Neurotrophic Regulation of Synapse Development and Plasticity

Bai Lu, PhD, Head, Section on Neural Development and Plasticity
Feng Yang, MD, PhD, Staff Scientist
Guhan Nagappan, PhD, Research Fellow
Sundar Ganeson, PhD, Visiting Fellow
Yuanyuan Ji, PhD, Visiting Fellow
Fan Mei, BS, Predoctoral Fellow

Our laboratory focuses on the role of neurotrophins, a class of secretory proteins critical for synapse development and plasticity. As one of the first laboratories to elucidate the synaptic functions of neurotrophins, we discovered that (1) brain-derived neurotrophic factor (BDNF) promotes the development of early-phase long-term potentiation (E-LTP) in the hippocampus mediated by an enhanced response to tetanic stimulation through vesicle docking; (2) BDNF elicits synapse-specific modulation by regulating TrkB receptor trafficking through activity-dependent insertion, endocytosis, synaptic localization, and so forth; (3) neurotrophic regulation occurs via two modes: acute modulation of synaptic transmission and plasticity and long-term alteration of the structure and function of synapses; (4) a single-nucleotide polymorphism (SNP) in the pro-domain of BDNF affects activity-dependent BDNF secretion, resulting in impairment in hippocampal function and short-term memory in humans; (5) extracellular conversion of proBDNF to mature BDNF by protease tPA/plasmin is essential for late-phase LTP (L-LTP), a cellular model for long-term memory; and (6) proBDNF, if uncleaved, facilitates hippocampal long-term depression (LTD) by activating the p75^NTR receptor. Over the past year, we made significant progress in revealing several new aspects of neurotrophin functions.

Distinct role of long 3′UTR BDNF mRNA in spine morphology and synaptic plasticity in hippocampal pyramidal neurons

Woo,¹ Lu; in collaboration with An, Gharami, Liao, Vanevski

A common and yet puzzling phenomenon in cell biology is the existence of transcripts that code for exactly the same protein but bear different 3′ untranslated regions (3′UTR). One such example is the messenger RNA for BDNF. There are two pools of BDNF mRNA in neurons with similar abundance: one with a short 3′UTR and the other with a long 3′UTR. It was not known why a neuron would need two mRNAs of different length encoding the same BDNF protein. We have now provided evidence suggesting that mRNAs of different 3′UTR confer different functions because of their distinct subcellular localization. We show that the short 3′UTR BDNF mRNAs are restricted to neuronal cell bodies, whereas the long 3′UTR BDNF mRNAs are also localized in dendrites. In a mouse mutant in which the long 3′UTR is truncated, dendritic targeting of BDNF mRNAs is impaired. We observed little BDNF in hippocampal dendrites despite normal levels of total BDNF protein. The mutant exhibits enlargement of dendritic spines and deficits in pruning as well as selective impairment in long-term potentiation in dendrites, but not in somata, of hippocampal neurons. These results are significant for several reasons. First, they suggest a cellular mechanism whereby BDNF, an important regulator for brain development and plasticity, might elicit diverse cellular functions ranging from cell survival to synaptic transmission. Second, the results provide clear experimental evidence that dendritically localized mRNAs play crucial roles in regulating spine morphology, a long-sought result in the field of local protein synthesis and synaptic plasticity. Finally, to our knowledge, we have for the first time demonstrated that different transcripts with the same coding sequence play distinct roles in a cell.

An J, Gharami K, Liao G-Y, Woo N, Lau AG, Vanevski F, Fu X, Torre ER, Jones K, Feng Y, Lu B, Xu B. Distinct role of long 3′UTR BDNF mRNA in spine morphology and synaptic plasticity in the apical dendrites of hippocampal pyramidal neurons. Cell 2008;134:175-187.
Lu Z, Je H-S, Young P, Gross J, Lu B, Feng G. Regulation of synaptic growth and maturation by a synapse-associated E3 ubiquitin ligase at the neuromuscular junction. J Cell Biol 2007;18:1077-1089.
Martinowich K, Lu B. BDNF and serotonin: role and relationships in mood disorders. Neuropsychopharmacology 2008;33:73-83.
Nagappan G, Woo NH, Lu B. Ama“Zinc” link between TrkB transactivation and synaptic plasticity. Neuron 2008;57:477-479.
Zhao WQ, Lu B. Expression of annexin A2 in GABAergic interneurons in the normal rat brain. J Neurochem 2007;100:1211-1223.

