Hyoung Kim

Synaptic Protein Degradation Underlies Destabilization of Retrieved Fear Memory

Reactivated memory undergoes a rebuilding process that depends on de novo protein synthesis. This suggests that retrieval is dynamic and serves to incorporate new information into preexisting memories. However, little is known about whether or not protein degradation is involved in the reorganization of retrieved memory. We found that postsynaptic proteins were degraded in the hippocampus by polyubiquitination after retrieval of contextual fear memory. Moreover, the infusion of proteasome inhibitor into the CA1 region immediately after retrieval prevented anisomycin-induced memory impairment, as well as the extinction of fear memory. This suggests that ubiquitin- and proteasome-dependent protein degradation underlies destabilization processes after fear memory retrieval. It also provides strong evidence for the existence of reorganization processes whereby preexisting memory is disrupted by protein degradation, and updated memory is reconsolidated by protein synthesis.

A cellular model of memory reconsolidation involves reactivation-induced destabilization and restabilization at the sensorimotor synapse in Aplysia

The memory reconsolidation hypothesis suggests that a memory trace becomes labile after retrieval and needs to be reconsolidated before it can be stabilized. However, it is unclear from earlier studies whether the same synapses involved in encoding the memory trace are those that are destabilized and restabilized after the synaptic reactivation that accompanies memory retrieval, or whether new and different synapses are recruited. To address this issue, we studied a simple nonassociative form of memory, long-term sensitization of the gill- and siphon-withdrawal reflex in Aplysia, and its cellular analog, long-term facilitation at the sensory-to-motor neuron synapse. We found that after memory retrieval, behavioral long-term sensitization in Aplysia becomes labile via ubiquitin/proteasome-dependent protein degradation and is reconsolidated by means of de novo protein synthesis. In parallel, we found that on the cellular level, long-term facilitation at the sensory-to-motor neuron synapse that mediates long-term sensitization is also destabilized by protein degradation and is restabilized by protein synthesis after synaptic reactivation, a procedure that parallels memory retrieval or retraining evident on the behavioral level. These results provide direct evidence that the same synapses that store the long-term memory trace encoded by changes in the strength of synaptic connections critical for sensitization are disrupted and reconstructed after signal retrieval.

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.

Overexpression and RNA interference of Ap-cyclic AMP-response element binding protein-2, a repressor of long-term facilitation, in Aplysia kurodai sensory-to-motor synapses.

cyclic AMP-response element binding protein-2 (CREB2) is a member of the CREB/transcription factor (CREB/ATF4) family. CREB2 is a transcription factor known to be involved in Aplysia long-term facilitation. To further examine the role of ApCREB2 on long-term synaptic facilitation, we isolated ApCREB2 from Aplysia kurodai in full-length cDNA library, and found that the overexpression of ApCREB2 blocked 5-hydroxytryptamine (5-HT)-induced long-term synaptic facilitation in Aplysia sensory-to-motor synapses. Furthermore, a single pulse of 5-HT, which normally induces only short-term facilitation, in the presence of ApCREB2 inhibition by RNA interference, induced long-term facilitation in Aplysia sensory-to-motor synapses. These results suggest that ApCREB2 is a functional repressor of long-term facilitation in Aplysia sensory-to-motor synapses.

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.

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.

Aggregate formation and the impairment of long-term synaptic facilitation by ectopic expression of mutant huntingtin in Aplysia neurons.

Huntingtons disease (HD) is caused by an expansion of a polyglutamine (polyQ) tract within huntingtin (htt) protein. To examine the cytotoxic effects of polyQ‐expanded htt, we overexpressed an enhanced green fluorescent protein (EGFP)‐tagged N‐terminal fragment of htt with 150 glutamine residues (Nhtt150Q‐EGFP) in Aplysia neurons. A combined confocal and electron microscopic study showed that Aplysia neurons expressing Nhtt150Q‐EGFP displayed numerous abnormal aggregates (diameter 0.5–5u2003µm) of filamentous structures, which were formed rapidly (approximately 2u2003h) but which were sustained for at least 18u2003days in the cytoplasm. Furthermore, the overexpression of Nhtt150Q‐EGFP in sensory cells impaired 5‐hydroxytryptamine (5‐HT)‐induced long‐term synaptic facilitation in sensori‐motor synapses without affecting basal synaptic strength or short‐term facilitation. This study demonstrates the stability of polyQ‐based aggregates and their specific effects on long‐term synaptic plasticity.

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.

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.

A transducible nuclear/nucleolar protein, mLLP, regulates neuronal morphogenesis and synaptic transmission

Cell-permeable proteins are emerging as unconventional regulators of signal transduction and providing a potential for therapeutic applications. However, only a few of them are identified and studied in detail. We identify a novel cell-permeable protein, mouse LLP homolog (mLLP), and uncover its roles in regulating neural development. We found that mLLP is strongly expressed in developing nervous system and that mLLP knockdown or overexpression during maturation of cultured neurons affected the neuronal growth and synaptic transmission. Interestingly, extracellular addition of mLLP protein enhanced dendritic arborization, demonstrating the non-cell-autonomous effect of mLLP. Moreover, mLLP interacts with CCCTC-binding factor (CTCF) as well as transcriptional machineries and modulates gene expression involved in neuronal growth. Together, these results illustrate the characteristics and roles of previously unknown cell-permeable protein mLLP in modulating neural development.