Tae H. Oh

Two distinct mechanisms are involved in 6-hydroxydopamine- and MPP+- induced dopaminergic neuronal cell death: Role of caspases, ROS, and JNK

In this study, we examined the possibility that MPTP and 6‐hydroxydopamine (6‐OHDA) act on distinct cell death pathways in a murine dopaminergic neuronal cell line, MN9D. First, we found that cells treated with 6‐OHDA accompanied ultrastructural changes typical of apoptosis, whereas MPP+ treatment induced necrotic manifestations. Proteolytic cleavage of poly(ADP‐ribose)polymerase by caspase was induced by 6‐OHDA, whereas it remained uncleaved up to 32 h after MPP+ treatment and subsequently disappeared. Accordingly, 6‐OHDA‐ but not MPP+‐induced cell death was significantly attenuated in the presence of a broad‐spectrum caspase inhibitor, N‐benzyloxy‐carbonyl‐Val‐Ala‐Asp‐fluomethylketone (Z‐VAD‐fmk). As measured by fluorometric probes, the level of reactive oxygen species (ROS) significantly increased after 6‐OHDA treatment. In contrast, the level of dihydroethidium‐sensitive ROS following MPP+ treatment remained unchanged while a slight increase in dichlorofluorescin‐sentive ROS was temporarily observed. As demonstrated by immunoblot analysis, the level of superoxide dismutase was down‐regulated following 6‐OHDA treatment, whereas it remained unchanged after MPP+ treatment. Cotreatment of cells with antioxidants such as N‐acetylcysteine or Mn(III)tetrakis(4‐benzoic acid)porphyrin chloride (MnTBAP, cell‐permeable superoxide dismutase mimetic) rescued 6‐OHDA‐ but not MPP+‐induced cell death, whereas inclusion of catalase or NG‐nitro‐l‐arginine had no effect in both cases. In addition, 6‐OHDA induced ROS‐mediated c‐Jun N‐terminal kinase (JNK) activation that was attenuated in the presence of N‐acetylcysteine or MnTBAP but not catalase or Z‐VAD‐fmk. In contrast, MPP+ has little effect on JNK activity, indicating that ROS and/or ROS‐induced cell death signaling pathway seems to play an essential role in 6‐OHDA–mediated apoptosis but not in MPP+‐induced necrosis in a mesencephalon‐derived, dopaminergic neuronal cell line. J. Neurosci. Res. 57:86–94, 1999.

Ginsenosides Rb1and Rg3protect cultured rat cortical cells from glutamate‐induced neurodegeneration

Certain natural products and Asian herbal remedies have been used in Asia to attenuate neurodegenerative diseases, including senile dementia. We have examined derivatives of several natural products for potential neuroprotective activity in an in vitro test system. In the present study, we assayed a number of compounds that were isolated from Panax ginseng C.A. Meyer (Araliaceae) for an ability to protect rat cortical cell cultures from the deleterious effects of the neurotoxicant, glutamate. We found that ginsenosides Rb1 and Rg3 significantly attenuated glutamate‐induced neurotoxicity. Brief exposure of cultures to excess glutamate caused extensive neuronal death. Glutamate‐induced neuronal cell damage was reduced significantly by pretreatment with Rb1 and Rg3. Ginsenosides Rb1 and Rg3 inhibited the overproduction of nitric oxide, which routinely follows glutamate neurotoxicity, and preserved the level of superoxide dismutase in glutamate‐treated cells. Furthermore, in cultures treated with glutamate, these ginsenosides inhibited the formation of malondialdehyde, a compound that is produced during lipid peroxidation, and diminished the influx of calcium. These results show that ginsenosides Rb1 and Rg3 exerted significant neuroprotective effects on cultured cortical cells. Therefore, these compounds may be efficacious in protecting neurons from oxidative damage that is produced by exposure to excess glutamate. J. Neurosci. Res. 53:426–432, 1998.

