Joo-Young Im

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.

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.

Cadmium-induced astroglial death proceeds via glutathione depletion.

Cadmium is a heavy metal that accumulates in the body, and its accumulation in the brain damages both neurons and glial cells. In the current study, we explored the mechanism underlying cadmium toxicity in primary cortical astroglia cultures. Chronic treatment with 10 μM cadmium was sufficient to cause 90% cell death in 18 hr. However, unlike that observed in neurons, cadmium‐induced astroglial toxicity was not attenuated by the antioxidants trolox (100 μM), caffeic acid (1 mM), and vitamin C (1 mM). In contrast, extracellular 100 μM glutathione (GSH; γ‐Glu‐Cys‐Gly) or 100 μM cysteine almost completely blocked cadmium‐induced astroglial death, whereas 300 μM oxidized GSH (GSSG) or 300 μM cystine, which do not have the free thiol group, were ineffective. In addition, cadmium toxicity was noticeably inhibited or enhanced when intracellular GSH was, respectively, increased by using the cell‐permeable glutathione ethyl ester (GSH‐EE) or depleted by using buthionine sulfoximine (BSO), an inhibitor of γ‐glutamylcysteine synthetase. In agreement with these data, intracellular GSH levels were found to be depressed in cadmium‐treated astrocytes. These results suggest that the toxic effect of cadmium on primary astroglial cells involves GSH depletion and, furthermore, that GSH administration can potentially be used to counteract cadmium‐induced astroglial cell death therapeutically.

Behavioral stress causes mitochondrial dysfunction via ABAD up-regulation and aggravates plaque pathology in the brain of a mouse model of Alzheimer disease

Basic and clinical studies have reported that behavioral stress worsens the pathology of Alzheimer disease (AD), but the underlying mechanism has not been clearly understood. In this study, we determined the mechanism by which behavioral stress affects the pathogenesis of AD using Tg-APPswe/PS1dE9 mice, a murine model of AD. Tg-APPswe/PS1dE9 mice that were restrained for 2h daily for 16 consecutive days (2-h/16-day stress) from 6.5months of age had significantly increased Aβ(1-42) levels and plaque deposition in the brain. The 2-h/16-day stress increased oxidative stress and induced mitochondrial dysfunction in the brain. Treatment with glucocorticoid (corticosterone) and Aβ in SH-SY5Y cells increased the expression of 17β-hydroxysteroid dehydrogenase (ABAD), mitochondrial dysfunction, and levels of ROS, whereas blockade of ABAD expression by siRNA-ABAD in SH-SY5Y cells suppressed glucocorticoid-enhanced mitochondrial dysfunction and ROS accumulation. The 2-h/16-day stress up-regulated ABAD expression in mitochondria in the brain of Tg-APPswe/PS1dE9 mice. Moreover, all visible Aβ plaques were costained with anti-ABAD in the brains of Tg-APPswe/PS1dE9 mice. Together, these results suggest that behavioral stress aggravates plaque pathology and mitochondrial dysfunction via up-regulation of ABAD in the brain of a mouse model of AD.

Repression of phospho-JNK and infarct volume in ischemic brain of JIP1-deficient mice

Mice lacking JIP1, a scaffold protein that organizes JNK pathway components, were constructed independently by two groups. The proposed in vivo function, however, remains contradictory; One study reported that targeted disruption of the jip1 caused embryonic death due to the requirement of JIP1 for fertilized eggs (Thompson et al. [2001] J. Biol. Chem. 276:27745–27748). In contrast, another group (Whitmarsh et al. [2001] Genes Dev. 15:2421–2432) demonstrated that JIP1‐deficient mice were viable and that the JIP1 null mutation inhibited the kainic acid‐induced JNK activation and neuronal death. The current study was undertaken to re‐elucidate the in vivo roles of JIP1 using newly generated JIP1 knockout mice. Our JIP1‐deficient mice were viable and healthy. The transient focal ischemic insult produced by middle cerebral artery occlusion (MCAO) strongly activated JNK in brain of jip1+/+, jip1+/−, and jip1−/− mice. Increased JNK activity was sustained for more than 22 hr in jip1+/+ and jip1+/−, whereas it was repressed rapidly in jip1−/−. Concomitantly, the infarct volume produced by the ischemic insult in jip1−/− was reduced notably compared to that in jip1+/+ brain. These results suggest that JIP1 plays a pivotal role in regulating the maintenance of phosphorylated JNK and neuronal survival in postischemic brain, but is not essential for JNK activation and early development.

Markedly attenuated acute and chronic pain responses in mice lacking adenylyl cyclase-5.

Chronic inflammatory and neuropathic pain is often difficult to manage using conventional remedies. The underlying mechanisms and therapeutic strategies required for the management of chronic pain need to be urgently established. The cyclic AMP (cAMP) second messenger system has been implicated in the mechanism of nociception, and the inhibition of the cAMP pathway by blocking the activities of adenylyl cyclase (AC) and protein kinase A has been found to prevent chronic pain in animal models. However, little is known regarding which of the 10 known isoforms of AC are involved in nociceptive pathways. Therefore, we investigated the potential pronociceptive function of AC5 in nociception using recently developed AC5 knockout mice (AC5−/−). We found that AC5−/− mice show markedly attenuated pain‐like responses in acute thermal and mechanical pain tests as compared with the wildtype control. Also, AC5−/− mice display hypoalgesic responses to inflammatory pain induced by subcutaneous formalin injection into hindpaws, and to non‐inflammatory and inflammatory visceral pain induced by injecting magnesium sulfate or acetic acid into the abdomen. Moreover, AC5−/− mice show strongly suppressed mechanical and thermal allodynia in two nerve injury‐induced neuropathic pain models. These results suggest that AC5 is essential for acute and chronic pain, and that AC5 knockout mice provide a useful model for the evaluation of the pathophysiological mechanisms of pain.

