Increased rat brain cytochrome c correlates with degree of perinatal copper deficiency rather than apoptosis

Reductions in copper due to dietary restriction or transporter deficiency in brindled mice or humans with Menkes disease lead to reduced cuproenzyme activities, mitochondrial abnormalities, neurodegeneration and early mortality. The mechanisms for observed neuropathology remain unknown. Some researchers studying mutant mice suggest brain apoptosis as a possible factor based on changes in transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and increased cytosolic cytochrome c and decreased Bcl-2 levels. Perinatal copper deficiency was induced in Holtzman rats during late gestation and lactation to investigate the role of apoptosis in the developing brain. Analysis of 13- and 24-d-old (P13 and P24) brains from male copper-deficient and copper-adequate rats revealed no difference in cytosolic cytochrome c or total Bcl-2 levels. Cerebellar TUNEL staining and caspase-3 activity were higher in the P12 copper-deficient than in the copper-adequate pups. However, TUNEL staining decreased and caspase-3 activity was not detected at P24 even though pups were more copper deficient based on cortex copper, Cu, Zn-superoxicle dismutase and cytochrome c oxiclase activities. This suggests that neuronal apoptosis is not enhanced by dietary copper deficiency in the brain. Lower Bcl-2 levels were detected in the copper-deficient rat hearts, consistent with apoptotic processes in mice reported by others. A robust enhancement of cytochrome c was observed in the total brain extracts and purified brain mitochondria of copper-deficient pups. Higher cytochrome c appeared to be correlated with the degree of copper deficiency and seemed to be associated with increased mitochondrial mass, because higher levels of voltage-dependent anion channel and mitochondrial complex I were also detected. The biochemical mechanisms for elevated cytochrome c are not known nor are the physiological consequences.