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Pre- and post-synaptic modification by neurotrophins
Neuroscience Research 43 (2002) 193 /199 www.elsevier.com/locate/neures
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Pre- and post-synaptic modification by neurotrophins Masami Kojima a,*, Ronald L. Klein b, Hiroshi Hatanaka c a
b
AIST, Midorigaoka, Ikeda, Osaka 563-8577, Japan Department of Pharmacology and Therapeutics, University of Florida, JHMHC Box 100267, Gainesville, FL 32610-0267, USA c Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan Received 18 January 2002; accepted 11 March 2002
Abstract Since the discovery of nerve growth factor, there has been accumulating evidence that neurotrophins (NTs) mediate various biological responses of peripheral and central neurons. NTs have been traditionally studied as the regulating factors of neuronal survival and differentiation. Recent data indicate that NTs can modify neuronal plasticity by specific changes in pre- and postsynaptic functions. Whether the NT action is pre- or post-synaptic, however, remains to be controversy. Here we review the recent advances of NTs involved in synaptic plasticity, as well as the pre- and post-synaptic arguments. We also review the recent discovery that proneurotrophins and mature NTs have the differential ability to bind selective receptors and mediate distinctive biological actions. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Neurotrophin; Brain-derived neurotrophic factor; Activity-dependent plasticity; Neurotrophin receptor; Green fluorescent protein
1. Introduction Neurotrophins (NTs) are a family of small, secreted proteins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, and NT-4/5 (Lewin and Barde, 1996). The primary event in NT signaling is the activation of receptor tyrosine kinases of the Trk family, which have ligand-binding specificities as follows: NGF to TrkA; BDNF and NT4/5 to TrkB; and NT-3 to TrkC (Lee et al., 2001a; Patapoutian and Reichardt, 2001). This specific binding leads to the receptor dimerization, activation, and recruitment of various adapter proteins to initiate signaling cascades (Kaplan and Miller, 1997). Another NT receptor is p75NTR, a member of tumor necrosis factor receptor family, which binds to all NTs with approximately equivalent affinities (Lee et al., 2001a). It is postulated that p75NTR-mediated signaling cascades are involved in apoptotic neuronal death (Majdan and Miller, 1999).
* Corresponding author. Tel.: /81-727-51-9081; fax: /81-727-519628 E-mail address: [email protected] (M. Kojima).
Although NTs have traditionally been studied as the regulating factors of neural survival and differentiation (Lewin and Barde, 1996; Kaplan and Miller, 1997), the field of synapse development and physiology recently opened a new area of NT research on neuronal plasticity (Poo, 2001). This research is supported by the study showing that mRNA encoding NTs and their receptors are strongly expressed in the adult brain in areas with a high degree of plasticity, the hippocampus and neocortex (Kokaia et al., 1993). The NT hypothesis for neuronal plasticity proposes that neural activity regulates the expression, secretion and signaling of NTs to induce specific changes in synaptic efficacy and synapse morphology (Thoenen, 1995; Poo, 2001).
2. Three types of regulation for the NT hypothesis The NT hypothesis regarding activity-dependent plasticity includes three kinds of regulation: (1) NTdependent modification of synaptic plasticity; (2) regulation of NT expression; and (3) regulation of NT secretion and transport. NT researchers have investigated these three facets of regulation both electrophysiologically and biochemically.
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 0 3 4 - 2
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2.1. Neurotrophin modification of synaptic transmission Since Lohof et al. (1993) clearly demonstrated that both spontaneous and evoked synaptic transmission were elevated a few minutes after the application of BDNF or NT-3 in Xenopus nerve /muscle cultures, a number of electrophysiological studies have suggested that NTs can modify synaptic transmission, long-term potentiation (LTP) and long-term depression in cell cultures, slice preparations, and intact brain (Schuman, 1999; Poo, 2001). At central synapses, NTs enhance excitatory transmission (Lessmann et al., 1994; Kang and Schuman, 1995) and suppress inhibitory transmission (Kim et al., 1994; Tanaka et al., 1997). Whether NT-induced synaptic potentiation occurs pre- or post-synaptically has been investigated. Most studies have indicated that the NT modification of synaptic transmission occurs presynaptically (Lohof et al., 1993; Lessmann et al., 1994; Kang and Schuman, 1995; Li et al., 1998; Schinder et al., 2000). Although the molecular mechanism is not fully understood, a recent study clearly demonstrated that the phosphorylation of synapsin I was involved in the NT modification of synaptic transmission (Jovanovic et al., 2000). Until now, there is little evidence indicating postsynaptic actions of NTs. BDNF enhanced the responses of NMDA, but not AMPA receptors in cultured cortical neurons (Levine et al., 1998), although this modification is not expected to produce the potentiation of the evoked actions by BDNF because AMPA receptors mainly mediate glutamate-exerting synaptic transmission (Poo, 2001). Genetically modified mice provided solid data implicating NTs in LTP. BDNF knockout mice were deficient for the induction of LTP in the hippocampal CA1 region, which could be reversed by application of BDNF (Korte et al., 1996; Patterson et al., 1996). Further, treatments with function-blocking TrkB antibody consistently reduced LTP (Kang et al., 1997; Chen et al., 1999). Curiously, BDNF maintained late phase LTP in the CA1 region after the LTP induction (Kang et al., 1997; Korte et al., 1998), suggesting that BDNF is a synaptic morphogen for memory formation. 2.2. Activity-dependent expression of neurotrophins The broad and strong expression pattern of NTs and their receptors in the adult brain (Kokaia et al., 1993) coupled with the modulation of expression by neural activity suggests that there is a link between NT function and activity-dependent plasticity. In addition to seizure activity (Gall and Isackson, 1989; Ernfors et al., 1991), LTP increases the expression of NTs (Patterson et al., 1992; Castren et al., 1993). Regulation of NT gene expression was also found in cultured neurons, where depolarization with glutamate or high potassium chlo-
