Elsevier

Developmental Brain Research

Volume 119, Issue 2, 7 February 2000, Pages 209-216
Developmental Brain Research

Research report
Glial-derived neurotrophic factor (GDNF) prevents ethanol-induced apoptosis and JUN kinase phosphorylation

https://doi.org/10.1016/S0165-3806(99)00171-6Get rights and content

Abstract

Ethanol exposure during neural development leads to substantial neuronal loss in multiple brain regions. Our previous research indicated that exogenous glial-derived neurotrophic factor (GDNF) attenuated ethanol-induced cerebellar Purkinje cell loss. Additionally, ethanol decreased GDNF release suggesting that ethanol disrupts GDNF-signaling pathways. The present experiments utilized a homogeneous GDNF-responsive neuroblastoma cell line (SK-N-SH) to test the hypothesis that exogenous GDNF could attenuate ethanol-induced cell loss by suppressing cytotoxic signaling pathways and cell suicide. We measured two independently regulated markers of apoptosis, DNA fragmentation and the externalization of phosphatidylserine to the outer cell membrane leaflet. Ethanol induced a dose-related increase in both apoptosis and necrosis. Lower concentrations of ethanol (34 and 68 mM) specifically increased DNA fragmentation, while all concentrations (up to 137 mM) increased phosphatidylserine translocation, suggesting that ethanol induction of apoptosis is not a unitary process. Furthermore, only higher concentrations of ethanol (103 and 137 mM) induced necrosis. Additionally, ethanol specifically induced phosphorylation of c-jun N-terminal-kinase (JNK), a mitogen-activated protein (MAP) kinase selectively associated with apoptosis. In contrast, ethanol did not alter the phosphorylation of another MAP kinase, the extracellular signal-regulated kinases (ERK) that mediate cell survival. Thus, ethanol activated specific intracellular cell death-associated pathways and induced cell death. GDNF, in turn, prevented both ethanol-induced apoptosis and the activation of the death-associated JNK cascade. Therefore, GDNF may regulate multiple pathways to prevent ethanol-induced cell loss.

Introduction

Ethanol consumption during fetal development can lead to a constellation of behavioral and anatomical defects collectively termed fetal alcohol syndrome or FAS 32, 33. FAS is characterized by a variety of deficits including craniofacial malformations, pre- and post-natal growth retardation as well as a variety of nervous system deficits including cerebellar ataxia and gait abnormalities 32, 33, 58, 59, 61. Although it has been 25 years since FAS was first characterized, the mechanisms of ethanol-induced brain damage in the developing nervous system are not well understood.

Substantial evidence from several laboratories indicates that ethanol exposure results in a loss of a variety of cell types including cerebellar Purkinje and granule cells 21, 53, olfactory mitral and granule cells [6], hippocampal pyramidal cells 4, 7, 53, 65, somatosensory cortical neurons [45], and cells in the retina [12]. Such cell loss may be the result of alterations in the cell cycle [44], abnormal cell migration patterns [43], phenotype alteration 34, 60, or cell death. Recent studies lend support to the hypothesis that ethanol exposure during development can result in cell death. For example, ethanol has been shown to induce apoptosis in cerebellar granule cells by inhibiting the trophic effect of NMDA or IGF-1 5, 52, 71. Ethanol has also been shown to induce apoptosis in a variety of other cell types including neural crest cells [9], astroglia [29]and promyelocytes [2].

Manipulation of trophic support may be one mechanism by which ethanol-induced cell death may be prevented. Previous studies have focused on the role of trophic factors, including the NGF and βFGF, in preventing ethanol-induced cell loss in culture models of the cerebellum, hippocampus, septum, dorsal root ganglia neurons or PC12 cells 3, 24, 25, 26, 39, 51. Our previous studies indicate that glial-derived neurotrophic factor (GDNF), a member of the TGF-β superfamily [38], protects neurons against ethanol-induced cell loss [42]. Furthermore, ethanol decreases GDNF release from cerebellar explant cultures [41]. These results suggest that ethanol may interfere with GDNF-activated trophic and survival mechanisms. Exogenous GDNF may, in turn, attenuate the mechanism(s) of ethanol-induced associated death signals.

In these experiments, we utilized a homogenous, GDNF-responsive human neuroblastoma cell line (SK-N-SH) [67]as a model for ethanol-induced cell death and to determine whether GDNF could prevent ethanol-induced apoptosis. We also examined the effect of ethanol on basal and GDNF-dependent phosphorylation of two divergent mitogen-activated protein (MAP) kinase signal cascades, the extracellular signal-regulated kinases (ERK) and c-jun N-terminal kinase (JNK) pathways. ERK activation has been primarily associated with cell survival [20]. In contrast, the JNK pathway has been associated with the induction of cell suicide 1, 22, 27, 37, 68, though JNK activation has other functions as well 23, 27, 30, 54, 57, 69.

Section snippets

Cell culture

SK-N-SH cells were obtained from ATCC and cultured in 24 well microtiter plates in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cultures were incubated at 37°C with 5% CO2. Cells were administered the appropriate ethanol treatment after reaching approximately 80% confluence.

DNA fragmentation

Exposure to the lower two concentrations of ethanol (34 and 68 mM) for 24 h significantly increased DNA fragmentation by 82% and 156%, respectively over control cultures. Changes in DNA fragmentation were not observed in cultures that were exposed to higher concentrations of ethanol. Concurrent administration of GDNF (5 and 75 ng; concentrations which can attenuate ethanol-induced cell loss [42]) significantly attenuated alcohol-induced DNA fragmentation. However, GDNF alone did not affect

Discussion

The results of our study indicate that ethanol induces apoptosis in neuroblastoma cells, and are consistent with previous reports in the literature 2, 5, 9, 29, 71. However, the mechanisms by which ethanol induces apoptosis are poorly understood. Some evidence indicates that ethanol can increase TNF-α 50, 56as well as Fas-Apo-1 expression (Z.F. Cheema, R.C. Miranda, unpublished observations). JNK, a MAP kinase associated with apoptosis and p38 66, 70is activated by receptor-mediated cell death

Acknowledgements

This study was funded in part by grants from NIH (AA05523 to J.R.W. and MH55724 to R.C.M). We thank Drs. Doug Dohrman, Gerald Frye and Wei-Jung Chen (Texas A&M University Health Science Center) for their critical review of this manuscript and Faisal Cheema and Jane Miller for technical assistance.

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