Chae-Seok Lim

Mutation in the Phosphorylation Sites of MAP Kinase Blocks Learning-Related Internalization of apCAM in Aplysia Sensory Neurons

The synaptic growth that accompanies 5-HT-induced long-term facilitation of the sensory to motor neuron connection in Aplysia is associated with the internalization of apCAM at the surface membrane of the sensory neuron. We have now used epitope tags to examine the fate of each of the two apCAM isoforms (membrane bound and GPI-linked) and find that only the transmembrane form is internalized. This internalization can be blocked by overexpression of transmembrane constructs with a single point mutation in the two MAPK consensus sites, as well as by injection of a specific MAPK antagonist into sensory neurons. These data suggest MAPK phosphorylation at the membrane is important for the internalization of apCAMs and, thus, may represent an early regulatory step in the growth of new synaptic connections that accompanies long-term facilitation.

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.

Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons

Adeno-associated virus (AAV) vectors can deliver transgenes to diverse cell types and are therefore useful for basic research and gene therapy. Although AAV has many advantages over other viral vectors, its relatively small packaging capacity limits its use for delivering large genes. The available transgene size is further limited by the existence of additional elements in the expression cassette without which the gene expression level becomes much lower. By using alternative combinations of shorter elements, we generated a series of AAV expression cassettes and systematically evaluated their expression efficiency in neurons to maximize the transgene size available within the AAV packaging capacity while not compromising the transgene expression. We found that the newly developed smaller expression cassette shows comparable expression efficiency with an efficient vector generally used for strong gene expression. This new expression cassette will allow us to package larger transgenes without compromising expression efficiency.

Neuronal mechanisms and circuits underlying repetitive behaviors in mouse models of autism spectrum disorder

Autism spectrum disorder (ASD) refers to a broad spectrum of neurodevelopmental disorders characterized by three central behavioral symptoms: impaired social interaction, impaired social communication, and restricted and repetitive behaviors. However, the symptoms are heterogeneous among patients and a number of ASD mouse models have been generated containing mutations that mimic the mutations found in human patients with ASD. Each mouse model was found to display a unique set of repetitive behaviors. In this review, we summarize the repetitive behaviors of the ASD mouse models and variations found in their neural mechanisms including molecular and electrophysiological features. We also propose potential neuronal mechanisms underlying these repetitive behaviors, focusing on the role of the cortico-basal ganglia-thalamic circuits and brain regions associated with both social and repetitive behaviors. Further understanding of molecular and circuitry mechanisms of the repetitive behaviors associated with ASD is necessary to aid the development of effective treatments for these disorders.

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 0.5–5 µm) of filamentous structures, which were formed rapidly (approximately 2 h) but which were sustained for at least 18 days 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.

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.

Flavonoid variation of theAconitum jaluense complex (Ranunculaceae) in Korea

Thirteen flavonoid compounds were isolated and identified from five Korean species in theA. jaluense complex; they were glycosylated derivatives of the flavonols kaempferol, quercetin, and isorhamnetin, and of the flavone apigenin. The flavonoid data revealed the presence of two entities in the complex in Korea; one includesA. jaluense s. str. and the other includes the remaining four species which have identical flavonoid profiles. Based on these results, in conjunction with evidence from the morphology, it is suggested that the taxa should be recognized as two sub-species ofA. jaluense s. l. The flavonoid data also provide strong evidence for the occurrence of hybridization betweenA. jaluense s. str. andA. japonicum subsp.napiforme at Mt. Chiri in southern Korea.

Intracellular Membrane Association of the Aplysia cAMP Phosphodiesterase Long and Short Forms via Different Targeting Mechanisms

