Anna A. Gybina

The modulation of oxygen radical production by nitric oxide in mitochondria.

Biological systems that produce or are exposed to nitric oxide (NO⋅) exhibit changes in the rate of oxygen free radical production. Considering that mitochondria are the main intracellular source of oxygen radicals, and based on the recently documented production of NO⋅ by intact mitochondria, we investigated whether NO⋅, produced by the mitochondrial nitric-oxide synthase, could affect the generation of oxygen radicals. Toward this end, changes in H2O2production by rat liver mitochondria were monitored at different rates of endogenous NO⋅ production. The observed changes in H2O2 production indicated that NO⋅affected the rate of oxygen radical production by modulating the rate of O2 consumption at the cytochrome oxidase level. This mechanism was supported by these three experimental proofs: 1) the reciprocal correlation between H2O2production and respiratory rates under different conditions of NO⋅ production; 2) the pattern of oxidized/reduced carriers in the presence of NO⋅, which pointed to cytochrome oxidase as the crossover point; and 3) the reversibility of these effects, evidenced in the presence of oxymyoglobin, which excluded a significant role for other NO⋅-derived species such as peroxynitrite. Other sources of H2O2 investigated, such as the aerobic formation of nitrosoglutathione and the GSH-mediated decay of nitrosoglutathione, were found quantitatively negligible compared with the total rate of H2O2 production.

VARIABLE RESPONSE OF SELECTED CUPROPROTEINS IN RAT CHOROID PLEXUS AND CEREBELLUM FOLLOWING PERINATAL COPPER DEFICIENCY

Recent immunohistochemical characterization of the copper transport protein, Ctr1, reported enriched levels in mouse choroid plexus, and enhancement by copper deficiency. To extend and confirm this, experiments were conducted with Holtzman rats. Following perinatal copper deficiency there was an 80% reduction in brain copper of 24–27 day old copper-deficient (Cu-) rat pups compared to copper-adequate (Cu+) controls. Choroid plexus immunoblot analysis with rabbit anti-hCtr1 demonstrated a 50% higher Ctr1 protein expression in Cu-samples. However, levels of copper chaperone for superoxide dismutase (CCS) were unchanged, suggesting that Ctr1 buffers the choroid plexus against copper deficiency, since CCS normally is much higher in Cu-tissues. There were 13% lower levels of cytochrome c oxidase subunit IV (COX IV) detected in Cuchoroid plexus. In contrast, in cerebellum of Cu-rats CCS was 2-fold higher and COXIV 1.7-fold lower than Cu+ rats consistent with severe copper deficiency. Brain mitochondria from Cu-rats had severe reductions in COXIV content and CCO activity and modest but significant elevations in CCS and reductions in Cu, Zn-superoxide dismutase. COXIV may be a more sensitive marker for copper deficiency than CCS and may prove useful to assess copper status.

Rat brain iron concentration is lower following perinatal copper deficiency.

Experiments performed with Holtzman rats demonstrated that brain iron (Fe) was lower by postnatal day 13 (P13) in pups born and nursed by dams that began copper‐deficient (–Cu) treatment at embryonic day 7. Transcardial perfusion of P24–P26 males and females to remove blood Fe contamination revealed that brain Fe was still 20% lower in –Cu than +Cu rats. Estimated blood content of brain for –Cu rats was greater than for +Cu rats; for all groups, values ranged between 0.43 and 1.03%. Using group‐specific data and regression analyses, r = 0.99, relating blood Fe to hemoglobin, brain Fe in non‐perfused rats in a replicate study was lower by 33% at P13 and 39% at P24 in –Cu rats. Brain extracts from these rats and from P50 rats from a post‐weaning model were compared by immunobloting for transferrin receptor (TfR1). P24 brain –Cu/+Cu TfR1 was 3.08, suggesting that brains of –Cu rats were indeed Fe deficient. This ratio in P13 rats was 1.44, p < 0.05. No change in P50 –Cu rat brain TfR1 or Fe content was detected despite a 50% reduction in plasma Fe. The results suggest that brain Fe accumulation depends on adequate Cu nutriture during perinatal development.

Copper deficiency alters the neurochemical profile of developing rat brain

Abstract Copper deficiency is associated with impaired brain development and mitochondrial dysfunction. Perinatal copper deficiency was produced in Holtzman rats. In vivo proton NMR spectroscopy was used to quantify 18 cerebellar and hippocampal metabolites on postnatal day 21 (P21). Copper status was evaluated in male copper-adequate (CuA) and copper-deficient (CuD) brothers at P19 and at P23, 2 days following NMR experiments, by metal and in vitro metabolite data. Compared to CuA pups, CuD pups had lower ascorbate concentration in both brain regions, confirming prior HPLC data. Both regions of CuD rats also had lower N-acetylaspartate levels consistent with delayed development or impaired mitochondrial function similar to prior work demonstrating elevated lactate and citrate. For other metabolites, the P21 neurochemical profile of CuD rats was remarkably similar to CuA rats but uniquely different from iron-deficient or chronic hypoxia models. Further research is needed to determine the neurochemical consequences of copper deficiency.

