Among the first to reveal their novel function, we study the role of neurotrophins, a class of secretory proteins that emerged in recent years as key regulators of synaptic transmission and plasticity. One of our key discoveries was that brain-derived neurotrophic factor (BDNF) enhances early-phase long-term potentiation (E-LTP) in the hippocampus by facilitating synaptic response to tetanic stimulation through enhanced synaptic vesicle docking. We also discovered that synapse-specific modulation by BDNF is achieved by regulating the trafficking of the receptor TrkB through activity-dependent insertion, endocytosis, synaptic localization, and so forth. The two modes of neurotrophic regulation are acute modulation of synaptic transmission and plasticity and long-term alteration of the structure and function of synapses. A single nucleotide polymorphism (SNP) in the pro-domain of BDNF affects activity-dependent BDNF secretion, leading to impairment in hippocampal function and short-term memory in humans. In addition, proteolytic conversion of proBDNF to mature BDNF by extracellular protease tPA/plasmin is essential for late-phase LTP (L-LTP), a form of plasticity implicated in long-term memory. In the past year, our most exciting finding was the discovery that, if uncleaved, proBDNF facilitates hippocampal long-term depression (LTD) by activating p75NTR. We have also made progress in understanding the molecular mechanisms underlying activity-dependent secretion of BDNF.
Conversion of pro- to mature BDNF by extracellular proteases and its role in L-LTP
Pang, Zaitsev, Woo, Sakata, Lu; in collaboration with Hempstead, Teng
Long-term memory is believed to be mediated by protein synthesis–dependent L-LTP. Two secretory proteins, tPA and BDNF, have been implicated in the process of long-term memory, but their relationship is unclear. We demonstrate 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 demonstrate that mBDNF, but not cleavage-resistant proBDNF, rescues L-LTP in tPA and plasminogen knockout mice. Biochemical experiments also show that, by converting plasminogen to plasmin, tPA cleaves proBDNF to form mBDNF. In addition, genetic and pharmacological experiments reveal that, in L-LTP expression, mBDNF acts downstream of plasmin, which in turn acts downstream of tPA. Moreover, application of mBDNF converts E-LTP to L-LTP and rescues L-LTP when the inhibitor anisomycin blocks protein synthesis. The present study identified tPA/plasmin as an endogenous enzyme system that converts proBDNF to mBDNF in the hippocampus. It also revealed a physiological role for such conversion in the brain. The results suggest that mBDNF is a key protein synthesis product responsible for L-LTP expression and provide a mechanistic link between two seemingly independent molecular systems in L-LTP expression.
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, Chang J. Regulation of neurogenesis by neurotrophins. Neural Glia Biol 2005 (in press).
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;4:407-430.
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.
Ren-Patterson RF, Cochran LW, Holmes A, Sherrill S, Huang S-J, Tolliver T, Lesch K-P, Lu B, Murphy DL. Loss of BDNF gene allele exacerbates brain monoamine deficiencies and increases stress abnormalities of serotonin transporter knockout mice. J Neurosci Res 2005;79:756-771.
Activity-dependent trafficking of BDNF receptor TrkB
Je, Pang, Nagappan, Lu; in collaboration with Kojima, Koshimizu, Numakawa, Shimazu, Suzuki
While 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 (the cholesterol- and sphingolipid-enriched membrane microdomains thought to be signaling platforms for extracellular stimuli) from non-raft 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 the translocation. Disruption of lipid rafts by depleting cholesterol from cell surface blocked the ligand-induced translocation. Moreover, disruption of lipid rafts prevented the potentiating effects of BDNF on transmitter release in cultured neurons as well as the 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 that is selective for synaptic modulation by BDNF in the central nervous system.
We next examined how cAMP controls BDNF/TrkB signaling. It has been hypothesized that cAMP “gates” the synaptic actions of BDNF, but the underlying molecular mechanisms remain unclear. We show that cAMP regulates BDNF function by modulating TrkB receptor signaling and trafficking. cAMP controls TrkB phosphorylation, with three features characteristic for cAMP gating: BDNF-induced TrkB phosphorylation was attenuated by inhibitors of cAMP signaling and potentiated by cAMP analogues while the 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 the synaptic scaffolding protein PSD-95. In cultured hippocampal neurons, cAMP gated long-term modulation of spine density by BDNF, but it did not gate dendritic branching. The 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.
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;8:164-172.
Lu B, Nagappan G. Activity-dependent trafficking of BDNF receptor TrkB: implications in synaptic plasticity. Trends Neurosci 2005 (in press).
Nagappan G, Lu B. PIKE/Homer complex: a novel kink between mGluR1 and PI3 kinase. Trends Neurosci 2004;27:645-648.
Suzuki S, Numakawa T, Shimazu K, Koshimizu H, Hatanaka H, Mei L, Lu B, Kojima M. BDNF-induced translocation of TrkB into lipid rafts: functional role in synaptic transmission. J Cell Biol 2004;167:1205-1215.
