The Effects of Copper and Manganese Status on Brain Copper and Manganese Concentrations, Prion Characteristics and Antioxidant Capacity in the Bovine[1]

 

L. R. Legleiter and J. W. Spears

 

Summary

Brain prion concentrations were higher for copper (Cu) adequate versus Cu deficient mature Angus cows.  Prion proteinase degradability and apparent molecular weight were not affected by Cu status.  Current ongoing research is being conducted to better describe the relationship between trace mineral status and prion protein biology in the bovine.     

 

Introduction

Research is continuing to support the Prusiner hypothesis (Prusiner, 1991), that in bovine spongiform encephalopathy (BSE) and similar neurodegenerative diseases such as scrapie and Cruetzfeldt-Jakob disease the infective agents are abnormal prion proteins, apparently mutated from the naturally occurring cellular prion.  While it is known that prions are involved in neurodegenerative diseases, the biological role of the cellular prion has yet to be elucidated.  Additionally, the cause of the mutation of normal prion proteins to their infective isoform is unclear. 

Several studies have indicated a functional role of Cu in the structure of the prion protein, as well as a relationship between Cu and prion antioxidant activity (Brown et al., 2001).  It has been hypothesized that a deficiency of Cu could lead to a change in the structure and function of the prion proteins, especially in the presence of high levels of manganese (Mn) which may replace the Cu (Thackray et al., 2002).  Whether prion protein metal imbalances are a cause or effect of prion diseases has yet to be determined.        

While extensive work in this area has been conducted using mouse models and cell cultures, limited work has been done in the bovine.  The preliminary study described here and current ongoing studies examine the effects of Cu and Mn status on brain Cu and Mn levels, prion protein concentrations, Cu and Mn binding, prion proteinase degradability, prion protein superoxide dismutase-like activity and oxidative stress markers in the bovine.

 

Preliminary study

Twelve copper-deficient multiparous Angus cows were used to determine the effects of Cu repletion on brain Cu concentration and brain prion protein characteristics.  Cows were considered Cu-deficient based on liver Cu concentrations (< 30 mg Cu/kg DM) after receiving a low Cu diet supplemented with 5 mg molybdenum/kg of diet DM and 0.3% sulfur for 216 d.  Copper-deficient cows received one of three treatments; 1) control (no supplemental Cu), 2) organic Cu (10 mg/kg DM), and 3) inorganic Cu (10 mg/kg DM), during a 159 d repletion phase. 

Liver and brain samples were taken immediately after euthanasia.  Liver Cu concentrations were greater (P = 0.001) in supplemented (treatments 2 and 3) than non-supplemented (control) cows.  Brain Cu concentrations tended (P = 0.17) to be greater for Cu-supplemented cows than control cows.  Brain prion proteins were extracted from brain tissue by homogenization followed by centrifugation.  Prion proteins were electrophoretically separated and transferred to polyvinylidene diflouride membranes.  Prions were probed with primary and secondary antibodies, visualized using chemiluminescence, and relative optical densities of bands quantified.  Relative optical densities of bands were greater (P = 0.02) for Cu-supplemented cows than non-supplemented cows which corresponds to increased levels of prion proteins.  Proteinase degradability was not affected by treatment as all prions were completely degraded after exposure to proteinase K.  The apparent molecular weight of prion proteins, as determined by comparison to a molecular weight standard, was not affected (P = 0.27) by treatment.  These data suggest that prion protein concentrations are affected by the Cu status of the animal. 

 

Ongoing research

Current research is being conducted to assess the effects of mineral status on prion proteins in mature cows, feedlot steers and yearling calves.  Treatments will generally consist of: control (complete and adequate mineral profile), Cu deficient, and Cu deficient in the presence of excess Mn.  While BSE and other prion diseases have largely been reported in mature animals (greater than 30 months of age) prion proteins may be altered earlier in life, thus these studies include animals less than 30 months of age.  Additionally, the yearling calves will have been on treatment since the third trimester, in utero, up to a year of age to determine if damn mineral status affects offspring prion protein biology.       

After euthanasia brain samples will be collected for prion protein analysis.  Homogenized brain samples and immunoprecipitated prions will be analyzed for Cu and Mn concentrations, proteinase degradability, and prion concentrations.  Polyacrylamide gel electrophoresis and western blotting will allow for comparison of prion molecular weights and glycoform distributions.  Brain oxidative markers, protein ubiquitination, lipid peroxidation and protein oxidation will also be measured.

 

Implications   

Results from the preliminary study indicated that Cu status affected brain prion protein concentration.  Ongoing research will enable us to better describe the relationship between trace minerals and brain prion protein biology in the bovine, which may enhance our understanding of BSE and other prion diseases. 

 

References

Brown, D. R., C. Clive, and S. J. Haswell.  2001.  Antioxidant activity related to copper binding of native prion protein.  J. Neurochem.  76:69-76.

Prusiner, S. B.  1991.  Molecular biology of prion diseases.  Science.  252:1515-1522.

Thackray, A. M., R. Knight, S. J. Haswell, R. Bojdoso, and D. R. Brown.  2002.  Metal imbalance and compromised antioxidant function are early changes in prion disease.  Biochem. J.  362:253-258.