurons were measured for each condition; ***P < 0.001, one-way ANOVA, Bonferroni's multiple comparison test). BDNF also increased the spine area in control neurons, but not in TrkbS478A/S478A neurons (bottom; **P < 0.01, Kolmogorov-Smirnov test). Scale bars represent 10 μm. (c) GFP-transfected hippocampal neurons (17–18 d in vitro (DIV)) cultured from TrkbWT/WT or TrkbS478A/S478A mutant mice were stimulated by uncaging MNI-glutamate, and the changes in spine size at different duration after glutamate receptor activation were monitored by time-lapse confocal microscopy. Representative images are shown (left). Quantification revealed a significant difference in the fold change in spine area between TrkbWT/WT and TrkbS478A/S478A neurons in response to uncaged glutamate (right; data were pooled from three experiments, 20 neurons were measured for each genotype; *P < 0.05, Student's t-test). Scale bar represents 5 μm. Data are expressed as mean ± s.e.m. for the histograms in ac.

  3. S478 phosphorylation regulates the interaction between TrkB and the Rac GEF TIAM1.
    Figure 3: S478 phosphorylation regulates the interaction between TrkB and the Rac GEF TIAM1.

    (a) BDNF-induced TrkB tyrosine phosphorylation and phosphorylation of downstream targets such as AKT and ERK was not affected in TrkbS478A/S478A cortical neurons (data were pooled from three experiments with neurons cultured from three mice for each genotype; P > 0.05 between TrkbWT/WT and TrkbS478A/S478A neurons, one-way ANOVA, Bonferroni's multiple comparison test). Full-length blots were presented in Supplementary Figure 11. (b) BDNF induced tyrosine phosphorylation of TIAM1 and co-immunoprecipitation of TrkB and TIAM1 in cultured cortical neurons. The specificity of the co-immunoprecipitation was confirmed by absence of TrkB and phospho-Y829 TIAM1 in the control immunoprecipitation with IgG. (c) Co-immunoprecipitation between TrkB and TIAM1 was performed in 293T cells that overexpressed Cdk5 and p35 together with either wild-type TrkB or TrkB S478A mutant. Mutation of TrkB at S478 substantially reduced its association with TIAM1. (d) TIAM1 was co-immunoprecipitated with TrkB in whole-brain lysate from control mice (TrkbWT/WT), but not the TrkbS478A/S478A mice (three pairs of mice for each genotype; ***P < 0.001, Student's t-test). Immunoprecipitation with IgG served as a control for the specificity of interaction. (e) Treatment of cortical neurons with BDNF triggered tyrosine phosphorylation of TIAM1 at Y829 in cortical neurons derived from control mice (TrkbWT/WT), but the induction was completely abolished in TrkbS478A/S478A cortical neurons. (f) TIAM1 is required for BDNF-induced spine morphogenesis. Coexpression of dominant-negative TIAM1 (TIAM1 PHCCEx) with GFP significantly reduced the area (*P < 0.05, Kolmogorov-Smirnov test) and density (*P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA, Bonferroni multiple comparison test) of dendritic spines before and after BDNF treatment. We measured 21–35 dendrites from 13–19 neurons for each condition. Scale bar represents 10 μm. Data are expressed as mean ± s.e.m. in the histograms of a, d and f.

  4. Cdk5-mediated phosphorylation of TrkB at S478 is required for BDNF-induced Rac1 activity and glutamate-induced PAK phosphorylation.
    Figure 4: Cdk5-mediated phosphorylation of TrkB at S478 is required for BDNF-induced Rac1 activity and glutamate-induced PAK phosphorylation.

    (a) BDNF induced Rac1 activity in cultured cortical neurons, as indicated by an increase in GTP-bound Rac1. The induction of Rac1 activity by BDNF was abolished in the presence of roscovitine. (b) The induction of Rac1 activity by BDNF was abolished in Cdk5−/− cortical neurons. (c) The enhanced Rac1 activity after BDNF stimulation was blocked in neurons expressing dominant-negative TIAM1. (d) BDNF-induced Rac1 activity was abolished in cortical neurons derived from TrkbS478A/S478A mice (data were pooled from three experiments for ac and four experiments for d; *P < 0.05, *** P < 0.001, one-way ANOVA, Bonferroni's multiple comparison test). (e) Cortical neurons (14 DIV) were silenced for 4 h by tetrodotoxin and NBQX before treatment with glutamate (50 μM) for 5 min. Glutamate increased phosphorylation of PAK in cortical neurons (**P < 0.01), and the increase was significantly attenuated in the presence of TrkB-IgG (1 μg ml−1) (pooled from three experiments, *P < 0.05, one-way ANOVA, Tukey multiple comparison test). (f) The induction of PAK phosphorylation by glutamate was observed in cortical neurons derived from the control mice TrkbWT/WT, but the increase in PAK phosphorylation was significantly reduced in TrkbS478A/S478A mutant cortical neurons (data were pooled from three experiments, with neurons cultured from three mice for each genotype; *P < 0.05, **P < 0.01, one-way ANOVA, Tukey multiple comparison test). Data were expressed as mean ± s.e.m. Full-length blots were presented in Supplementary Figure 11.

  5. Cdk5-mediated phosphorylation of TrkB at S478 is required for increased S6 phosphorylation and PSD-95 expression following NMDA receptor activation.
    Figure 5: Cdk5-mediated phosphorylation of TrkB at S478 is required for increased S6 phosphorylation and PSD-95 expression following NMDA receptor activation.

    (a) Treatment of cultured cortical neurons (14 DIV) with bicuculline (20 μM) and glycine (200 μM, 15 or 30 min) increased TrkB phosphorylation at both Y490 and S478 (pooled from three experiments; *P < 0.05, **P < 0.01, Student's t-test). (b) The induction of TrkB phosphorylation at S478 was abolished in the presence of the NMDA receptor antagonist AP5 (200 μM, 30min; pooled from four experiments, *P < 0.05, one-way ANOVA, Bonferroni's multiple comparison test). (c) The Cdk5 inhibitor roscovitine (Ros, 10 μM) abolished the NMDA receptor–induced TrkB phosphorylation at S478 (pooled from three experiments, *P < 0.05, one-way ANOVA, Newman-Keuls multiple comparison test). (d) The induced TrkB phosphorylation was absent in cortical neurons derived from Cdk5−/− mice (pooled from four experiments, and neurons were cultured from four mice for each genotype, *P < 0.05, ***P < 0.001, one-way ANOVA, Tukey's multiple comparison test). (e) Treatment of control (TrkbWT/WT) cortical neurons with bicuculline and glycine (15 min for phospho-Akt; 30 min for phospho-S6 and PSD-95) significantly increased phosphorylation of Akt and S6 ribosomal protein, and expression of PSD-95. The induction was absent in TrkbS478A/S478A mutant neurons (for phospho-Akt and phospho-S6, data were pooled from three experiments and neurons were cultured from three mice for each genotype; for PSD-95, data were pooled from five experiments with neurons being cultured from five mice for each genotype; **P < 0.01, ***P < 0.001, one way ANOVA, Tukey multiple comparison test). Full-length blots were presented in Supplementary Figure 11. Data were expressed as mean ± s.e.m.

  6. Impaired hippocampal LTP in TrkbS478A/S478A mutant mice.
    Figure 6: Impaired hippocampal LTP in TrkbS478A/S478A mutant mice.

    (a) BDNF-facilitated LTP in hippocampal slices was abolished in p35−/− mice (top) and TrkbS478A/S478A mice (bottom). Early phase LTP (E-LTP) was induced by a train of 100 Hz in the presence or absence of recombinant BDNF, and the fEPSP amplitude was recorded for 1 h after the tetanus. Exogenous BDNF significantly increased the fEPSP amplitude in wild-type p35+/+ (control, n = 15 slices, 11 mice; BDNF, n = 9 slices, 3 mice; **P < 0.01, one-way ANOVA; top) or the TrkbWT/WT control mice (control, n = 10 slices, 6 mice; BDNF, n = 9 slices, 5 mice; *P < 0.05, one-way ANOVA; bottom). The facilitation effect of BDNF was abolished in both the p35−/− mice (control, n = 10 slices, 4 mice; BDNF, n = 9 slices, 3 mice; top) and TrkbS478A/S478A mutant mice (control, n = 7 slices, 5 mice; BDNF, n = 6 slices, 4 mice; bottom). (b) TBS-induced LTP at the CA3-CA1 synapses of hippocampal slices from wild-type (Trkb+/+) (n = 16 slices, 7 mice) or TrkbS478A/S478A mutant (n = 17 slices, 8 mice). Quantification indicated that the fold change in average fEPSP amplitude 170–180 min after TBS stimulation was significantly reduced in the TrkbS478A/S478A mice when compared with wild-type mice (**P < 0.01, Student's t-test). Data were expressed as mean ± s.e.m.

  7. TrkbS478A/S478A mice display impaired memory in Morris water maze and novel object recognition, but have normal fear memory.
    Figure 7: TrkbS478A/S478A mice display impaired memory in Morris water maze and novel object recognition, but have normal fear memory.

    (a) Wild-type (Trkb+/+, n = 16) and TrkbS478A/S478A (n = 15) littermates were tested in a Morris water maze. Trkb+/+ mice spent significantly more time in the target quadrant (Q2) than any of the other quadrants in the probe trial (P < 0.001, one-way ANOVA, Newman-Keuls multiple comparison test). For TrkbS478A/S478A mice, the time spent in the target quadrant was similar to that spent in the other quadrants (P > 0.05, one-way ANOVA, Newman-Keuls multiple comparison test). Compared with wild-type mice, the target quadrant occupancy of TrkbS478A/S478A mice was also significantly reduced (*P < 0.05, two-way ANOVA, Bonferroni multiple comparison test). The dotted line indicates the chance level (25%). Swimming velocity was similar between Trkb+/+ and TrkbS478A/S478A mice (Trkb+/+ = 21.73 ± 0.57 cm s−1, TrkbS478A/S478A = 21.76 ± 0.64 cm s−1; P > 0.05, Student's t-test). (b) Escape latency between Trkb+/+ and TrkbS478A/S478A mice in the Morris water maze was similar on any given training day (P > 0.05, two-way ANOVA, Bonferroni multiple comparison test). (c) In the novel object recognition task, Trkb+/+ mice (n = 11), but not TrkbS478A/S478A mice (n = 10), preferred to explore the novel object during test day (three experiments, ***P < 0.001; one-way ANOVA, Tukey multiple comparison test). (d) In the passive avoidance task, the step through latency on day 3 (24 h after foot-shock) was similar between Trkb+/+ (n = 6) and TrkbS478A/S478A mice (n = 5) (P > 0.05, one-way ANOVA, Newman-Keuls multiple comparison test). (e) There was no significant difference in percent freezing between Trkb+/+ (n = 8) and TrkbS478A/S478A mice (n = 13) in contextual fear conditioning (P > 0.05, one-way ANOVA, Tukey multiple comparison test). Data are expressed as mean ± s.e.m. in all panels.

References

  1. Huang, E.J. & Reichardt, L.F. Trk receptors: roles in neuronal signal transduction. Annu. Rev. Biochem. 72, 609642 (2003).
  2. Kandel, E.R. The molecular biology of memory storage: a dialogue between genes and synapses. Science 294, 10301038 (2001).
  3. Lu, Y., Christian, K. & Lu, B. BDNF: A key regulator for protein synthesis–dependent LTP and long-term memory? Neurobiol. Learn. Mem. 89, 312323 (2008).
  4. Engert, F. & Bonhoeffer, T. Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399, 6670 (1999).
  5. Maletic-Savatic, M., Malinow, R. & Svoboda, K. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283, 19231927 (1999).
  6. Matsuzaki, M., Honkura, N. & Ellis-Davies, G.C.R. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761766 (2004).
  7. Fukazawa, Y. et al. Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP maintenance in vivo. Neuron 38, 447460 (2003).
  8. Tanaka, J. et al. Protein synthesis and neurotrophin-dependent structural plasticity of single dendritic spines. Science 319, 16831687 (2008).
  9. Chen, Z.Y. et al. Genetic variant BDNF (Val66Met) polymorphism alters anxiety-related behavior. Science 314, 140143 (2006).
  10. An, J.J. et al. Distinct role of long 3' UTR BDNF mRNA in spine morphology and synaptic plasticity in hippocampal neurons. Cell 134, 175187 (2008).
  11. Ninan, I. et al. The BDNF Val66Met polymorphism impairs NMDA receptor–dependent synaptic plasticity in the hippocampus. J. Neurosci. 30, 88668870 (2010).
  12. Tyler, W.J. & Pozzo-Miller, L.D. BDNF enhances quantal neurotransmitter release and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses. J. Neurosci. 21, 42494258 (2001).
  13. Ji, Y., Pang, P.T., Feng, L. & Lu, B. Cyclic AMP controls BDNF-induced TrkB phosphorylation and dendritic spine formation in mature hippocampal neurons. Nat. Neurosci. 8, 164172 (2005).
  14. Rex, C.S. et al. Brain-derived neurotrophic factor promotes long-term potentiation–related cytoskeletal changes in adult hippocampus. J. Neurosci. 27, 30173029 (2007).
  15. Minichiello, L. et al. Essential role for TrkB receptors in hippocampus-mediated learning. Neuron 24, 401414 (1999).
  16. Minichiello, L. et al. Mechanism of TrkB-mediated hippocampal long-term potentiation. Neuron 36, 121137 (2002).
  17. Cheung, Z.H., Chin, W.H., Chen, Y., Ng, Y.P. & Ip, N.Y. Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons. PLoS Biol. 5, e63 (2007).
  18. Lai, K.O. & Ip, N.Y. Recent advances in understanding the roles of Cdk5 in synaptic plasticity. Biochim. Biophys. Acta 1792, 741745 (2009).
  19. Matsuzaki, M. et al. Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 10861092 (2001).
  20. Harvey, C.D. & Svoboda, K. Locally dynamic synaptic learning rules in pyramidal neuron dendrites. Nature 450, 11951200 (2007).
  21. Korkotian, E. & Segal, M. Morphological constraints on calcium-dependent glutamate receptor trafficking into individual dendritic spine. Cell Calcium 42, 4157 (2007).
  22. Nakayama, A.Y. & Luo, L.Q. Intracellular signaling pathways that regulate dendritic spine morphogenesis. Hippocampus 10, 582586 (2000).
  23. Tashiro, A., Minden, A. & Yuste, R. Regulation of dendritic spine morphology by the Rho family of small GTPases: antagonistic roles of Rac and Rho. Cereb. Cortex 10, 927938 (2000).
  24. Miyamoto, Y., Yamauchi, J., Tanoue, A., Wu, C. & Mobley, W.C. TrkB binds and tyrosine-phosphorylates Tiam1, leading to activation of Rac1 and induction of changes in cellular morphology. Proc. Natl. Acad. Sci. USA 103, 1044410449 (2006).
  25. Tolias, K.F. et al. The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines. Neuron 45, 525538 (2005).
  26. Xie, Z. et al. Kalirin-7 controls activity-dependent structural and functional plasticity of dendritic spines. Neuron 56, 640656 (2007).
  27. Saneyoshi, T. et al. Activity-dependent synaptogenesis: regulation by a CaM-kinase kinase/CaM-kinase I/beta PIX signaling complex. Neuron 57, 94107 (2008).
  28. Barco, A. et al. Gene expression profiling of facilitated L-LTP in VP16-CREB mice reveals that BDNF is critical for the maintenance of LTP and its synaptic capture. Neuron 48, 123137 (2005).
  29. Buchsbaum, R.J., Connolly, B.A. & Feig, L.A. Regulation of p70 S6 kinase by complex formation between the rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin. J. Biol. Chem. 278, 1883318841 (2003).
  30. Lu, W. et al. Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 29, 243254 (2001).
  31. Richter, J.D. & Klann, E. Making synaptic plasticity and memory last: mechanisms of translational regulation. Genes Dev. 23, 111 (2009).
  32. Todd, P.K., Mack, K.J. & Malter, J.S. The fragile X mental retardation protein is required for type-I metabotropic glutamate receptor-dependent translation of PSD-95. Proc. Natl. Acad. Sci. USA 100, 1437414378 (2003).
  33. Figurov, A., Pozzo-Miller, L.D., Olafsson, P., Wang, T. & Lu, B. Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature 381, 706709 (1996).
  34. Ohshima, T. et al. Impairment of hippocampal long-term depression and defective spatial learning and memory in p35 mice. J. Neurochem. 94, 917925 (2005).
  35. Kang, H., Welcher, A.A., Shelton, D. & Schuman, E.M. Neurotrophins and time: different roles for TrkB signaling in hippocampal long-term potentiation. Neuron 19, 653664 (1997).
  36. Patterson, S.L. et al. Some forms of cAMP-mediated long-lasting potentiation are associated with release of BDNF and nuclear translocation of phospho-MAP kinase. Neuron 32, 123140 (2001).
  37. Abel, T. et al. Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory. Cell 88, 615626 (1997).
  38. Miller, S. et al. Disruption of dendritic translation of CaMKIIalpha impairs stabilization of synaptic plasticity and memory consolidation. Neuron 36, 507519 (2002).
  39. Kelleher, R.J. III, Govindarajan, A., Jung, H.Y., Kang, H. & Tonegawa, S. Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 116, 467479 (2004).
  40. Heldt, S.A., Stanek, L., Chhatwal, J.P. & Ressler, K.J. Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories. Mol. Psychiatry 12, 656670 (2007).
  41. Rampon, C. et al. Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice. Nat. Neurosci. 3, 238244 (2000).
  42. Wei, F.Y. et al. Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability. J. Neurochem. 93, 502512 (2005).
  43. Fischer, A., Sananbenesi, F., Pang, P.T., Lu, B. & Tsai, L.H. Opposing roles of transient and prolonged expression of p25 in synaptic plasticity and hippocampus-dependent memory. Neuron 48, 825838 (2005).
  44. Hawasli, A.H. et al. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation. Nat. Neurosci. 10, 880886 (2007).
  45. Li, B.S. et al. Regulation of NMDA receptors by cyclin-dependent kinase-5. Proc. Natl. Acad. Sci. USA 98, 1274212747 (2001).
  46. Morabito, M.A., Sheng, M. & Tsai, L.H. Cyclin-dependent kinase 5 phosphorylates the N-terminal domain of the postsynaptic density protein PSD-95 in neurons. J. Neurosci. 24, 865876 (2004).
  47. Bramham, C.R. Local protein synthesis, actin dynamics and LTP consolidation. Curr. Opin. Neurobiol. 18, 524531 (2008).
  48. Bolton, M.M., Pittman, A.J. & Lo, D.C. Brain-derived neurotrophic factor differentially regulates excitatory and inhibitory synaptic transmission in hippocampal cultures. J. Neurosci. 20, 32213232 (2000).
  49. van der Geer, P., Hunter, T. & Lindberg, R.A. Receptor protein-tyrosine kinases and their signal transduction pathways. Annu. Rev. Cell Biol. 10, 251337 (1994).
  50. Fu, A.K. et al. Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction. Nat. Neurosci. 4, 374381 (2001).
  51. Liu, P. et al. Bcl11a is essential for normal lymphoid development. Nat. Immunol. 4, 525532 (2003).
  52. Southon, E. & Tessarollo, L. Manipulating mouse embryonic stem cells. Methods Mol. Biol. 530, 165185 (2009).
  53. Reid, S.W. & Tessarollo, L. Isolation, microinjection and transfer of mouse blastocysts. Methods Mol. Biol. 530, 269285 (2009).
  54. Xie, H. et al. Brain-derived neurotrophic factor rescues and prevents chronic intermittent hypoxia-induced impairment of hippocampal long-term synaptic plasticity. Neurobiol. Dis. 40, 155162 (2010).
  55. Fu, W.Y. et al. Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism. Nat. Neurosci. 10, 6776 (2007).

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Author information

Affiliations

  1. Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China.

    • Kwok-On Lai,
    • Alan S L Wong,
    • Man-Chun Cheung,
    • Pei Xu,
    • Zhuoyi Liang,
    • Ka-Chun Lok,
    • Zelda H Cheung &
    • Nancy Y Ip
  2. Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China.

    • Kwok-On Lai,
    • Alan S L Wong,
    • Man-Chun Cheung,
    • Pei Xu,
    • Zhuoyi Liang,
    • Ka-Chun Lok,
    • Zelda H Cheung &
    • Nancy Y Ip
  3. State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China.

    • Kwok-On Lai,
    • Alan S L Wong,
    • Man-Chun Cheung,
    • Pei Xu,
    • Zhuoyi Liang,
    • Ka-Chun Lok,
    • Zelda H Cheung &
    • Nancy Y Ip
  4. School of Biomedical Science, The Chinese University of Hong Kong, Hong Kong, China.

    • Hui Xie &
    • Wing-Ho Yung
  5. Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA.

    • Mary E Palko &
    • Lino Tessarollo

Contributions

K.-O.L. and N.Y.I. supervised the project. K.-O.L., A.S.L.W., Z.H.C. and N.Y.I. designed the experiments. K.-O.L., A.S.L.W., M.-C.C., P.X., Z.L. and K.-C.L. conducted the experiments. K.-O.L., A.S.L.W., M.-C.C., P.X., Z.L., K.-C.L. and N.Y.I. carried out the data analyses. W.-H.Y. designed and carried out the data analyses of the electrophysiology experiments and H.X. performed the electrophysiology experiments and data analysis. L.T. and M.E.P designed and generated the knock-in mice. K.-O.L., Z.H.C. and N.Y.I. wrote the manuscript.

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The authors declare no competing financial interests.

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