The overall hypothesis of this exploratory proposal is that a key mechanism underlying ethanol-mediated opening of the mitochondrial permeability transition pore complex and possibly apoptosis activation, is perturbation of adenine nucleotide translocase (ANT) function by 4- hydroxynonenal (HNE) adduct formation. Key responses to HNE are, as previously documented, inhibition of ANT activity, activation of the mitochondrial permeability transition pore, and ultimate release of pro-apoptotic proteins. It is proposed that this occurs as a multifactorial event which includes direct modification of specific amino acid residues of ANT and its impaired interactions with cyclophilin D (CyP-D). Significance: This proposal address a new and novel, mitochondrially-mediated, fundamental mechanism by which ethanol may elicit apoptotic death of neurons. Neuron survival control points such as this are prime targets for the ultimate development of new clinical interventions.
Specific Aim 1 will address the hypothesis that modifications of adenine nucleotide translocase by 4- hydroxynonenal adduct formation is a mechanism underlying ethanol-related effects on the mitochondrial permeability transition pore complex and ultimately on induction of apoptosis.
Specific Aim 2 will address the hypothesis that direct modifications of cyclophilin-D by 4- hydroxynonenal adduct formation alter critical interactions with adenine nucleotide translocase and that this is one mechanism underlying ethanol-related effects on the mitochondrial permeability transition pore complex. Experimental Approaches: The experimental approaches will utilize Mass Spectrometry to determine specific amino acid residues on ANT and CyP-D that are modified and point-mutational manipulations to establish the functional significance of these ethanol-related modifications. Studies will also establish effects of these specific ANT and CyP-D modifications on CyP-D binding to ANT, on permeability transition regulation, and on sensitivity to ethanol-mediated neuron death. The experimental model will be cultured fetal rat cerebral cortical neurons exposed to clinically relevant ethanol treatment regimens.
Exposure of cultured fetal rat cerebral cortical neurons to ethanol rapidly elicits oxidative stress and increased formation of 4-hydroxynonenal (HNE) in mitochondria, which is followed by mitochondrial leakage of pro-apoptotic proteins and apoptotic death. This exploratory proposal addresses the hypothesis that a mechanism underlying ethanol-mediated opening of the permeability transition pore complex (PTPC) is connected to HNE modifications of the pore components, adenine nucleotide translocase (ANT) and cyclophilin D (CyP-D). Thus, the proposed studies will provide insight into basic mechanisms underlying mitotoxic responses to ethanol as well as fundamental mechanisms of PTPC function.