Background: Phosphodiesterases play a role in cAMP regulation through specific targeting. Results: Membrane targeting of the Aplysia phosphodiesterase long and short forms is mediated hydrophobically and electrostatically, respectively. Conclusion: The Aplysia phosphodiesterase long and short forms are targeted to the intracellular membranes by different mechanisms. Significance: This is the first report demonstrating that phosphodiesterase is targeted to the membranes by hydrophobic or electrostatic interactions. Phosphodiesterases (PDEs) play key roles in cAMP compartmentalization, which is required for intracellular signaling processes, through specific subcellular targeting. Previously, we showed that the long and short forms of Aplysia PDE4 (ApPDE4), which are localized to the membranes of distinct subcellular organelles, play key roles in 5-hydroxytryptamine-induced synaptic facilitation in Aplysia sensory and motor synapses. However, the molecular mechanism of the isoform-specific distinct membrane targeting was not clear. In this study, we further investigated the molecular mechanism of the membrane targeting of the ApPDE4 long and short forms. We found that the membrane targeting of the long form was mediated by hydrophobic interactions, mainly via 16 amino acids at the N-terminal region, whereas the short form was targeted solely to the plasma membrane, mainly by nonspecific electrostatic interactions between their N termini and the negatively charged lipids such as the phosphatidylinositol polyphosphates PI4P and PI(4,5)P2, which are embedded in the inner leaflet of the plasma membrane. Moreover, oligomerization of the long or short form by interaction of their respective upstream conserved region domains, UCR1 and UCR2, enhanced their plasma membrane targeting. These results suggest that the long and short forms of ApPDE4 are distinctly targeted to intracellular membranes through their direct association with the membranes via hydrophobic and electrostatic interactions, respectively.

Enhancing inhibitory synaptic function reverses spatial memory deficits in Shank2 mutant mice

ABSTRACT Autism spectrum disorders (ASDs) are a group of developmental disorders that cause variable and heterogeneous phenotypes across three behavioral domains such as atypical social behavior, disrupted communications, and highly restricted and repetitive behaviors. In addition to these core symptoms, other neurological abnormalities are associated with ASD, including intellectual disability (ID). However, the molecular etiology underlying these behavioral heterogeneities in ASD is unclear. Mutations in SHANK2 genes are associated with ASD and ID. Interestingly, two lines of Shank2 knockout mice (e6–7 KO and e7 KO) showed shared and distinct phenotypes. Here, we found that the expression levels of Gabra2, as well as of GABA receptor‐mediated inhibitory neurotransmission, are reduced in Shank2 e6–7, but not in e7 KO mice compared with their own wild type littermates. Furthermore, treatment of Shank2 e6–7 KO mice with an allosteric modulator for the GABAA receptor reverses spatial memory deficits, indicating that reduced inhibitory neurotransmission may cause memory deficits in Shank2 e6–7 KO mice. This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’. HIGHLIGHTSExpression of Gabra2 gene coding GABAA receptor &agr;2 subunit was reduced in Shank2 e6–7 KO mice, but not in Shank2 e7 KO mice.GABAA receptor‐mediated synaptic transmission was impaired in Shank2 e6–7 KO mice, resulting in reduced I/E ratio.A GABAA receptor &agr;2 agonist L838,417 restored the impaired I/E ratio in Shank2 e6–7 KO mice.L838,417 treatment reversed spatial memory deficits in Shank2 e6–7 KO mice without affecting the social deficit.

Involvement of cAMP-guanine nucleotide exchange factor II in hippocampal long-term depression and behavioral flexibility

BackgroundGuanine nucleotide exchange factors (GEFs) activate small GTPases that are involved in several cellular functions. cAMP-guanine nucleotide exchange factor II (cAMP-GEF II) acts as a target for cAMP independently of protein kinase A (PKA) and functions as a GEF for Rap1 and Rap2. Although cAMP-GEF II is expressed abundantly in several brain areas including the cortex, striatum, and hippocampus, its specific function and possible role in hippocampal synaptic plasticity and cognitive processes remain elusive. Here, we investigated how cAMP-GEF II affects synaptic function and animal behavior using cAMP-GEF II knockout mice.ResultsWe found that deletion of cAMP-GEF II induced moderate decrease in long-term potentiation, although this decrease was not statistically significant. On the other hand, it produced a significant and clear impairment in NMDA receptor-dependent long-term depression at the Schaffer collateral-CA1 synapses of hippocampus, while microscopic morphology, basal synaptic transmission, and depotentiation were normal. Behavioral testing using the Morris water maze and automated IntelliCage system showed that cAMP-GEF II deficient mice had moderately reduced behavioral flexibility in spatial learning and memory.ConclusionsWe concluded that cAMP-GEF II plays a key role in hippocampal functions including behavioral flexibility in reversal learning and in mechanisms underlying induction of long-term depression.