Kyoung-Shim Kim

Optimization of chronic stress paradigms using anxiety‐ and depression‐like behavioral parameters

Chronic stress is a risk factor for psychiatric illnesses, such as anxiety and depression disorders. To understand the underlying mechanism regarding how chronic stress triggers such psychiatric dysfunctions, restraint‐based chronic stress models have been attempted in the past. However, total durations of repeated restraint stress and the evaluation time points used after the last restraint application vary from experiment to experiment. One reason for these methodological heterogeneities is related to considerable ambiguity concerning the definition of chronic stress, particularly in animal models. In the present study, we used behavioral traits, anxiety and depression, as stress‐assessment parameters that meet operationally useful requirements for the definition of the chronic stress state. We demonstrate that restraint treatment for 2 or 8 hr daily for 14 days is enough to produce anxiety‐ and depression‐like behaviors, whereas a 2 hr‐10 days restraint was marginally effective. cDNA microarray analysis identified 34 genes in the hippocampus and 72 genes in the amygdala with expression levels that were up‐ or down‐regulated by >2.0‐fold. Among the wide range of genes identified in this analysis, genes required for energy metabolism, signal transduction, transcription, synaptic plasticity, and remodeling of the brain architecture were notable. Our results suggest that the psychiatric criteria of anxiety and depression can be used as chronic stress‐assessment parameters and that a restraint stress paradigm consisting of restraint treatment for 2 or 8 hr daily for 14 days could be used as a prototype paradigm for chronic stress studies.

Behavioral stress accelerates plaque pathogenesis in the brain of Tg2576 mice via generation of metabolic oxidative stress

Alzheimer’s disease (AD) is a progressive neurodegenerative disease caused by genetic and non‐genetic factors. Most AD cases may be triggered and promoted by non‐genetic environmental factors. Clinical studies have reported that patients with AD show enhanced baseline levels of stress hormones in the blood, but their physiological significance with respect to the pathophysiology of AD is not clearly understood. Here we report that AD mouse models exposed to restraints for 2 h daily on 16 consecutive days show increased levels of β‐amyloid (Aβ) plaque deposition and commensurable enhancements in Aβ(1–42), tau hyperphosphorylation, and neuritic atrophy of cortical neurons. Repeated restraints in Tg2576 mice markedly increased metabolic oxidative stress and down‐regulated the expression of MMP‐2, a potent Aβ‐degrading enzyme, in the brain. These stress effects were reversed by blocking the activation of the hypothalamus‐pituitary‐adrenal gland axis with the corticotropin‐releasing factor receptor antagonist NBI 27914, further suggesting that over‐activation of the hypothalamic‐pituitary‐adrenal axis is required for stress‐enhanced AD‐like pathogenesis. Consistent with these findings, corticosteroid treatments to cultured primary cortical neurons increased metabolic oxidative stress and down‐regulated MMP‐2 expression, and MMP‐2 down‐regulation was reversed by inhibition of oxidative stress. These results suggest that behavioral stress aggravates AD pathology via generation of metabolic oxidative stress and MMP‐2 down‐regulation.

Adenylyl cyclase-5 activity in the nucleus accumbens regulates anxiety-related behavior.

Type 5 adenylyl cyclase (AC5) is highly concentrated in the dorsal striatum and nucleus accumbens (NAc), two brain areas which have been implicated in motor function, reward, and emotion. Here we demonstrate that mice lacking AC5 (AC5−/−) display strong reductions in anxiety‐like behavior in several paradigms. This anxiolytic behavior in AC5−/− mice was reduced by the D1 receptor antagonist SCH23390 and enhanced by the D1 dopamine receptor agonist, dihydrexidine (DHX). DHX‐stimulated c‐fos induction in AC5−/− mice was blunted in the dorso‐lateral striatum, but it was overactivated in the dorso‐medial striatum and NAc. The siRNA‐mediated inhibition of AC5 levels within the NAc was sufficient to produce an anxiolytic‐like response. Microarray and RT‐PCR analyses revealed an up‐regulation of prodynorphin and down‐regulation of cholecystokinin (CCK) in the NAc of AC5−/− mice. Administration of nor‐binaltorphimine (a kappa opioid receptor antagonist) or CCK‐8s (a CCK receptor agonist) reversed the anxiolytic‐like behavior exhibited by AC5−/− mutants. Taken together, these results suggest an essential role of AC5 in the NAc for maintaining normal levels of anxiety.

Protective effects of extracellular glutathione against Zn2+‐induced cell death in vitro and in vivo

The central nervous system reserves high concentrations of free Zn2+ in certain excitatory synaptic vesicles. In pathological conditions such as transient cerebral ischemia, traumatic brain injury, and kainic acid (KA)‐induced seizure, free Zn2+ is released in excess at synapses, which causes neuronal and glial death. We report here that glutathione (GSH) can be used as an effective means for protection of neural cells from Zn2+‐induced cell death in vitro and in vivo. Chronic treatment with 35 μM Zn2+ led to death of primary cortical neurons and primary astrocytes. The Zn2+ toxicity of cortical neurons was partially protected by 1 mM of GSH, whereas the Zn2+ toxicity of primary astrocyte cultures was blocked completely by 100 μM of GSH. To evaluate the beneficial effects of GSH in vivo, an excitotoxin‐induced neural cell death model was established by intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 μg) KA, which produced selective neuronal death, especially in CA1 and CA3 hippocampal regions. The i.c.v. co‐injection of 200 pmol of GSH significantly attenuated KA‐induced neuronal cell death and reactive gliosis in hippocampus. The results of this study suggest the contribution of Zn2+ in the excitotoxin‐induced neural cell death model and a potential value of GSH as a therapeutic means against Zn2+‐induced pathogenesis in brain.

