PKA-activated ApAF-ApC/EBP heterodimer is a key downstream effector of ApCREB and is necessary and sufficient for the consolidation of long-term facilitation.

Long-term memory requires transcriptional regulation by a combination of positive and negative transcription factors. Aplysia activating factor (ApAF) is known to be a positive transcription factor that forms heterodimers with ApC/EBP and ApCREB2. How these heterodimers are regulated and how they participate in the consolidation of long-term facilitation (LTF) has not, however, been characterized. We found that the functional activation of ApAF required phosphorylation of ApAF by PKA on Ser-266. In addition, ApAF lowered the threshold of LTF by forming a heterodimer with ApCREB2. Moreover, once activated by PKA, the ApAF-ApC/EBP heterodimer transactivates enhancer response element-containing genes and can induce LTF in the absence of CRE- and CREB-mediated gene expression. Collectively, these results suggest that PKA-activated ApAF-ApC/EBP heterodimer is a core downstream effector of ApCREB in the consolidation of LTF.

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.

A nucleolar protein ApLLP induces ApC/EBP expression required for long-term synaptic facilitation in aplysia neurons.

In Aplysia, long-term synaptic plasticity is induced by serotonin (5-HT) or neural activity and requires gene expression. Here, we demonstrate that ApLLP, a novel nucleolus protein, is critically involved in both long-term facilitation (LTF) and behavioral sensitization. Membrane depolarization induced ApLLP expression, which activated ApC/EBP expression through a direct binding to CRE. LTF was produced by a single pulse of 5-HT 30 min after the membrane depolarization. This LTF was blocked when either ApLLP or ApC/EBP were blocked by specific antibodies. In contrast, ApLLP overexpression induced LTF in response to a single 5-HT treatment. Simultaneously, a siphon noxious stimulus (SNS) to intact Aplysia induced ApLLP and ApC/EBP expression, and single tail shock 30 min after SNS transformed short-term sensitization to long-term sensitization of siphon withdrawal reflex. These results suggest that ApLLP is an activity-dependent transcriptional activator that switches short-term facilitation to long-term facilitation.

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.

Two interdependent TRPV channel subunits, inactive and Nanchung, mediate hearing in Drosophila.

Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that a Drosophila protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.

Thiol oxidation-mediated cell death in Aplysia cultured sensory neurons.

Cellular thiol groups modulate various aspects of cellular function, including cell death. In this study, we found that a thiol oxidant, diamide, induced morphological changes such as cell swelling, membrane blebbing, and chromatin condensation in Aplysia cultured sensory neurons. Furthermore, diamide induced biphasic changes in the membrane potential, where hyperpolarization was followed by depolarization. Moreover, these diamide-induced cytotoxic effects were completely blocked by the equimolar addition of the disulfide reducing agent dithiothreitol (DTT). We also found that during H(2)O(2)-induced cell death, DTT attenuated cell swelling and membrane blebbing, but not DNA breakage, whereas the vitamin E analogue trolox attenuated DNA breakage, but not cell swelling and membrane blebbing. These results demonstrate that during H(2)O(2)-induced cell death, apoptotic features such as DNA breakage are mediated in part by free radical generation, whereas necrotic features such as cell swelling and membrane blebbing are primarily mediated by the oxidation of cellular thiol groups.

Impairment of a parabolic bursting rhythm by the ectopic expression of a small conductance Ca2+-activated K+ channel in Aplysia neuron R15.

The electrical properties of neurons are produced by the coordinated activity of ion channels. K+ channels play a key role in shaping action potentials and in determining neural firing patterns. Small conductance Ca2+-activated K+ (SK(Ca)) channels are involved in modulating the slow component of afterhyperpolarization (AHP). Here we examine whether rat type 2 SK(Ca) (rSK2) channels can affect the shape of the action potential and the neural firing pattern, by overexpressing rat SK2 channels in Aplysia neuron R15. Our results show that rSK2 overexpression decreased the intra-burst frequency and changed the regular bursting activity of neurons to an irregular bursting or beating pattern in R15. Furthermore, the overexpression of rSK2 channels increased AHP and reduced the duration of the action potential. Thus, our results suggest that ectopic SK(Ca) channels play an important role in regulating the firing pattern and the shape of the action potential.

Beta-amyloid peptide binding protein does not couple to G protein in a heterologous Xenopus expression system.

Alzheimer's disease is a neurodegenerative disorder related to the formation of protein aggregates. beta-Amyloid protein (A beta), generated by enzymatic cleavage of amyloid precursor protein (APP), can cause such aggregation, and these aggregates may cause neuronal cell death by inducing apoptosis. However, A beta-induced intracellular signaling pathways involved in the neuronal death are not well understood. Recently it was shown that A beta aggregates induce neuronal cell death via beta-amyloid peptide-binding protein (BBP), a receptor for A beta in BBP-transfected cells, which is known to be sensitive to pertussis toxin, a G alpha(i/o) family inhibitor. However, the actual coupling of BBP to the pertussis-sensitive G protein was not demonstrated. In this study, we performed electrophysiological recordings using the two-electrode voltage-clamp technique to test whether human or Drosophila BBPs, singly or in combination with APP, are coupled to a specific type of G protein. Our results suggest that BBP is not directly coupled to G alpha(i/o), G alpha(s), or G alpha(q) proteins and that BBP may need a component other than APP to exert its toxic effect in concert with A beta.

