Copper is an essential micronutrient, which plays a key biochemical role in cellular respiration, neurotransmitter production, peptide biogenesis and antioxidant defense. While nutritional and genetic diseases clearly indicate a critical role for copper in the development of the human central nervous system, the precise mechanisms underlying this process are not well understood. The long-term objective of these studies is to define the cellular and molecular determinants of nutritional copper homeostasis in the developing central nervous system. Recent studies reveal that the delivery of copper to specific targets within the cell is mediated by a unique class of proteins termed metallochaperones. The studies in this application are intended to elucidate the function of the copper chaperone Atox1 in brain copper homeostasis utilizing mice with a germline interruption of the Atox1 gene. These animals demonstrate marked alterations in copper metabolism and a profound neurological phenotype resulting in early neonatal mortality. Analysis of Atox1 function and gene expression in the developing central nervous system will be accomplished using mice with a targeted insertion of the LacZ gene in the Atox1 allele. The role of Atox1 in central nervous system development will be determined by analyzing the temporal and spatial expression of Atox1 and by histological examination of the developing brain of Atox1-/- mice. The molecular and cellular mechanisms of copper trafficking by Atox1 will be evaluated by examining alterations in copper metabolism following 64Cu metabolic labeling of primary neuronal and glial cell cultures from Atox1+/+ and Atox1-/-mice. Novel cuproproteins in the Atox1 delivery pathway detected in this analysis will be isolated and identified by mass spectrometry. The results from these studies will provide novel mechanistic insights into copper homeostasis in the developing central nervous system and may aid in development of nutritional strategies to prevent brain injury in human diseases.
