Differential effects of ethanol on bid, tBid, and Bax:tBid interactions in postnatal day 4 and postnatal day 7 rat cerebellum.

BACKGROUND: Exposure to ethanol (EtOH) during central nervous system (CNS) development can lead to a wide array of neuroanatomical, behavioral, and cognitive abnormalities, broadly subsumed under the fetal alcohol spectrum disorder classification. One mode of EtOH-induced interference in the normal developmental program appears to be through induction of apoptotic processes mediated by the Bcl-2 family of survival-regulatory proteins. The present series of studies investigated the role of the Bcl-2-related, pro-apoptotic Bid protein, and its truncated, apoptotically active fragment, tBid, in developmental EtOH neurotoxicity. METHODS: Protein analyses were made via enzyme-linked immunosorbent assays (ELISA) in neonatal rat cerebellum, of basal Bid, and of Bid and tBid, following EtOH exposure via vapor inhalation, at an age of peak EtOH sensitivity in this region (postnatal day 4 [P4]) and a later age of relative resistance (P7). ELISA analyses were also made of Bax:tBid heterodimers, a process which activates Bax, essential for its apoptotic functioning. Finally, in vitro assessments of the importance of tBid to EtOH neurotoxicity were made in cultured cerebellar granule cells, using a specific tBid inhibitor. RESULTS: Basal levels of Bid were higher at P4 compared to P7, possibly contributing to the differential sensitivity. EtOH exposure elicited further increases in cytosolic Bid and mitochondrial tBid when administration was at P4, but not at P7. Bax:tBid heterodimers were markedly increased by EtOH exposure on P4, an increase which persisted even 2 hours after termination of treatment. Similar effects were not seen at P7. The in vitro analyses revealed that tBid inhibition provided complete protection against EtOH-induced cell death and depressed EtOH-mediated cytochrome-c release. CONCLUSIONS: These results suggest that Bid/tBid may be important elements in EtOH-mediated neurotoxicity during CNS development. The molecular processes and interactions revealed may represent critical points which can be targeted in studies concerned with designing possible therapeutic strategies for minimizing these devastative effects.

Ethanol influences on Bax translocation, mitochondrial membrane potential, and reactive oxygen species generation are modulated by vitamin E and brain-derived neurotrophic factor.

BACKGROUND: This study investigated ethanol influences on intracellular events that predispose developing neurons toward apoptosis and the capacity of the antioxidant α-tocopherol (vitamin E) and the neurotrophin brain-derived neurotrophic factor (BDNF) to modulate these effects. Assessments were made of the following: (i) ethanol-induced translocation of the pro-apoptotic Bax protein to the mitochondrial membrane, a key upstream event in the initiation of apoptotic cell death; (ii) disruption of the mitochondrial membrane potential (MMP) as a result of ethanol exposure, an important process in triggering the apoptotic cascade; and (iii) generation of damaging reactive oxygen species (ROS) as a function of ethanol exposure. METHODS: These interactions were investigated in cultured postnatal day 8 neonatal rat cerebellar granule cells, a population vulnerable to developmental ethanol exposure in vivo and in vitro. Bax mitochondrial translocation was analyzed via subcellular fractionation followed by Western blot, and mitochondrial membrane integrity was determined using the lipophilic dye, JC-1, that exhibits potential-dependent accumulation in the mitochondrial membrane as a function of the MMP. RESULTS: Brief ethanol exposure in these preparations precipitated Bax translocation, but both vitamin E and BDNF reduced this effect to control levels. Ethanol treatment also resulted in a disturbance of the MMP, and this effect was blunted by the antioxidant and the neurotrophin. ROS generation was enhanced by a short ethanol exposure in these cells, but the production of these harmful free radicals was diminished to control levels by cotreatment with either vitamin E or BDNF. CONCLUSIONS: These results indicate that both antioxidants and neurotrophic factors have the potential to ameliorate ethanol neurotoxicity and suggest possible interventions that could be implemented in preventing or lessening the severity of the damaging effects of ethanol in the developing central nervous system seen in the fetal alcohol syndrome (FAS).

Effects of acute ethanol exposure on regulatory mechanisms of Bcl-2-associated apoptosis promoter, bad, in neonatal rat cerebellum: differential effects during vulnerable and resistant developmental periods.