ride increased the expression of NGF and BDNF mRNAs (Zafra et al., 1990; Lindholm et al., 1994). The expression of NT was also induced by more physiological stimuli in vivo, in the visual system. Visual experience regulated the expression of BDNF, whereas blockade of visual input led to the downregulation of BDNF mRNA (Castren et al., 1992). This regulation is also supported by the genomic analyses of the BDNF gene, which consists of five exons and four distinct promoters. The four transcribed BDNFs are polyadenylated at either of two distinct sites within the 3? untranslated region, by which mechanism a total of eight distinct BDNF transcripts are synthesized (Timmusk et al., 1993). All eight mRNAs, however, encode the identical BDNF protein. Interestingly, the study with exon-specific probes indicated that four first exons have different roles in neural activity (Timmusk et al., 1993). Ca2 influx through L-type voltage-sensitive Ca2 channels (VSCCs) was shown to increase the expression of exon I-, exon II-, and exon III-containing BDNF mRNAs (Ghosh et al., 1994; Timmusk et al., 1995; Tao et al., 1998; Shieh et al., 1998). It was also demonstrated that VSCCs-dependent transcription from exon III promoter was mediated via the CREB family of transcription factors (Tao et al., 1998) and regulated by CaM kinase IV (Shieh et al., 1998). The involvement of CREB and CaM kinase IV suggests that BDNF gene expression is regulated by signaling cascades underlying activitydependent neuronal plasticity. 2.3. Activity-dependent secretion of neurotrophins NT secretion has been investigated by sensitive ELISA and immunoprecipitation assays. In hippocampal slices or cultured cells overexpressing NGF, glutamate or high potassium chloride stimulated the rapid secretion of NGF (Blochl and Thoenen, 1995). Similar secretion was observed in hippocampal slices (Canossa et al., 1997) or cultured cells overexpressing BDNF (Goodman et al., 1996; Griesbeck et al., 1999). In cultured sensory neurons, activity-dependent secretion of endogenous BDNF was measured by an in situ ELISA assay (Balkowiec and Katz, 2000). Interestingly, the release was dependent on the pattern or frequency of the nerve impulse, suggesting that BDNF secretion resembles that of neuropeptides. The results together suggest that neural activity is involved in NT modifcation of synaptic functions. Secretion of growth factors is classified as constitutive or regulated. Several studies showed the constitutive secretion of BDNF and NGF in overexpressing cultured neurons and slice preparations (Blochl and Thoenen, 1995; Griesbeck et al., 1999). However, a recent study using cDNAs encoding proNGF and proBDNF demonstrated that NGF secretion is constitutive while BDNF
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secretion is activity-dependent (Mowla et al., 1999). One explanation for this discrepancy may be the different methodologies. In any case, the mechanism of NT release and from which sites remains to be an essential question to understand how NTs may regulate activitydependent neuronal plasticity. 2.4. Pre- and post-synaptic secretion of NTs To test the effect of NTs on activity-dependent plasticity, most experiments have used bath application of NTs by superfusion, which does not mimic their natural actions in vivo and has produced some discrepant results (Schuman, 1999). For example, some studies showed rapid actions of BDNF on excitatory synaptic transmission while others did not. One explanation for this discrepancy is the different perfusion rates of the applied BDNF because BDNF does not penetrate tissues easily. This discussion motivated researchers to study the modes of NT actions, and especially, whether NT secretion is pre- or post-synaptic. Electrophysiological studies indicate both pre- and post-synaptic modification induced by NTs. One model is that NT-containing vesicles are anterogradely transported to presynaptic terminals, released, and then taken up by postsynaptic neurons. Biochemical studies showed the presence of BDNF in the vesicular fraction of brain synaptosomes (Fawcett et al., 1997). Immunocytochemical studies also indicated that BDNF was transported to presynaptic terminals, even in regions lacking the expression of BDNF mRNA, and this anterograde transport was blocked by the microtubule inhibitor colchicine (Conner et al., 1997; Altar et al., 1997). More interestingly, 125Ilabeled NT-3 was anterogradely transported to axon terminals after injection into the chick eye and taken up trans-synaptically in retinal ganglion cells (von Bartheld et al., 1996). Whether this anterograde transport is linked to neuronal activity was addressed in a recent study discussed below. It was also shown that NTs are secreted from dendrites of postsynaptic neurons and modify presynaptic functions. In a study of Xenopus nerve/muscle cultures, a higher frequency of spontaneous acetylcholine secretion was found in the terminals contacting myocytes overexpressing NT-4 than in controls (Wang and Poo, 1997). The effect was blocked by TrkB-IgG, a NT-4 scavenger protein. This study strongly suggests that postsynaptic NTs may elicit presynaptic transmitter release. Studies with cultured CNS neurons indicate the postsynaptic distribution and activity-dependent secretion of NTs (Blochl and Thoenen, 1996; Goodman et al., 1996; Kojima et al., 2001; Hartmann et al., 2001). However, whether NTs secreted from postsynaptic neurons alter the presynaptic function of CNS neurons remains to be fully answered and green fluorescent
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protein (GFP)-based imaging will help to understand these actions.
