Effects of Chronic LY341495 on Hippocampal mTORC1 Signaling in Mice with Chronic Unpredictable Stress-Induced Depression.

In several rodent models, acute administration of the metabotropic glutamate 2/3 (mGlu2/3) receptor antagonist LY341495 induced antidepressant-like effects via a mechanism of action similar to that of ketamine. However, the effects of chronic mGlu2/3 antagonism have not yet been explored. Therefore, we investigated the effects of chronic LY341495 treatment on the mechanistic target of rapamycin complex 1 (mTORC1) signaling and the levels of synaptic proteins in mice subjected to chronic unpredictable stress (CUS). LY341495 (1 mg/kg) was administered daily for 4 weeks to mice with and without CUS exposure. After the final treatment, the forced swimming test (FST) was used to assess antidepressant-like effects. The hippocampal levels of mTORC1-related proteins were derived by Western blotting. Chronic LY341495 treatment reversed the CUS-induced behavioral effects of FST. CUS significantly reduced the phosphorylation of mTORC1 and downstream effectors [eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP-1) and small ribosomal protein 6 (S6)], as well as the expression of synaptic proteins postsynaptic density-95 (PSD-95) and AMPA receptor subunit GluR1 (GluA1) in the hippocampus. However, chronic LY341495 treatment rescued these deficits. Our results suggest that the activation of hippocampal mTORC1 signaling is related to the antidepressant effect of chronic LY341495 treatment in an animal model of CUS-induced depression.

Early Enriched Environment Prevents Epigenetic p11 Gene Changes Induced by Adulthood Stress in Mice.

Positive experiences in early life may improve the capacity to cope with adulthood stress through epigenetic modification. We investigated whether an enriched environment (EE) in the postnatal period affected epigenetic changes in the p11 gene induced by chronic unpredictable stress (CUS) in adult C57BL/6J mice. EE was introduced for 5 weeks during postnatal days 21-55. After EE, the mice were subjected to CUS for 4 weeks. EE prevented depression-like behavior induced by adult CUS. EE prevented a decrease in p11 mRNA and histone H3 acetylation induced by CUS, with changes in the expression of histone deacetylase 5. Moreover, EE prevented changes in trimethylation of histone H3 lysine 4 (H3K4) and H3K27 induced by CUS. Furthermore, EE had positive effects on behavior and epigenetic alterations in adult mice without CUS. These results suggest that one of the underlying mechanisms of early-life EE may involve epigenetic modification of the hippocampal p11 gene promoter.

Effects of Early Life Stress on Epigenetic Changes of the Glucocorticoid Receptor 17 Promoter during Adulthood.

Growing evidence suggests that early life stress (ELS) has long-lasting effects on glucocorticoid receptor (GR) expression and behavior via epigenetic changes of the GR exon 17 promoter. However, it remains unclear whether ELS regulates histone modifications of the GR exon 17 promoter across the life span. We investigated the effects of maternal separation (MS) on histone acetylation and methylation of GR exon 17 promoter in the hippocampus, according to the age of adults. Depression-like behavior and epigenetic regulation of GR expression were examined at young and middle adulthood in mice subjected to MS from postnatal day 1 to 21. In the forced swimming test, young adult MS mice showed no effect on immobility time, but middle-aged MS mice significantly increased immobility time. Young adult and middle-aged MS mice showed decreased GR expression. Their two ages showed decreased histone acetylation with increased histone deacetylases (HDAC5) levels, decreased permissive methylation, and increased repressive methylation at the GR exon 17 promoter. The extent of changes in gene expression and histone modification in middle adulthood was greater than in young adulthood. These results indicate that MS in early life causes long-term negative effects on behavior via histone modification of the GR gene across the life span.

N-Acetyl-D-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration.

Recently, we showed that N-acetylglucosamine kinase (NAGK), an enzyme of amino sugar metabolism, interacts with dynein light chain roadblock type 1 (DYNLRB1) and promotes the functions of dynein motor. Here, we report that NAGK interacts with nuclear distribution protein C (NudC) and lissencephaly 1 (Lis1) in the dynein complex. Yeast two-hybrid assays, pull-down assays, immunocytochemistry, and proximity ligation assays revealed NAGK-NudC-Lis1-dynein complexes around nuclei, at the leading poles of migrating HEK293T cells, and at the tips of migratory processes of cultured rat neuroblast cells. The exogenous expression of red fluorescent protein (RFP)-tagged NAGK accelerated HEK293T cell migration during in vitro wound-healing assays and of neurons during in vitro neurosphere migration and in utero electroporation assays, whereas NAGK knockdown by short hairpin RNA (shRNA) delayed migration. Finally, a small NAGK peptide derived from the NudC interacting domain in in silico molecular docking analysis retarded the migrations of HEK293T and SH-SY5Y cells. These data indicate a functional interaction between NAGK and dynein-NudC-Lis1 complex at the nuclear envelope is required for the regulation of cell migration.

Dynamin-1-like protein (Dnm1L) interaction with kinesin light chain 1 (KLC1) through the tetratricopeptide repeat (TPR) domains.

Kinesin light chain 1 (KLC1) mediates binding of KIF5 motor to specific cargo. Using the yeast two-hybrid screening, we found that mitochondrial fission protein dynamin-1-like protein (Dnm1L) interacted with KLC1, but not KIF5. Dnm1L and KLC1 were co-localized in cultured cells. These results suggest that KLC1 may play a potential role in post-fission mitochondrial transport.

Neuronal cell-surface protein neurexin 1 interaction with multi-PDZ domain protein MUPP1.

Location of membrane proteins is often stabilized by PDZ domain-containing scaffolding proteins. Using the yeast two-hybrid screening, we found that neurexin 1 interacted with multi-PDZ domain protein 1 (MUPP1) through PDZ domain. Neurexin 2 and 3 also interacted with MUPP1. MUPP1 and neurexin 1 were co-localized in cultured cells. These results suggest a novel mechanism for localizing neurexin 1 to synaptic sites.

The heterotrimeric kinesin-2 family member KIF3A directly binds to disabled-1 (Dab1).

The heterotrimeric molecular motor kinesin-2 is involved in the microtubule-dependent transport of intracellular cargo. It consists of two distinct motor subunits (KIF3A, and KIF3B) and a non-motor subunit, kinesin-associated protein 3 (KAP3). The cargo-binding domain (CBD) at the carboxyl (C)-terminus of KIF3s plays an important role in the interaction with several different binding proteins. To identify the binding proteins for heterotrimeric kinesin-2, we performed a yeast two-hybrid screen and found a new interaction with Disables-1 (Dab1), the intracellular adaptor protein of reelin receptors. Dab1 bound to the CBD of KIF3A, but did not interact with the C-terminal domain of KIF3B, KIF5B, KIF17 or KAP3. The phosphotyrosine binding (PTB) domain-containing region of Dab1 is essential for the interaction with KIF3A. KIF3A interacted with GST-Dab1, and GST-CaMKIIα, but did not interact with GST-apolipoprotein E receptor 2 (ApoER2)-C or with GST alone. When co-expressed in HEK-293T cells, KIF3A co-precipitated with Dab1, but not with KIF5B. Dab1 and KIF3A were co-localized in cultured cells. We also identified deduced cell surface expression of ApoER2 in KIF3A dominant-negative cells. These results suggest that the KIF3A plays a role in the intracellular trafficking of ApoER2 to the cell surface.

KIF21A-mediated axonal transport and selective endocytosis underlie the polarized targeting of NCKX2.

We have previously shown that K(+)-dependent Na(+)/Ca(2+) exchanger (NCKX) is a major calcium clearance mechanism at the large axon terminals of central neurons, whereas their somata display little NCKX activity. We investigated mechanisms underlying the axonal polarization of NCKX2 in rat hippocampal neurons. We identified NCKX2 as the first neuron-specific cargo molecule of kinesin family member 21A (KIF21A). The intracellular loop of NCKX2 specifically interacted with the WD-40 repeats, a putative cargo-binding domain, of KIF21A. Dominant-negative mutant or depletion of KIF21A inhibited the transport of NCKX2-GFP to axon fibers. Knockdown of KIF21A caused calcium dysregulation at axonal boutons but not at somatodendritic regions. Despite the axonal polarization of the NCKX activity, both somatodendritic and axonal regions were immunoreactive to NCKX2. The surface expression of NCKX2 revealed by live-cell immunocytochemistry, however, displayed highly polarized distribution to the axon. Inhibition of endocytosis increased the somatodendritic surface NCKX2 and thus abolished the axonal polarization of surface NCKX2. These results indicate that KIF21A-mediated axonal transport and selective somatodendritic endocytosis underlie the axonal polarized surface expression of NCKX2.

Effects of liraglutide on depressive behavior in a mouse depression model and cognition in the probe trial of Morris water maze test.

BACKGROUND: We investigated the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) agonist, on a depression-like phenotype in mice exposed to chronic unpredictable stress (CUS). Learning and memory were also assessed using the Morris water maze (MWM) test. METHODS: Liraglutide (0.3 mg/kg/day for 21 days) was administered to mice with or without exposure to CUS. After 21 days of CUS, the forced swim test (FST) was performed to assess its antidepressant effect. To evaluate cognitive function, liraglutide was administered to mice under stress-free conditions for 21 days, and then the MWM test was performed on 6 consecutive days. RESULTS: Chronic liraglutide treatment reduced FST immobility in mice with and without CUS. In the probe trial of the Morris water maze test, the search error rate was reduced and the time spent and path length in the target quadrant and the number of platform crossings were increased. LIMITATION: Additional animal model experiments and molecular level studies are needed to support the results obtained in this study. CONCLUSIONS: Liraglutide appears to exert antidepressant effects and could improve cognitive function. Based on these results, GLP-1 agonists could have potential as novel antidepressants.

Neuromolecular Etiology of Bipolar Disorder: Possible Therapeutic Targets of Mood Stabilizers.

Bipolar disorder is a mental illness that causes extreme mood swings and has a chronic course. However, the mechanism by which mood episodes with completely opposite characteristics appear repeatedly, or a mixture of symptoms appears, in patients with bipolar disorder remains unknown. Therefore, mood stabilizers are indicated only for single mood episodes, such as manic episodes and depressive episodes, and no true mood-stabilizing drugs effective for treating both manic and depressive episodes currently exist. Therefore, in this review, therapeutic targets that facilitate the development of mood stabilizers were examined by reviewing the current understanding of the neuromolecular etiology of bipolar disorder.

AMPA receptor-mTORC1 signaling activation is required for neuroplastic effects of LY341495 in rat hippocampal neurons.

The group II metabotropic glutamate 2/3 (mGlu2/3) receptor antagonist LY341495 produces antidepressant-like effects by acting on mammalian target of rapamycin complex 1 (mTORC1) signaling and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors in rodent. We investigated whether LY341495 affects neuroplasticity via these mechanisms in rat primary hippocampal cultures under conditions of dexamethasone (DEX)-induced neurotoxicity. Ketamine was used for comparison. Hippocampal cultures were treated with LY341495 under conditions of DEX-induced toxicity. Changes in mTORC1-mediated proteins were determined by Western blotting analyses. Changes in dendritic outgrowth and spine density were evaluated via immunostaining. LY341495 significantly prevented DEX-induced decreases in the levels of mTORC1, 4E-BP1, and p70S6K phosphorylation as well as the levels of the synaptic proteins. These effects were blocked by pretreatment with the AMPA receptor inhibitor 2,3-dihydroxy-6-nitro-7sulfamoyl-benzo(f)quinoxaline (NBQX) and the mTORC1 inhibitor rapamycin. LY341495 significantly attenuated DEX-induced decreases in dendritic outgrowth and spine density. Pretreatment with rapamycin and NBQX blocked these effects of LY341495. Further analyses indicted that induction of BDNF expression produced by LY341495 was blocked by pretreatment with NBQX and rapamycin. LY341495 has neuroplastic effects by acting on AMPA receptor-mTORC1 signaling under neurotoxic conditions. Therefore, activation of AMPA receptor and mTORC1 signaling, which enhance neuroplasticity, may be novel targets for new antidepressants.

The Neuroprotective Effects of Melatonin: Possible Role in the Pathophysiology of Neuropsychiatric Disease.

Melatonin is a hormone that is secreted by the pineal gland. To date, melatonin is known to regulate the sleep cycle by controlling the circadian rhythm. However, recent advances in neuroscience and molecular biology have led to the discovery of new actions and effects of melatonin. In recent studies, melatonin was shown to have antioxidant activity and, possibly, to affect the development of Alzheimer's disease (AD). In addition, melatonin has neuroprotective effects and affects neuroplasticity, thus indicating potential antidepressant properties. In the present review, the new functions of melatonin are summarized and a therapeutic target for the development of new drugs based on the mechanism of action of melatonin is proposed.

Erratum: Lee, J.G., et al. The Neuroprotective Effects of Melatonin: Possible Role in the Pathophysiology of Neuropsychiatric Disease. Brain Sciences 2019, 9(10), 285.

The authors wish to make an erratum to the published version of their paper [...].

Upregulation by KCl treatment of eukaryotic translation elongation factor 1A (eEF1A) mRNA in the dendrites of cultured rat hippocampal neurons.

Activity-dependent local translation in the dendrites of brain neurons plays an important role in the synapse-specific provision of proteins necessary for strengthening synaptic connections. In this study we carried out combined fluorescence in situ hybridization (FISH) and immunocytochemistry (IC) and showed that more than half of the eukaryotic elongation factor 1A (eEF1A) mRNA clusters overlapped with or were immediately adjacent to clusters of PSD-95, a postsynaptic marker, in the dendrites of cultured rat hippocampal neurons. Treatment of the neurons with KCl increased the density of the dendritic eEF1A mRNA clusters more than two-fold. FISH combined with IC revealed that the KCl treatment increased the density of eEF1A mRNA clusters that overlapped with or were immediately adjacent to PSD-95 clusters. These results indicate that KCl treatment increases both the density of eEF1A mRNA clusters and their synaptic association in dendrites of cultured neurons.

Suppression of interleukin-2 gene expression by isoeugenol is mediated through down-regulation of NF-AT and NF-kappaB.

Isoeugenol is a naturally occurring methoxyphenol found in a variety of foods and essential oils. We investigated the effect of isoeugenol on T-cell function and the regulatory mechanism underlying its effect. Isoeugenol and its structural analog eugenol suppressed the lymphoproliferative response to concanavalin A stimulation in B6C3F1 mouse splenocyte cultures. Isoeugenol inhibited phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io)-induced IL-2 mRNA expression and protein secretion in B6C3F1 mouse splenocytes, and in EL4.IL-2 mouse T-cells, as determined by real-time RT-PCR and ELISA, respectively. To further characterize the inhibitory mechanism of isoeugenol at the transcriptional level, we examined the DNA binding activity of the transcription factors for IL-2 using an electrophoretic mobility shift assay. Isoeugenol decreased the binding activity of NF-AT and NF-kappaB in PMA/Io-stimulated EL4.IL-2 cells, but no significant effect was observed for AP-1 or Oct binding activity. Western blot analysis showed that isoeugenol also decreased the nuclear translocation of cytoplasmic NF-AT and NF-kappaB. These results suggest that isoeugenol suppresses IL-2 production through a decrease of IL-2 mRNA expression and that the inhibition is mediated, at least in part, through the down-regulation of NF-AT and NF-kappaB.

Interaction of FUN14 domain containing 1, a mitochondrial outer membrane protein, with kinesin light chain 1 via the tetratricopeptide repeat domain.

Kinesin 1 is a member of the kinesin superfamily proteins (KIFs) of microtubule-dependent molecular motor proteins that transport organelles and protein complexes in cells. Kinesin 1 consists of a homo- or hetero-dimer of kinesin heavy chains (KHCs), often, although not always, associated with two kinesin light chains (KLCs). KLCs are non-motor proteins that associate with many different binding proteins and cargoes, but their binding partners have not yet been fully identified. In the present study, a yeast two-hybrid system was used to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. The results of the current study revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin 1) and KIF3A (a motor subunit of kinesin 2) in the yeast two-hybrid assay. The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. Collectively, these results indicate that KLC1 may potentially compete with LC3, a key component for autophagosome formation, to interact with FUNDC1.

Antidepressant effect of Chaihu-Shugan-San extract and its constituents in rat models of depression.

Herbal preparations may be effective alternatives in the treatment of depression, which remains difficult to manage. Chaihu-Shugan-San (CSS), an oriental traditional medicine, has been used as a remedy for Hwa-Byung, a Korean culture-bound syndrome resembling depression. We examined whether aqueous extracts of CSS and its constituent herbs exert antidepressant-like effects in two experimental animal models: the forced swimming test (FST) and the chronic mild stress (CMS) model. The herbal extracts were administered orally for 7 days in the FST and for 21 days during the CMS model; imipramine at 20 mg/kg/day was injected intraperitoneally as a positive control. CSS, Radix Bupleuri (one of the most important constituent plants in CSS), and imipramine had significant anti-immobility effects in the FST and reversed the CMS-induced reduction in sucrose consumption. Rhizoma Cyperi, another constituent of CSS, had antidepressant activity in the FST, while it failed in the CMS model. In conclusion, our results suggest that CSS and its constituent herbs exert antidepressant-like effects comparable to those of imipramine in experimental animal models.

N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points.

N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74-96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching.

N-acetyl-D-glucosamine kinase is a component of nuclear speckles and paraspeckles.

Protein O-GlcNAcylation, dictated by cellular UDP-N-acetylglucosamine (UDP-GlcNAc) levels, plays a crucial role in posttranslational modifications. The enzyme GlcNAc kinase (NAGK, E.C. 2.7.1.59) catalyzes the formation of GlcNAc-6-phosphate, which is a major substrate for the biosynthesis of UDP-GlcNAc. Recent studies have revealed the expression of NAGK in different types of cells especially in neuronal dendrites. Here, by immunocytochemistry (ICC) and immunonucleochemistry (INC) of cultured rat hippocampal neurons, HEK293T and GT1-7 cells, we have showed that NAGK immuno-reactive punctae being present in the nucleoplasm colocalized with small nuclear ribonucleoprotein-associated protein N (snRNPN) and p54NRB, which are speckle and paraspeckle markers, respectively. Furthermore, NAGK IR cluster was also found to be colocalized with GTF2H5 (general transcription factor IIH, polypeptide 5) immuno reactive punctae. In addition, relative localization to the ring of nuclear lamin matrix and to GlcNAc, which is highly enriched in nuclear pore complexes, showed that NAGK surrounds the nucleus at the cytoplasmic face of the nuclear outer membrane. By in situ proximity ligation assay (PLA) we confirmed the colocalization of NAGK with snRNPN in the nucleus and in dendrites, while we also verified the interactions of NAGK with p54NRB, and with GTF2H5 in the nucleus. These associations between NAGK with speckle, paraspeckle and general transcription factor suggest its regulatory roles in gene expression.

Opening of mitochondrial ATP-sensitive potassium channels evokes oxygen radical generation in rabbit heart slices.

The purpose of this study was to determine whether ATP-sensitive potassium channel (K(ATP) channel) activation generates oxygen free radicals in the rabbit heart. We assayed malondialdehyde (MDA) in rabbit heart slices in vitro as an indicator of oxygen free radical generation. The K(ATP) channel openers, pinacidil and cromakalim, significantly increased MDA production in a concentration-dependent manner. MDA formation also increased linearly with incubation time in the presence of K(ATP) channel openers. The K(ATP) channel blockers, glibenclamide and 5-hydroxydecanoate (5-HD), decreased K(ATP) channel opener-induced MDA formation in a concentration-dependent manner. When Fe(2+) was administered to heart slices that had been pretreated with K(ATP) channel openers, a marked elevation in MDA was observed, compared to heart slices that were treated with Fe(2+) alone. A positive linear correlation between Fe(2+) and MDA level was observed. The MDA levels of heart slices subjected to anoxia for 15 min remained unchanged until reperfusion. When the heart slices were reoxygenated for 30 min, a marked increase in MDA formation was observed. However, in the presence of glibenclamide and 5-HD, reperfusion following anoxia did not result in increased MDA. These results suggest that the opening of mitochondrial K(ATP) channels in rabbit heart slices evokes oxygen free radical generation via a Fenton-type reaction.