Joseph R. Prohaska - US grants

The long-range objective of this research is to elucidate the biochemical roles of copper in immune function and to determine when during development copper is most critical to the immune system. Selected biochemical and immunological techniques will be used. To accomplish these goals and to test certain hypotheses, animal models will be employed. We will induce dietary copper deficiency during perinatal development in C57BL and C58 mice during several discrete periods from gestation to early adulthood. Repletion studies will be conducted. Copper status during depletion and following repletion will be assessed by determining blood hematocrit and plasma ceruloplasmin activity. Organ (spleen and thymus) and serum levels of total copper and iron will be measured by atomic absorption spectroscopy (AAS). Changes in functional copper pools will be evaluated by assaying the activity of copper-zinc superoxide dismutase or cytochrome c oxidase. Immunological evaluation will include investigating humoral immunity by determining the number of antibody-producing cells in spleen in response to sheep erythrocyte challenge. Cell-mediated immunity will be studied by quantifying mitogen reactivity of cultured thymocytes and splenocytes and by mixed lymphocyte reaction. Lymphocyte subpopulations from spleen will be determined by immuno-fluorescent procedures using monoclonal antibodies to surface antigens. Plant lectins (PNA and SBA) will be used to evaluate maturational development of lymphocytes from thymus, spleen and bone marrow. Serum IgM and IgG will be measured by ELISA techniques. Additional biochemical measurements will be conducted to elucidate the mechanisms of impaired immunity while testing specific hypotheses. Lymphocytes from spleen and thymus will be isolated, and the plasma membrane will be characterized for changes in lipid and protein composition using gas chromatography and gel electrophoresis, respectively. Antioxidant status will be evaluated in lymphoid organs by quantifying specific metabolites. Repletion studies will be conducted comparing cupric or ferrous gluconate and purified mouse ceruloplasmin or transferrin to determine the extent, if any, that altered immunity is iron-dependent. Plasma, brain and spleen levels of norepinephrine will be quantified by HPLC with electrochemical detection. Copper metabolism will be studied in developing lymphoid organs using 67Cu and AAS.

biomedical equipment resource; biomedical equipment purchase;

DESCRIPTION (provided by applicant): Insufficient copper (Cu) during perinatal development of rodents has a major impact on the central nervous system leading to altered neurochemistry and behavior even after long-term Cu repletion. Human intakes of Cu during pregnancy and lactation may be suboptimal but current RDA recommendations do not encourage the need for supplements. The long-range goal of this research is to identify the biochemical roles of Cu responsible for severe long-term neurochemical and behavioral consequences of perinatal Cu deficiency. Four specific aims will test the overall hypothesis that altered cuproenzymes are responsible for the phenotype observed. Research will utilize nutritional and genetic models with Holtzman rats and transgenic mice. AIM 1: We will test the hypothesis that limitation in the Cu-dependent enzyme dopamine beta- monooxygenase (DBM) is responsible by treating rats with L-3.4-dihydroxyphenvlserine (L-DOPS) to bypass the DBM step and restore low brain norepinephrine. AIM 2: We will test the hypothesis that limitation in Cu.Zn-superoxide dismutase (SOD) is responsible by comparing features of Cu deficiency in SOD -/- mice to wild-type controls Secondly, we will compare these mice to mice with altered SOD activity, Ctrl +/- mice, due to lower copper transport capacity. Thirdly, we will restore the deficit in SOD activity following these treatments with TEMPOL a membrane permeable SOD mimetic. AIM 3: We will test the hypothesis that limitation in mitochondrial cytochrome C oxidase (CCO) is responsible by characterizing brain energy metabolism in Cu deficient rats with a rat model of chronic CCO inhibition that uses cyanide. AIM 4: We will test the hypothesis that limitation in Cu-dependent ferroxidases lead to lower brain iron and are responsible by comparing rats reared on an iron-fortified diet to restore brain iron.

DESCRIPTION (provided by applicant): Normal brain development requires appropriate levels of nutrients, hormones, and other signaling molecules presented to the brain at precise developmental timepoints. For example, iodine deficiency results in reduced thyroid hormone (TH) production and severe developmental abnormalities. Similarly, copper (Cu) and iron (Fe) deficiencies during late brain development result in strikingly similar derangements in brain development. The long-term goal of the proposed research program is to understand the molecular basis of Cu, Fe, and TH action in the developing brain. To this end, the investigators have developed a model that seeks to identify a shared molecular mechanism causative of the aberrancies in brain development associated with these three deficiencies. Recent data have revealed that TH synthesis is reliant on adequate Fe levels. Fe is likely required for normal function of the TH synthesizing enzyme thyroid peroxidase. Interestingly, Cu deficiency is also associated with reduced TH levels. The investigators'preliminary data may provide an explanation for this latter finding. They have observed that Cu deficient rodents become Fe deficient. The Fe deficiency in these animals likely results in TH deficiency. Thus, they have formulated the following hypothesis: that Cu and Fe deficiencies lead to reductions in brain TH levels. The associated reduction in TH levels deleteriously affects brain development and therefore, contributes to the derangements in brain development and function observed in Cu and Fe deficient animals. Three specific aims are proposed to test these hypotheses: Aim 1 is to assess the effects of Cu and Fe deficiency during late brain development on circulating and brain TH levels. Aim 2 is to compare the molecular abnormalities associated with Cu, Fe, and TH deficiencies during late brain development. Aim 3 is to assess the effects of TH repletion on molecular abnormalities associated with Cu and Fe deficiencies during late brain development. These data will reveal whether reduced TH levels mediate some of the pathophysiological effects of Cu and Fe deficiency during neonatal brain development. These studies will further provide the preliminary data necessary to conduct mechanistic studies designed to reveal the precise contributions of each constituent towards brain development and function. If the hypotheses prove correct in model animal studies, it will be important to assess the TH status of Cu and Fe deficient infants to ensure that adequate TH status is obtained during neonatal development. Such findings may have a direct and immediate clinical impact, as even transient TH deprivation during late brain development is associated with reduced cognitive abilities later in life.