3. Imaging studies of neurotrophin action The attachment of a target protein with a fluorescent protein, such as GFP, makes it possible to image the intracellular location and dynamics of the target protein in living cells. This technology was applied to current neuroscience to visualize the molecular dynamics at synapses and in dendrites (Umeda and Okabe, 2001). 3.1. Studies with GFP-tagged BDNF With a similar strategy, BDNF was tagged with GFP and gene transfer was used to express the BDNF /GFP fusion protein in cultured cells, slice preparations, and the intact brain (Fig. 1). Kojima et al. (2001) expressed BDNF/GFP in dissociated hippocampal neurons and maintained them for more than 3 weeks. The BDNF/ GFP formed fluorescence clusters and colocalized with PSD-95 immunoreactivity, providing a visual suggestion of postsynaptic location of BDNF in mature neurons. In a time-lapse imaging study, BDNF/GFP fluorescence
Fig. 1. Expression of BDNF /GFP in the rat brain. The GFP-tagged BDNF, which mimics the biological function and release kinetics of native BDNF (Kojima et al., 2001), was expressed in the adult rat brain using the adeno-associated virus (AAV) vector system (see Peel and Klein, 2000, for review). One month after injecting the BDNF / GFP AAV vector into either neocortex (A and B) or hippocampus (C and D) at a dose of 1/1010 particles, the transgene product was detected in brain sections by GFP native fluorescence (not shown) and GFP immunohistochemistry (A /D). The immunoreactivity filled the axons and dendrites of the cortical (A and B) and CA1 pyramidal (C and D) neurons, and did not accumulate in nuclei, similar to a BDNFmyc fusion protein, and different than unfused GFP, which gets expressed in nuclei (Klein et al., 1998, 1999). Bar in A/100 mm; bar in B /60 mm. A, C; B, D, same magnification.
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rapidly disappeared in response to depolarizing stimulation. The BDNF /GFP could therefore allow us to visualize activity-dependent dynamics of BDNF in living neurons. Using this construct, Kohara et al. (2001) clearly showed visual data of activity-dependent transfer of BDNF to postsynaptic neurons. In this study, BDNF / GFP was anterogradely transported to axonal terminals and taken up by postsynaptic neurons. Interestingly, the interneuronal transfer was remarkably reduced by the treatment of tetrodotoxin, a blocker of synaptic currents. 3.2. Tagged BDNF and Trk receptors for trafficking and signaling studies GFP-tagging provided a new strategy of NT research for three reasons. First, as well as the conventional neurotrophic actions of BDNF, novel spatial dynamics of BDNF can be observed. With similar strategies, the dynamics of other NTs can now be compared to one another. Probes based on fluorescent energy transfer technology have recently been developed that can directly visualize intracellular signaling cascades in vivo (Miyawaki and Tsien, 2000; Mochizuki et al., 2001). This technology may allow visualization of the interaction between NTs and Trk receptors. Second, we showed the postsynaptic location of BDNF /GFP and depolarization-induced reduction in BDNF /GFP intensity in cultured hippocampal neurons. Recently, Hartmann et al. (2001) showed that similar reduction occurred upon high-frequency synaptic stimulation. These studies together indicate that the postsynaptic BDNF is secreted in response to synaptic stimuli (Fig. 2). The restricted distribution of BDNF / GFP suggests that NT regulation of synaptic functions could occur at a single synapse level. This possibility is also suggested by an immunoelectron microscope study with the antibody specific for the full-length TrkB receptor (Drake et al., 1999). They demonstrated TrkB
Fig. 2. Pre- and post-synaptic mechanisms of NTs. Data indicate that the transport and secretion of NTs could occur both pre-synaptically and post-synaptically (Section 2.4). GFP-based imaging technology may permit the visualization of novel and conventional actions NTs (Section 3.1), and address the significance of both pre- and postsynaptic pathways.
