Bai Lu, PhD, Head, Section on Neural Development and Plasticity
Feng Yang, MD, PhD, Research Fellow
Eugene Zaitsev, PhD, Research Fellow
Hyun-Soo Je, PhD, Postdoctoral Fellow
Cristina Chiaruttini, PhD, Visiting Fellow
Sundar Ganesan, PhD, Visiting Fellow
Yuanyuan Ji, PhD, Visiting Fellow
Guhan Nagappan, PhD, Visiting Fellow
Kazuko Sakata, PhD, Visiting Fellow
Newton Woo, PhD, Visiting Fellow

We mainly 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. We found that (1) brain-derived neurotrophic factor (BDNF) promotes the development of early-phase long-term potentiation (E-LTP) in the hippocampus, which is mediated by an enhanced response to tetanic stimulation through vesicle docking; (2) BDNF elicits synapse-specific modulation by regulating activity-dependent insertion, endocytosis, and synaptic localization of the TrkB receptor; (3) 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; (4) a single nucleotide polymorphism, or SNP, in the pro-domain of BDNF affects activity-dependent BDNF secretion, resulting in impairment in hippocampal function and short-term memory in human; (5) 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; and (6) proBDNF, when uncleaved, facilitates hippocampal long-term depression (LTD) by activating the p75NTR receptor. Over the past year, we made significant progress in understanding the molecular mechanisms underlying long-term synaptic modulation by neurotrophins.

Distinct mechanisms for NT-3-induced acute and long-term synaptic potentiation

Je, Yang, Lu; in collaboration with Zhou

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 acute modulation in three distinct ways. First, 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, which 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 long-term but not acute effects. Third, long-term effects depend on new protein synthesis. Inhibition of protein translation prevented NT3-induced long-term structural and functional changes at the NMJ without affecting acute potentiation of synaptic transmission by NT3. Taken together, our results support a model in which endocytosis of NT3-TrkC complex activates the 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 our study is the ability to separate acute- from long-term synaptic modulation by neurotrophins based on their distinct molecular mechanisms rather than relying exclusively on temporal scales of the neurotrophin's actions.

We next investigated 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 transcription mediated by cAMP response element binding protein (CREB) could block the enhancement of transmitter release elicited by NT-3 without affecting the synaptic varicosity of the presynaptic terminals. Further analysis indicated that CREB is activated through the Ca2+/calmodulin-dependent kinase IV (CaMKIV) pathway rather than through 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 showed that the small GTPase Rap1, but not Ras, acts upstream of MAPK activation by NT-3. Taken together, our findings indicate that NT-3 uses 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 synaptic transmission. Our findings may have general implications for understanding cell-biological mechanisms underlying synapse development and plasticity.

Functional significance of adult neurogenesis in learning and memory

Shimazu, ¹ Zhao, ² Lu; in collaboration with Akbarian, Bates, Deng, Jaenisch, Li

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 suggested that FGF-2 stimulates proliferation but not differentiation of neuronal progenitor cells (NPCs) while NT3 enhances differentiation of these cells without affecting proliferation. We used a gene knockout approach to study whether and how FGF-2 and NT3 control neurogenesis in vivo as well as to identify their impact on learning and memory. To determine 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 and generation of new neurons in the adult dentate gyrus (DG). Moreover, deficits in neurogenesis in FGFR1 mutant mice were accompanied by severe impairment of long-term potentiation (LTP) at the medial perforant path-granule neuron synapses in the hippocampal dentate gyrus. Water-maze experiments further demonstrated that the FGFR1 mutant mice exhibited significant deficits in memory consolidation, but not in formation of new memory. Spatial learning in these mice was normal. Our findings show that proliferative neurogenesis in the adult DG is important for memory consolidation.

To study the role of NPC differentiation, we generated a conditional mutant line in which the NT3 gene was deleted in the brain. Unlike the FGFR1 mutant, NT3 knockout mice exhibited significant deficits in the survival and differentiation, but not proliferation, of NPCs in the DG. 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 selective impairment in LTP in the lateral, but not in medial, perforant path-granule neuron synapses. As a consequence, 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, our 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.