BACKGROUND: Prenatal alcohol exposure produces anatomical and behavioral abnormalities associated with fetal alcohol syndrome (FAS). Animal FAS models have demonstrated temporal windows of vulnerability in the developing cerebellum, with substantial ethanol (EtOH)-mediated apoptotic activation during these periods. In rodents, the cerebellum is most sensitive to EtOH on postnatal days 4 to 6 (P4 to P6). At slightly later ages (P7 and later), this region is less vulnerable to EtOH. The present study investigated EtOH effects on mechanisms related to activities of Bad, a proapoptotic member of the Bcl-2 gene family, to further characterize processes underlying these disparate EtOH sensitivities. In healthy cells, Bad is retained in the cytosol by association with 14-3-3, a primarily cytosolic protein. Bad promotes apoptosis by disassociating from 14-3-3 and sequestering Bcl-xL through heterodimerization. This dimerization prevents the neutralizing association of Bcl-xL with Bax, freeing Bax to perform in a prodeath manner. Caspase-dependent cleavage of Bad to a 15-kDa fragment increases its proapoptogenic capacity. METHODS: Two hours following EtOH exposure of P4 and P7 animals via inhalation, we determined how exposure affects intracellular localization and proteolytic cleavage of Bad and expression of cerebellar 14-3-3, using subcellular fractionation and Western blot techniques. Ethanol effects on interactions between Bad and 14-3-3 or Bcl-xL at the more vulnerable and less vulnerable ages were determined using an enzyme-linked immunosorbent assay-based technique to detect native protein-protein interactions. RESULTS: At P4, EtOH increased mitochondrial localization of Bad, expression of a 15-kDa fragment recognized by Bad antibody, and formation of Bad:Bcl-xL complexes. At that more vulnerable age, EtOH also decreased formation of Bad:14-3-3 complexes. At P7, EtOH increased Bad:14-3-3 complexes and reduced Bad:Bcl-xL complexes. Cytosolic 14-3-3 remained unchanged by EtOH at P4 and P7. CONCLUSIONS: Ethanol-induced alterations of Bad-related mechanisms at P4 favor a prodeath response. EtOH does not influence these same mechanisms in a manner that promotes cell death at P7. Divergent Bad-related responses at these 2 developmental ages likely contribute to their differential EtOH vulnerability.

Functional mechanisms of apoptosis-related proteins in neonatal rat cerebellum are differentially influenced by ethanol at postnatal days 4 and 7.

Exposure of the developing nervous system to ethanol (EtOH) produces neurological aberrations associated with fetal alcohol syndrome. During a well-defined period, cerebellar neurons are highly susceptible to EtOH-induced death, primarily through apoptosis. Neonatal rodent cerebellum is exquisitely sensitive to EtOH on postnatal days 4-6 (P4-6); however, at slightly later developmental ages (P7 and later), EtOH effects are minimal. We have previously shown that EtOH differentially influences expression of apoptosis-related proteins of the Bcl-2 survival-regulatory gene family in P4 and P7 cerebellum. In the present study, the effects of EtOH on multiple functional mechanisms of Bcl-2, Bcl-xL, and Bax were investigated to characterize further the processes underlying these disparate EtOH sensitivities. For these analyses, we addressed the following questions, by using P4 and P7 cerebellar tissue following in vivo exposure: 1) Are there differential patterns of expression of antiapoptotic Bcl-2 or proapoptotic Bax in EtOH-vulnerable Purkinje cells that could contribute to the different degrees of temporal EtOH vulnerability? 2) How does EtOH affect intracellular localization of apoptosis-related proteins? 3) Does cleavage of Bax contribute to EtOH sensitivity? 4) Does EtOH differentially modulate cerebellar protein-protein interactions of Bcl-2, Bcl-xL, and Bax at the vulnerable vs. the resistant ages? Overall, we show that, at P4, the EtOH-mediated effects on Bcl-2, Bcl-xL, and Bax favor a prodeath response, whereas most of the intracellular responses to EtOH exposure at P7 promote survival. Such differential responsiveness likely plays a major role in the disparate ethanol vulnerability at these two postnatal ages.

Mitochondrially targeted vitamin E and vitamin E mitigate ethanol-mediated effects on cerebellar granule cell antioxidant defense systems.