immunoreactivity both presynaptically at axonal terminals and postsynaptically at dendritic spines and shafts. The recent paper by Kovalchuk et al. (2002) found postsynaptic actions of BDNF on Ca2 signaling in dendritic spines and a rapid induction of LTP. Although whether this regulation is induced by presynaptic BDNF or postsynaptic BDNF remains unclear, synaptically distributed BDNF and TrkB could exert it. Third, although GFP-tagged BDNF could visualize the anterograde transport of BDNF (Fig. 2), the retrograde transport of BDNF has not been seen. However, a recent study of compartmented cultures of DRG neurons, GFP-tagged TrkB was retrogradely transported to the cell body after the addition of BDNF to the axonal terminals (Watson et al., 1999). In this culture system, drugs can be applied selectively to either the cell body or the neurites. Further, a recent study with this culture technique suggested that NT activated different MAP kinase signaling pathways depending on whether they were added to axonal terminals or cell bodies (Watson et al., 2001). The location of the activated Trk receptors in the neurons might therefore determine which signaling cascades are activated in response to NTs. The unique combination of GFPbased fluorescent probes with primary culture techniques may identify the spatial and temporal distinctions of signaling cascades in fully differentiated neurons.
4. Extracellular protease activity regulates neurotrophinbinding affinity for their receptors A novel mechanism of extracellular NT processing was recently discovered (Lee et al., 2001b), outlined in Fig. 2. It is well known that NTs are first produced as 30 /35 kDa precursor proteins and processed to be the biologically active 12/15 kDa proteins. Precursor NTs contain a signal sequence and pairs of basic amino acids recognized by proteases. The intracellular enzymes, serine protease furin and prohormone convertase, cleave in the middle of proNTs to produce the mature forms (Seidah et al., 1996). The prodomain sequences are highly conserved across species (Heinrich and Lum, 2000), noteworthy as this indicates that they have additional functions. The prodomain is thought to determine either the constitutive or regulated secretory pathway and assist in the folding process (Rattenholl et al., 2001). Lee et al. (2001b) first suggested that proNTs were secreted and cleaved extracellularly by various extracellular proteases. Using recombinant proNGF and mature NGF, they tested the binding affinity for NGF receptors. Surprisingly, proNGF bound with a higher affinity to p75NTR, and with reduced affinity for TrkA, compared to mature NGF. Consistent with its greater affinity for p75NTR, proNGF induced p75-mediated
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5. Concluding remarks
Fig. 3. A novel regulation of NT action. Lee et al. (2001b) demonstrated that proNGF binds with higher affinity than mature NGF to p75NTR, leading to the signaling cascade of apoptosis. Mature NGF binds to TrkA receptor, leading to a signaling cascade of survival. They also showed that proNTs were secreted and cleaved extracellularly by matrix metalloproteases and serine protease. These findings are certain to lead to new models of NT action.
apoptosis to a greater extent than mature NGF. ProNGF may therefore be a highly selective ligand for p75NTR that signals for apoptosis (Fig. 3). These findings provide a chance to explore unsolved questions in the NT field, for example, a model of functional antagonism between TrkA and p75NTR during the period of naturally occurring cell death and target innervation (Majdan and Miller, 1999). How extracellular protease activity converts proNTs to the mature forms during this period is an intriguing question. Matrix metalloproteases are positioned as either transmembrane proteins or extracellular matrix proteins, so that they could process secreted proNTs at suitable domains. In addition to the three other known NT receptors, TrkA, TrkB, and TrkC, numerous adaptor proteins that bind to p75NTR have been reported (Lee et al., 2001a). NADE and NRAGE contribute to cell death. RhoA GTPase, Schwann cell factor-1 and NRAGE exert nonapoptotic activities such as neurite elongation and growth arrest. Considering these interactions with p75NTR, unprocessed NTs might exert various biological activities in nervous system. Do unprocessed NTs participate in NT-dependent modification of plasticity? Synaptic secretion of BDNF occurs through the regulated secretory pathway, which is mutually linked to the enhancement of synaptic activity (Poo, 2001). ProBDNF is secreted even by hippocampal neurons (Mowla et al., 1999; Kojima et al., 2001), suggesting that proteolytic cleavage of proBDNF may be essential for the activation of TrkB receptors. Because extracellular protease activity is modified during the formation of LTP (Luthi et al., 1997; Baranes et al., 1998), proteolytic cleavage of proNTs may be involved in the NT modification of plasticity.
Since NGF was discovered by Levi-Montalcini, Hamburger, and Cohen, experimental evidence has indicated that NTs are responsible for mediating diverse cellular responses from neuronal survival to neuronal plasticity, suggesting that they are biologically significant over the entire lifespan. This review focused on two areas of NT research: (1) the NT-dependent modification of neuronal plasticity; and (2) the current molecular advances achieved via novel model systems. The application of imaging techniques to this field allowed us to examine the spatial and temporal range of neurotrophic actions. Considering the localized activation of Trk receptors and MAP kinase signaling cascades (Watson et al., 2001), spatially restricted effects of NTs could lead to input-specific modification of synaptic functions (Schinder et al., 2000). The recent findings about the functionality of proNTs (Lee et al., 2001b) have added a new perspective for NT researchers. Because NT research has yielded a number of surprising advances, it will likely contribute to our understanding of general principles of cell biology.
