Volkmar Lessmann

Neurotrophin secretion: current facts and future prospects

The proteins of the mammalian neurotrophin family (nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5)) were originally identified as neuronal survival factors. During the last decade, evidence has accumulated implicating them (especially BDNF) in addition in the regulation of synaptic transmission and synaptogenesis in the CNS. However, a detailed understanding of the secretion of neurotrophins from neurons is required to delineate their role in regulating synaptic function. Some crucial questions that need to be addressed include the sites of neurotrophin secretion (i.e. axonal versus dendritic; synaptic versus extrasynaptic) and the neuronal and synaptic activity patterns that trigger the release of neurotrophins. In this article, we review the current knowledge in the field of neurotrophin secretion, focussing on activity-dependent synaptic release of BDNF. The modality and the site of neurotrophin secretion are dependent on the processing and subsequent targeting of the neurotrophin precursor molecules. Therefore, the available data regarding formation and trafficking of neurotrophins in the secreting neurons are critically reviewed. In addition, we discuss existing evidence that the characteristics of neurotrophin secretion are similar (but not identical) to those of other neuropeptides. Finally, since BDNF has been proposed to play a critical role as an intercellular synaptic messenger in long-term potentiation (LTP) in the hippocampus, we try to reconcile this possible role of BDNF in LTP with the recently described features of synaptic BDNF secretion.

Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses.

The protein brain‐derived neurotrophic factor (BDNF) has been postulated to be a retrograde or paracrine synaptic messenger in long‐term potentiation and other forms of activity‐dependent synaptic plasticity. Although crucial for this concept, direct evidence for the activity‐dependent synaptic release of BDNF is lacking. Here we investigate secretion of BDNF labelled with green fluorescent protein (BDNF–GFP) by monitoring the changes in fluorescence intensity of dendritic BDNF–GFP vesicles at glutamatergic synaptic junctions of living hippocampal neurons. We show that high‐frequency activation of glutamatergic synapses triggers the release of BDNF–GFP from synaptically localized secretory granules. This release depends on activation of postsynaptic ionotropic glutamate receptors and on postsynaptic Ca2+ influx. Release of BDNF–GFP is also observed from extrasynaptic dendritic vesicle clusters, suggesting that a possible spatial restriction of BDNF release to specific synaptic sites can only occur if the postsynaptic depolarization remains local. These results support the concept of BDNF being a synaptic messenger of activity‐dependent synaptic plasticity, which is released from postsynaptic neurons.

Postsynaptic Secretion of BDNF and NT-3 from Hippocampal Neurons Depends on Calcium–Calmodulin Kinase II Signaling and Proceeds via Delayed Fusion Pore Opening

The mammalian neurotrophins (NTs) NGF, BDNF, NT-3, and NT-4 constitute a family of secreted neuronal growth factors. In addition, NTs are implicated in several forms of activity-dependent synaptic plasticity. Although synaptic secretion of NTs has been described, the intracellular signaling cascades that regulate synaptic secretion of NTs are far from being understood. Analysis of NT secretion at the subcellular level is thus required to resolve the role of presynaptic and postsynaptic NT secretion for synaptic plasticity. Here, we transfected cultures of dissociated rat hippocampal neurons with green fluorescent protein-tagged versions of BDNF and NT-3, respectively, and identified NT vesicles at glutamatergic synapses by colocalization with the cotransfected postsynaptic marker PSD-95 (postsynaptic density-95)-DsRed. Depolarization-induced secretion of BDNF and NT-3 was monitored with live cell imaging. Direct postsynaptic depolarization with elevated K+ in the presence of blockers of synaptic transmission allowed us to investigate the signaling cascades that are involved in the postsynaptic NT vesicle secretion process. We show that depolarization-induced postsynaptic NT secretion is elicited by Ca2+ influx, either via L-type voltage-gated calcium channels or via NMDA receptors. Subsequent release of Ca2+ from internal stores via ryanodine receptors is required for the secretion process. Postsynaptic NT secretion is inhibited in the presence of KN-62 ([4(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester) and KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide), indicating a critical dependence on the activation of α-calcium–calmodulin-dependent protein kinase II (CaMKII). The cAMP/protein kinase A (PKA) signaling inhibitor Rp-cAMP-S impaired NT secretion, whereas elevation of intracellular cAMP levels was without effect. Using the Trk inhibitor k252a, we show that NT-induced NT secretion does not contribute to the NT release process at synapses, and BDNF does not induce its own secretion at postsynaptic sites. Release experiments in the presence of the fluorescence quencher bromphenol blue provide evidence for asynchronous and prolonged fusion pore opening of NT vesicles during secretion. Because fusion pore opening is fast compared with compound release, the speed of NT release seems to be limited by diffusion of NTs out of the vesicle. Together, our results reveal a strong dependence of activity-dependent postsynaptic NT secretion on Ca2+ influx, Ca2+ release from internal stores, activation of CaMKII, and intact PKA signaling, whereas Trk signaling and activation of Na+ channels is not required.

