Jae-Hyung Bach

The involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein (MAP) kinase in TRAIL/Apo2L-induced apoptosis

To determine the apoptotic signaling pathway which tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL/Apo2L) induced, we investigated the contribution of reactive oxygen species (ROS), p38 mitogen‐activated protein (MAP) kinase and caspases in human adenocarcinoma HeLa cells. Here we show that upon TRAIL/Apo2L exposure there was pronounced ROS accumulation and activation of p38 MAP kinase, and that activation of caspases and apoptosis followed. Pretreatment with antioxidants such as glutathione or estrogen attenuated TRAIL/Apo2L‐induced apoptosis through a reduction of ROS generation and diminished p38 MAP kinase and caspase activation. The p38 MAP kinase inhibitor SB203580 prevented apoptosis through a blockage of caspase activation, although ROS generation was not attenuated. Furthermore, the pan‐caspase inhibitor Z‐Val‐Ala‐DL‐Asp‐fluoromethyl ketone fully prevented apoptosis, while neither ROS accumulation nor p38 MAP kinase activation were affected. Therefore, our results suggest that TRAIL/Apo2L‐induced apoptosis is mediated by ROS‐activated p38 MAP kinase followed by caspase activation in HeLa cells.

Mitogen‐activated protein kinase/extracellular signal‐regulated kinase attenuates 3‐hydroxykynurenine‐induced neuronal cell death

3‐Hydroxykynurenine (3‐HK), an endogenous tryptophan metabolite, is known to have toxic effects in brain. However, the molecular mechanism of the toxicity has not been well identified. In this study, we investigated the involvement of MAPK/extracellular signal‐regulated kinase (ERK) in the 3‐HK‐induced neuronal cell damage. Our results showed that 3‐HK induced apoptotic neuronal cell death and ERK phosphorylation occurred during cell death. Inhibition of ERK activation using PD98059 considerably increased cell death. Furthermore, cell death was preceded by mitochondrial malfunction including collapse of mitochondrial membrane potential (ΔΨm) and cytochrome c release from mitochondria to the cytosol. Interestingly, inhibition of ERK dramatically increased mitochondrial malfunction, and enhanced caspase activation, resulting in enhanced neuronal cell death. Thus, our results show that ERK plays a protective role by maintaining mitochondrial function and regulating caspase activity under conditions of cellular stress.

C-terminal fragment of amyloid precursor protein induces astrocytosis

One of the pathophysiological features of Alzheimers disease is astrocytosis around senile plaques. Reactive astrocytes may produce proinflammatory mediators, nitric oxide, and subsequent reactive oxygen intermediates such as peroxynitrites. In the present study, we investigated the possible role of the C‐terminal fragment of amyloid precursor protein (CT‐APP), which is another constituent of amyloid senile plaque and an abnormal product of APP metabolism, as an inducer of astrocytosis. We report that 100 nm recombinant C‐terminal 105 amino acid fragment (CT105) of APP induced astrocytosis morphologically and immunologically. CT105 exposure resulted in activation of mitogen‐activated protein kinase (MAPK) pathways as well as transcription factor NF‐κB. Pretreatment with PD098059 and/or SB203580 decreased nitric oxide (NO) production and nuclear factor‐kappa B (NF‐κB) activation. But inhibitors of NF‐κB activation did not affect MAPKs activation whereas they abolished NO production and attenuated astrocytosis. Furthermore, conditioned media derived from CT105‐treated astrocytes enhanced neurotoxicity and pretreatment with NO and peroxynitrite scavengers attenuated its toxicity. These suggest that CT‐APP may participate in Alzheimers pathogenesis through MAPKs‐ and NF‐κB‐dependent astrocytosis and iNOS induction.