Acknowledgements This work was supported by a grant to M.K. from NEDO and a grant to H.H. from CREST.
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Update Article
Pre- and post-synaptic modification by neurotrophins Masami Kojima a,*, Ronald L. Klein b, Hiroshi Hatanaka c a
b
AIST, Midorigaoka, Ikeda, Osaka 563-8577, Japan Department of Pharmacology and Therapeutics, University of Florida, JHMHC Box 100267, Gainesville, FL 32610-0267, USA c Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan Received 18 January 2002; accepted 11 March 2002
Abstract Since the discovery of nerve growth factor, there has been accumulating evidence that neurotrophins (NTs) mediate various biological responses of peripheral and central neurons. NTs have been traditionally studied as the regulating factors of neuronal survival and differentiation. Recent data indicate that NTs can modify neuronal plasticity by specific changes in pre- and postsynaptic functions. Whether the NT action is pre- or post-synaptic, however, remains to be controversy. Here we review the recent advances of NTs involved in synaptic plasticity, as well as the pre- and post-synaptic arguments. We also review the recent discovery that proneurotrophins and mature NTs have the differential ability to bind selective receptors and mediate distinctive biological actions. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Neurotrophin; Brain-derived neurotrophic factor; Activity-dependent plasticity; Neurotrophin receptor; Green fluorescent protein
1. Introduction Neurotrophins (NTs) are a family of small, secreted proteins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, and NT-4/5 (Lewin and Barde, 1996). The primary event in NT signaling is the activation of receptor tyrosine kinases of the Trk family, which have ligand-binding specificities as follows: NGF to TrkA; BDNF and NT4/5 to TrkB; and NT-3 to TrkC (Lee et al., 2001a; Patapoutian and Reichardt, 2001). This specific binding leads to the receptor dimerization, activation, and recruitment of various adapter proteins to initiate signaling cascades (Kaplan and Miller, 1997). Another NT receptor is p75NTR, a member of tumor necrosis factor receptor family, which binds to all NTs with approximately equivalent affinities (Lee et al., 2001a). It is postulated that p75NTR-mediated signaling cascades are involved in apoptotic neuronal death (Majdan and Miller, 1999).
* Corresponding author. Tel.: /81-727-51-9081; fax: /81-727-519628 E-mail address: [email protected] (M. Kojima).
Although NTs have traditionally been studied as the regulating factors of neural survival and differentiation (Lewin and Barde, 1996; Kaplan and Miller, 1997), the field of synapse development and physiology recently opened a new area of NT research on neuronal plasticity (Poo, 2001). This research is supported by the study showing that mRNA encoding NTs and their receptors are strongly expressed in the adult brain in areas with a high degree of plasticity, the hippocampus and neocortex (Kokaia et al., 1993). The NT hypothesis for neuronal plasticity proposes that neural activity regulates the expression, secretion and signaling of NTs to induce specific changes in synaptic efficacy and synapse morphology (Thoenen, 1995; Poo, 2001).
2. Three types of regulation for the NT hypothesis The NT hypothesis regarding activity-dependent plasticity includes three kinds of regulation: (1) NTdependent modification of synaptic plasticity; (2) regulation of NT expression; and (3) regulation of NT secretion and transport. NT researchers have investigated these three facets of regulation both electrophysiologically and biochemically.
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 0 3 4 - 2
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2.1. Neurotrophin modification of synaptic transmission Since Lohof et al. (1993) clearly demonstrated that both spontaneous and evoked synaptic transmission were elevated a few minutes after the application of BDNF or NT-3 in Xenopus nerve /muscle cultures, a number of electrophysiological studies have suggested that NTs can modify synaptic transmission, long-term potentiation (LTP) and long-term depression in cell cultures, slice preparations, and intact brain (Schuman, 1999; Poo, 2001). At central synapses, NTs enhance excitatory transmission (Lessmann et al., 1994; Kang and Schuman, 1995) and suppress inhibitory transmission (Kim et al., 1994; Tanaka et al., 1997). Whether NT-induced synaptic potentiation occurs pre- or post-synaptically has been investigated. Most studies have indicated that the NT modification of synaptic transmission occurs presynaptically (Lohof et al., 1993; Lessmann et al., 1994; Kang and Schuman, 1995; Li et al., 1998; Schinder et al., 2000). Although the molecular mechanism is not fully understood, a recent study clearly demonstrated that the phosphorylation of synapsin I was involved in the NT modification of synaptic transmission (Jovanovic et al., 2000). Until now, there is little evidence indicating postsynaptic actions of NTs. BDNF enhanced the responses of NMDA, but not AMPA receptors in cultured cortical neurons (Levine et al., 1998), although this modification is not expected to produce the potentiation of the evoked actions by BDNF because AMPA receptors mainly mediate glutamate-exerting synaptic transmission (Poo, 2001). Genetically modified mice provided solid data implicating NTs in LTP. BDNF knockout mice were deficient for the induction of LTP in the hippocampal CA1 region, which could be reversed by application of BDNF (Korte et al., 1996; Patterson et al., 1996). Further, treatments with function-blocking TrkB antibody consistently reduced LTP (Kang et al., 1997; Chen et al., 1999). Curiously, BDNF maintained late phase LTP in the CA1 region after the LTP induction (Kang et al., 1997; Korte et al., 1998), suggesting that BDNF is a synaptic morphogen for memory formation. 2.2. Activity-dependent expression of neurotrophins The broad and strong expression pattern of NTs and their receptors in the adult brain (Kokaia et al., 1993) coupled with the modulation of expression by neural activity suggests that there is a link between NT function and activity-dependent plasticity. In addition to seizure activity (Gall and Isackson, 1989; Ernfors et al., 1991), LTP increases the expression of NTs (Patterson et al., 1992; Castren et al., 1993). Regulation of NT gene expression was also found in cultured neurons, where depolarization with glutamate or high potassium chlo-