Mice lacking adenylyl cyclase‐5 cope badly with repeated restraint stress

Physiological responses to acute stress proceed with the activation of the hypothalamus‐pituitary‐adrenal gland (HPA) system. Many brain regions are known to modulate the HPA axis activation in stress responses, but the detailed neural circuits and signaling system in the upstream of the HPA axis have to be explored further. Type 5 adenylyl cyclase (AC5) is highly concentrated in the dorsal striatum and nucleus accumbens, which are implicated in reward and stress‐related behavior. AC5–/– mice exposed to daily 2‐hr restraint stress for only 3–5 days showed poor stress‐coping responses, including severe body weight loss, poor coat condition, respiratory difficulties, and freezing behavior. Plasma corticosterone levels during 2‐hr stress sessions increased in AC5–/– mice compared with those of AC5+/+ mice. However, neither the corticosterone receptor antagonist RU486 nor the CRH receptor antagonist NBI27914 blocked their poor stress coping, whereas the administration of the GABAA receptor allosteric modulator diazepam or the D1 dopamine receptor antagonist SCH23390 prior to restraint stress sessions changed their stress‐coping response to the stressed AC5+/+ mouse level. Stress‐triggered c‐Fos expression was completely blunted in the dorsal striatum of AC5–/–. These results suggest that the AC5‐associated signal system and neural network are involved in the regulation of anxiety and stress‐coping response.

SK-PC-B70M confers anti-oxidant activity and reduces Aβ levels in the brain of Tg2576 mice

SK-PC-B70M is an oleanolic-glycoside saponin-enriched fraction derived from the root of Pulsatilla koreana. Recently, it was reported that hederacolchiside-E is an active ingredient of SK-PC-B70M that confers a neuroprotective effect against the cytotoxicity induced by Abeta(1-42) in SK-N-SH neuroblastoma cells. SK-PC-B70M improves scopolamine-induced impairments of spatial working memory in rats. In the present study, we investigated whether SK-PC-B70M has a beneficial effect on the Tg2576 murine model of Alzheimers disease. ELISA analysis revealed that the levels of soluble and insoluble forms of Abeta(1-42) in Tg2576 mice fed SK-PC-B70M (2000 ppm) from 11 months to 16 months of age were reduced to, respectively, 66% and 79% of the control Tg2576 mice. Anti-Abeta antibody-stained brain sections of Tg2576 mice with SK-PC-B70M (2000 ppm) consistently showed a reduction in plaque formation in the brain. Western blot analyses showed altered expressions of various cellular factors, such as up-regulation of transthyretin, phospho-ERK, and phospho-CREB in the brain treated with SK-PC-B70M. SK-PC-B70M suppressed the neuronal toxicity induced by H(2)O(2) in primary cortical culture. Moreover, biochemical and immunohistochemical analyses showed that the levels of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), oxidized by-products of lipid peroxidation, were notably reduced in the hippocampus of Tg2576 mice treated with SK-PC-B70M compared with the Tg2576 control. These results suggest that SK-PC-B70M attenuates AD-like pathology in the brain of Tg2576 mice.

Morphogenetic lung defects of JSAP1-deficient embryos proceeds via the disruptions of the normal expressions of cytoskeletal and chaperone proteins

Recent studies have shown that JNK/stress‐activated protein kinase‐associated protein 1 (JSAP1)‐deficient mice die from respiratory failure shortly after birth. To understand the underlying mechanism, we investigated the histological appearances and cell type changes in developing jsap1−/− lungs between E12.5 and E18.5. At the light microscopic level, no overt abnormality was detected in jsap1−/− until E16.5. However, alveoli and airway formations that normally occur after E16.5 were poorly advanced in jsap1−/−. Despite these morphological defects, surfactant secreting cells labeled by anti‐SP‐B or anti‐SP‐C were present in normal ranges in jsap1−/− lungs. Smooth muscle α‐actin expressing cells were also developed in jsap1−/− lungs, although actin expression was decreased. The expressions of transcriptional factors, such as, nuclear factor Ib (Nfib), N‐myc, and octamer transcriptional factor 1 (Oct‐1), which play a critical role in lung morphogenesis, were found to be down‐regulated, whereas signal transducer and activator of transcription 3 (Stat3), sonic hedgehog (Shh), and smoothened (Smo) were up‐regulated, in jsap1−/− lungs at E17.5‐E18.5 compared with those in jsap1+/+ lungs. Proteomics analysis of E17.5 lung identified 39 proteins with altered expressions, which included actin, tropomyosin, myosin light chain, vimentin, heat shock protein (Hsp27), and Hsp84. These results suggest that JSAP1 is required for the normal expressions of cytoskeletal and chaperone proteins in the developing lung, and that impaired expressions of these proteins might cause morphogenetic defects observed in jsap1−/− lungs.

Label-free fluorescent real-time monitoring of adenylyl cyclase.

In cellular signaling, adenylyl cyclase plays a key role in the hydrolysis of ATP to cyclic AMP and pyrophosphate. Using a synthetic fluorescent chemosensor (PyDPA) which binds strongly to the pyrophosphate group, we have developed a label-free fluorescent real-time detection system for adenylyl cyclase. This assay would be the first adenylyl cyclase assay based on chemosensing the production of pyrophosphate.

STEP signaling pathway mediates psychomotor stimulation and morphine withdrawal symptoms, but not for reward, analgesia and tolerance

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEPC230X−/− mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEPC230X−/− mice displayed reduced hyperlocomotion in response to intrastriatal injection of the μ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.