Human Noxin is an anti‐apoptotic protein in response to DNA damage of A549 non‐small cell lung carcinoma

Human Noxin (hNoxin, C11Orf82), a homolog of mouse noxin, is highly expressed in colorectal and lung cancer tissues. hNoxin contains a DNA‐binding C‐domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. Expression of hNoxin is associated with S phase in cancer cells and in normal cells. Expression of hNoxin was induced by ultraviolet (UV) irradiation. Knockdown of hNoxin caused growth inhibition of colorectal and lung cancer cells. The comet assay and western blot analysis revealed that hNoxin knockdown induced apoptosis through activation of p38 mitogen‐activated protein kinase (MAPK)/p53 in non‐small cell lung carcinoma A549 cells. Furthermore, simultaneous hNoxin knockdown and treatment with DNA‐damaging agents, such as camptothecin (CPT) and UV irradiation, enhanced apoptosis, whereas Trichostatin A (TSA) did not. However, transient overexpression of hNoxin rescued cells from DNA damage‐induced apoptosis but did not block apoptosis in the absence of DNA damage. These results suggest that hNoxin may be associated with inhibition of apoptosis in response to DNA damage. An adenovirus expressing a short hairpin RNA against hNoxin transcripts significantly suppressed the growth of A549 tumor xenografts, indicating that hNoxin knockdown has in vivo anti‐tumor efficacy. Thus, hNoxin is a DNA damage‐induced anti‐apoptotic protein and potential therapeutic target in cancer.

DNA damage-induced apoptosis suppressor (DDIAS), a novel target of NFATc1, is associated with cisplatin resistance in lung cancer

In a previous study, we reported that DNA damage induced apoptosis suppressor (DDIAS; hNoxin), a human homolog of mouse Noxin, functions as an anti-apoptotic protein in response to DNA repair. Here we reveal that DDIAS is a target gene of nuclear factor of activated T cells 2 (NFATc1) and is associated with cisplatin resistance in lung cancer cells. In the DDIAS promoter analysis, we found that NFATc1 activated the transcription of DDIAS through binding to NFAT consensus sequences in the DDIAS promoter. In addition, tissue array immunostaining revealed a correlation between DDIAS and NFATc1 expression in human lung tumors. NFATc1 knockdown or treatment with the NFAT inhibitor cyclosporine A induced apoptosis and led to growth inhibition of lung cancer cells, indicating the functional relevance of both the proteins. In contrast, DDIAS overexpression overcame this NFATc1 knockdown-induced growth inhibition, supporting the cancer-specific role of DDIAS as a target gene of NFATc1. NFATc1 or DDIAS inhibition clearly enhanced apoptosis induced by cisplatin in NCI-H1703 and A549 cells. Conversely, DDIAS overexpression rescued NCI-H1703 cells from cisplatin-mediated cell death and caspase-3/7 activation. These results suggest that NFATc1-induced DDIAS expression contributes to cisplatin resistance, and targeting DDIAS or NFATc1 impairs the mechanism regulating cisplatin resistance in lung cancer cells. Taken together, DDIAS is a target of NFATc1 and is associated with cisplatin resistance in lung cancer cells.

Nordihydroguaiaretic acid induces astroglial death via glutathione depletion

Nordihydroguaiaretic acid (NDGA) is known to cause cell death in certain cell types that is independent of its activity as a lipoxygenase inhibitor; however, the underlying mechanisms are not fully understood. In the present study, we examined the cellular responses of cultured primary astroglia to NDGA treatment. Continuous treatment of primary astroglia with 30 μM NDGA caused >85% cell death within 24 hr. Cotreatment with the lipoxygenase products 5‐HETE, 12‐HETE, and 15‐HETE did not override the cytotoxic effects of NDGA. In assays employing the mitochondrial membrane potential‐sensitive dye JC‐1, NDGA was found to induce a rapid and almost complete loss of mitochondrial membrane potential. However, the mitochondrial permeability transition pore inhibitors cyclosporin A and bongkrekic acid did not block NDGA‐induced astroglial death. We found that treatment with N‐acetyl cysteine (NAC), glutathione (GSH), and GSH ethyl ester (GSH‐EE) did inhibit NDGA‐induced astroglial death. Consistently, NDGA‐induced astroglial death proceeded in parallel with intracellular GSH depletion. Pretreatment with GSH‐EE and NAC did not block NDGA‐induced mitochondrial membrane potential loss, and there was no evidence that reactive oxygen species (ROS) production was involved in NDGA‐induced astroglial death. Together, these results suggest that NDGA‐induced astroglial death occurs via a mechanism that involves GSH depletion independent of lipoxygenase activity inhibition and ROS stress.