Activity-dependent BDNF transcription and secretion

Sakata,² Woo,¹ Nagappan, Zaitsev,³ Lu; in collaboration with Martinowich, Shen, Enfors

In one study, we address the role of activity-dependent BDNF gene expression. Transcription of the BDNF gene is controlled by several promoters that drive the expression of several transcripts encoding the same protein. Promoter-IV contributes significantly to activity-dependent BDNF transcription. We generated promoter-IV–mutant mice (BDNF-KIV) by inserting the GFP gene followed by a stop codon into exon IV. This genetic manipulation resulted in disruption of promoter-IV–mediated BDNF expression, particularly in the prefrontal cortex (PFC), an area involved in working memory and other executive functions. Interestingly, the BDNF-KIV exhibited a selective impairment in parvalbumin-positive GABAergic neurons and inhibitory but not excitatory postsynaptic currents in the PFC, leading to an aberrant spike-timing–dependent synaptic potentiation (STDP). Behaviorally, BDNF-KIV mice are significantly impaired in PFC-mediated reversal learning and fear memory extinction, but not in working memory. Our results demonstrated the importance of promoter-IV–dependent BDNF transcription in GABAergic function and revealed an unexpected role of BDNF in behavioral perseverance. Our study may shed light on the pathogenesis of several cognitive disorders in which perseverance is prominent, including schizophrenia and post-traumatic stress disorder (PTSD).

In another study, we addressed the role of activity-dependent BDNF gene expression. We previously showed that pro- and mature BDNF often elicit opposing biological effects. For instance, mature BDNF (mBDNF) is critical for LTP induced by high-frequency stimulation (HFS), whereas proBDNF facilitates LTD induced by low-frequency stimulation (LFS). Given that mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect proBDNF or mBDNF, we showed that LFS induced predominantly proBDNF secretion in cultured hippocampal neurons. In contrast, HFS preferentially increased extracellular mBDNF. Inhibition of extracellular, but not intracellular, cleavage of proBDNF greatly reduced HFS-induced extracellular mBDNF. Moreover, HFS, but not LFS, selectively induced the secretion of tPA, an important protease involved in extracellular conversion of proBDNF to mBDNF. Thus, high-frequency neuronal activity controls the ratio of extracellular proBDNF to mBDNF by regulating the secretion of extracellular proteases. Our study demonstrated activity-dependent control of extracellular proteolytic cleavage of a secretory protein and revealed an important mechanism that controls diametrically opposed functions of BDNF isoforms. In addition to revealing for first time how neuronal activity can control the cleavage of a secreted protein, our work yielded useful tools for studying the distinct functions of proBDNF and mBDNF.

Galloway E, Woo NH, Lu B. Persistent neural activity in the prefrontal cortex: a mechanism by which BDNF regulates working memory? Prog Brain Res 2008;169:251-266.
Lu B, Woo NH. Trophic factors in synaptic plasticity and memory. In: Squire LR, ed. Encyclopedia of Neuroscience Volume 2. Elsevier, 2008;135-143.
Lu Y, Christian K, Lu B. BDNF: a key mediator protein synthesis-dependent L-LTP and long-term memory? Neurobiol Learn Mem 2008;89:312-323.
Nagappan G, Zaitsev E, Senatorov VV, Yang J, Hempstead BL, Lu B. Control of extracellular cleavage of proBDNF by high frequency neuronal activity. Proc Natl Acad Sci USA 2008, in press.
Sakata K, Woo NH, Martinowich K, Enfors P, Shen L, Lu B. Promoter-IV driven BDNF transcription regulates prefrontal cortex parvalbumin-interneurons and behavioral perseverance. Proc Natl Acad Sci USA, in press.

