Section on Neural Development and Plasticity

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neurotrophic regulation of

synapse development and plasticity

Bai Lu, PhD, Head, Section on Neural Development and Plasticity

Feng Yang, MD, PhD, Research Fellow

Eugene Zaitsev, PhD, Research Fellow

Guhan Naggapan, PhD, Visiting Fellow

Kazuko Sakata, PhD, Visiting Fellow

Newton Woo, PhD, Visiting Fellow

Mingrui Zhao, PhD, Visiting Fellow

Hyun-Soo Je, BS, Predoctoral Fellow

Petti Pang, BS, Graduate Student

We study molecular mechanisms underlying synapse development and function, with particular emphasis on the role of neurotrophic factors. Traditionally, neurotrophic factors are defined as secretory proteins that regulate neuronal survival and differentiation. Recent studies indicate that a major function of neurotrophic factors in the brain is the regulation of synaptic transmission and plasticity. Our laboratory was among the first to reveal this novel function. Using rodent models, we demonstrated that brain-derived neurotrophic factor (BDNF) plays a key role in hippocampal long-term potentiation (LTP), a cellular model for learning and memory. Using the Xenopus neuromuscular synapse as a model, we identified two modes of regulation by neurotrophins: acute modulation of synaptic transmission and plasticity and long-term alteration of the structure and function of synapses. In the past year, we discovered the mechanistic link between tissue plasminogen activator (tPA), an extracellular protease implicated in long-term plasticity and memory, and BDNF in late-phase LTP (L-LTP) in the hippocampus. We have also made progress in understanding how BDNF signaling through TrkB receptor is controlled. In addition, we discovered a dynamin-independent form of synaptic vesicle endocytosis.

Long-term regulation of synaptic structures and function by BDNF

Pang, Zaistev, Woo, Sakata, Lu; in collaboration with Hempstead, Welker

The rodent barrel cortex offers an excellent model system in which to study activity-dependent synaptogenesis. Mechanical stimulation in adult whisker induces the expression of BDNF as well as synapse formation in layer IV of the barrel cortex. We used serial section electron microscopy to study the ultrastructural alterations of synaptic connectivity in the barrel cortex, including measurement of synapse density, spine morphology, and synaptic vesicle distribution. BDNF heterozygote (+/−) mice, with a reduced level of BDNF mRNA and protein, had a barrel neuropil indistinguishable from that of wild-type (+/+) controls. After 24 hours of whisker stimulation, however, the +/+ mice, but not the +/− mice, exhibited a significant increase in the number of synapses formed. The balance between excitatory and inhibitory synapses was modified in the +/+ mice, but not in the +/− mice. The distribution of synaptic vesicles in excitatory synapses was the same in +/+ and +/− mice and was not influenced by the stimulation paradigm. Moreover, the spine volume was significantly more increased by stimulation in +/− mice than in +/+ mice. These results provide direct evidence that, in vivo, BDNF plays a crucial role in the structural rearrangement of cortical circuitry as a consequence of an enhanced sensory stimulation.

Long-term memory is believed to be mediated by protein synthesis–dependent L-LTP. Two secretory proteins, tPA and BDNF, have been implicated in L-LTP, but their relationship is unclear. We demonstrated that, by activating the extracellular protease plasmin, tPA converts the precursor proBDNF to the mature BDNF (mBDNF) in the hippocampus and that such conversion is critical for L-LTP expression. Our electrophysiological studies demonstrated that mBDNF, but not the cleavage-resistant proBDNF, rescues L-LTP in tPA- and plasminogen-knockout mice. Biochemical experiments also showed that tPA, by converting plasminogen to plasmin, cleaves proBDNF to form mBDNF. In addition, genetic and pharmacological experiments revealed that, in L-LTP expression, mBDNF is downstream of plasmin, which is downstream of tPA. Moreover, application of mBDNF converts early-phase LTP (E-LTP) to L-LTP and rescues L-LTP when protein synthesis is blocked by the inhibitor anisomycin. These results suggest that mBDNF is a key protein synthesis product responsible for L-LTP expression. Taken together, the present study has identified both tPA/plasmin as an endogenous enzyme system that converts proBDNF to mBDNF in the hippocampus and a physiological role of such conversion in the brain. Further, the results have provided a mechanistic link between these two seemingly independent molecule systems in L-LTP expression.