ride increased the expression of NGF and BDNF mRNAs (Zafra et al., 1990; Lindholm et al., 1994). The expression of NT was also induced by more physiological stimuli in vivo, in the visual system. Visual experience regulated the expression of BDNF, whereas blockade of visual input led to the downregulation of BDNF mRNA (Castren et al., 1992). This regulation is also supported by the genomic analyses of the BDNF gene, which consists of five exons and four distinct promoters. The four transcribed BDNFs are polyadenylated at either of two distinct sites within the 3? untranslated region, by which mechanism a total of eight distinct BDNF transcripts are synthesized (Timmusk et al., 1993). All eight mRNAs, however, encode the identical BDNF protein. Interestingly, the study with exon-specific probes indicated that four first exons have different roles in neural activity (Timmusk et al., 1993). Ca2 influx through L-type voltage-sensitive Ca2 channels (VSCCs) was shown to increase the expression of exon I-, exon II-, and exon III-containing BDNF mRNAs (Ghosh et al., 1994; Timmusk et al., 1995; Tao et al., 1998; Shieh et al., 1998). It was also demonstrated that VSCCs-dependent transcription from exon III promoter was mediated via the CREB family of transcription factors (Tao et al., 1998) and regulated by CaM kinase IV (Shieh et al., 1998). The involvement of CREB and CaM kinase IV suggests that BDNF gene expression is regulated by signaling cascades underlying activitydependent neuronal plasticity. 2.3. Activity-dependent secretion of neurotrophins NT secretion has been investigated by sensitive ELISA and immunoprecipitation assays. In hippocampal slices or cultured cells overexpressing NGF, glutamate or high potassium chloride stimulated the rapid secretion of NGF (Blochl and Thoenen, 1995). Similar secretion was observed in hippocampal slices (Canossa et al., 1997) or cultured cells overexpressing BDNF (Goodman et al., 1996; Griesbeck et al., 1999). In cultured sensory neurons, activity-dependent secretion of endogenous BDNF was measured by an in situ ELISA assay (Balkowiec and Katz, 2000). Interestingly, the release was dependent on the pattern or frequency of the nerve impulse, suggesting that BDNF secretion resembles that of neuropeptides. The results together suggest that neural activity is involved in NT modifcation of synaptic functions. Secretion of growth factors is classified as constitutive or regulated. Several studies showed the constitutive secretion of BDNF and NGF in overexpressing cultured neurons and slice preparations (Blochl and Thoenen, 1995; Griesbeck et al., 1999). However, a recent study using cDNAs encoding proNGF and proBDNF demonstrated that NGF secretion is constitutive while BDNF
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secretion is activity-dependent (Mowla et al., 1999). One explanation for this discrepancy may be the different methodologies. In any case, the mechanism of NT release and from which sites remains to be an essential question to understand how NTs may regulate activitydependent neuronal plasticity. 2.4. Pre- and post-synaptic secretion of NTs To test the effect of NTs on activity-dependent plasticity, most experiments have used bath application of NTs by superfusion, which does not mimic their natural actions in vivo and has produced some discrepant results (Schuman, 1999). For example, some studies showed rapid actions of BDNF on excitatory synaptic transmission while others did not. One explanation for this discrepancy is the different perfusion rates of the applied BDNF because BDNF does not penetrate tissues easily. This discussion motivated researchers to study the modes of NT actions, and especially, whether NT secretion is pre- or post-synaptic. Electrophysiological studies indicate both pre- and post-synaptic modification induced by NTs. One model is that NT-containing vesicles are anterogradely transported to presynaptic terminals, released, and then taken up by postsynaptic neurons. Biochemical studies showed the presence of BDNF in the vesicular fraction of brain synaptosomes (Fawcett et al., 1997). Immunocytochemical studies also indicated that BDNF was transported to presynaptic terminals, even in regions lacking the expression of BDNF mRNA, and this anterograde transport was blocked by the microtubule inhibitor colchicine (Conner et al., 1997; Altar et al., 1997). More interestingly, 125Ilabeled NT-3 was anterogradely transported to axon terminals after injection into the chick eye and taken up trans-synaptically in retinal ganglion cells (von Bartheld et al., 1996). Whether this anterograde transport is linked to neuronal activity was addressed in a recent study discussed below. It was also shown that NTs are secreted from dendrites of postsynaptic neurons and modify presynaptic functions. In a study of Xenopus nerve/muscle cultures, a higher frequency of spontaneous acetylcholine secretion was found in the terminals contacting myocytes overexpressing NT-4 than in controls (Wang and Poo, 1997). The effect was blocked by TrkB-IgG, a NT-4 scavenger protein. This study strongly suggests that postsynaptic NTs may elicit presynaptic transmitter release. Studies with cultured CNS neurons indicate the postsynaptic distribution and activity-dependent secretion of NTs (Blochl and Thoenen, 1996; Goodman et al., 1996; Kojima et al., 2001; Hartmann et al., 2001). However, whether NTs secreted from postsynaptic neurons alter the presynaptic function of CNS neurons remains to be fully answered and green fluorescent
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protein (GFP)-based imaging will help to understand these actions.
