Pyung-Lim Han

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.

Glycyrrhizic acid affords robust neuroprotection in the postischemic brain via anti-inflammatory effect by inhibiting HMGB1 phosphorylation and secretion

High mobility group box 1 (HMGB1) is an endogenous danger signal molecule. In a previous report, we showed that HMGB1 is massively released during NMDA-induced acute damaging process in the postischemic brain and triggers inflammatory processes, like microglial activation. siRNA-mediated HMGB1 knockdown markedly reduced infarct volumes, confirming the crucial role played by HMGB1 in the postischemic brain. In the present study, we showed neuroprotective effects of glycyrrhizin (GL) in the postischemic rat brain after middle cerebral artery occlusion (MCAO). GL, a triterpene present in the roots and rhizomes of licorice, Glycyrrhiza glabra, has been shown to have anti-inflammatory and anti-viral effects. It has been reported that GL binds directly to HMGB1, and inhibits its chemoattractant and mitogenic activities. The administration of GL (10mg/kg) intravenously at 3 or 6h after MCAO reduced infarct volumes to 12.9±4.2% and 46.2±9.9%, respectively, of untreated control. This neuroprotective effect was accompanied by improvements in motor impairment and neurological deficits and suppressions of microglia activation and proinflammatory cytokine induction. Interestingly, GL almost completely blocked HMGB1 secretion in the postischemic brain and in lipopolysaccharide (LPS)-treated microglia cells. Furthermore, HMGB1 phosphorylation, which is the initial step for HMGB1 secretion, and the interaction between HMGB1 and protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase IV (CaMKIV) were suppressed dose-dependently by GL. Here, we hypothesized that the blockage for the putative phosphorylation sites in HMGB1 by GL might be attributed to this suppression. In addition to the anti-inflammatory effects, we found that GL has anti-excitotoxic and anti-oxidative effects in neurons. Together these results indicate that GL has neuroprotective efficacy in the postischemic brain via its anti-inflammatory, anti-excitotoxic, and anti-oxidative effects and in particular, it exerts anti-inflammatory effect, at least in part, by inhibiting HMGB1 secretion.

Progressive cognitive impairment and anxiety induction in the absence of plaque deposition in C57BL/6 inbred mice expressing transgenic amyloid precursor protein

Numerous transgenic mouse models for Alzheimers disease (AD) have been generated to recapitulate the histological pathogenesis and behavioral phenotypes of AD brain. However, none of the existing models exhibits the full spectrum of AD symptoms, nor have all of the traits mimicked by the developed animal models been successfully represented within a single mouse line, indicating that the development of transgenic lines showing new features of the AD‐like brain should be explored. Here we report on a transgenic mouse line, named Tg‐APP (Sw, V717F)/B6, that expresses the human amyloid precursor protein (APP) containing the Swedish and the V717F Indiana mutations in the brains of inbred C57BL/6 mice, designed to eliminate the potential phenotypic variations attributed to the compound genetic backgrounds adopted in most AD mouse models. The Tg‐APP (Sw, V717F)/B6 mice expressed the transgene transcript, in the heterozygote state, at a level of 2.6 ± 0.1 fold higher than that of endogenous mouse APP. However, no Aβ‐plaque deposition was produced in the brain of the Tg‐APP (Sw, V717F)/B6 mice up to 18 months of age. The Tg‐APP(Sw, V717F)/B6 mice at 13–15 months showed reduced expression of calbindin and c‐Fos in the brain. The Tg‐APP (Sw, V717F)/B6 mice at 11–14 months displayed decreased motor coordination, learning and memory deficits, and severely increased anxiety. These phenotypes were not observed in the Tg‐APP (Sw, V717F)/B6 mice at 5–7 months. Microarray analysis revealed altered expression, in the amygdala of the Tg‐APP (Sw, V717F)/B6 mice, of genes previously implicated in anxiety. Taken together, these results suggest that the transgenic APP, or its derivatives, produces the age‐dependent pathophysiology of the AD‐like brain and that the progressive cognitive impairment and anxiety induction can proceed in the absence of visible Aβ‐plaque deposition.

Intranasal Delivery of HMGB1 siRNA Confers Target Gene Knockdown and Robust Neuroprotection in the Postischemic Brain

Noninvasive intranasal drug administration has been noted to allow direct delivery of drugs to the brain. In the present study, the therapeutic efficacy of intranasal small interfering RNA (siRNA) delivery was investigated in the postischemic rat brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA was delivered intranasally in normal adult rats using e-PAM-R, a biodegradable PAMAM dendrimer, as gene carrier. Florescence-tagged siRNA was found in the cytoplasm and processes of neurons and of glial cells in many brain regions, including the hypothalamus, amygdala, cerebral cortex, and striatum, in 1 hour after infusion, and the FITC-fluorescence was continuously detected for at least 12 hours. When siRNA for high mobility group box 1 (HMGB1), which functions as an endogenous danger molecule and aggravates inflammation, was delivered intranasally, the target gene was significantly depleted in many brain regions, including the prefrontal cortex and striatum. More importantly, intranasal delivery of HMGB1 siRNA markedly suppressed infarct volume in the postischemic rat brain (maximal reduction to 42.8 ± 5.6% at 48 hours after 60 minutes middle cerebral artery occlusion (MCAO)) and this protective effect was manifested by recoveries from neurological and behavioral deficits. These results indicate that the intranasal delivery of HMGB1 siRNA offers an efficient means of gene knockdown-mediated therapy in the ischemic brain.

