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Principal Investigator: Peter J. Syapin
Co-Investigator: Alma C. Sanchez
Glia, also called neuroglia, are dynamic CNS cells, responding to the environment along side neurons. Both astrocytes and neurons need to function properly for normal behavior (Frisch et al., 2003), and data indicates astrocytes can modulate rewarding effects of drugs (Narita et al., 2006). Thus, there is no a priori reason to believe that glial cells are not important targets for drugs that alter behavior, including alcohol. Far too little is understood about acute and chronic effects of alcohol on glial cells. This is unfortunate since they serve important function such as sensors of local and global activity and regulators of host defense signaling mechanisms and neuroinflammation. Astrocytes also modulate neuronal survival and synaptic plasticity. The long‐term objective of this research is to investigate ethanol effects on signal generation and transduction by human astrocytes. There are several reasons for this focus. First and foremost is the paucity of mechanistic data regarding effects of ethanol on human astrocytes (see Background). Second, astrocytes are a cellular phenotype where speciation has produced significant changes in cellular interactions and functional capacity (Oberheim et al., 2006; Oberheim et al., 2009, see Background also), thus they may respond differently to alcohol compared to their rodent counterparts. Third, pure preparations of human astrocytes are now readily available, allowing sufficient material for in‐depth investigations. Finally, human astrocyte cultures are genetically‐unique since they are derived from individual donors. This allows gender and genotypic differences to be explored and analyzed at a molecular and cellular level. We have data to suggest that alcohol alters key signal generation and transduction mechanisms in human astrocytes, which can have far reaching consequences. Therefore, we hypothesize that alcohol exposure disrupts intracellular mechanisms in human astrocytes driving in part a new homeostasis during alcohol intoxication that contributes to reduced neuroprotection via modified host defense and other responses. This R01 application will facilitate a better mechanistic understanding of alcohol‐induced changes in human astrocytes through three primary aims:
Aim 1 will test the hypothesis that alcohol interacts with transmitter and cytokine signaling to change the activity of the NF-kB family of transcription factors. Studies on RELA (p65) observed enhanced nuclear location in human astroglia with ethanol exposure (Davis and Syapin, 2004c). This aim addresses how and why this occurs. The approach is to treat primary human astrocytes with and without ethanol (25 – 150 mM) and cytokines (TNF‐α, IL‐1β, IFNγ and combinations thereof) or transmitters (ATP, noradrenaline, and glutamate or analogs) for up to 6 h to examine acute effects. Chronic effects will use astrocytes expose to 25 – 75 mM for up to 7 days prior to cytokine or transmitter stimulation. Effects on protein expression, nuclear translocation, and DNA binding activity of NFKB1 (p50/p105), NFKB2 (p52/p100), REL (c-Rel) and RELA (p65) will be characterized, as will interactions with NF‐κB activator (NKAP) and inhibitor (BCL-3) proteins. These studies will provide a more complete picture of ethanol effects on NF-kB signaling.
Aim 2 will test the hypothesis that alcohol alters astrocyte host defense systems and will investigate the underlying molecular mechanisms. Generation of reactive nitrogen and oxygen species is a central mechanism of host defense. Ethanol alters reactive nitrogen generation by effects on inducible nitric‐oxide synthase (iNOS) expression in human astrocytes (Davis et al., 2002; Davis and Syapin, 2004a, see also Preliminary Studies). This aim addresses how expression is changed. To do so, we will identify regulatory cis‐acting elements of the NOS2A promoter and their trans‐acting binding partners that mediate acute (25 – 150 mM) and chronic (25 – 75 mM) ethanol effects. Effects on iNOS mRNA stability will be considered also, as will potential regulation by the antioxidant protein hemeoxygenase‐1 (HO‐1), the latter based on recent data from the PI's laboratory (Parhizgar, 2007). Aim 2 also will characterize ethanol effects on the superoxide-generating NADPH oxidase (NOX) enzyme complex, since not much is known about ethanol effects on reactive oxygen generation by human brain cells, including astrocytes. An effect can be inferred, however, based on its involvement in alcoholic liver disease (De Minicis and Brenner, 2008). The approach is to characterize acute and chronic ethanol effects on superoxide generation by unstimulated human astrocytes and those stimulated by cytokines, transmitters, and PKC activation. To understand NOX expression by human astrocytes and any ethanol effects thereon, the presence and quantity of protein and/or mRNA for NOX1, CYBB (Nox2, gp91phox), NOX3, NOX4, NOX5, DUOX1, and DUOX2 will be characterized, as well as the presence and activation of CYBA (p22phox), NCF1 (p40phox), NCF2 (NOXA2, p67phox), NCF4 (p40phox), and NOXA1, key protein regulators of NOX activity (see Leto and Geiszt, 2006; Nauseef, 2008).
Aim 3 will test the hypothesis that alcohol alters intercellular signaling between human astrocytes and microglia. Cytokines, including chemokines, are soluble proteins that participate in intercellular communication during host defense and at other times. Chemokines may represent a third major signaling system in the brain (Adler et al., 2005). Studies show alcohol changes chemokine production by human astroglia (Davis and Syapin, 2004b) and their levels in alcoholic brain tissue (He and Crews, 2008). This aim addresses potential functional consequences of alcohol on intercellular signaling, and characterizes further those cytokines susceptible to change by alcohol in human astrocytes. The approach is to treat primary human astrocytes with and without ethanol (25 – 150 mM) and cytokines (TNF‐α, IL‐1β, IFNγ and combinations thereof) or transmitters (ATP, noradrenaline, and glutamate or analogs) for up to 24 h to examine acute effects. Chronic effects will use astrocytes expose to 25 – 75 mM for up to 7 days prior to cytokine or transmitter stimulation. Effects on functional signals secreted by astrocytes will be assessed by the migration response of immortalized human CHME-5 microglia to astrocyte conditioned media (see Preliminary Studies). To identify the signals released and their susceptibility to alcohol exposure, conditioned media will be collected from treated and untreated astrocyte cultures for cytokines protein arrays and ELISAs. mRNA will be collected also to characterize those cytokines transcriptionally activated or whose mRNA has been stabilized, using RNase protection assays.Taken together, results from these 3 aims will provide vital information on alcohol effects on important signal generation and transduction systems in human astrocytes and the underlying molecular mechanisms involved.