Hyungju Park

Vascular endothelial growth factor (VEGF) signaling regulates hippocampal neurons by elevation of intracellular calcium and activation of calcium/calmodulin protein kinase II and mammalian target of rapamycin.

The present study was undertaken to characterize neuronal activity-dependent expression and release of vascular endothelial growth factor (VEGF) from rat hippocampal neurons and its contribution to neuronal functions. Increased levels of VEGF164 mRNA were evident both in cultured neurons and slices, but not astrocytes, following membrane depolarization with KCl. Activity-dependent expression of VEGF, as well as its release, was dependent on the activation of the N-methyl-d-aspartate receptors or L-type voltage-activated calcium channels. A brief (10 min) application of recombinant VEGF165 to neurons elicited a slow rise in cytosolic Ca2+ in a VEGFR2 dependent manner. The VEGF-induced Ca2+ responses required Ca2+ influx, phospholipase Cgamma and Ca2+ stores. An inhibitor of transient receptor potential canonical channels reduced the VEGF-induced Ca2+ responses by 50%, suggesting the involvement of transient receptor potential canonical channels in the VEGF-mediated responses. The same brief stimulus with VEGF led to long-term synaptic enhancement dependent on protein synthesis. VEGF had prominent effects on the activation calcium/calmodulin protein kinase II and cAMP responsive element binding protein as well as extracellular signal-regulated protein kinase and mammalian target of rapamycin-all in a VEGFR2 dependent manner. Our findings suggest that VEGF released from neuronal cells plays a local role in Ca2+ influx and synaptic transmission that may influence the generation of long-term changes in synaptic efficacy.

Nuclear Translocation of CAM-Associated Protein Activates Transcription for Long-Term Facilitation in Aplysia

Repeated pulses of serotonin (5-HT) induce long-term facilitation (LTF) of the synapses between sensory and motor neurons of the gill-withdrawal reflex in Aplysia. To explore how apCAM downregulation at the plasma membrane and CREB-mediated transcription in the nucleus, both of which are required for the formation of LTF, might relate to each other, we cloned an apCAM-associated protein (CAMAP) by yeast two-hybrid screening. We found that 5-HT signaling at the synapse activates PKA which in turn phosphorylates CAMAP to induce the dissociation of CAMAP from apCAM and the subsequent translocation of CAMAP into the nucleus of sensory neurons. In the nucleus, CAMAP acts as a transcriptional coactivator for CREB1 and is essential for the activation of ApC/EBP required for the initiation of LTF. Combined, our data suggest that CAMAP is a retrograde signaling component that translocates from activated synapses to the nucleus during synapse-specific LTF.

Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia

Serotonin (5-HT) plays a critical role in modulating synaptic plasticity in the marine mollusc Aplysia and in the mammalian nervous system. In Aplysia sensory neurons, 5-HT can activate several signal cascades, including PKA and PKC, presumably via distinct types of G protein-coupled receptors. However, the molecular identities of these receptors have not yet been identified. We here report the cloning and functional characterization of a 5-HT receptor that is positively coupled to adenylyl cyclase in Aplysia neurons. The cloned receptor, 5-HTapAC1, stimulates the production of cAMP in HEK293T cells and in Xenopus oocytes. Moreover, the knockdown of 5-HTapAC1 expression by RNA interference blocked 5-HT-induced cAMP production in Aplysia sensory neurons and blocked synaptic facilitation in nondepressed or partially depressed sensory-to-motor neuron synapses. These data implicate 5-HTapAC1 as a major modulator of learning related synaptic facilitation in the direct sensory to motor neuron pathway of the gill withdrawal reflex.

An Aplysia Type 4 Phosphodiesterase Homolog Localizes at the Presynaptic Terminals of Aplysia Neuron and Regulates Synaptic Facilitation

The cAMP-dependent signaling pathway is critically involved in memory-related synaptic plasticity. cAMP-specific type 4 phosphodiesterases (PDE4) play a role in this process by regulating the cAMP concentration. However, it is unclear how PDE4 is involved in regulating synaptic plasticity. To address this issue in Aplysia sensory-to-motor synapses, we identified a long isoform of the PDE4 homolog in Aplysia kurodai (apPDE), with genetic and biochemical properties similar to those of mammalian PDE4s. Furthermore, apPDE is localized to the membrane and presynaptic region. Both apPDE overexpression and knock-down impaired short- and long-term facilitation, indicating that an appropriate expression level of apPDE in synaptic regions is required for normal synaptic facilitation. By using fluorescence resonance energy transfer-based measurement of in vivo protein kinase A (PKA) activation, we found that the PKA activation by 5-hydroxytryptamine (5-HT) was impaired in both apPDE-overexpressed and knock-down synapses. Analogous to the inhibition of apPDE by RNA interference, chronic rolipram treatment before 5-HT stimulation also impaired the PKA activation by 5-HT, suggesting that regulation of the synaptic cAMP level by PDE4 is critical for normal synaptic facilitation. Together, we suggest that PDE4s localized in the synapses play a critical role in regulating the optimum cAMP level required for normal synaptic plasticity.

