Jungsoo Lee

Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disablility. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2−/−) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-d-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with d-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2−/− mice. Furthermore, treatment of Shank2−/− mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2−/− mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.

Ethanol preference in C. elegans

Caenorhabditis elegans senses multiple environmental stimuli through sensory systems and rapidly changes its behaviors for survival. With a simple and well‐characterized nervous system, C. elegans is a suitable animal model for studying behavioral plasticity.

The Caenorhabditis elegans homologue of Down syndrome critical region 1, RCN-1, inhibits multiple functions of the phosphatase calcineurin.

A conserved family of calcineurin-regulating proteins whose members have been implicated in several disease models such as Down syndrome, Alzheimers disease, and cardiac hypertrophy has been identified in several organisms including yeast, mice, and humans. We have characterized Caenorhabditis elegans rcn-1, which belongs to this family of calcineurin regulators, and shows approximately 40% identity with the human homologue DSCR-1. rcn-1 is expressed in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail. rcn-1 expression is upregulated by calcineurin activity. RCN-1 binds to calcineurin A from C.elegans lysate in a calcium-dependent manner, and inhibits bovine calcineurin phosphatase activity dose-dependently. In addition, overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertility defects, slow growth, and serotonin-resistant egg-laying defects. Moreover, phenotypes observed in gain-of-function calcineurin mutant animals were restored to normal by RCN-1 overexpression. These results demonstrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1.

Conserved Role of unc-79 in Ethanol Responses in Lightweight Mutant Mice

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

SEB-3, a CRF receptor-like GPCR, regulates locomotor activity states, stress responses and ethanol tolerance in Caenorhabditis elegans.

The CRF (corticotropin‐releasing factor) system is a key mediator of the stress response. Alterations in CRF signaling have been implicated in drug craving and ethanol consumption. The development of negative reinforcement via activation of brain stress systems has been proposed as a mechanism that contributes to alcohol dependence. Here, we isolated a gain‐of‐function allele of seb‐3, a CRF receptor‐like GPCR in Caenorhabditis elegans, providing an in vivo model of a constitutively activated stress system. We also characterized a loss‐of‐function allele of seb‐3 and showed that SEB‐3 positively regulates a stress response that leads to an enhanced active state of locomotion, behavioral arousal and tremor. SEB‐3 also contributed to acute tolerance to ethanol and to the development of tremor during ethanol withdrawal. Furthermore, we found that a specific CRF1 receptor antagonist reduced acute functional tolerance to ethanol in mice. These findings demonstrate functional conservation of the CRF system in responses to stress and ethanol in vertebrates and invertebrates.

SHN‐1, a Shank homologue in C. elegans, affects defecation rhythm via the inositol‐1,4,5‐trisphosphate receptor

Protein localization in the postsynaptic density (PSD) of neurons is mediated by scaffolding proteins such as PSD‐95 and Shank, which ensure proper function of receptors at the membrane. The Shank family of scaffolding proteins contain PDZ (PSD‐95, Dlg, and ZO‐1) domains and have been implicated in the localizations of many receptor proteins including glutamate receptors in mammals. We have identified and characterized shn‐1, the only homologue of Shank in Caenorhabditis elegans. The shn‐1 gene shows approximately 40% identity over 1000 amino acids to rat Shanks. SHN‐1 protein is localized in various tissues including neurons, pharynx and intestine. RNAi suppression of SHN‐1 did not cause lethality or developmental abnormality. However, suppression of SHN‐1 in the itr‐1 (sa73) mutant, which has a defective inositol‐1,4,5‐trisphosphate (IP3) receptor, resulted in animals with altered defecation rhythm. Our data suggest a possible role of SHN‐1 in affecting function of IP3 receptors in C. elegans.

βPix Up-regulates Na+/H+ Exchanger 3 through a Shank2-mediated Protein-Protein Interaction

Na+/H+ exchanger 3 (NHE3) plays an important role in neutral Na+ transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of βPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and βPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, βPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of βPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in βPix-mediated NHE3 regulation. Knockdown of endogenous βPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that βPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.

A deubiquitinating enzyme, UCH/CeUBP130, has an essential role in the formation of a functional microtubule‐organizing centre (MTOC) during early cleavage in C. elegans

Background Deubiquitinating enzymes generate monomeric ubiquitin in protein degradation pathways and are known to be important for the early development in many organisms.