A common thread for pain and memory synapses? Brain-derived neurotrophic factor and trkB receptors

Recent evidence indicates that trophic factors can exert fast effects on neurones and so alter synaptic plasticity. Here, we focus on brain-derived neurotrophic factor (BDNF), which exerts a modulatory action at hippocampal synapses that are involved in learning and memory, and at the first pain synapse between primary sensory neurones and dorsal horn neurones. Hippocampal and sensory neurones share some properties for the release of endogenous BDNF. In the Schaffer collateral pathway of the hippocampus, binding of BDNF to high-affinity trkB receptors is essential for the induction of long-term potentiation, a specific type of synaptic plasticity. However, the consequences of BDNF binding to trkB receptors in the dorsal horn in relation to pain mechanisms are less well established and are considered in detail.

Differential vesicular targeting and time course of synaptic secretion of the mammalian neurotrophins.

Neurotrophins are a family of secreted neuronal survival and plasticity factors comprising NGF, BDNF, neurotrophin-3 (NT-3), and NT-4. Whereas synaptic secretion of BDNF has been described, the routes of intracellular targeting and secretion of NGF, NT-3, and NT-4 in neurons are poorly understood. To allow for a direct comparison of intracellular targeting and release properties, all four mammalian neurotrophins were expressed as green fluorescent protein fusion proteins in cultured rat hippocampal neurons. We show that BDNF and NT-3 are targeted more efficiently to dendritic secretory granules of the regulated pathway of secretion (BDNF, in 98% of cells; NT-3, 85%) than NGF (46%) and NT-4 (23%). For all NTs, the remaining cells showed targeting to the constitutive secretory pathway. Fusing the BDNF pre-pro sequence to NT-4 directed NT-4 more efficiently to the regulated pathway of secretion. All neurotrophins, once directed to the regulated secretion pathway, were detected near synapsin I-positive presynaptic terminals and colocalized with PSD-95-DsRed (postsynaptic density-95-Discosoma red), suggesting postsynaptic targeting of the neurotrophins to glutamatergic synapses. Depolarization-induced release of all neurotrophins from synaptic secretory granules was slow (delay in onset, 10-30 s; τ = 120-307 s) compared with transmitter release kinetics monitored with FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] destaining (onset, <5 s; τ = 13 ± 2 s). Among the neurotrophins, NT-4 secretion was most rapid but still proceeded 10 times more slowly than transmitter secretion. Preincubation of neurons with monensin (neutralizing intragranular pH, thus solubilizing the peptide core) increased the speed of secretion of BDNF, NGF, and NT-3 to the value of NT-4. These data suggest that peptide core dissolution in secretory granules is the critical determinant of the speed of synaptic secretion of all mammalian NTs and that the speed of release is not compatible with fast transmitter-like actions of neurotrophins.

Activity-Dependent Dendritic Release of BDNF and Biological Consequences

Network construction and reorganization is modulated by the level and pattern of synaptic activity generated in the nervous system. During the past decades, neurotrophins, and in particular brain-derived neurotrophic factor (BDNF), have emerged as attractive candidates for linking synaptic activity and brain plasticity. Thus, neurotrophin expression and secretion are under the control of activity-dependent mechanisms and, besides their classical role in supporting neuronal survival neurotrophins, modulate nearly all key steps of network construction from neuronal migration to experience-dependent refinement of local connections. In this paper, we provide an overview of recent findings showing that BDNF can serve as a target-derived messenger for activity-dependent synaptic plasticity and development at the single cell level.

