Symposium 8, Sunday Sept. 23rd 4 pm–5.30 pm; Room: Lecture Hall 4
Intracellular actions of ethanol: Chairpersons: Rommelspacher H (Germany), Bakalkin G (Sweden)
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Presentation S8-1
Molecular adaptations counteract alcohol neurotoxicity in the prefrontal cortex of human chronic alcoholics: biochemical analysis
Bakalkin G, Johansson S, Marinova Z, Oekvist A, Sheedy D, Garrick T, Harper C, Ekstroem TJ, Kuzmin A, Yakovleva T (Sweden)
Aims. Behavioral, imaging and neuropathological studies suggest that neurons and glial cells are abnormal and that their density is decreased in the brain of human chronic alcoholics. To identify molecular mechanisms underlying these adaptations, we examined expression of several genes essential for execution of cell death and for cell protection, at the mRNA and protein levels. We also related these levels to content of neuronal and glial markers.
Methods. Proteins and mRNAs of cell suicide (including Bax and active caspase 3) and protection (including Bcl-2 and Bcl-XL) machineries and protein markers of neurons (class III β-tubulin and 
-enolase) and glia (GFAP) were analyzed by quantitative immunoblotting or quantitative RT-PCR in the prefrontal (PFC) and motor (MC) cortices of 14 chronic human alcoholics and 14 control subjects. The PFC was chosen because it is impaired in alcoholics and MC was studied for comparison.
Results. In the PFC of human alcoholics, we observed up- and downregulation of key components of cell protection and suicide machineries, respectively, but no alterations in levels neuronal and glial biochemical markers. No molecular changes were evident in the MC, which was characterized by different homeostatic levels of expression of anti- and proapoptotic genes.
Conclusions. Molecular analysis suggests that vulnerability of the PFC to chronic alcohol consumption is associated with the activation of cell protective mechanisms along with inhibition of cell death pathways. These allostatic adaptations may counteract alcohol neurotoxicity selectively in the PFC.
Presentation S8-2
Ethanol induces apoptosis by anoikis in astrocytes: Role of the RHOA/ROCCK-1/MLC pathway
Guerri C, Miñambres R, Perez-Arago MA, Talens-Visconti R, Guasch RM (Spain)
Aims. Glial cells are very sensitive to the effects of ethanol during brain development. We have demonstrated that alcohol exposure induces astroglial death and causes reorganization of both the actin cytoskeleton and focal adhesions. These changes mimic some of the features observed during the initial steps of an anoikis process, a programmed cell death induced by loss of anchorage. Since small Rho GTPases have been implicated in apoptosis, we asked whether ethanol induces an apoptosis by anoikis in astrocytes and whether the RhoA signalling pathway participates in the morphological changes associated with anoikis.
Methods. Astrocytes in primary culture were treated with 100 mM ethanol for 3, 6, 14 or 24 hours. Apoptosis was quantified by the determinations of sub-G0/G1 population, caspase-3 activity and annexin-V/7-AAD. Immunofluorescence was used to evaluate the actin-myosin changes. Transfection, immunoprecipitation, western blotting and kinase assay were also used to evaluate RhoA, ROCCK-1 and MLC.
Results. We demonstrated that astrocytes exposed to ethanol undergo morphological changes associated with anoikis, including the peripheral reorganization of focal adhesions and actin-myosin system, cell contraction, membrane blebbing and chromatin condensation. We found that either the small GTPase RhoA or its effector ROCK-I (Rho kinase), promotes membrane blebbing in astrocytes. Ethanol induces a ROCK-I activation that is mediated by RhoA, rather than by caspase-3 cleavage. Accordingly, the RhoA inhibitor C3, completely abolishes the ethanol-induced ROCK-I activation. Inhibition of both RhoA and ROCK prevents the membrane blebbing induced by ethanol. Ethanol also promotes myosin light chain (MLC) phosphorylation, which might be involved in the actin-myosin contraction. Changes in actin reorganization associated with anoikis were not observed in neurons. These results strongly support that ethanol-exposed astrocytes undergo apoptosis by anoikis and also that the RhoA/ROCK-I/MLC pathway participates in this process.
