Ethanol impairs activation of retinoic acid receptors in cerebellar granule cells in a rodent model of fetal alcohol spectrum disorders.

EXPOSURE OF THE developing fetus to ethanol is associated with a pattern of birth defects collectively known as Fetal Alcohol Spectrum Disorder (FASD). The most devastating effects of FASD occur in the central nervous system. In children with FASD, these effects often manifest as mental retardation, hyperactivity, epilepsy, ataxia, and impairment of executive function (Guerri et al., 2009). Epidemiological studies indicate that FASD is a major public health problem in all industrialized countries. Women who consume alcohol during pregnancy place themselves at risk of having a child with FASD. Approximately, 40,000 infants in the United States are born with FASD per year, costing an estimated $6 billion in health, education, and social service expenses (Monsen, 2009). Although the development of all brain areas is affected by ethanol, the cerebellum is particularly targeted (Bauer-Moffett and Altman, 1977). However, the signaling mechanisms and molecular targets by which ethanol exerts its effects on the cerebellum have not been identified. Neurons are particularly sensitive to ethanol-related toxicity during the synaptogenesis phase of brain development. Synaptogenesis is characterized by neurite elaboration, synapse formation, and the onset of neuronal signaling (Olney et al., 2000). In humans, synaptogenesis begins during the third trimester of pregnancy and continues through the first few years of life (Dobbing and Sands, 1979). In rodents, this period corresponds to postnatal days 4 to 9 (P4 to P9). A single exposure to ethanol during this period depletes neurons (West et al., 1990). The third trimester is also an important period for cerebellar development. Prenatal alcohol exposure during the third trimester damages the human cerebellum (Bookstein et al., 2006). Likewise, exposure of rat pups to ethanol during P4–9 reduces cerebellar size (Bauer-Moffett and Altman, 1977). The cerebellum of the newborn rat develops during the postnatal period and presents a unique model for investigating the impacts of ethanol on the developing brain. Although ethanol toxicity affects both cerebellar Purkinje cells and cerebellar granule neurons (CGNs) (Maier and West, 2001), susceptibility to ethanol-associated damage peaks during P4 to P6 in Purkinje cells (Pierce et al., 1999). Alternatively, at P7, granule neurons, which are the predominant cell type in the external germinal granule layer (EGL), remain susceptible to ethanol until they are fully differentiated at later stages of brain development (Altman, 1972a; Oberdoerster and Rabin, 1999). While CGNs begin the process of maturation postnatally, Purkinje cells originate during the prenatal period (embryonic days 13 to 16) and continue differentiation in postnatal periods (Altman, 1972b; Light et al., 2002). Thus, CGNs are typically used to study the effects of ethanol on survival and neuronal differentiation. Previous studies from our laboratory have shown that exposure of rat pups (P7) to ethanol inhibit differentiation and causes excessive death of CGNs. This indicates that CGNs are important targets of ethanol toxicity (Joshi et al., 2006b). Understanding the mechanisms by which ethanol exerts its damaging effects on the immature cerebellum could guide the development of therapies for alcohol-induced neurotoxicity. Retinoic acid (RA) was first recognized as essential for the control of patterning and neuronal differentiation in the developing embryonic brain (Maden, 2002). The cellular effects of RA are mediated by two classes of receptors: retinoid receptors (RARs) and rexinoid receptors (RXRs). RARs and RXRs each have three major subtypes (α, β, and γ), all of which function as transcription factors (Kliewer et al., 1992; Maden, 2002). Together with their nuclear retinoid (RAR) and rexinoid (RXR) receptors, RA controls genetic programs that are essential for embryonic development, organogenesis, organ homeostasis, cell growth, differentiation, and apoptosis (Kastner et al., 1995). Defective RA signaling causes neurodegeneration (Corcoran et al., 2002b; Goodman and Pardee, 2003). Likewise, RA has been shown to control brain patterning (Maden, 2000, 2002). Hypo-functioning of retinoid signaling causes Alzheimer’s disease (Corcoran et al., 2004). Experiments in primary cultured neurons have shown that RA promotes the formation of neurites in cortical neurons. Moreover, an antagonist of RARβ regulates neurite outgrowth in dorsal root ganglionic neurons (Corcoran et al., 2002a; Guleria et al., 2006; Santillano et al., 2005). Each of these studies highlights the importance of RA during normal and disease conditions. As the cerebellum expresses a high number of RXR receptors, and RXR targets genes predominantly involved in apoptosis (Szondy et al., 1998), it is possible that ethanol activates RXRs to induce apoptosis of granule cells (Yamamoto et al., 1999; Zhao et al., 2004). To test this hypothesis, we studied the effects of ethanol on the expression and activation of RA receptors. Our studies have demonstrated that high dose of ethanol reduces RAR expression and inhibits activation of RARs in the cerebellum and in CGNs. Alternatively, high-dose ethanol increases the expression and activation of RXRs in the cerebellum and in CGNs. These studies indicate that high-dose ethanol may promote harmful effects in CGNs by targeting RA receptors.