3. Imaging studies of neurotrophin action The attachment of a target protein with a fluorescent protein, such as GFP, makes it possible to image the intracellular location and dynamics of the target protein in living cells. This technology was applied to current neuroscience to visualize the molecular dynamics at synapses and in dendrites (Umeda and Okabe, 2001). 3.1. Studies with GFP-tagged BDNF With a similar strategy, BDNF was tagged with GFP and gene transfer was used to express the BDNF /GFP fusion protein in cultured cells, slice preparations, and the intact brain (Fig. 1). Kojima et al. (2001) expressed BDNF/GFP in dissociated hippocampal neurons and maintained them for more than 3 weeks. The BDNF/ GFP formed fluorescence clusters and colocalized with PSD-95 immunoreactivity, providing a visual suggestion of postsynaptic location of BDNF in mature neurons. In a time-lapse imaging study, BDNF/GFP fluorescence
Fig. 1. Expression of BDNF /GFP in the rat brain. The GFP-tagged BDNF, which mimics the biological function and release kinetics of native BDNF (Kojima et al., 2001), was expressed in the adult rat brain using the adeno-associated virus (AAV) vector system (see Peel and Klein, 2000, for review). One month after injecting the BDNF / GFP AAV vector into either neocortex (A and B) or hippocampus (C and D) at a dose of 1/1010 particles, the transgene product was detected in brain sections by GFP native fluorescence (not shown) and GFP immunohistochemistry (A /D). The immunoreactivity filled the axons and dendrites of the cortical (A and B) and CA1 pyramidal (C and D) neurons, and did not accumulate in nuclei, similar to a BDNFmyc fusion protein, and different than unfused GFP, which gets expressed in nuclei (Klein et al., 1998, 1999). Bar in A/100 mm; bar in B /60 mm. A, C; B, D, same magnification.
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rapidly disappeared in response to depolarizing stimulation. The BDNF /GFP could therefore allow us to visualize activity-dependent dynamics of BDNF in living neurons. Using this construct, Kohara et al. (2001) clearly showed visual data of activity-dependent transfer of BDNF to postsynaptic neurons. In this study, BDNF / GFP was anterogradely transported to axonal terminals and taken up by postsynaptic neurons. Interestingly, the interneuronal transfer was remarkably reduced by the treatment of tetrodotoxin, a blocker of synaptic currents. 3.2. Tagged BDNF and Trk receptors for trafficking and signaling studies GFP-tagging provided a new strategy of NT research for three reasons. First, as well as the conventional neurotrophic actions of BDNF, novel spatial dynamics of BDNF can be observed. With similar strategies, the dynamics of other NTs can now be compared to one another. Probes based on fluorescent energy transfer technology have recently been developed that can directly visualize intracellular signaling cascades in vivo (Miyawaki and Tsien, 2000; Mochizuki et al., 2001). This technology may allow visualization of the interaction between NTs and Trk receptors. Second, we showed the postsynaptic location of BDNF /GFP and depolarization-induced reduction in BDNF /GFP intensity in cultured hippocampal neurons. Recently, Hartmann et al. (2001) showed that similar reduction occurred upon high-frequency synaptic stimulation. These studies together indicate that the postsynaptic BDNF is secreted in response to synaptic stimuli (Fig. 2). The restricted distribution of BDNF / GFP suggests that NT regulation of synaptic functions could occur at a single synapse level. This possibility is also suggested by an immunoelectron microscope study with the antibody specific for the full-length TrkB receptor (Drake et al., 1999). They demonstrated TrkB
Fig. 2. Pre- and post-synaptic mechanisms of NTs. Data indicate that the transport and secretion of NTs could occur both pre-synaptically and post-synaptically (Section 2.4). GFP-based imaging technology may permit the visualization of novel and conventional actions NTs (Section 3.1), and address the significance of both pre- and postsynaptic pathways.
