Tanja Brigadski

Mechanisms, locations, and kinetics of synaptic BDNF secretion: An update

Brain-derived neurotrophic factor (BDNF) and other members of the protein family of neurotrophins have been implicated in a multitude of processes that are important for neuronal development and synaptic plasticity in the rodent central nervous system. In comparison to the wealth of information available with respect to the biological functions of neurotrophins, our knowledge regarding the processes that govern synaptic secretion of neurotrophins is scarce. Using live cell imaging of GFP-tagged neurotrophins in primary neurons, immunocytochemical detection of endogenous BDNF in fixed cells, and by blocking the action of endogenously released BDNF by means of TrkB receptor bodies in living neurons, several studies in recent years have allowed to better understand the time course and the mechanisms of synaptic secretion of neurotrophins. This review will summarize the current knowledge regarding the intracellular processing of proneurotrophins, the targeting of neurotrophin vesicles to axons and dendrites, and the mechanisms of activity-dependent secretion of BDNF at synapses. Since these processes are known to be cell type dependent, special emphasis is given to observations gained from experiments in primary 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.

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.

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.

Truncated TrkB receptor-induced outgrowth of dendritic filopodia involves the p75 neurotrophin receptor

The Trk family of receptor tyrosine kinases and the p75 receptor (p75NTR) mediate the effects of neurotrophins on neuronal survival, differentiation and synaptic plasticity. The neurotrophin BDNF and its cognate receptor tyrosine kinase, TrkB.FL, are highly expressed in neurons of the central nervous system. At later stages in postnatal development the truncated TrkB splice variants (TrkB.T1, TrkB.T2) become abundant. However, the signalling and function of these truncated receptors remained largely elusive. We show that overexpression of TrkB.T1 in hippocampal neurons induces the formation of dendritic filopodia, which are known precursors of synaptic spines. The induction of filopodia by TrkB.T1 occurs independently of neurotrophin binding and of kinase activity of endogenous TrkB.FL. Coexpression of a p75NTR lacking an intracellular domain inhibits the TrkB.T1-induced effect in a dominant negative manner. Steric hindrance of extracellular p75NTR interactions with a specific antibody, or absence of p75NTR with an intact extracellular domain also inhibit this TrkB.T1-induced effect. We thus propose a novel signalling pathway initiated by neurotrophin-independent extracellular or intramembrane interaction of TrkB.T1 with the p75NTR receptor, which modulates dendritic growth via p75NTR signalling cascades.

Neuronal precursor‐specific activity of a human doublecortin regulatory sequence

The doublecortin (DCX) gene encodes a 40‐kDa microtubule‐associated protein specifically expressed in neuronal precursors of the developing and adult CNS. Due to its specific expression pattern, attention was drawn to DCX as a marker for neuronal precursors and neurogenesis, thereby underscoring the importance of its promoter identification and promoter analysis. Here, we analysed the human DCX regulatory sequence and confined it to a 3.5‐kb fragment upstream of the ATG start codon. We demonstrate by transient transfection experiments that this fragment is sufficient and specific to drive expression of reporter genes in embryonic and adult neuronal precursors. The activity of this regulatory fragment overlapped with the expression of endogenous DCX and with the young neuronal markers class III β‐tubulin isotype and microtubule‐associated protein Map2ab but not with glial or oligodendroglial markers. Electrophysiological data further confirmed the immature neuronal nature of these cells. Deletions within the 3.5‐kb region demonstrated the relevance of specific regions containing transcription factor‐binding sites. Moreover, application of neurogenesis‐related growth factors in the neuronal precursor cultures suggested the lack of direct signalling of these factors on the DCX promoter construct.

Relationships of peripheral IGF-1, VEGF and BDNF levels to exercise-related changes in memory, hippocampal perfusion and volumes in older adults