Ethanol (EtOH) disrupts the structure and function of the developing nervous system, sometimes leading to birth defects associated with fetal alcohol syndrome (FAS). Animal FAS models indicate that cellular membrane peroxidation, intracellular oxidant accumulation, and suppression of endogenous antioxidant enzymes contribute to the toxic effects of EtOH. Mitochondrially targeted vitamin E (MitoVit E), a chemically engineered form of vitamin E (VE) designed to accumulate in the mitochondria, has been shown to inhibit intracellular oxidant accumulation and cell death more effectively than VE. In previous investigations, we have shown that, in vivo, VE reduces neuronal death in the developing cerebellum of EtOH-exposed animals, and, in vitro, VE prevents apoptotic and necrotic death of EtOH-exposed cerebellar granule cells (CGCs). The present investigation shows that, in a FAS CGC model, 1 nM MitoVit E renders significant neuroprotection against EtOH concentrations as high as 1600 mg/dL. The present study also demonstrates that, in this same model, MitoVit E mitigates EtOH-induced accumulation of intracellular oxidants and counteracts suppression of glutathione peroxidase/glutathione reductase (GSH-Px/GSSG-R) functions, protein expression of gamma-glutamylcysteine synthetase (gamma-GCS), and total cellular glutathione (GSH) levels. In the presence and absence of EtOH, VE amplifies the protein expression levels of gamma-GCS, an enzyme that performs the rate-limiting step for GSH synthesis, and total GSH levels. These results suggest that MitoVit E and VE ameliorate EtOH toxicity through non-oxidant mechanisms-modulations of endogenous cellular proteins-and antioxidant means.

Protective mechanisms of pycnogenol in ethanol-insulted cerebellar granule cells.

Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), inhibits apoptosis and necrosis of developing neurons exposed acutely to ethanol (EtOH). The present study shows that the protective mechanisms of PYC in EtOH-exposed postnatal day 9 cerebellar granule cells (P9 CGCs) include (1) reduction of reactive oxygen species (ROS) production; (2) counteraction of suppressed copper/zinc superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase/reductase (GSH-Px/GSSG-R) system activities; (3) upregulation of Cu/Zn SOD protein expression; (4) mitigation of the EtOH-mediated exacerbation of catalase (CAT) activity; and, (5) specific binding and inhibition of active caspase-3. These results indicate that the mechanisms by which PYC antagonizes EtOH-induced oxidative stress include oxidant scavenging and modulation of endogenous, cellular proteins. Using findings from the present and previous studies, a model delineating the mechanisms of EtOH effects on the system of antioxidant enzymes in developing CGCs is presented.

Pycnogenol and vitamin E inhibit ethanol-induced apoptosis in rat cerebellar granule cells.

Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol-induced activation of caspase-3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure.

Nerve growth factor and chronic ethanol treatment alter calcium homeostasis in developing rat septal neurons.

Chronic ethanol treatment (CET) during development produces cellular adaptations resulting in tolerance to the acute effects of ethanol (EtOH). The objectives of this study were to determine whether CET during the prenatal period (PCET) followed by a period of in vitro CET (PCET-CET) altered intracellular calcium [Ca(2+)](i) and produced tolerance to acute EtOH treatment (AET), and whether nerve growth factor (NGF) modulated the effects of PCET-CET in cultured developing rat septal neurons. Fetuses were obtained from EtOH-fed and sucrose-fed (diet-control) female rats. Neurons from PCET fetuses were cultured in the presence of NGF (+NGF) and 200 mg/dl (mg %) EtOH and diet-control cultures received NGF and no EtOH. PCET and diet-control cultures were then divided into two groups, +NGF and -NGF (withdrawn from NGF), and exposed acutely to one of five doses of EtOH during stimulation with potassium (K(+)) chloride. [Ca(2+)](i) was measured using fura-2. PCET-CET did not affect resting [Ca(2+)](i). PCET-CET decreased and acute EtOH withdrawal increased overall K(+)-stimulated changes in [Ca(2+)](i), but only in +NGF PCET neurons. Reducing the level of EtOH from 200 to 100 mg % decreased overall K(+)-stimulated [Ca(2+)](i) in -NGF PCET neurons. The effects of PCET-CET or PCET-CET combined with NGF on overall K(+)-stimulated changes in [Ca(2+)](i) occurred mostly in the early and middle phases of the K(+)-response. NGF reduced overall K(+)-stimulated changes in [Ca(2+)](i) in PCET neurons during EtOH withdrawal and during AET with 200 mg % EtOH and increased overall K(+)-stimulated changes in [Ca(2+)](i) during AET with 400 and 800 mg % EtOH. There was no effect of NGF on overall K(+)-stimulated changes in [Ca(2+)](i) in diet-control neurons with the exception that NGF-treatment decreased overall K(+)-stimulated changes in [Ca(2+)](i) during AET with 400 mg % EtOH. The effects of AET on overall K(+)-stimulated changes in [Ca(2+)](i) mostly occurred in +NGF PCET neurons. In conclusion, CET during development of the brain could adversely affect Ca(2+)-dependent functions such as neuronal migration, neurite outgrowth, and synaptogenesis in neurons even in the presence of neurotrophin support.