Studies of genes involved in schizophrenia

Yang, Lu; in collaboration with Chang, Chen, Huffaker, Sambataro, Lipska, Kleinman, Weinberger

Conventional genetic linkage and association studies have confirmed that complex human diseases, such as diabetes and schizophrenia, involve several genes and that each gene elicits very small effects across populations. Moreover, given the complexity of genetic factors and their interaction with the environment, it is often difficult to link genetic findings with important biological or mechanistic aspects of an illness. To overcome these obstacles, we took a powerful translational approach that dissects the complex phenomenology of psychosis into several neural system and molecular components and maps genetic association onto several levels of analysis. Using this approach, we identified what may constitute a new class of schizophrenia susceptibility genes—KCNH2, which encodes a potassium channel and, more important, a novel isoform of this type of potassium channel that is primate- and brain-specific and whose levels are 2.5-fold elevated in schizophrenia brain tissue. Expression of this novel isoform is predicted by risk-associated SNPs in the gene, even in normal brain tissue; moreover, risk SNPs predict cognition and related brain physiology in normal subjects. Postmortem expression analysis shows a 2.5-fold increase in Isoform 3.1 over KCNH2-1A in the schizophrenic hippocampus. Structurally, Isoform 3.1 lacks most of the PAS (Per-ARNT-Sim) domain critical for slow channel deactivation. Electrophysiological characterization in primary cortical neurons revealed that overexpression of Isoform 3.1 results in a rapidly deactivating K⁺ current and a high-frequency, non-adapting firing pattern. These results demonstrated a novel KCNH2 channel involved in cortical physiology, cognition, and psychosis, providing a potential new target for psychotherapeutic drugs.

Duan X, Chang JH, Ge S, Faulkner RL, Kim JY, Kitabatake Y, Liu X, Yang C, Jordan JD, Ma D, Liu CY, Ganesan S, Cheng H-J, Ming G, Lu B, Song H. Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain. Cell 2007;134:175-187.
Lu B, Martinowich K. Cell biology of BDNF and its relevance to schizophrenia. Novartis Found Symp 2008;289:119-135.
Martinowich K, Manji H, Lu B. New insights into BDNF function in depression and anxiety. Nat Neurosci 2007;10:1089-1093.
Zhao M, Li D, Shimazu K, Zhou YX, Lu B, Deng C-X. Fibroblast growth factor receptor-1 is required for long-term potentiation, memory consolidation, and neurogenesis. Biol Psychiatry 2007;62:381-390.

¹ Newton Woo, PhD, former Postdoctoral Fellow
² Kazuko Sakata, PhD, former Visiting Fellow
³ Eugene Zaitsev, PhD, former Research Fellow

Collaborators

Juan Ji An, PhD, Georgetown University School of Medicine, Washington, DC
Jay H. Chang, PhD, Genes, Cognition and Psychosis Program, NIMH, Bethesda, MD
Jingshan Chen, PhD, Genes, Cognition and Psychosis Program, NIMH, Bethesda, MD
Kusumika Gharami, PhD, Georgetown University School of Medicine, Washington, DC
Steve Huffaker, PhD, Genes, Cognition and Psychosis Program, NIMH, Bethesda, MD
Joel Kleinman, MD, Genes, Cognition and Psychosis Program, NIMH, Bethesda, MD
Guey-Ying Liao, PhD, Georgetown University School of Medicine, Washington, DC
Barbara Lipska, PhD, Genes, Cognition and Psychosis Program, NIMH, Bethesda, MD
Keri Martinowich, PhD, Mood and Anxiety Program, NIMH, Bethesda, MD
Fabio Sambataro, PhD, Genes, Cognition and Psychosis Program, NIMH, Bethesda, MD
Liya Shen, PhD, Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, Bethesda, MD
Filip Vanevski, BA, Georgetown University School of Medicine, Washington, DC
Daniel Weinberger, MD, Clinical Brain Disorders Branch, NIMH, Bethesda, MD
Baoji Xu, PhD, Georgetown University Medical Center, Washington, DC

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