P53 mediates ceramide-induced apoptosis in SKN-SH cells

Ceramide induces apoptotic cell death in a dose- and time-dependent manner in neuroblastoma SKN-SH cells. Pretreatment with caspase inhibitors blocks cell death, suggesting that a set of caspase activities including caspase 1, as well as caspase 3, are involved in ceramide-induced apoptosis in SKN-SH cells. Treatment with a caspase inhibitor 3 h after ceramide addition did not inhibit cell death, although caspase activity was substantially reduced. Ceramide-induced apoptosis is accompanied by accumulation of p53 followed by an increase of Bax and decrease of Bcl-2 levels. Inhibition of p53 expression with p53 antisense oligonucleotides inhibits apoptosis and prevents the increase in Bax and decrease in Bcl-2. Furthermore, pretreatment with p53 antisense oligonucleotides markedly inhibits the induction of caspase activity. These results suggest that p53 regulates the ratio Bcl-2/Bax and the expression/activation of caspases during ceramide-induced apoptosis in SKN-SH cells. Caspase inhibition did not alter the expression of p53, Bcl-2 and Bax. Thus ceramide-induced reduction in the Bcl-2/Bax ratio, increase in caspase activity, and apoptosis is dependent upon increases in cellular p53 levels which play a critical role in the regulation of apoptotic cell death.

Estrogen attenuates cell death induced by carboxy-terminal fragment of amyloid precursor protein in PC12 through a receptor-dependent pathway.

In the present study, we investigated effects of estrogen on cell death induced by carboxy‐terminal fragment of amyloid precursor protein (CT), a candidate causative substance in the pathogenesis of Alzheimers disease. 17β‐Estradiol attenuated CT‐induced cell death in PC12 cells, whereas 17α‐estradiol, nonestrogenic stereoisomer, did not exert any significant protective effect on CT‐induced cell death. These results suggest that protective effects of estrogen may be mediated by estrogen receptor (ER) in PC12 cells. To confirm the results, we determined the effects of tamoxifen, an estrogen receptor antagonist. Tamoxifen blocked the protective effects of 17β‐estradiol, although it did not affect those of 17α‐estradiol. Overall, it might be thought that the protective effect of estradiol on CT‐induced cell death is achieved by hormonal properties mediated through the estrogen receptor rather than the structural properties as a reducing agent. J. Neurosci. Res. 65:403–407, 2001.

Effects of carboxyl-terminal fragment of Alzheimer’s amyloid precursor protein and amyloid β-peptide on the production of cytokines and nitric oxide in glial cells

In a brain with Alzheimers disease (AD), activated glial cells are observed surrounding amyloid plaque. In this study, to investigate the roles of carboxyl‐terminal fragments (CTs) of amyloid precursor protein (APP) and amyloid β‐peptide (Aβ) in inflammatory processes possibly linked to neurodegeneration associated with AD, we examined the effects of the CT of APP with 105 amino acid residues (CT105) on the alteration of inflammatory mediators in rat cortical astrocytes and microglial cells and compared these effects to those of Aβ. We found that cytokines including interleukin 1β (IL‐1β) and tumor necrosis factor a and chemokines such as macrophage inflammatory protein 1a, monocyte chemoattractant protein 1, and RANTES (regulated on activation, normal T expressed and secreted) were highly induced by 100 nM CT105 in a time‐dependent manner, whereas the same amount of Aβ1‐42 did not significantly induce these mediators. In addition, inducible nitric oxide synthase (iNOS) expression and nitrite accumulation induced by CT105 were inhibited by IL‐1 receptor antagonist (IL‐1ra). Furthermore, the addition of conditioned media from CT105‐treated astrocytes to rat cortical neurons caused a decrease in cell viability. Pretreatment with IL‐1ra, NG‐nitro‐L‐arginine methyl ester (a specific inhibitor of NOS), and pyrollidine dithiocarbamate [an inhibitor of nuclear factor‐κB (NF‐κB)] prevented neuronal cell death caused by conditioned media from CT105‐ treated astrocytes. Our results indicate that the activation of astrocytes by CT105 leads to neuronal cell death in vitro, presumably caused by the increase in nitric oxide derived from the induction of iNOS through the activation of cytokines and NF‐κB. Thus, CT105 may contribute to stimulation of inflammatory processes linked to delayed neurodegeneration in AD.