Studies of genes involved in cognition and psychosis

Pang,3 Lu; in collaboration with Fischer, Murphy, Ren-Patterson, Sartorius, Tsai, Weinberger

We recently launched a new line of research to study genes involved in cognitive brain function and psychiatric disorders. In one study, we investigated the genetic interaction between BDNF and serotonin transporter (SERT) genes by generating double-mutant mice that were homozygous null for SERT and heterozygous for BDNF. Male, but not female, double-mutant mice exhibited more pronounced anxiety-like behaviors than single SERT homozygotes as well as an increase in serotonin concentrations (5HT) in the hypothalamus and an enhanced ACTH response to stress. In contrast, female double mutants exhibited very little reduction in hypothalamic 5HT and no increase in anxiety behavior. Interestingly, estrogen treatment of the male mutants could reverse the reduction in hypothalamic 5HT. Our findings support the hypothesis that estrogen enhances BDNF function, leading to alterations in the serotonin circuits that modulate anxiety-like behaviors.

In another study, we characterized a novel splice variant of the metabotropic glutamate receptor 3 gene (GRM3), a schizophrenia susceptibility gene. The new transcript is expressed in the prefrontal cortex, hippocampus, and cerebellum and is translated into a truncated protein with a conserved extracellular ligand-binding domain, no seven-transmembrane domain, and a unique 96-amino acid C-terminus. Immunostaining and cell fractionation studies showed that the truncated protein is primarily membrane-associated.

In collaboration with Li-Huei Tsai's group, we studied the role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity and memory. The gene encoding Cdk5 is implicated in several neurodegenerative diseases. Proteolytic cleavage of p35, a regulatory subunit of Cdk5, by calpain results in the generation of truncated p25 protein, which causes aberrant activation of Cdk5. Using region-specific and inducible transgenic mice, we showed that a brief increase in p25 expression in the hippocampus enhanced LTP and facilitated hippocampus-dependent memory. Moreover, overexpression of p25 resulted in an increase in the number of dendritic spines and synapses. Importantly, transient production of p25, achieved by an induction of p25 expression followed by its repression, enhanced memory without causing neurodegeneration. In contrast, prolonged p25 production caused severe cognitive deficits accompanied by synaptic and neuronal loss and impairment in LTP. Our data suggest a role for p25 in synaptic plasticity, synaptogenesis, and learning and memory in the adult brain and provide a model whereby dysregulation of a plasticity factor can contribute to neurodegeneration.

¹ Kazuhiro Shimazu, MD, PhD, former Visiting Fellow
² Mingrui Zhao, PhD, former Visiting Fellow
³ Petti T. Pang, BS, former Graduate Student

COLLABORATORS

Schahram Akbarian, MD, University of Massachusetts Medical School, Worcester, MA
Brian Bates, PhD, Wyeth Research, Applied Genomics, Cambridge, MA
Chuxia Deng, PhD, Genetics of Development and Disease Branch, NIDDK, Bethesda, MD
Andre Fischer, PhD, Massachusetts Institute of Technology, Cambridge, MA
Rudolph Jaenisch, MD, Whitehead Institute for Biomedical Research, Cambridge, MA
Dan Li, PhD, Genetics of Development and Disease Branch, NIDDK, Bethesda, MD
Dennis Murphy, MD, Laboratory of Clinical Science, NIMH, Bethesda, MD
Renee Ren-Patterson, MD, PhD, Laboratory of Clinical Science, NIMH, Bethesda, MD
Leah Sartorius, PhD, Clinical Brain Disorders Branch, NIMH, Bethesda, MD
Li-Huei Tsai, PhD, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA
Daniel Weinberger, MD, Clinical Brain Disorders Branch, NIMH, Bethesda, MD
Zhuan Zhou, PhD, Institute of Neuroscience, Shanghai, China

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