Backpropagating Action Potentials Trigger Dendritic Release of BDNF during Spontaneous Network Activity

Brain-derived neurotrophic factor (BDNF) is a major regulator of activity-dependent synapse development and plasticity. Because BDNF is a secreted protein, it has been proposed that BDNF is released from target neurons in an activity-dependent manner. However, direct evidence for postsynaptic release of BDNF triggered by ongoing network activity is still lacking. Here we transfected cultures of dissociated hippocampal neurons with green fluorescent protein (GFP)-tagged BDNF and combined whole-cell recording, time-lapse fluorescent imaging, and immunostaining to monitor activity-dependent dendritic release of BDNF. We found that spontaneous backpropagating action potentials, but not synaptic activity alone, led to a Ca2+-dependent dendritic release of BDNF-GFP. Moreover, we provide evidence that endogenous BDNF released from a single neuron can phosphorylate CREB (cAMP response element-binding protein) in neighboring neurons, an important step of immediate early gene activation. Therefore, together, our results support the hypothesis that BDNF might act as a target-derived messenger of activity-dependent synaptic plasticity and development.

N-Cadherin Transsynaptically Regulates Short-Term Plasticity at Glutamatergic Synapses in Embryonic Stem Cell-Derived Neurons

The cell adhesion molecule N-cadherin has been proposed to regulate synapse formation in mammalian central neurons. This is based on its synaptic localization enabling alignment of presynaptic and postsynaptic specializations by an adhesion mechanism. However, a potential role of N-cadherin in regulating synaptic transmission has remained elusive. In this paper, a functional analysis of N-cadherin knock-out synapses was enabled by in vitro neuronal differentiation of mouse embryonic stem cells circumventing the early embryonic lethality of mice genetically null for N-cadherin. In our in vitro system, initial synapse formation was not altered in the absence of N-cadherin, which might be attributable to compensatory mechanisms. Here, we demonstrate that N-cadherin is required for regulating presynaptic function at glutamatergic synapses. An impairment in the availability of vesicles for exocytosis became apparent selectively during high activity. Short-term plasticity was strongly altered with synaptic depression enhanced in the absence of N-cadherin. Most intriguingly, facilitation was converted to depression under specific stimulation conditions. This indicates an important role of N-cadherin in the control of short-term plasticity. To analyze, whether N-cadherin regulates presynaptic function by a transsynaptic mechanism, we studied chimeric cultures consisting of wild-type neocortical neurons and ES cell-derived neurons. With N-cadherin absent only postsynaptically, we observed a similar increase in short-term synaptic depression as found in its complete absence. This indicates a retrograde control of short-term plasticity by N-cadherin. In summary, our results revealed an unexpected involvement of a synaptic adhesion molecule in the regulation of short-term plasticity at glutamatergic synapses.

Essential cooperation of N-cadherin and neuroligin-1 in the transsynaptic control of vesicle accumulation

Cell adhesion molecules are key players in transsynaptic communication, precisely coordinating presynaptic differentiation with postsynaptic specialization. At glutamatergic synapses, their retrograde signaling has been proposed to control presynaptic vesicle clustering at active zones. However, how the different types of cell adhesion molecules act together during this decisive step of synapse maturation is largely unexplored. Using a knockout approach, we show that two synaptic adhesion systems, N-cadherin and neuroligin-1, cooperate to control vesicle clustering at nascent synapses. Live cell imaging and fluorescence recovery after photobleaching experiments at individual synaptic boutons revealed a strong impairment of vesicle accumulation in the absence of N-cadherin, whereas the formation of active zones was largely unaffected. Strikingly, also the clustering of synaptic vesicles triggered by neuroligin-1 overexpression required the presence of N-cadherin in cultured neurons. Mechanistically, we found that N-cadherin acts by postsynaptically accumulating neuroligin-1 and activating its function via the scaffolding molecule S-SCAM, leading, in turn, to presynaptic vesicle clustering. A similar cooperation of N-cadherin and neuroligin-1 was observed in immature CA3 pyramidal neurons in an organotypic hippocampal network. Moreover, at mature synapses, N-cadherin was required for the increase in release probability and miniature EPSC frequency induced by expressed neuroligin-1. This cooperation of two cell adhesion systems provides a mechanism for coupling bidirectional synapse maturation mediated by neuroligin-1 to cell type recognition processes mediated by classical cadherins.

The functional role of the second NPXY motif of the LRP1 beta-chain in tissue-type plasminogen activator-mediated activation of N-methyl-D-aspartate receptors.

The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-d-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors.