Kazuyuki Kiyosue

Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival

BackgroundProneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75NTR) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood.ResultsBased on two rare single nucleotide polymorphisms (SNPs) of the human brain-derived neurotrophic factor (BDNF) gene, we generated R125M-, R127L- and R125M/R127L-BDNF, which have amino acid substitution(s) near the cleavage site between the pro- and mature-domain of BDNF. Western blot analyses demonstrated that these BDNF variants are poorly cleaved and result in the predominant secretion of proBDNF. Using these cleavage-resistant proBDNF (CR-proBDNF) variants, the molecular and cellular roles of proBDNF on the CNS neurons were examined. First, CR-proBDNF showed normal intracellular distribution and secretion in cultured hippocampal neurons, suggesting that inhibition of proBDNF cleavage does not affect intracellular transportation and secretion of BDNF. Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS neurons. Treatment with CR-proBDNF elicited apoptosis of cultured cerebellar granule neurons (CGNs), while treatment with mature BDNF (matBDNF) promoted cell survival. Third, we examined the effects of CR-proBDNF on neuronal morphology using more than 2-week cultures of basal forebrain cholinergic neurons (BFCNs) and hippocampal neurons. Interestingly, in marked contrast to the action of matBDNF, which increased the number of cholinergic fibers and hippocampal dendritic spines, CR-proBDNF dramatically reduced the number of cholinergic fibers and hippocampal dendritic spines, without affecting the survival of these neurons.ConclusionThese results suggest that proBDNF has distinct functions in different populations of CNS neurons and might be responsible for specific physiological cellular processes in the brain.

Brain-Derived Neurotrophic Factor Regulates Cholesterol Metabolism for Synapse Development

Brain-derived neurotrophic factor (BDNF) exerts multiple biological functions in the CNS. Although BDNF can control transcription and protein synthesis, it still remains open to question whether BDNF regulates lipid biosynthesis. Here we show that BDNF elicits cholesterol biosynthesis in cultured cortical and hippocampal neurons. Importantly, BDNF elicited cholesterol synthesis in neurons, but not in glial cells. Quantitative reverse transcriptase-PCR revealed that BDNF stimulated the transcription of enzymes in the cholesterol biosynthetic pathway. BDNF-induced cholesterol increases were blocked by specific inhibitors of cholesterol synthesis, mevastatin and zaragozic acid, suggesting that BDNF stimulates de novo synthesis of cholesterol rather than the incorporation of extracellular cholesterol. Because cholesterol is a major component of lipid rafts, we investigated whether BDNF would increase the cholesterol content in lipid rafts or nonraft membrane domains. Interestingly, the BDNF-mediated increase in cholesterol occurred in rafts, but not in nonrafts, suggesting that BDNF promotes the development of neuronal lipid rafts. Consistent with this notion, BDNF raised the level of the lipid raft marker protein caveolin-2 in rafts. Remarkably, BDNF increased the levels of presynaptic proteins in lipid rafts, but not in nonrafts. An electrophysiological study revealed that BDNF-dependent cholesterol biosynthesis plays an important role for the development of a readily releasable pool of synaptic vesicles. Together, these results suggest a novel role for BDNF in cholesterol metabolism and synapse development.

Optical microscopic observation of fluorescence enhanced by grating-coupled surface plasmon resonance

On the substrate carrying a sub-wavelength grating covered with a thin metal layer, a fluorescent dye-labeled cell was observed by fluorescence microscope. The fluorescence intensity was more than 20 times greater than that on an optically flat glass substrate. Such a great fluorescence enhancement from labeled cells bound to the grating substrate was due to the excitation by grating coupled surface plasmon resonance. The application of a grating substrate to two-dimensional detection and fluorescence microscopy appears to offer a promising method of taking highly sensitive fluorescence images.

Diminished Neuronal Activity Increases Neuron-Neuron Connectivity Underlying Silent Synapse Formation and the Rapid Conversion of Silent to Functional Synapses

Neuronal activity regulates the synaptic strength of neuronal networks. However, it is still unclear how diminished activity changes connection patterns in neuronal circuits. To address this issue, we analyzed neuronal connectivity and relevant mechanisms using hippocampal cultures in which developmental synaptogenesis had occurred. We show that diminution of network activity in mature neuronal circuit promotes reorganization of neuronal circuits via NR2B subunit-containing NMDA-type glutamate receptors (NR2B-NMDARs), which mediate silent synapse formation. Simultaneous double whole-cell recordings revealed that diminishing neuronal circuit activity for 48 h increased the number of synaptically connected neuron pairs with both silent and functional synapses. This increase was accompanied by the specific expression of NR2B-NMDARs at synaptic sites. Analysis of miniature EPSCs (mEPSCs) showed that the frequency of NMDAR-mediated, but not AMPAR-mediated, mEPSCs increased, indicating that diminished neuronal activity promotes silent synapse formation via the surface delivering NR2B-NMDARs in mature neurons. After activation of neuronal circuit by releasing from TTX blockade (referred as circuit reactivation), the frequency of AMPAR-mediated mEPSCs increased instead, and this increase was prevented by ifenprodil. The circuit reactivation also caused an increased colocalization of glutamate receptor 1-specfic and synaptic NR2B-specific puncta. These results indicate that the circuit reactivation converts rapidly silent synapses formed during activity suppression to functional synapses. These data may provide a new example of homeostatic circuit plasticity that entails the modulation of neuron-neuron connectivity by synaptic activity.

Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.

LONG-LASTING ENHANCEMENT OF SYNAPTIC ACTIVITY IN DISSOCIATED CEREBRAL NEURONS INDUCED BY BRIEF EXPOSURE TO MG2+ -FREE CONDITIONS

The long-lasting enhancement of periodic clusters of spontaneous excitatory postsynaptic currents (SEPSCs) was examined in dissociated chick cerebral neurons that had been transiently exposed to Mg2+-free solution for 15 min. Since the enhancement was diminished by blockade of synaptic transmission, it clearly depended on synaptic activities. A specific antagonist of N-methyl-D-aspartate receptors (NMDARs) also inhibited the potentiations. Furthermore, the presence of inhibitors of protein and RNA synthesis in the Mg2+-free solution blocked the potentiation. In the potentiated neurons, the frequency of miniature excitatory postsynaptic currents (mEPSPs) increased. In addition, a diffusible molecule(s) that promoted the potentiation appeared to be involved in this phenomenon, since the conditioned medium of Mg2+-free treated neurons enhanced synaptic activity in other dish.

PKC and CaMKII dependent synaptic potentiation in cultured cerebral neurons.

We have reported that the long-lasting potentiation of spontaneous excitatory postsynaptic currents (SEPSCs) was induced by a Mg(2+)-free treatment in cultured chick cerebral neurons and a factor(s) extracellularly released during the treatment could induce the potentiation by itself. In this paper, protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase type II (CaMKII) but not protein kinase A (PKA) were reported to contribute to the potentiation mechanism during a step between the activation of the N-methyl-D-aspartate receptors by the Mg(2+)-free treatment and the secretion of the protein factor(s).

Selective formation of silent synapses on immature postsynaptic cells in cocultures of chick neurons of different ages

Glutamatergic synapses usually contain two types of ionotropic glutamate receptor, N-methyl-D-aspartate receptors (NMDARs) and non-NMDA receptors (non-NMDARs), and the ratio of these receptors is thought to be critical for synaptic plasticity. To determine whether or not the ratio of these receptors at synaptic sites is controlled by the developmental stage of postsynaptic neurons, we applied a dual whole-cell recording technique to a culture of dissociated chick cerebral neurons of different ages. We found that formation of synapses that contained both types of receptor required maturation of postsynaptic neurons. Moreover, during the early development of postsynaptic neurons, NMDARs were selectively present at synaptic sites prior to the presence of non-NMDARs, even though both types of receptor were expressed in functional form in the neuronal membranes.

Re-expression of NR2B-containing NMDA receptors in vitro by suppression of neuronal activity.

N‐methyl‐d‐aspartate receptors (NMDARs) are known to play critical roles in the development of the nervous system, and their expression is regulated in an activity‐dependent fashion during development. However, the regulation of NMDAR expression after circuit formation is less well understood. To examine this, we performed patch‐clamp recordings from chick cerebral neurons in an activity‐controlled culture. Analysis of NMDAR channels from neurons before synapse formation showed that there are two components in channel open kinetics. The major slow component is clearly blocked by ifenprodil, a specific inhibitor of NR2B‐containing NMDARs. In contrast, slow component of NMDAR channel opening from neurons after synapse formation became minor and ifenprodil had little effect on the NMDAR channel openings. Furthermore, this change is reversibly regulated by neuronal activity, in that suppression induces the re‐expression of NR2B‐containing NMDARs, even after circuit formation.

Two modes of activity-dependent synaptogenesis of cerebral neurons in vitro

Effects of transduction activity and transmission activity on synaptogenesis of chick cerebral neurones in dissociated cell culture were studied electrophysio-logically using two blockers for these activities, tetrodotoxin (TTX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. CNQX inhibited the increase of evoked EPSCs more effectively than TTX, whereas both blockers similarly reduced the increase of miniature EPSCs (Minis). These data indicated that not only transduction-dependent transmission activity but also transduction-independent spontaneous activity regulate the synaptic efficiency. These two activities are suggested to change the quanta) amplitude and the number of synaptic sites, respectively.