Fluoxetine attenuates kainic acid-induced neuronal cell death in the mouse hippocampus

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) and one of the commonly prescribed antidepressants. Numerous clinical observations and animal studies indicate that fluoxetine enhances the anticonvulsant potencies of several antiepileptic drugs. In the previous report, we showed that fluoxetine strongly protects against delayed cerebral ischemic injury. In the present study, the authors investigated whether fluoxetine has a beneficial effect on KA-induced neuronal cell death. An intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 microg) of KA produced typical neuronal cell death both in CA1 and CA3 regions of the hippocampus. Although, there was no significant difference in the time course or severity of epileptic behavior, the systemic administration of fluoxetine 30 min before KA administration significantly attenuated this neuronal cell death. Fluoxetine was found to suppress neuronal cell loss when injected at 10 mg/kg and the effect was enhanced at 50 mg/kg. Furthermore, this fluoxetine-induced neuroprotection was accompanied by marked improvements in memory impairment, as determined by passive avoidance tests. KA-induced gliosis and proinflammatory marker (COX-2, IL-1beta, and TNF-alpha) inductions were also suppressed by fluoxetine administration. It is interesting to note here that fluoxetine treatment suppressed NF-kappaB activity dose-dependently in KA-treated mouse brains, suggesting that this explains in part its anti-inflammatory effect. Together, these results suggest that fluoxetine has therapeutic potential in terms of suppressing KA-induced pathogenesis in the brain, and that these neuroprotective effects are associated with its anti-inflammatory effects.

Repression of Tau Hyperphosphorylation by Chronic Endurance Exercise in Aged Transgenic Mouse Model of Tauopathies

The present study was undertaken to investigate whether chronic endurance exercise affects tau phosphorylation levels in the brain with Alzheimers disease (AD)‐like pathology. To address this, the transgenic (Tg) mouse model of tauopathies, Tg‐NSE/htau23, which expresses human tau23 in the brain, was chosen. Animals were subjected to chronic exercise for 3 months from 16 months of age. The exercised Tg mouse groups were treadmill run at speeds of 12 m/min (intermediate exercise group) or 19 m/min (high exercise group) for 1 hr/day, 5 days/week, during the 3‐month period. Chronic endurance exercise in Tg mice increased the expression of Cu/Zn‐superoxide dismutase (SOD) and catalase, and also their enzymatic activities in the brain. In parallel, chronic exercise in Tg mice up‐regulated the expression of phospho‐PKCα, phospho‐AKT, and phospho‐PI3K, and down‐regulated the expressions of phospho‐PKA, phosphor‐p38, phospho‐JNK, and phospho‐ERK. Moreover, chronic exercise up‐regulated both cytosolic and nuclear levels of β‐catenin, and the expression of T‐cell factor‐4 (Tcf‐4) and cyclin D1 in the brain. As a consequence of such changes, the levels of phospho‐tau in the brain of Tg mice were markedly decreased after exercise. Immunohistochemical analysis showed an exercised‐induced decrease of the phospho‐tau levels in the CA3 subregion of the hippocampus. These results suggest that chronic endurance exercise may provide a therapeutic potential to alleviate the tau pathology.

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.

Progressive neuronal loss and behavioral impairments of transgenic C57BL/6 inbred mice expressing the carboxy terminus of amyloid precursor protein

The beta-secretase cleaved Abeta-bearing carboxy-terminal fragments (betaCTFs) of amyloid precursor protein (APP) in neural cells have been suggested to be cytotoxic. However, the functional significance of betaCTFs in vivo remains elusive. We created a transgenic mouse line Tg-betaCTF99/B6 expressing the human betaCTF99 in the brain of inbred C57BL/6 strain. Tg-betaCTF99/B6 mouse brain at 12-16 months showed severely down-regulated calbindin, phospho-CREB, and Bcl-xL expression and up-regulated phospho-JNK, Bcl-2, and Bax expression. Neuronal cell density in the Tg-betaCTF99/B6 cerebral cortex at 16-18 months was lower than that of the non-transgenic control, but not at 5 months. At 11-14 months, Tg-betaCTF99/B6 mice displayed cognitive impairments and increased anxiety, which were not observed at 5 months. These results suggest that increased betaCTF99 expression is highly detrimental to the aging brain and that it produces a progressive and age-dependent AD-like pathogenesis.

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.