Hippocampal long-term depression regulated by proBDNF/p75 NTR signaling
Woo, Pang, Lu; in collaboration with Hempstead, Milner, Siao
Pro- and mature brain-derived neurotrophic factor activates two distinct receptors: p75 neurotrophin receptor (p75NTR) and TrkB. Mature BDNF facilitates hippocampal synaptic potentiation through TrkB. We demonstrated that, by activating p75NTR, proBDNF facilitates hippocampal LTD. Electron microscopy localized p75NTR in dendritic spines in addition to the afferent terminals of CA1 neurons. Genetic deletion of p75NTR in mice selectively impaired the NMDA receptor–dependent LTD without affecting other forms of synaptic plasticity. p75NTR–/–-mutant mice also exhibited a decrease in the expression of NR2B, an NMDA receptor subunit uniquely involved in LTD. Activation of p75NTR by proBDNF enhanced NR2B-dependent LTD and NR2B-mediated synaptic currents. The results demonstrate a critical role for proBDNF-p75NTR signaling in LTD and its potential mechanism and, together with the finding that mature BDNF promotes synaptic potentiation, suggest a bidirectional regulation of synaptic plasticity by pro- and mature BDNF.
Based on the observation that proneurotrophins stimulate apoptosis while mature neurotrophins promote cell survival in various cell populations and that proBDNF facilitates LTD while mature BDNF enhances LTP, we proposed a “yin-yang” model in which pro- and mature neurotrophins elicit diametrically opposing effects through activation of two distinct receptors: p75NTR and Trk receptor tyrosine kinases. Thus, the proteolytic cleavage of proneurotrophins represents a new mechanism that controls the direction of action of neurotrophins. Our model may have profound implications for understanding the role of neurotrophins in a wide range of cellular processes.
Lu B, Chang J. Regulation of neurogenesis by neurotrophins. Neural Glia Biology 2005 (in press).
Lu B, Woo NH, Pang PT. The yin and yang of neurotrophin action. Nat Rev Neurosci 2005;8:603-614.
Ren-Patterson RF, Cochran LW, Holmes A, Sherrill S, Huang S-J, Tolliver T, Lesch K-P, Lu B, Murphy DL. Loss of BDNF gene allele exacerbates brain monoamine deficiencies and increases stress abnormalities of serotonin transporter knockout mice. J Neurosci Res 2005;79:756-771.
Woo NH, Siao C-J, Pang PT, Teng HK, Milner TA, Hempstead BL, Lu B. ProBDNF/p75 NTR signaling is necessary for hippocampal long-term depression. Nat Neurosci 2005;8:1069-1077.
Interaction of the sorting motif on mature BDNF with the sorting receptor CPE required for activity-dependent secretion of BDNF
Zaitsev, Lu; in collaboration with Kim, Loh, Lou, Snell
Although activity-dependent secretion of BDNF is thought to be an important mechanism that mediates synaptic plasticity and short-term memory, it is unclear how BDNF achieves regulated secretion. We have previously shown that the pro-domain, particularly the region that contains val66, is critical for activity-dependent secretion of BDNF. In collaboration with the laboratory of Peng Loh, we provided evidence that a sorting motif localized in the mature domain of BDNF interacts with a sorting receptor and that such interaction is important in targeting BDNF to the regulated secretory pathway. X-ray crystal structure analysis of BDNF revealed a putative sorting motif, I16E18I105D106, for regulated secretion. Substitution mutation of the acidic residues in the motif resulted in missorting of proBDNF to the constitutive pathway in AtT20 cells. Introduction of an acidic residue to the relevant position in nerve growth factor (NGF), which is largely secreted constitutively, redirected a significant proportion of the factor into the regulated pathway. Modeling and binding studies indicated that the acidic residues in the BDNF sorting motif interact with two basic residues in the sorting receptor carboxypeptidase E (CPE). 35S pulse–chase experiments showed that activity-dependent secretion of endogenous BDNF from cortical neurons was obliterated in CPE knockout mice. Thus, we have identified a mechanism whereby the specific motif I16E18I105D106 interacts with CPE to sort proBDNF into regulated pathway vesicles for activity-dependent secretion. Further studies are necessary to delineate the relationships between the pro-domain and mature domains in activity-dependent secretion of BDNF.
Lou H, Kim SK, Zaitsev E, Sneil CR, Lu B, Loh YP. Sorting and activity dependent secretion of BDNF requires an interaction with the sorting receptor carboxypeptidase E. Neuron 2005;45:245-255.
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, PhD, Weill Medical College of Cornell University, New York, NY
Soo-Kyung Kim, PhD, Laboratory of Bioorganic Chemistry, NIDDK, Bethesda, MD
Masami Kojima, PhD, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
Hisatsugu Koshimizu, PhD, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan
Y. Peng Loh, PhD, Laboratory of Developmental Neurobiology, NICHD, Bethesda, MD
Teresa A. Milner, PhD, Weill Medical College of Cornell University, New York, NY
Chia-Jen Siao, PhD, Weill Medical College of Cornell University, New York, NY
Chris R. Snell, PhD, Medivir UK Ltd., Chesterford, UK
Henry K. Teng, PhD, Weill Medical College of Cornell University, New York, NY
For further information, contact bailu@mail.nih.gov.