N Termini of apPDE4 Isoforms Are Responsible for Targeting the Isoforms to Different Cellular Membranes.

Phosphodiesterases (PDEs) are known to play a key role in the compartmentalization of cAMP signaling; however, the molecular mechanisms underlying intracellular localization of different PDE isoforms are not understood. In this study, we have found that each of the supershort, short, and long forms of apPDE4 showed distinct localization in the cytoplasm, plasma membrane, and both plasma membrane and presynaptic terminals, respectively. The N-terminal 20 amino acids of the long form of apPDE4 were involved in presynaptic terminal targeting by binding to several lipids. In addition, the N terminus of the short form of apPDE4 bound to several lipids including phosphoinositols, thereby targeting the plasma membrane. Overexpression of the long and the short forms, but not the supershort form attenuated 5-HT-induced membrane hyperexcitability. Finally, the knockdown of apPDE4s in sensory neurons impaired both short-term and long-term facilitation. Thus, these results suggest that apPDE4s can participate in the regulation of cAMP signaling through specific subcellular localization by means of lipid binding activities.

Regulation of ApC/EBP mRNA by the Aplysia AU-rich element-binding protein, ApELAV, and its effects on 5-hydroxytryptamine-induced long-term facilitation.

Aplysia CCAAT enhancer‐binding protein (ApC/EBP), a key molecular switch in 5‐hydroxytryptamine (5‐HT)‐induced long‐term facilitation of Aplysia, is quickly and transiently expressed in response to a 5‐HT stimulus, but the mechanism underlying this dynamic expression profile remains obscure. Here, we report that the dynamic expression of ApC/EBP during long‐term facilitation is regulated at the post‐transcriptional level by AU‐rich element (ARE)‐binding proteins. We found that the 3′UTR of ApC/EBP mRNA contains putative sequences for ARE, which is a representative post‐transcriptional cis‐acting regulatory element that modulates the stability and/or the translatability of a distinct subset of labile mRNAs. We cloned the Aplysia homologue of embryonic lethal abnormal visual system homologue (ELAV/Hu) protein, one of the best‐studied RNA‐binding proteins that associate with ARE, and elucidated the involvement of Aplysia ELAV/Hu protein in ApC/EBP gene expressional regulation. Cloned Aplysia ELAV/Hu protein, Aplysia embryonic lethal abnormal visual system (ApELAV), bound to an AU‐rich region within the 3′UTR of ApC/EBP mRNA. Additionally, ApELAV controlled the expression of ApC/EBP 3′UTR‐containing reporter gene by functioning as a stability‐enhancing factor. In particular, 5‐HT‐induced long‐term facilitation was impaired when the AU‐rich region within the 3′UTR of ApC/EBP was over‐expressed, which suggests the significance of this region in 5‐HT‐induced ApC/EBP expression, and in the resultant formation of long‐term facilitation. Our results imply that the Aplysia ARE‐binding protein, ApELAV, can regulate ApC/EBP gene expression at the mRNA level, and accordingly, ARE‐mediated post‐transcriptional mechanism may serve a crucial function in regulating the expression of ApC/EBP in response to a 5‐HT stimulus.

The artificial AT-rich block enhanced the production of bovine growth hormone in Escherichia coli

Abstract In order to increase the synthesis of bovine growth hormone (bGH) using T7 promoter system in E. coli, the artificial AT-rich block was introduced into the upstream region of a consensus Shine-Dalgarno (SD) sequence and the spacer region (between SD and ATG codon) was enriched with A and T nucleotides. The cells harboring pTAJ plasmids with AT-rich block produced bGH in the range of 3% to 25% and the cells harboring pTBJ plasmids with AT-rich sequence in the spacer region from 0.8% to 20% of total cell proteins. This result suggests that AT rich block and AT nucleotides in the spacer region destabilize mRNA secondary structure, depending on the downstream coding information of bGH gene and also, implying that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.

Adenomatous polyposis coli-stimulated GEF 1 (Asef1) is a negative regulator of excitatory synaptic function

Guanine nucleotide exchange factors (GEFs) play important roles in many cellular processes, including regulation of the structural plasticity of dendritic spines. A GEF protein, adenomatous polyposis coli-stimulated GEF 1 (Asef1, ARHGEF4) is highly expressed in the nervous system. However, the function of Asef1 has not been investigated in neurons. Here, we present evidence showing that Asef1 negatively regulates the synaptic localization of postsynaptic density protein 95 (PSD-95) in the excitatory synapse by inhibiting Staufen-mediated synaptic localization of PSD-95. Accordingly, Asef1 expression impairs synaptic transmission in hippocampal cultured neurons. In addition, neuronal activity facilitates the dissociation of Asef1 from Staufen in a phosphoinositide 3 kinase (PI3K)-dependent manner. Taken together, our data reveal Asef1 functions as a negative regulator of synaptic localization of PSD-95 and synaptic transmission.