Egan MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, Zaistev E, Bertolino A, Gold B, Goldman D, Dean M, Lu B, Weinberger DR. The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 2003;112:257-269.

Genoud C, Knott GW, Sakata K, Lu B, Welker E. Reduced BDNF expression prevents synapse formation in the adult somatosensory cortex. J Neurosci 2004;24:2394-2400.

Lu B. Pro-region of neurotrophins: role in synaptic modulation. Neuron 2003;38:735-738.

Pang PT, Lu B. Regulation of late-phase LTP and long-term memory in normal and aging hippocampus: role of secreted proteins tPA and BDNF. Aging Res Rev 2004;3:407-430.

Pang PT, Teng H, Zaitsev E, Woo KN, Sakata K, Zhen S, Teng K, Yung W-H, Hempstead B, Lu B. Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 2004;306:487-491.

Mechanisms controlling BDNF signaling through the TrkB receptor

Sakata, Lu; in collaboration with Kojima, Zheng

Although BDNF plays a key role in synapse development and plasticity, the underlying signaling mechanisms remain largely unknown. We found that BDNF rapidly recruits full-length TrkB (TrkB-FL) receptor into lipid rafts, cholesterol- and sphingolipid-enriched membrane microdomains thought to be signaling platforms for extracellular stimuli, from nonraft regions of neuronal plasma membranes. Truncated TrkB lacking the intracellular kinase domain was not translocated, and Trk inhibitors blocked the translocation of TrkB-FL, suggesting that phosphorylation by TrkB tyrosine kinase is required for such translocation. Disruption of lipid rafts by depleting cholesterol from the cell surface blocked the ligand-induced translocation. Moreover, disruption of lipid rafts prevented potentiating effects of BDNF on transmitter release in cultured neurons and synaptic response to tetanus in hippocampal slices. In contrast, lipid rafts are not required for BDNF regulation of neuronal survival. Thus, ligand-induced TrkB translocation into lipid rafts may represent a signaling mechanism selective for synaptic modulation by BDNF in the central nervous system.

We further tested the role of lipid rafts in BDNF signaling during axon growth, which requires signal transduction of extracellular cues for directional motility. We showed that lipid raft disruption abolished growth cone attraction and repulsion in gradients of BDNF and netrin-1, respectively, but exerted no effects on glutamate-induced attraction. Interestingly, local raft disruption on one side of the growth cone exposed to uniform BDNF or netrin-1 produced opposite turning responses to that induced by the gradients. Lipid raft manipulation also blocked semaphorin 3A–induced growth cone repulsion, inhibition, and collapse. Finally, we found that guidance cue–elicited MAP kinase activation depended on raft integrity and that specific guidance receptors were associated with lipid rafts. The results demonstrate an essential role for lipid rafts in growth cone guidance and suggest that localized signaling through these dynamic microdomains underlies specific actions of extracellular cues on developing axons.

We next examined how cAMP controls BDNF/TrkB signaling. While it has been hypothesized that the synaptic actions of BDNF are “gated” by cAMP, the underlying molecular mechanisms remain unclear. We showed that cAMP regulates BDNF function by modulating TrkB receptor signaling and trafficking. TrkB phosphorylation is controlled by cAMP, with three features characteristic for cAMP gating: first, inhibitors of cAMP signaling attenuated BDNF-induced TrkB phosphorylation; second, cAMP analogs potentiated such phosphorylation; and third, activation of cAMP pathway alone had no effect on TrkB phosphorylation. cAMP also facilitated trafficking of TrkB to dendritic spines, possibly by promoting its interaction with synaptic scaffolding protein PSD-95, and gated BDNF’s long-term modulation of spine density, but not dendritic branching, in cultured hippocampal neurons. These results reveal a specific role of cAMP in controlling BDNF actions in the brain and provide new insights into the molecular mechanism underlying cAMP gating.

