The effect of ethanol on glial cell functions has been recently identified by the Division of Neuroscience and Behavior of the National Institute of Alcohol Abuse and Alcoholism as a priority scientific area. This proposal focuses on the role of astrocytes as mediators of the teratogenic effect of ethanol on the central nervous system. Astrocytes are able to modulate neuritogenesis and synaptogenesis by secreting neuroactive proteins and peptides and by regulating the composition of the extracellular matrix. What controls astrocytes secretion is still unknown. We hypothesize that muscarinic receptor stimulation in astrocytes by neuron- derived acetylcholine shifts astrocyte secretion toward a permissive environment for neuronal differentiation. We have indeed observed that muscarinic receptor stimulation in astrocytes induces neuritogenesis and synaptogenesis in hippocampal neurons co-cultured with astrocytes or exposed to astrocyte conditioned medium. We also found that ethanol inhibits this effect, thus revealing a potentially new and important mechanism contributing to the deleterious effects of alcohol on fetal brain. The overarching hypothesis of this proposal is that the inhibition by ethanol of muscarinic signaling in astrocytes, which has been characterized during the previous funding period, would hamper their ability to stimulate neuronal differentiation, by affecting astrocyte secretion.
The specific aims of this proposal are: 1: To characterize the intracellular mechanisms involved in carbachol-treated astrocyte promotion of neuritogenesis and synaptogenesis in hippocampal neurons by means of biochemical, immunocytochemical and morphological techniques, and to assess the inhibitory effect of ethanol on this action. 2: To investigate factors released by carbachol- and/or- ethanol-treated astrocytes using a quantitative proteomic approach. 3: To investigate the effect of carbachol and ethanol on the transcription and release by astrocytes of fibronectin, laminin, plasminogen activator inhibitor 1, thrombospondin 1, neurocan and brevican;these factors are released by astrocytes, and have been shown, in preliminary experiments, to be affected by carbachol or ethanol. In addition, the role of all these factors on hippocampal neuron differentiation will be investigated by silencing their expression or by selectively removing them from astrocyte-conditioned medium. 4: To substantiate our observations of astrocyte-neuron interactions in isolated cell preparations with experiments carried out in a more complex system, the hippocampal slice. Altogether, these studies are aimed at elucidating novel aspects of the communication between astrocytes and neurons and at characterizing new mechanisms involved in the developmental neurotoxicity of ethanol.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Research Project (R01)
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Neurotoxicology and Alcohol Study Section (NAL)
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Regunathan, Soundar
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University of Washington
Public Health & Prev Medicine
Schools of Public Health
United States
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Roqué, Pamela J; Guizzetti, Marina; Costa, Lucio G (2014) Synaptic structure quantification in cultured neurons. Curr Protoc Toxicol 60:12.22.1-32
Guizzetti, Marina; Kavanagh, Terrance J; Costa, Lucio G (2011) Measurements of astrocyte proliferation. Methods Mol Biol 758:349-59
Guizzetti, Marina; Moore, Nadia H; VanDeMark, Kathryn L et al. (2011) Muscarinic receptor-activated signal transduction pathways involved in the neuritogenic effect of astrocytes in hippocampal neurons. Eur J Pharmacol 659:102-7
Giordano, Gennaro; Guizzetti, Marina; Dao, Khoi et al. (2011) Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: Role of astrocytes and extracellular matrix proteins. Biochem Pharmacol 82:1792-9
Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro et al. (2010) Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors. Glia 58:1395-406
Moore, Nadia H; Costa, Lucio G; Shaffer, Scott A et al. (2009) Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation. J Neurochem 108:891-908
VanDeMark, Kathryn L; Guizzetti, Marina; Giordano, Gennaro et al. (2009) The activation of M1 muscarinic receptor signaling induces neuronal differentiation in pyramidal hippocampal neurons. J Pharmacol Exp Ther 329:532-42
VanDemark, Kathryn L; Guizzetti, Marina; Giordano, Gennaro et al. (2009) Ethanol inhibits muscarinic receptor-induced axonal growth in rat hippocampal neurons. Alcohol Clin Exp Res 33:1945-55
Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro et al. (2008) Modulation of neuritogenesis by astrocyte muscarinic receptors. J Biol Chem 283:31884-97
Guizzetti, Marina; Chen, Jing; Oram, John F et al. (2007) Ethanol induces cholesterol efflux and up-regulates ATP-binding cassette cholesterol transporters in fetal astrocytes. J Biol Chem 282:18740-9

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