Minocycline Alleviates Death of Oligodendrocytes by Inhibiting Pro-Nerve Growth Factor Production in Microglia after Spinal Cord Injury

Spinal cord injury (SCI) causes a permanent neurological disability, and no satisfactory treatment is currently available. After SCI, pro-nerve growth factor (proNGF) is known to play a pivotal role in apoptosis of oligodendrocytes, but the cell types producing proNGF and the signaling pathways involved in proNGF production are primarily unknown. Here, we show that minocycline improves functional recovery after SCI in part by reducing apoptosis of oligodendrocytes via inhibition of proNGF production in microglia. After SCI, the stress-responsive p38 mitogen-activated protein kinase (p38MAPK) was activated only in microglia, and proNGF was produced by microglia via the p38MAPK-mediated pathway. Minocycline treatment significantly reduced proNGF production in microglia in vitro and in vivo by inhibition of the phosphorylation of p38MAPK. Furthermore, minocycline treatment inhibited p75 neurotrophin receptor expression and RhoA activation after injury. Finally, minocycline treatment inhibited oligodendrocyte death and improved functional recovery after SCI. These results suggest that minocycline may represent a potential therapeutic agent for acute SCI in humans.

Role of Tumor Necrosis Factor-α in Neuronal and Glial Apoptosis after Spinal Cord Injury

We investigated the role of tumor necrosis factor (TNF)-alpha in the onset of neuronal and glial apoptosis after traumatic spinal cord crush injury in rats. A few TUNEL-positive cells were first observed within and surrounding the lesion area 4 h after injury, with the largest number observed 24-48 h after injury. Double-labeling of cells using cell type-specific markers revealed that TUNEL-positive cells were either neurons or oligodendrocytes. One hour after injury, an intense immunoreactivity to TNF-alpha was observed in neurons and glial cells in the lesion area, but also seen in cells several mm from the lesion site rostrally and caudally. The level of nitric oxide (NO) also significantly increased in the spinal cord 4 h after injury. The injection of a neutralizing antibody against TNF-alpha into the lesion site several min after injury significantly reduced both the level of NO observed 4 h thereafter as well as the number of apoptotic cells observed 24 h after spinal cord trauma. An inhibitor of nitric oxide synthase (NOS), N(G)-monomethyl-l-arginine acetate (l-NMMA), also reduced the number of apoptotic cells. This reduction of apoptotic cells was associated with a decrease in DNA laddering on agarose gel electrophoresis. These results suggest that: (i) TNF-alpha may function as an external signal initiating apoptosis in neurons and oligodendrocytes after spinal cord injury; and (ii) TNF-alpha-initiated apoptosis may be mediated in part by NO as produced by a NOS expressed in response to TNF-alpha.

Cleavage of Bax is mediated by caspase‐dependent or ‐independent calpain activation in dopaminergic neuronal cells: protective role of Bcl‐2

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress‐specific protease activation pathway exists, and to what extent Bcl‐2 plays a role in preventing drug‐induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase‐dependent apoptosis while a dopaminergic neurotoxin, MPP+ largely induced caspase‐independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS‐ and MPP+‐induced cell death, Bax was cleaved into an 18‐kDa fragment. This 18‐kDa fragment appeared only in the mitochondria‐enriched heavy membrane fraction of STS‐treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP+‐treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [35S]methionine‐labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP+‐ and STS‐induced Bax cleavage. Intriguingly, overexpression of baculovirus‐derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS‐ but not MPP+‐induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP+‐induced but not from STS‐induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl‐2 prevented both STS‐ and MPP+‐induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl‐2 may play a protective role by primarily blocking drug‐induced caspase or calpain activity in dopaminergic neuronal cells.

Systemic administration of 17β-estradiol reduces apoptotic cell death and improves functional recovery following traumatic spinal cord injury in rats

Recent evidence indicates that estrogen exerts neuroprotective effects in both brain injury and neurodegenerative diseases. We examined the protective effect of estrogen on functional recovery after spinal cord injury (SCI) in rats. 17β-estradiol (3, 100, or 300 μg/kg) was administered intravenously 1-2 h prior to injury (pre-treatment), and animals were then subjected to a mild, weight-drop spinal cord contusion injury. Estradiol treatment significantly improved hind limb motor function as determined by the Basso-Beattie-Bresnahan (BBB) locomotor open field behavioral rating test. Fifteen to 30 days after SCI, BBB scores were significantly higher in estradiol-treated (100 μg/kg) rats when compared to vehicle-treated rats. Morphological analysis showed that lesion sizes increased progressively in either vehicle-treated or 17β-estradiol-treated spinal cords. However, in response to treatment with 17β-estradiol, the lesion size was significantly reduced 18-28 days after SCI when compared to vehicle-treated ...

