Involvement of Permeability Transition Pore in Developmental Alcohol Neurotoxicit

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DESCRIPTION (provided by applicant): Animal fetal alcohol syndrome (FAS) models have demonstrated a temporal window during the brain growth spurt in which cerebellum is particularly vulnerable to the neurotoxic effects of ethanol (EtOH). In rodents, the brain growth spurt period occurs during postnatal day 4 (P4) through P10. In humans, this developmental stage occurs during the third trimester of pregnancy when neurobehavioral abnormalities associated with FAS are introduced. Rat cerebellum is particularly sensitive to EtOH during P4-6. At a slightly later neonatal period (P7 and later), the effects of comparable exposure are minimal. During the brain growth spurt, many neurons undergo apoptosis. This EtOH-mediated neuronal death is primarily Bax-dependent. There are two known general mechanisms by which Bax induces mitochondria to release apoptotic death effectors: 1) Bax can directly interact with the mitochondrial permeability transition pore (PTP) to prolong its opening [PTP-dependent]; or, 2) Bax molecules can homo-oligomerize to create a channel in the mitochondrial membrane [PTP-independent]. Under most apoptotic conditions, apoptotic activation is PTP-independent. Under physiological conditions (e.g., no interaction with Bax), regulatory proteins connect oxidative phosphorylation to glucose metabolism and maintain membrane integrity. The mechanism by which EtOH mediates mitochondrial-activated apoptosis at this pivotal point is unknown. However, based on previous studies and preliminary data, we hypothesize that during a period of maximum EtOH susceptibility (P4), EtOH-mediated apoptosis in the rat cerebellum is activated by the PTP-dependent Bax mechanism. At a slightly later more EtOH resistant period (P7), we hypothesize that EtOH effects on PTP regulatory proteins are minimal. This proposed investigation uniquely addresses the role of PTP regulatory proteins specific to alcohol neurotoxicity in developing neurons. Additionally, it tests a novel, potential regulatory mechanism of Bad-a Bax-supporting protein-which we speculate promotes apoptosis in cerebellum of P4 EtOH-exposed animals by binding and sequestering mitochondrial hexokinase, a glucose metabolizing enzyme. For the proposed research, Long-Evans rats are exposed to a single dose of EtOH on P4 and P7. Novel elements used for this in vivo investigation include: 1) isoelectric focusing to identify the mechanism of Bax-dependent apoptotic activation induced by EtOH; and, 2) an ELISA-based technique recently developed in our laboratory (Siler-Marsiglio et al., 2005a, 2006) that detects and quantifies potential competing native protein-protein interactions. Analyses of differential regulatory protein-protein interactions in cerebellum at EtOH-sensitive compared to EtOH-resistant ages are important for revealing mechanisms critical to developmental EtOH neurotoxicity, and will be particularly important in identifying possible sites for eventual therapeutic intervention.