Conclusions. These findings provide the first evidence that ethanol can cause cell death by anoikis and suggest that alterations in actin cytoskeleton organization may underlie the ethanol-induced astroglial death during brain development. (Supported by SAF2003-06217, SAF2006- 02178, FIS PI105/1205, Red RTA).
Presentation S8-3
Ethanol alterations of transcription factors regulate neuronal vitality
Crews FT, Zou J (USA)
Aims. Glutamate-NMDA receptor excitotoxicity and oxidative stress are two common mechanisms associated with most neurodegenerative diseases. We hypothesize that the vital state of neurons is regulated in part by two key transcription factors, CREB and NF-
B. Ethanol is hypothesized to increase sensitivity to insults by shifting transcription away from CREB, towards the proinflammatory NF-B transcription factor.
Methods. To test this hypothesis we used hippocampal-entorhinal cortex slice cultures and in-vivo binge treatment of rats. EMSA was used to assess transcription factor DNA binding. Neurotoxicity was assessed using propidium iodide and histochemistry, ELISA and RTPCR for protein and mRNA respectively.
Results. Glutamate and oxidative stress neurotoxicity, using H2O2, are both associated with a decrease in CREB DNA binding and an increase in NF-B DNA binding. Ethanol decreased CREB and increased NF-B DNA binding and increased neurotoxicity to both glutamate and H2O2. Agents reverse ethanol effects on CREB and NF-B DNA binding activity alter ethanol enhanced neurotoxicity. Rolipram, a phosphodiesterase IV inhibitor, increased CREB DNA binding activity and decreased toxicity, whereas TNF 
, increased NF-B DNA binding activity and increased neurotoxicity to both glutamate and H2O2. BHT, blocked ethanol-TNF , increased NF-B DNA binding activity and reversed the increase in neurotoxicity. BDNF expression follows sensitivity to toxicity suggesting it is one factor that contributes to neuronal vitality secondary to the balance of CREB-NF-B activated transcription.
Conclusions. These studies suggest that ethanol alters CREB and NF-B DNA transcription in a manner that sensitizes neurons to toxic insults.
Presentation S8-4
Effects of chronic ethanol exposure on differentiating SH-SY5Y human neuroblastome cells
Hellmann J, Wernicke C, Rommelspacher H (Germany)
Aims. Long-term effects of ethanol on human SH-SY5Y neuroblastoma cells were analyzed, focusing on proliferation, differentiation and the responsiveness of the ERK cascade to brain derived nerve growth factor (BDNF).
Methods. The cells were differentiated with retinoic acid (RA) to a functional and morphological neuronal phenotype with largely suppressed proliferation, where two main stages of differentiation were observed. Initially, a diffuse neuritic network was formed by scattered cells, while after 2 weeks, migration of the cells led to the formation of cell clusters connected by large bundles of parallel neurites. Cells were exposed to RA and 100 mM ethanol for up to 4 weeks.
Results. Despite the presence of RA, ethanol exposure increased proliferation, inhibited cell cluster formation and impaired the expression of both SNAP-25 synaptic protein and Raf-1 kinase inhibitor protein (RKIP), both of which are known to be associated with differentiation. Another crucial step in neuronal differentiation, BDNF signaling, was found to be impaired at the level of ERK activity. To elucidate the mechanisms possibly involved in this impairment, several steps in the signal transduction cascade were investigated using quantitative RT-PCR and Western blot analysis. qRT-PCR revealed ethanol-related changes in the Trk-B receptor, but also in Raf-1, MEK and especially RKIP. Furthermore, the protein kinase C (), which is known to mediate some effects of RKIP on ERK signaling, was impaired by ethanol. Finally, several transcripts were also affected by ethanol withdrawal.
Conclusions. The findings suggest several mechanisms for the impairment of neuronal differentiation and the organisation of neuronal networks by chronic ethanol exposure.