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* Systemic Proteomics Research Center and
The center of Functional Analysis of Human Genome, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea;
Department of Biology, Chungnam National University, Daejeon, Korea;
Department of Biological Science, Sung Kyun Kwan University, Suwon, Korea; and
|| Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, Korea
2Correspondence: Jeong Heon Ko, Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon 305–333, Korea. E-mail: jhko{at}kribb.re.kr
SPECIFIC AIMS
Oxidative glutamate toxicity has been implicated in the pathology of neuronal cell death. However, the underlying mechanism responsible for this is not fully understood. Gangliosides have been specifically identified as modulators of cell growth and differentiation in the central nervous system (CNS). This study aimed to examine changes in ganglioside levels during glutamate-induced neuronal cell death and to elucidate the relationships between these changes and neuronal cell death.
PRINCIPAL FINDINGS
1. GM3 is up-regulated in glutamate-induced neuronal cell death
GM3 is the major ganglioside in the mouse hippocampal cell line HT22, as evidenced by HPTLC analysis, and its cellular concentration increased in a time-dependent manner after glutamate treatment (Fig. 1A
). Although inhibitors of massive ROS production and Ca2+ influx, typical features associated with oxidative glutamate toxicity, prevented glutamate-induced cell death, had no measurable effect on GM3 levels (Fig. 1B
), suggesting that an increment in GM3 levels is either an independent event on or upstream from ROS production or Ca2+ influx.
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2. RNA interference-mediated silencing of GM3 synthase rescues glutamate-induced neuronal cell death, preventing 12-LOX activation, massive ROS production, and an increase in intracellular Ca2+ levels
Glutamate was found to induce cell death in mock-transfectant cells (Mock) but not in cells stably expressing siRNA for GM3 synthase (siST3GalV) (Fig. 2
A, B), suggesting that the up-regulation of GM3 is required for glutamate-induced neuronal cell death. The glutamate-induced increase in ROS or Ca2+ was blocked in siST3GalV cells but not in mock cells (Fig. 2C
, 2D
). GSH depletion induced by glutamate causes ROS production via 12-LOX activation in neuronal cell death. 12-LOX activation was significantly blocked in siST3GalV cells compared to mock control cells after glutamate treatment (Fig. 2F
), but intracellular GSH depletion was not affected by a decrease in endogenous GM3 levels (Fig. 2E
). Collectively, these results suggest that GM3 may regulate downstream events such as ROS production and Ca2+ influx, and GSH depletion cooperates with GM3 to activate 12-LOX in the signaling pathway of glutamate-induced cell death.
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3. 12-LOX is activated by being recruited to GEM
A significant subset of the 12-LOX pool was colocalized in glycosphingolipid-enriched microdomains (GEM) in a GM3-dependent manner, and treatment with glutamate led to higher amounts of 12-LOX being tethered in GEM regions. Likewise, a negligible amount of GM3 in GEM failed to tether 12-LOX in the domains. The activation of 12-LOX resulted in an overproduction of ROS and an increased Ca2+ influx.
4. An increase in GM3 levels induces neuronal cell death
Exogenously added GM3, but not other gangliosides such as GD3 or GT1b, induced cell death in a dose-dependent manner in HT22 cells. The endogenous accumulation of GM3 as the result of the overexpression of the gene coding for mouse GM3 synthase (ST3GalV) in HT22 cells, which is responsible for GM3 synthesis, was also sufficient to trigger cell death. To explore the in vivo role of GM3, zebrafish was used as an animal model system. When cell death was examined using an acridine orange staining method, a dramatic change in the number of neuronal cell deaths in embryos was observed, especially in the CNS of embryos that overexpress GM3 synthase. These results strongly suggest that the overexpression of GM3 causes neuronal cell death in the in vivo zebrafish animal model system.
CONCLUSIONS AND SIGNIFICANCE
The findings herein demonstrate that GM3 levels are increased in immortalized mouse hippocampal HT22 cells after exposure to glutamate. Unlike cumulative studies indicating that GD3 induces death in non-neuronal cells, our study concluded that GD3 plays no role in the death of neuronal cells. Altered GM3 levels were instead connected to the neuronal cell death. GD3 contributes to apoptotic cell death by disrupting the mitochondrial transmembrane potential leading to the release of cytochrome c and the activation of caspase 3. In contrast, GM3 mediated the glutamate-induced oxidative death of neuronal cells, in which apoptotic elements such as Bax and caspase-3 activation, mitochondrial membrane depolarization, DNA fragmentation, and cytochrome c release is not observed. The in vitro GM3 effect on neuronal cell death was also observed in a zebrafish in vivo animal system, in which the overexpression of GM3 was stimulated by microinjecting mRNA of GM3 synthase. The increased GM3 concentration led to the death of tissues near the CNS at an early developmental stage, when the most of the tissue consists of neurons and neuronal precursor cells.
GEM is enriched with gangliosides and signaling proteins, such as protein phosphatase, and is considered to act as "glycosignaling domain", either by recruiting molecules involved in signaling pathways, allowing their molecular interaction, or by modulating signaling functions. However, relationships between gangliosides and signaling proteins in GEM have not been intensively investigated. Although the activation of 12-LOX is thought to include the translocation of soluble enzymes to the nuclear or plasma membranes, little information on the cellular distribution of 12-LOX is available. Our functional study revealed that a subset of the 12-LOX pool was colocalized with GM3 in GEM and the concentration of 12-LOX in GEM was dependent on that of GM3. The GM3-governed translocation of 12-LOX to GEM was responsible for an overproduction of 12-HETE, which in turn led to the overproduction of ROS and an increase in Ca2+ influx. GSH depletion, a common feature of glutamate-induced oxidative toxicity, means a loss of defensive power against oxidative stresses and also a trigger of 12-LOX activation, peroxide production, and Ca2+ influx. GSH depletion appears to involve a pathway that is independent from an increase in GM3 and to cooperate with the increased GM3 to activate 12-LOX and to stimulate ensuing oxytosis during glutamate-induced cell death. The proposed mechanism underlying HT22 neuronal cell death by oxidative glutamate toxicity is given in Fig. 3
.
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In this report, we provide the first evidence to demonstrate that ganglioside GM3 is directly connected to the neuronal cell death by stimulating machinery in the oxidative pathway. The activation of 12-LOX was closely associated with an increase in GM3 levels in GEM. GM3 not only mediates the effect of glutamate on the oxidative death of HT22 cells but also acts, in itself, as a modulator of in vivo neuronal cell death. We propose that a method for controlling GM3 levels could be a potent therapeutic approach in a variety of neurodegenerative diseases.
FOOTNOTES
1 These authors contributed equally to this work. 
3 These authors share senior authorship.
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4911fje
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