Glial-derived neurotrophic factor (GDNF) prevents ethanol (EtOH) induced B92 glial cell death by both PI3K/AKT and MEK/ERK signaling pathways
Introduction
Glial cells are essential for the development and maintenance of the nervous system: they guide migrating neurons to their final location [35], [66], and promote both neuronal and their own glial development and survival through the production of growth factors [69]. They also participate in several brain functions such as neurogenesis [20], [23], [74], synapses formation [58], [80], pain [84], and neuroprotection against toxic insults [4] (for a review see [83]). However, glial functions can be affected by early exposure to ethanol and have been proposed as potential targets in the fetal alcohol syndrome [31], [32]. Ethanol exposure during brain development leads to substantial cell loss in multiple regions of the nervous system, such as cerebellar Purkinje and granule cells [34], [62], olfactory mitral and granule cells [10], hippocampal pyramidal cells [7], [62], somatosensory cortical neurons [52], retinal cells [14], as well as the induction of apoptosis in a variety of other cell types including neural crest [12], [68], astroglia [31], [37] and promyelocytes [2]. Clinical and experimental evidence indicates that alcohol exposure in uterus induces structural and functional abnormalities in gliogenesis and glial–neuronal interactions, suggesting a potential role for glial cells in ethanol-induced brain abnormalities [30]. Despite that this mechanism is not clear, a recent study suggests a role for JNK in ethanol-induced toxicity in neural cells [43]. JNK signaling pathway is required for neuronal apoptosis in response to excitotoxic stress [89], and plays a crucial role in the responses stimulated by pro-inflammatory cytokines, environmental stress, and apoptotic agents [13], [19], [42], [52], [71]. Considerable progress has been achieved in identifying the upstream kinases of the JNK cascade. However, the role of JNK in the apoptotic response of glial cells is unclear. Thus, we have focused on JNK pathway and defined the requirement of JNK in ethanol-induced apoptosis.
On the other hand, growth factors play an important role in the control of neural homeostasis. The manipulation of trophic support may constitute a viable strategy for the prevention of ethanol-induced abnormalities. GNDF, a TGF-β related growth factor that is expressed in human and rat glial cell lines [86], has recently gained attention for its many effects upon the nervous system, which range from regulation of cell proliferation and differentiation, to neuroprotection of dopaminergic neurons [9], [70], [76], retinal photoreceptors [63], and protection from neurotoxics agents, including ethanol-induced neuron loss [11], [49], [50]. The neuroprotective mechanisms of trophic factors are still a matter of controversy, and despite the evidence that GDNF targets include glial cells [22]; mechanisms of action of this neurotrophic factor upon glial responses are still unknown. GDNF signals through the RET/GFR alpah1 receptor [39], [78]. It has been observed to induce the activation of PI3K, and ERK pathways in several cell lines [15], [79], [82], [87]. Further evidences also suggests, that it plays a protective role by controlling the activation of AKT [38], [47], and ERK pathways [51], [56]. Of these, PI3K/AKT and ERK are likely candidates for mediating GDNF-induced cell survival. In this study we tested the effect of ethanol, the action of GDNF upon glial cell survival and death, and potential pathways involved.
Section snippets
Cells and culture procedure
The rat glioma cell line B92, established by Schubert et al. [73], was grown in D-MEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gen S.A., Argentina), penicillin 60 U/ml, streptomycin and gentamicyn 50 mg/l (Richet, Argentina) at 37 °C in a humidified chamber with 5% CO2. GDNF was obtained dried-lyophilized from Peprotech (USA). Briefly, cells were plated over four glass coverslips placed in 35 mm dish of Nunc® 6 plastic multiwells, at a density of 30,000 cells/cm2, covered with 1000 μl
Apoptosis in glial cultures. Short time exposure to EtOH and GDNF protective action
B92 glial cells plated at low density is an excellent short term assay to study cell growth. Under these assay conditions, cells expressed typical markers and were positive for GFAP, S100 and vimentine, which are specific for astrocyte lineages. On the other hand, immunocytochemistry (ICQ) was negative for the oligodendrocyte-specific marker A2B5 as well as for neurofilament (NF) 160 and NF 200 neuron markers (data not shown). Ethanol-induced cell death was measured by counting DAPI stained
GDNF protects glial cells
Ethanol effects on the nervous system include significant alterations on glial cells [5], [29], [40]. Though, knowledge on glial response to ethanol-induced cell loss is still scarce. Several studies have focused on the action of trophic factors in preventing injury-induced cell loss in neuronal models [8], [27]. However, growth factor actions in responses to ethanol are still not well understood, despite increasing evidence for their neuroprotective potential in ethanol-induced neurotoxicity
Acknowledgements
This work was supported by Scholarships from CICPBA and CONICET, and grants from CONAPLU and CONICET. Dr. Rafael Linden work's was supported by grants from CNPq, FAPERJ, and Pronex. We thank Beatriz Tosti for her help in the correction of manuscript and Veronica Labarta for cell line preservation at the Cell Culture Bank.
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