Alcohol use disorders (AUD) occur commonly in Veterans and produce serious morbidity and mortality. Alcohol damages the nervous system across the lifespan, leading to crippling neurological disorders in adults and fetal alcohol spectrum disorders in children with gestational exposure to alcohol. Both stages of life are relevant to Veterans, since returning Veterans comprise a growing proportion of women of childbearing age with AUDs. This proposal extends ongoing work directed at identifying medications that will prevent or reverse the toxic effects of alcohol on the nervous system at all stages of life. Alcohol causes developmental toxicity in the brain partly by blocking cell adhesion mediated by the L1 neural cell adhesion molecule. Alcohol blocks diverse actions of L1 that follow L1 adhesion, including L1-mediated axon outgrowth (L1MAO) and L1 activation of Src Family Kinase (SFK) signaling. Alcohol also blocks signaling through SFK for two other important growth factors: netrin-1 and GDNF. Alcohol interacts with a binding pocket on the extracellular domain of L1 (L1-ECD) - the portion of the molecule that protrudes from the cell membrane and binds to L1 molecules on other cells. Phosphorylation of specific residues in the L1 cytoplasmic domain (L1-CD) does not block L1 adhesion, but abolishes ethanol inhibition of L1 adhesion, presumably by altering the conformation of the alcohol binding pocket in the L1-ECD. Importantly, certain small molecules and peptides also block alcohol inhibition of L1 adhesion, and these alcohol antagonists prevent alcohol-induced damage to the developing brain. The small peptide NAPVSIPQ (NAP) protects the nervous system against a wide array of insults, including prenatal alcohol exposure. NAP blocks the effects of alcohol on L1 adhesion, L1MAO, and L1 signaling at femtomolar concentrations (as low as one part per quadrillion). The mechanism by which NAP acts so potently is unknown. Here we will investigate how NAP blocks the effects of alcohol in cultures of cerebellar granule neurons (CGN) and slices of cerebellar cortex from postnatal, adolescent, and adult rats [and in an in vivo model of early postnatal alcohol exposure.] There are three objectives: 1. to determine whether NAP blocks ethanol inhibition of L1 adhesion by modulating phosphorylation of the cytoplasmic domain; 2. to learn whether NAP blocks ethanol inhibition of SFK activation by L1, netrin-1, and GDNF; 3. to determine whether NAP prevents or reverses alcohol cerebellar neurotoxicity across the lifespan. Cultures will be exposed to alcohol for varying periods of time in the absence and presence of alcohol, L1, netrin-1, and GDNF. We will systematically mutate phosphorylation sites in the L1-CD to simulate permanent phosphorylation or dephosphorylation of these sites. These constructs will then be treated with alcohol and NAP to identify specific mutations that render NAP incapable of blocking the effects of alcohol. We will then test whether NAP modulates the activity of kinases or phosphatases that act at the identified sites. Alcohol inhibits axon outgrowth in CGNs by blocking the activatio of SFK by diverse growth factors. We will determine whether NAP blocks the effects of alcohol on the activation of SFK by L1, netrin-1, and GDNF in mature CGNs and cerebellar slices from postnatal day 7 (P7) rats. We will then determine whether NAP antagonizes the effects of alcohol on neuronal survival, neuronal networks, and growth factor signaling in slices from early postnatal (P7), adolescent (P40), and adult (P80) cerebellum and [in cerebellum from rats and mice exposed to ethanol during the early postnatal period.] These studies will help characterize the mechanism of action of NAP, a potent alcohol antagonist, and will lay the groundwork for the development of treatments to prevent and reverse alcohol neurotoxicity in the brain.
Alcohol use disorders are highly prevalent in the Veteran population and are a major cause of morbidity and mortality. Moreover, Veterans returning from the Iraq and Afghanistan wars have particularly high rates of PTSD, anxiety, depression, and drug and alcohol use disorders. Importantly, an increasing number of returning Veterans with alcohol use disorders are women of childbearing age, whose drinking endangers both themselves and their unborn children. The development of medications to reduce or reverse alcohol toxicity in the brain may decrease the burden of neurological disease in Veterans with alcohol use disorders.
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