immunoreactivity both presynaptically at axonal terminals and postsynaptically at dendritic spines and shafts. The recent paper by Kovalchuk et al. (2002) found postsynaptic actions of BDNF on Ca2 signaling in dendritic spines and a rapid induction of LTP. Although whether this regulation is induced by presynaptic BDNF or postsynaptic BDNF remains unclear, synaptically distributed BDNF and TrkB could exert it. Third, although GFP-tagged BDNF could visualize the anterograde transport of BDNF (Fig. 2), the retrograde transport of BDNF has not been seen. However, a recent study of compartmented cultures of DRG neurons, GFP-tagged TrkB was retrogradely transported to the cell body after the addition of BDNF to the axonal terminals (Watson et al., 1999). In this culture system, drugs can be applied selectively to either the cell body or the neurites. Further, a recent study with this culture technique suggested that NT activated different MAP kinase signaling pathways depending on whether they were added to axonal terminals or cell bodies (Watson et al., 2001). The location of the activated Trk receptors in the neurons might therefore determine which signaling cascades are activated in response to NTs. The unique combination of GFPbased fluorescent probes with primary culture techniques may identify the spatial and temporal distinctions of signaling cascades in fully differentiated neurons.
4. Extracellular protease activity regulates neurotrophinbinding affinity for their receptors A novel mechanism of extracellular NT processing was recently discovered (Lee et al., 2001b), outlined in Fig. 2. It is well known that NTs are first produced as 30 /35 kDa precursor proteins and processed to be the biologically active 12/15 kDa proteins. Precursor NTs contain a signal sequence and pairs of basic amino acids recognized by proteases. The intracellular enzymes, serine protease furin and prohormone convertase, cleave in the middle of proNTs to produce the mature forms (Seidah et al., 1996). The prodomain sequences are highly conserved across species (Heinrich and Lum, 2000), noteworthy as this indicates that they have additional functions. The prodomain is thought to determine either the constitutive or regulated secretory pathway and assist in the folding process (Rattenholl et al., 2001). Lee et al. (2001b) first suggested that proNTs were secreted and cleaved extracellularly by various extracellular proteases. Using recombinant proNGF and mature NGF, they tested the binding affinity for NGF receptors. Surprisingly, proNGF bound with a higher affinity to p75NTR, and with reduced affinity for TrkA, compared to mature NGF. Consistent with its greater affinity for p75NTR, proNGF induced p75-mediated
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5. Concluding remarks
Fig. 3. A novel regulation of NT action. Lee et al. (2001b) demonstrated that proNGF binds with higher affinity than mature NGF to p75NTR, leading to the signaling cascade of apoptosis. Mature NGF binds to TrkA receptor, leading to a signaling cascade of survival. They also showed that proNTs were secreted and cleaved extracellularly by matrix metalloproteases and serine protease. These findings are certain to lead to new models of NT action.
apoptosis to a greater extent than mature NGF. ProNGF may therefore be a highly selective ligand for p75NTR that signals for apoptosis (Fig. 3). These findings provide a chance to explore unsolved questions in the NT field, for example, a model of functional antagonism between TrkA and p75NTR during the period of naturally occurring cell death and target innervation (Majdan and Miller, 1999). How extracellular protease activity converts proNTs to the mature forms during this period is an intriguing question. Matrix metalloproteases are positioned as either transmembrane proteins or extracellular matrix proteins, so that they could process secreted proNTs at suitable domains. In addition to the three other known NT receptors, TrkA, TrkB, and TrkC, numerous adaptor proteins that bind to p75NTR have been reported (Lee et al., 2001a). NADE and NRAGE contribute to cell death. RhoA GTPase, Schwann cell factor-1 and NRAGE exert nonapoptotic activities such as neurite elongation and growth arrest. Considering these interactions with p75NTR, unprocessed NTs might exert various biological activities in nervous system. Do unprocessed NTs participate in NT-dependent modification of plasticity? Synaptic secretion of BDNF occurs through the regulated secretory pathway, which is mutually linked to the enhancement of synaptic activity (Poo, 2001). ProBDNF is secreted even by hippocampal neurons (Mowla et al., 1999; Kojima et al., 2001), suggesting that proteolytic cleavage of proBDNF may be essential for the activation of TrkB receptors. Because extracellular protease activity is modified during the formation of LTP (Luthi et al., 1997; Baranes et al., 1998), proteolytic cleavage of proNTs may be involved in the NT modification of plasticity.
Since NGF was discovered by Levi-Montalcini, Hamburger, and Cohen, experimental evidence has indicated that NTs are responsible for mediating diverse cellular responses from neuronal survival to neuronal plasticity, suggesting that they are biologically significant over the entire lifespan. This review focused on two areas of NT research: (1) the NT-dependent modification of neuronal plasticity; and (2) the current molecular advances achieved via novel model systems. The application of imaging techniques to this field allowed us to examine the spatial and temporal range of neurotrophic actions. Considering the localized activation of Trk receptors and MAP kinase signaling cascades (Watson et al., 2001), spatially restricted effects of NTs could lead to input-specific modification of synaptic functions (Schinder et al., 2000). The recent findings about the functionality of proNTs (Lee et al., 2001b) have added a new perspective for NT researchers. Because NT research has yielded a number of surprising advances, it will likely contribute to our understanding of general principles of cell biology.
Acknowledgements This work was supported by a grant to M.K. from NEDO and a grant to H.H. from CREST.
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