Minocycline inhibits apoptotic cell death via attenuation of TNF‐α expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co‐cultures

We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory‐mediated neurotoxicity using primary neuron/glia co‐cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule‐associated protein‐2 (MAP‐2) positive cells. Through terminal transferase deoxyuridine triphosphate‐biotin nick end labeling (TUNEL)‐staining and by measuring caspase‐3 activity, we found that LPS‐induced neuronal cell death was mediated by apoptosis. We determined that pre‐treatment with minocycline significantly inhibited LPS‐induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (NG‐monomethyl‐l‐arginine monoacetate (l‐NMMA) and S‐methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co‐cultures. Pre‐treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor‐alpha (TNF‐α) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS‐mediated iNOS induction and NO production was mediated by TNF‐α by using neutralizing antibodies to TNF‐α. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS‐induced production of TNF‐α.

Decursin from Angelica gigas mitigates amnesia induced by scopolamine in mice.

We previously reported that a total methanolic extract of the underground part of Angelica gigas Nakai (Umbelliferae) (here-in-after abbreviated AG) significantly inhibited acetylcholinesterase (AChE) activity. We characterized 12 coumarin derivatives including both decursin and decursinol from extracts of AG. In this study, we evaluated the anti-amnestic activity of decursin, a major coumarin constituent isolated from AG, in vivo using ICR mice with amnesia induced by scopolamine (1 mg/kg body weight, s.c.). Decursin, when administered to mice at 1 and 5 mg/kg body weight i.p., significantly ameliorated scopolamine-induced amnesia as measured in both the passive avoidance test and the Morris water maze test. Moreover, decursin significantly inhibited AChE activity by 34% in the hippocampus of treated mice. These results indicate that decursin may exert anti-amnestic activity in vivo through inhibition of AChE activity in the hippocampus.

Increased Production of Tumor Necrosis Factor-α Induces Apoptosis after Traumatic Spinal Cord Injury in Rats

We showed previously that, after spinal cord injury (SCI), tumor necrosis factor-alpha (TNF-alpha) may serve as an external signal, initiating apoptosis in neurons and oligodendrocytes. To further characterize the apoptotic cascade initiated by TNF-alpha after SCI, we examined the expression of TNF-alpha, inducible nitric oxide (NO) synthase (iNOS), and the level of NO after SCI. Western blots and reverse transcription polymerase chain reactions showed an early upregulation of TNF-alpha after injury. A peak TNF-alpha expression was observed within 1 h of injury. By 4 h after injury, the expression of iNOS and the level of NO were markedly increased in the injured spinal cord. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive cells were also first observed in the lesioned area 4 h after SCI. The largest number of TUNEL-positive cells was observed between 24-48 h after SCI. Injecting a neutralizing antibody against TNF-alpha into the lesion site after injury significantly reduced the expression of iNOS, the level of NO and the number of TUNEL-positive cells in the injured spinal cord. Injecting the NOS inhibitors, N(G)-monomethyl-L-arginine monoacetate and S-methylisothiourea sulfate, or an NO scavenger, carboxy-PTIO, into the lesion site also significantly reduced the level of NO and the degree of DNA laddering in the injured spinal cord. These data suggest that after SCI, apoptosis induced by TNF-alpha may be mediated in part by NO via upregulation of iNOS, induced in response to TNF-alpha.

Oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of Parkinson disease

The aim of this study was to investigate changes in protein profiles during the early phase of dopaminergic neuronal death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were identified whose expression was significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). In particular, we detected oxidative modification of thioredoxin-dependent peroxidases (peroxiredoxins; PRX) in treated MN9D cells. Oxidative modification of PRX induced by 6-OHDA was blocked in the presence of N-acetylcysteine, suggesting that reactive oxygen species (ROS) generated by 6-OHDA induce oxidation of PRX. These findings were confirmed in primary cultures of mesencephalic neurons and in rat brain injected stereotaxically. Overexpression of PRX1 in MN9D cells (MN9D/PRX1) exerted neuroprotective effects against death induced by 6-OHDA through scavenging of ROS. Consequently, generation of both superoxide anion and hydrogen peroxide following 6-OHDA treatment was decreased in MN9D/PRX1. Furthermore, overexpression of PRX1 protected cells against 6-OHDA-induced activation of p38 MAPK and subsequent activation of caspase-3. In contrast, 6-OHDA-induced apoptotic death signals were enhanced by RNA interference-targeted reduction of PRX1 in MN9D cells. Taken together, our data suggest that the redox state of PRX may be intimately involved in 6-OHDA-induced dopaminergic neuronal cell death and also provide a molecular mechanism by which PRX1 exerts a protective role in experimental models of Parkinson disease.