The long term objective of this research is to clarify the role of mitochondrial DNA (mtDNA) in pathogenesis of malformations induced by environmental agents. The research proposed here is directed at embryonic mtDNA expression in the mechanism of mercury-induced neural tube defects (NTDs) in mice. Mouse embryos make an essential switch from anaerobic to aerobic metabolism between the 8-somite and 28-somite stages of development. The oxidative transition can be modeled through the 16S ribosomal RNA (16S rRNA). Encoded in the mtDNA genome, the 16S rRNA transcript is an essential structural component of the mitochondrial ribosome. It is up regulated as the oxidative transition advances between the 8-somite and 28-somite stages of development, and de-regulated in embryos lacking the p53 tumor suppressor gene and in embryos exposed to low levels of mercury. These observations form the basis for a postulated sub-apoptotic function of p53 in the control of embryonic metabolism. Hypothesis one is that the developmental changes in 16S rRNA expression partition between p53-independent and p53-dependent controls, and these controls are differentially accessed in the developing prosencephalon and heart during oxidative transition. Hypothesis two is that mercury interferes with the p53-dependent control of 16S RNA biogenesis during induction of prosencephalic malformations, blocking a direct signaling effect of p53 on the mtDNA genome.
Specific aim 1 will measure the p53-sensitive and insensitive 16S RNA pools in the prosencephalon and heart between the 8-somite and 28-somite stages of development, and during pathogenesis of malformations induced by low concentrations of mercury.
Specific aim 2 will correlate changes in p53 sensitive 16S RNA pools of mitochondrial p53 protein and mtDNA-binding during the critical exposure period in mercury-induced malformation.
Specific aim 3 will determine the extent to which chronic changes in 16S RNA pools and mtDNA genomic stability are functionally dependent on mitochondrial p53 using embryonic cells stably transfected with mitochondrially targeted transdominant-negative p53 miniprotein. These experiments will set the stage for future studies to explore functional relationships in vivo using mitochondrially-targeted p53 transgenic mice, as well as provide insights on a novel mechanism of mercury-induced birth defects that may also be applicable to other environmental agents that cause neural tube defects.
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