Activity-dependent neuroprotective protein-derived peptide, NAP, preventing alcohol-induced apoptosis in fetal brain of C57BL/6 mouse.

Brain growth restriction is a hallmark of fetal alcohol exposure (FAE) and fetal alcohol syndrome (FAS) models (Roebuck et al., 1998). Alcohol exposure impedes cerebrum and cerebellum growth (Bauer-Moffett and Altman, 1975, 1977; Kornguth et al., 1979; Samson and Diaz, 1981; Sulik et al., 1981; Barron et al., 1988; Bonthius and West, 1990; Sari and Gozes, 2006). Our and other studies have demonstrated that the effects of prenatal alcohol exposure depend on the dose and timing of alcohol treatment (Webster et al., 1983; Sari and Gozes, 2006). During the period between embryonic day 7 (E7) and E14, the developing brain exhibited the highest susceptibility to alcohol exposure. Deficits in fetal brain growth might be induced possibly through an apoptotic mechanism (Ikonomidou et al., 2000). Mitochondrial activation of apoptotic pathways is one of the major players in ethanol-mediated neuronal death. Although studies have demonstrated that prenatal alcohol exposure induces mitochondrial dysfunction, little is known about its signaling pathways. The mitochondrion is a target organelle in ethanol-induced organ-system toxicity (Hoek et al., 2002). Prenatal alcohol exposure–induced mitochondrial dysfunction includes decreased mitochondrial glutathione concentration, decreased activities of respiratory chain complex IV and ATP synthase, and increased mitochondrial permeability transition (Ramachandran et al., 2001; Spong et al., 2001; Xu et al., 2005a; Green et al., 2006). We have recently found that prenatal alcohol exposure induced decreases in ATP synthase, ADP/ATP translocase, NADH dehydrogenase, ubiquinol-cytochrome-c reductase and prohibitin at E13 (Sari and Mechref, 2008). Together, these findings indicate that mitochondrial dysfunction is a key factor in alcohol-induced apoptosis. E13 age was chosen as an end-point of alcohol exposure because this developmental age holds several interests in my ongoing research projects in FAE model. Most of the developmental regulatory proteins are highly expressed at this age of development and altered as a consequence of alcohol exposure (Sari and Gozes, 2006; Sari and Mechref, 2008). In addition, at E13, the neural tube has undergone five major divisions to form a fetal brain composed of telencephalon, diencephalon, midbrain, hind-brain and spinal cord. Moreover, at this developmental stage, the 5-HT neurotransmitter system has formed and initiated its differentiation. Our previous study reported that prenatal alcohol exposure induces neural tube defects early at E13 (Zhou et al., 2003) and similarly alters early 5-HT neurons [for review see ref. (Sari and Gozes, 2006)]. Studies investigating alcohol exposure during development in animal models may provide important information for the identification of possible mechanisms of neuroprotection and allow for the development of intervention procedures, which may protect or attenuate the deleterious effects of alcohol exposure during pregnancy. Several studies have investigated the possible prevention of the effects of prenatal alcohol exposure by the treatment of pregnant mice with peptides that have been shown to be involved in neuroprotection (Spong et al., 2001; Poggi et al., 2003; Brenneman et al., 2004; Zhou et al., 2004; Sari and Gozes, 2006). Among these peptides, SALLRSIPA, known as SAL or ADNF-9, is derived from activity-dependent neurotrophic factor (ADNF) (Brenneman and Gozes, 1996; Brenneman et al., 1998). Another peptide with the sequence NAPVSIPQ peptide, termed NAP, derived from activity-dependent neuroprotective protein (ADNP) (Bassan et al., 1999; Zamostiano et al., 2001), demonstrated a potent protective effect against oxidative stress. In the present study, using our established FAE model in C57BL/6, we tested the neuroprotective effects of NAP at early stage of development E13. The ADNP is released from astroglia (Bassan et al., 1999) and found to be regulated by vasoactive intestinal peptide, VIP (Furman et al., 2004). High ADNP mRNA expression was found in the hippocampus, cerebral cortex and cerebellum in the mouse (Bassan et al., 1999) and in the human brain (Zamostiano et al., 2001), indicating a potential role in the central nervous system (Gozes et al., 1999). It has been further shown that knocking out the ADNP gene is lethal to the embryo and the ADNP-deficient embryos die at the time of neural tube closure (Pinhasov et al., 2003). Our and other studies have focused on the role of NAP in neuroprotection against prenatal alcohol exposure [for review see ref. (Sari and Gozes, 2006)]. A recent study demonstrated a significant decrease in the ADNP mRNA in the cerebral cortex of 7-day-old pups that were exposed prenatally to alcohol (Pascual and Guerri, 2007). Using an established model for FAS, it has been demonstrated at National Institutes of Health–National Institute of Child Health and Human Development (NIH-NICHD) that pretreatment with NAP prevented the fetal death and abnormalities induced by prenatal alcohol exposure (Spong et al., 2001). Importantly, the effects of enantiomer conformation (l- and d-forms) of this peptide against the insult of alcohol exposure was also investigated in FAS model (Brenneman et al., 2004). The d-form of NAP resulted in a potent effect with no loss of protective response regardless of increases in the doses. We have demonstrated previously the effects of d-NAP for its protective properties against fetal alcohol–related brain growth restriction in FAE model in C57BL/6 mice (Sari et al., 2001, 2003; Sari and Zhou, 2004; Zhou et al., 2004; Sari and Gozes, 2006). Administration of d-NAP alongside alcohol exposure, from E7 to E15, antagonized the effects of alcohol on brain weight, the size of several forebrain regions, and neural tube development [recently reviewed by us (Sari and Gozes, 2006)]. In the present study, using a similar liquid diet drinking paradigm, we investigated the cellular mechanism of NAP in neuroprotection with more focus on the apoptotic mechanisms that may involve extrinsic and intrinsic mitochondrial signaling pathways at E13.