The main focus of this laboratory is to study the role of neurotrophins, a class of secretory proteins critical for synapse development and plasticity. We were among the first to elucidate the synaptic functions of neurotrophins. Our key discoveries include: i) Brain derived neurotrophic factor (BDNF) promotes the development of early phase long-term potentiation (E-LTP) in the hippocampus. This is mediated by an enhanced response to tetanic stimulation through vesicle docking. ii) BDNF elicits synapse-specific modulation by regulating activity-dependent insertion, endocytosis, and synaptic localization of the TrkB receptor. iii) Neurotrophins regulate synaptic plasticity via two distinct modes: acute modulation of synaptic transmission and plasticity, and long-term alteration of the structure and function of synapses. iv) A single nucleotide polymorphism, or SNP, in the pro-domain of BDNF impacts activity-dependent BDNF secretion, resulting in impairment in hippocampal function and short-term memory in human. v) The extracellular conversion of proBDNF to mature BDNF by the protease tPA/plasmin is essential for late-phase LTP (L-LTP), a cellular model for long-term memory. vi) When uncleaved, proBDNF facilitates hippocampal long-term depression (LTD) by activating the p75NTR receptor. This year we have made significant progress towards the understanding of molecular mechanisms underlying long-term synaptic modulation by neurotrophins. 1) Distinct mechanisms for NT-3 Induced Acute and Long-term Synaptic Potentiation. While neurotrophins elicit both acute and long-term effects, it is unclear whether the two modes of action are mediated by the same or different mechanisms. Using the neuromuscular junction (NMJ) as a model system, we showed that long-term synaptic modulation by neurotrophin-3 (NT3) differs from the acute one in three distinct ways. First, the long-term effects require endocytosis of the NT3-TrkC receptor complex. The long-term physiological and morphological changes at NMJ were completely blocked by presynaptic expression of dominant negative dynamin, which prevents endocytosis of the NT3-TrkC complex. Moreover, bead-conjugated NT3 that is too large to be endocytosed, still induced acute effects but failed to elicit the long-term changes. Second, Akt, the downstream target of PI3 kinase, is necessary for the long-term but not acute effects. Third, the long-term effects depends new protein synthesis. Inhibition of protein translation prevented NT3-induced long-term structural and functional changes at the NMJ, without affecting the acute potentiation of synaptic transmission by NT3. Taken together, these results support a model in which the endocytosis of NT3-TrkC complex activates PI3 kinase/Akt pathway, which in turn triggers mTOR-mediated synthesis of new proteins necessary for long-term changes at neuromuscular synapses. A unique contribution of this study is to separate acute- from long-term synaptic modulation by neurotrophins based on their distinct molecular mechanisms, rather than purely on the temporal scales their actions. (J. Neurosci.) We next asked whether the structural and functional changes at the NMJ induced by long-term NT3 treatment are mediated by the same or different mechanisms. We found that inhibition of cAMP response element binding protein (CREB)-mediated transcription could block the enhancement of transmitter release elicited by NT-3, without affecting the synaptic varicosity of the presynaptic terminals. Further analysis indicates that CREB is activated through the Ca2+/calmodulin dependent kinase IV (CaMKIV) pathway, rather than the mitogen-activated protein kinase (MAPK) or cAMP pathways. In contrast, inhibition of the MAPK pathway prevented NT-3-induced structural but not functional changes. Genetic and imaging experiments indicate that the small GTPase Rap1, but not Ras, acts upstream of MAPK activation by NT-3. Taken together, our findings indicate that NT-3 utilizes two parallel but distinct molecular pathways to elicit long term changes in synaptic function and structure. NT3 activates the CaMKIV-CREB pathway to enhance synaptic efficacy but not synaptic growth. In addition, NT3 activates the Rap1-MAPK pathway to increase the number of synaptic sites, but not in synaptic transmission. These findings may have general implications in understanding the cell biological mechanisms underlying synapse development and plasticity. (J. Cell Biol.) 2) Functional significance of adult neurogenesis in learning and memory. A long-standing issue in neural stem cell biology is the functional significance of adult neurogenesis. Although substantial experimental data support the view that adult neurogenesis in the dentate gyrus participates in some forms of hippocampus-dependent learning or memory, direct evidence is still lacking. In vitro experiments suggest that FGF-2 stimulates the proliferation but not differentiation of neuronal progenitor cells (NPCs), while NT3 enhances the differentiation of these cells without affecting proliferation. Gene knockout approach was used to study whether and how FGF-2 and NT3 control neurogenesis in vivo, and their impact on learning and memory. To study the role of NPC proliferation, we generated a line of conditional knockout mice that lack FGFR1, a major receptor for FGF-2, in the brain. BrdU labeling experiments demonstrated that FGFR1 regulates the proliferation of NPCs, as well as generation of new neurons, in the adult dentate gyrus (DG). Moreover, deficits in neurogenesis in FGFR1 mutant mice were accompanied by a severe impairment of long-term potentiation (LTP) at the medial perforant path-granule neuron synapses in the hippocampal dentate. Water maze experiments further demonstrated that the FGFR1 mutant mice exhibited significant deficits in memory consolidation, but not formation of new memory. Spatial learning in these mice was normal. These findings showed that proliferative neurogenesis in adult dentate is important for memory consolidation. (Learning and Memory). To study the role of NPC differentiation, we generated a conditional mutant line in which the NT3 gene is deleted in the brain. Unlike the FGFR1 mutant, the NT3 knockout mice exhibited significant deficits in the survival and differentiation, but not proliferation, of NPCs in the dentate. Triple labeling for BrdU, the neuronal marker NeuN, and the glial marker GFAP showed that NT-3 affects the number of newly differentiated neurons, but not glia, in the DG. Field recordings revealed a selective impairment in LTP in the lateral, but not medial perforant path-granule neuron synapses. As a consequence, the NT-3 mutant mice exhibited severe deficits in hippocampus-dependent spatial memory. In addition to identifying a novel role of NT3 in adult NPC differentiation in vivo, this study provides a critical link between neurogenesis and dentate LTP, and suggests that adult neurogenesis contributes to spatial memory by regulating LTP at the dentate synapses. (Biological Psychiatry).
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