Copper deficiency results in AMP-activated protein kinase activation and acetylCoA carboxylase phosphorylation in rat cerebellum.

Copper (Cu) deficiency impairs cerebellar development including biosynthetic processes like myelination and synaptogenesis. The activity of cerebellar mitochondrial cuproenzyme cytochrome c oxidase is markedly lower in Cu deficient rat pups and is accompanied by higher lactate levels indicating mitochondrial inhibition. Cu deficiency impaired energy metabolism is thought to contribute to developmental delays, but specific mechanisms linking these phenomena have remained unexplored. AMP-activated protein kinase (AMPK) is a cellular energy sensor that is activated during mitochondrial inhibition and shuts down biosynthetic processes to help conserve cellular ATP levels. Activated AMPK phosphorylates and inhibits acetylCoA carboxylase (ACC), the first enzyme in fatty acid biosynthesis. We hypothesize that AMPK is activated and ACC inhibited in Cu deficient cerebella. Perinatal copper deficiency was studied in young rats in rapidly frozen cerebella. Compared to copper-adequate (Cu+) pups, copper-deficient (Cu-) pups were hypothermic, had lower brain copper levels and markedly higher cerebellar lactate. Concentration of phosphorylated AMPK (pAMPK), indicating AMPK activation, was robustly higher in Cu- cerebella of rat pups at two ages and in two separate experiments. Compared to Cu+ cerebella, pACC content was significantly higher in all Cu- samples. Mechanisms leading to AMPK activation remain elusive. Higher AMP/ATP ratios and increased reactive nitrogen species (RNS) can lead to AMPK activation. ATP and AMP concentrations were unaltered and nitric oxide metabolites and 3-nitrotyrosine peptide levels remained unchanged in Cu- cerebella. AMPK activation may explain how ATP levels can be maintained even with a severe mitochondrial loss of CCO function.

Fructose-2,6-bisphosphate is lower in copper deficient rat cerebellum despite higher content of phosphorylated AMP-activated protein kinase.

Limitation in copper (Cu) leads to pathophysiology in developing brain. Cu deficiency impairs brain mitochondria and results in high brain lactate suggesting augmented anaerobic glycolysis. AMP activated protein kinase (AMPK) is a cellular energy “master-switch” that is thought to augment glycolysis through phosphorylation and activation phosphofructokinase 2 (PFK2) resulting in increases of the glycolytic stimulator fructose-2,6-bisphosphate (F2,6BP). Previously, Cu deficiency has been shown to augment cerebellar AMPK activation. Cerebella of Cu-adequate (Cu+) and Cu-deficient (Cu−) rat pups were assessed to evaluate if AMPK activation in Cu− cerebella functioned to enhance PFK2 activation and increase F2,BP concentration. Higher levels of pAMPK were detected in Cu− cerebella. However, PFK2 activity, mRNA, and protein abundance were not affected by Cu deficiency. Surprisingly, F2,6BP levels were markedly lower in Cu− cerebella. Lower F2,6BP may be due to inhibition of PFK2 by citrate, as citrate concentration was significantly higher in Cu− cerebella. Data suggest AMPK activation in Cu− cerebellum does not augment glycolysis through a PFK2 mechanism. Furthermore, other metabolite data suggest that glycolysis may actually be blunted, since levels of glucose and glucose-6-phosphate were higher in Cu− cerebella than controls.

Augmented cerebellar lactate in copper deficient rat pups originates from both blood and cerebellum

Copper (Cu) is essential for proper brain development, particularly the cerebellum, and functions as a cofactor for enzymes including mitochondrial cytochrome c oxidase (CCO). Cu deficiency severely limits CCO activity. Augmented lactate in brain of Cu deficient (Cu-) humans and cerebella of Cu- rats is though to originate from impaired mitochondria. However, brain lactate may also originate from elevated blood lactate. The hypothesis that cerebellar lactate originates from elevated blood lactate in Cu- rat pups was tested. Analysis of Cu- and Cu adequate (Cu+) rat pups (experiment I) revealed blood lactate was elevated in Cu- rat pups and cerebellar lactate levels were closely correlated to blood lactate concentration. A second rat experiment (experiment II) assessed Cu- cerebellar lactate without the confounding factor of elevated blood lactate. Blood lactate levels of Cu- rat pups in experiment II were equal to those of controls; however, Cu- cerebellar lactate was still elevated, suggesting mitochondrial impairment by Cu deficiency. Treatment of rat pups with dichloroacetate (DCA), an activator of mitochondrial pyruvate dehydrogenase complex (PDC), lowered Cu- cerebellar lactate to control levels suggesting PDC inhibition is a site of mitochondrial impairment in Cu- cerebella. Results suggest Cu- cerebellar lactate originates from blood and cerebellum.