Synaptic facilitation by ectopic octopamine and 5-HT receptors in Aplysia.

The cAMP pathway plays a critical role in synaptic plasticity. We assessed using the ectopic expression of octopamine (OA) receptor, the contribution of the cAMP pathway to short-term facilitation of sensory-motor synapses in Aplysia. When synaptic connections were depressed to 20-30% of their initial EPSP amplitude, the application of OA to sensory cells expressing OA receptor showed significant synaptic facilitation, but this was less than the synaptic facilitation shown by 5-HT treatment. We also found that synaptic facilitation was further enhanced when OA was treated in the presence of 5-HT at non-depressed synapses, but not at depressed synapses. These results imply that the role of cAMP in synaptic facilitation is reduced as the synapse becomes depressed due to repeated activity.

Identification of nuclear/nucleolar localization signal in Aplysia learning associated protein of slug with a molecular mass of 18 kDa homologous protein.

We isolated a learning associated protein of slug with a molecular mass of 18 kDa (LAPS18) homologue from the expressed sequence tag database of Aplysia kurodai and named it Aplysia LAPS18-like protein (ApLLP). ApLLP encodes 120 amino acids and has 57% identity with LAPS18. To examine the subcellular expression pattern of ApLLP we constructed an EGFP-tagged ApLLP fusion protein and overexpressed it in both Aplysia neurons and COS-7 cells. In contrast to the previous findings, which showed that LAPS18 is secreted by COS-7 cells, ApLLP-EGFP was localized to the nucleus, and most of it to nucleoli. Analysis of deletion mutants of ApLLP-EGFP showed that the N-terminal and the C-terminal nucleolar and nucleus localization signal sequences are important for localization to the nucleus and the nucleoli.

Hydrogen peroxide modulates K+ ion currents in cultured Aplysia sensory neurons.

Hydrogen peroxide (H(2)O(2)) causes oxidative stress and is considered a mediator of cell death in various organisms. Our previous studies showed that prolonged (>6 h) treatment of Aplysia sensory neurons with 1 mM H(2)O(2) produced hyperpolarization of the resting membrane potential, followed by apoptotic morphological changes. In this study, we examined the effect of H(2)O(2) on the membrane conductance of Aplysia sensory neurons. Hyperpolarization was induced by 10 mM H(2)O(2) within 1 h, and this was attributed to increased membrane conductance. In addition, treatment with 10 mM H(2)O(2) for 3 min produced immediate depolarization, which was due to decreased membrane conductance. The H(2)O(2)-induced hyperpolarization and depolarization were completely blocked by dithiothreitol, a disulfide-reducing agent. The later increase of membrane conductance induced by H(2)O(2) was completely blocked by 100 mM TEA, a K(+) channel blocker, suggesting that H(2)O(2)-induced hyperpolarization is due to the activation of K(+) conductance. However, the inhibition of K(+) efflux by TEA did not protect against H(2)O(2)-induced cell death in cultured Aplysia sensory neurons, which indicates that the signal pathway leading to H(2)O(2)-induced cell death is more complicated than expected.

Hydrogen peroxide-induced cell death in cultured Aplysia sensory neurons.

Widespread neuronal cell death occurs during normal development and as a result of pathological conditions in the nervous system of many organisms. In this study, we investigated the cytotoxicity induced by H(2)O(2) in Aplysia mechanosensory neurons, which serve as a useful model in the study of learning and memory. Treatment with hydrogen peroxide (10(-2)-10 mM) for 3 h produced a nuclear DNA breakage in Aplysia sensory neurons, as revealed by TdT-mediated dUTP nick end labeling (TUNEL) staining, in a dose-dependent manner. Prolonged treatment (6-18 h) of Aplysia sensory neurons with 1 mM hydrogen peroxide produced dramatic morphological changes, such as neurite fragmentation, disintegration of the cell body, and a change in the resting membrane potential. This change in the resting potential was biphasic, and was initially hyperpolarized about 6 h after hydrogen peroxide treatment, but this later shifted to a depolarization some 13-18 h after treatment. Electron microscopic analysis also showed that this hydrogen peroxide-induced cell death was associated with apoptotic nuclear shrinkage, chromatin condensation, and necrotic swelling of organelles. Our results demonstrate that Aplysia sensory neurons show both apoptotic and necrotic characteristics as well as biphasic changes in resting potential during hydrogen peroxide-induced cell death.