Animal models point towards a key role of brain-derived neurotrophic factor (BDNF), insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in mediating exercise-induced structural and functional changes in the hippocampus. Recently, also platelet derived growth factor-C (PDGF-C) has been shown to promote blood vessel growth and neuronal survival. Moreover, reductions of these neurotrophic and angiogenic factors in old age have been related to hippocampal atrophy, decreased vascularization and cognitive decline. In a 3-month aerobic exercise study, forty healthy older humans (60 to 77years) were pseudo-randomly assigned to either an aerobic exercise group (indoor treadmill, n=21) or to a control group (indoor progressive-muscle relaxation/stretching, n=19). As reported recently, we found evidence for fitness-related perfusion changes of the aged human hippocampus that were closely linked to changes in episodic memory function. Here, we test whether peripheral levels of BDNF, IGF-I, VEGF or PDGF-C are related to changes in hippocampal blood flow, volume and memory performance. Growth factor levels were not significantly affected by exercise, and their changes were not related to changes in fitness or perfusion. However, changes in IGF-I levels were positively correlated with hippocampal volume changes (derived by manual volumetry and voxel-based morphometry) and late verbal recall performance, a relationship that seemed to be independent of fitness, perfusion or their changes over time. These preliminary findings link IGF-I levels to hippocampal volume changes and putatively hippocampus-dependent memory changes that seem to occur over time independently of exercise. We discuss methodological shortcomings of our study and potential differences in the temporal dynamics of how IGF-1, VEGF and BDNF may be affected by exercise and to what extent these differences may have led to the negative findings reported here.

Theta Burst Firing Recruits BDNF Release and Signaling in Postsynaptic CA1 Neurons in Spike-Timing-Dependent LTP

Timing-dependent LTP (t-LTP) is a physiologically relevant type of synaptic plasticity that results from repeated sequential firing of action potentials (APs) in pre- and postsynaptic neurons. t-LTP can be observed in vivo and is proposed to be a cellular correlate of memory formation. While brain-derived neurotrophic factor (BDNF) is essential to high-frequency stimulation-induced LTP in many brain areas, the role of BDNF in t-LTP is largely unknown. Here, we demonstrate a striking change in the expression mechanism of t-LTP in CA1 of the hippocampus following two distinct modes of synaptic activation. Single postsynaptic APs paired with presynaptic stimulation activated a BDNF-independent canonical t-LTP. In contrast, a theta burst of postsynaptic APs preceded by presynaptic stimulation elicited BDNF-dependent postsynaptic t-LTP that relied on postsynaptic BDNF secretion. This suggests that BDNF release during burst-like patterns of activity typically observed in vivo may play a crucial role during memory formation.

Chronic BDNF deficiency leads to an age-dependent impairment in spatial learning.

Brain-derived neurotrophic factor (BDNF) is a crucial mediator of neural plasticity and, consequently, of memory formation. In hippocampus-dependent learning tasks BDNF also seems to play an essential role. However, there are conflicting results concerning the spatial learning ability of aging BDNF(+/-) mice in the Morris water maze paradigm. To evaluate the effect of chronic BDNF deficiency in the hippocampus on spatial learning throughout life, we conducted a comprehensive study to test differently aged BDNF(+/-) mice and their wild type littermates in the Morris water maze and to subsequently quantify their hippocampal BDNF protein levels as well as expression levels of TrkB receptors. We observed an age-dependent learning deficit in BDNF(+/-) animals, starting at seven months of age, despite stable hippocampal BDNF protein expression and continual decline of TrkB receptor expression throughout aging. Furthermore, we detected a positive correlation between hippocampal BDNF protein levels and learning performance during the probe trial in animals that showed a good learning performance during the long-term memory test.

Evolution of Neuroplasticity in Response to Physical Activity in Old Age: The Case for Dancing.

From animal research, it is known that combining physical activity with sensory enrichment has stronger and longer-lasting effects on the brain than either treatment alone. For humans dancing has been suggested to be analogous to such combined training. Here we assessed whether a newly designed dance training program that stresses the constant learning of new movement patterns is superior in terms of neuroplasticity to conventional fitness activities with repetitive exercises and whether extending the training duration has additional benefits. Twenty-two healthy seniors (63–80 years) who had been randomly assigned to either a dance or a sport group completed the entire 18-month study. MRI, BDNF and neuropsychological tests were performed at baseline and after 6 and 18 months of intervention. After 6 months, we found a significant increase in gray matter volume in the left precentral gyrus in the dancers compared to controls. This neuroplasticity effect may have been mediated by the increased BDNF plasma levels observed in the dancers. Regarding cognitive measures, both groups showed significant improvements in attention after 6 months and in verbal memory after 18 months. In addition, volume increases in the parahippocampal region were observed in the dancers after 18 months. The results of our study suggest that participating in a long-term dance program that requires constant cognitive and motor learning is superior to engaging in repetitive physical exercises in inducing neuroplasticity in the brains of seniors. Therefore, dance is highly promising in its potential to counteract age-related gray matter decline.