Du J, Feng L, Zaitsev E, Je H-S, Liu X, Lu B. Regulation of TrkB receptor tyrosine kinase and its internalization by neuronal activity and Ca²⁺ influx. J Cell Biol 2003;163:385-395.

Guirland C, Suzuki S, Kojima M, Lu B, Zheng JQ. Lipid rafts mediate chemotropic guidance of nerve growth cones. Neuron 2004;42:51-62.

Ji Y, Pang PT, Feng L, Lu B. Cyclic AMP controls BDNF-induced TrkB phosphorylation and dendritic spine formation in hippocampal neurons. Nat Neurosci 2004, in press.

Lu B, Je H-S. Neurotrophic regulation of the development and function of the neuromuscular synapses. J Neurocytol 2004;32:931-941.

Suzuki S, Numakawa T, Shimazu K, Koshimizu H, Hara T, Hatanaka H, Mei L, Lu B, Kojima M. BDNF-induced recruitment of TrkB into lipid rafts: roles in synaptic transmission. J Cell Biol 2004;167:1205-1215.

A novel mechanism for synaptic vesicle endocytosis

Lu; in collaboration with Zheng

In addition to studying neurotrophic regulation, we are interested in the basic mechanisms for the development and function of synapses. One of the essential mechanisms is the activity-dependent endocytosis of synaptic vesicles. Synaptic vesicle endocytosis is believed to require Ca²⁺ and the small GTPase dynamin. We have recently identified a novel form of rapid endocytosis (RE) in dorsal root ganglion (DRG) neurons that, unlike previously described forms of endocytosis, is completely independent of Ca²⁺ and the GTPase dynamin. The RE is tightly coupled to a Ca²⁺-independent but voltage-dependent secretion (CIVDS). Using FM dye and capacitance measurements, we showed that membrane depolarization induces RE in the absence of Ca²⁺. Inhibition of dynamin function does not affect RE. Inhibitors of protein kinase A (PKA) suppress RE induced by high-frequency depolarization while PKA activators enhance RE induced by low-frequency depolarization. Biochemical experiments demonstrated that depolarization directly upregulates PKA activity in Ca²⁺-free medium. The results reveal a Ca²⁺- and dynamin-independent form of endocytosis in DRG neurons, with endocytosis controlled by neuronal activity and PKA-dependent phosphorylation.

Bamji SX, Shimazu K, Kimes N, Huelsken J, Birchmeier W, Lu B, Reichardt LF. Role of beta-catenin in synaptic vesicle localization and presynaptic assembly. Neuron 2003;40:719-731.

Guhan N, Lu B. PIKE/Homer complex: a novel link between mGluR1 and PI3 kinase. Trends Neurosci 2004;27:645-648.

Luo ZG, Je H-S, Yang F, Xiong WC, Lu B, Mei L. Implication of geranylgeranyltransferase I in synapse formation. Neuron 2003;40:703-717.

Yang F, He X, Russell J, Lu B. Ca²⁺ influx-independent synaptic potentiation mediated by mitochondrial Na⁺-Ca²⁺ exchanger and protein kinase C. J Cell Biol 2003;163:511-523.

Zhang C, Xiong W, Zheng H, Wang LC, Lu B, Zhou Z. Activity-dependent but Ca²⁺-independent endocytosis in dorsal root ganglion neurons. Neuron 2004;42:225-236.

COLLABORATORS

Barbara Hempstead, MD, Cornell University Medical School, New York, NY

Masami Kojima, PhD, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

Louis F. Reichardt, PhD, Herbert Boyer Program in Biological Science, University of California, San Francisco, CA

Egbert Welker, PhD, University of Lausanne, Switzerland

James Zheng, PhD, The University of Medicine and Dentistry of New Jersey, Piscataway, NJ

Zhuan Zhou, PhD, Institute of Neuroscience, Shanghai, China

For further information, contact lub@mail.nih.gov