Discovery of a neuroprotective mechanism, mediated by nitric oxide (NO), which mitigates alcohol-induced neuronal death. Damage to the nervous system is one of the most serious consequences of fetal alcohol syndrome (FAS). Animal studies have shown that alcohol exposure during development of the nervous system induces neuronal death in the cerebellum, hippocampus, and cortex. The neuronal losses, which are induced by alcohol, are temporally-dependent, and alcohol causes the greatest neuronal loss during a specific developmental time period, the “window of alcohol vulnerability”. After these brain regions have matured beyond this temporal window, alcohol-induced neuronal loss is much less severe. The molecular events responsible for this window are unclear. We hypothesize that a developmentally-regulated neuroprotective mechanism against alcohol toxicity may explain the differential toxicity of alcohol over time. During the window of alcohol vulnerability, this neuroprotective mechanism may not be functional, and the developing nervous system is highly vulnerable to alcohol-induced neuronal death. With maturation, a neuroprotective mechanism may become functional, thereby reducing alcohol vulnerability. Our research goals are to determine whether a neuroprotective mechanism exists, to identify its molecular components, and to determine whether temporal regulation of this neuroprotective mechanism offers an explanation for the window of alcohol vulnerability. Utilizing primary cultures of cerebellar granule neurons, we identified a neuroprotective-signaling pathway, which ameliorated alcohol-induced neuronal death in these cultures. This neuroprotective pathway (See diagram above) required the synthesis of nitric oxide (NO) as well as the downstream signaling events initiated by NO, such as activation of guanylyl cyclase, thereby increasing cGMP levels, and activation of cGMP-dependent protein kinase (PKG). Because in vitro studies suggested a key role for NO in neuroprotection, our in vivo studies utilized a mutant mouse strain, which lacked neuronal nitric oxide synthase (nNOS), the synthetic enzyme for NO in neurons. With this animal model, we discovered that NO mediates neuroprotective effects against alcohol toxicity in the cerebellum, hippocampus, and cortex. We are investigating the hypothesis that during the window of alcohol vulnerability, the immature nervous system lacks adequate NO-mediated neuroprotection and is vulnerable to alcohol neurotoxicity. With further development, NO-mediated neuroprotection may become functional thereby preventing the damaging effects of alcohol. Our studies have yielded several key discoveries. We have discovered a previously unknown neuroprotective mechanism against alcohol toxicity, which is mediated by NO, and functions both in vivo and in vitro. The lack of NO-mediated neuroprotection may be responsible for the high alcohol vulnerability of the immature brain during the window of alcohol vulnerability, and acquisition of functional NO-mediated neuroprotection may be responsible for the alcohol resistance of the mature nervous system. Our studies raise the exciting possibility that NO-mediated neuroprotection may be utilized in a gene therapy approach to counteract alcohol damage to the developing nervous system. We have found that neurons, which lack nNOS, are highly vulnerable to alcohol-induced death. However, introduction of the nNOS gene into these neurons reverses this alcohol vulnerability, and neurons are now alcohol resistant. Our exciting results suggest that neuroprotective genes protect neurons against alcohol toxicity and may foster a gene therapy approach to prevent alcohol damage to the developing nervous system.

Our research is the collaborative efforts of the following investigators: Nicholas J. Pantazis, Professor, Anatomy and Cell Biology; Daniel J. Bonthius, Associate Professor, Pediatrics; Bahri Karacay, Research Scientist, Pediatrics; Guiying Li, Postdoctoral Associate, Anatomy and Cell Biology; Ana Hutton-Kehrberg, Graduate Student, Neuroscience program; Ross McKim, Graduate Student, Neuroscience program; Jolonda Mahoney, Research Assistant, Pediatrics