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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Department of Animal Science and Interdepartmental Nutrition Program, North Carolina State University, Raleigh 27695-7621
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: bovine • copper • manganese • prion
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) that is resistant to proteinase K (PK) degradation (Prusiner et al., 1998).
Prions cooperatively bind Cu ions (Brown et al., 1997; Brown, 1999; Kramer et al., 2001), resulting in a stabilized structure (Hornshaw et al., 1995) and an acquired Cu-dependent superoxide dismutase (SOD)-like activity (Brown et al., 1997; Brown et al., 1999; Wong et al., 2000).
Prions may also bind Mn at the same octapeptide repeats that bind Cu ions (Brown et al., 2000; Brown, 2001). Large increases in brain Mn, coupled with decreases in brain Cu, have been associated with TSE (Wong et al., 2001a,b; Thackray et al., 2002). An imbalance in Cu and Mn that allows Mn ions to replace Cu on the octapeptide repeats may impair the function of PrPc as an antioxidant molecule (Lehmann, 2002; Deloncle et al., 2006) and allow for structural changes (Brown, 2001), resulting in proteinase-resistant prions (Brown et al., 2000; Deloncle et al., 2006). As proposed by Sulkowski (1992), these findings implicate metal ions in the pathogenesis of prion diseases, particularly sporadic TSE.
The hypothesis that brain Cu and Mn perturbations are responsible for key changes in prion characteristics is largely based on in vitro techniques and rodent model-based research. We recently reported that Cu deficiency in the mature bovine reduced brain Cu concentrations but had no detectable effects on brain prion proteins (Legleiter et al., 2007).
The current study was conducted to determine the effects of Cu deficiency, alone or coupled with high dietary Mn, on brain Cu and Mn concentrations and bovine prion protein biochemical characteristics.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Twelve mature Angus cows (6.1 ± 0.7 yr, 640.9 ± 28.4 kg) were used in this study. Initial plasma Cu was similar for all animals, so the cows were randomly assigned (4 cows per treatment) to 1 of 3 treatments: 1) control (adequate in Cu and Mn), 2) Cu-deficient (–Cu), and 3) Cu-deficient plus high dietary Mn (–Cu+Mn). Supplemental Cu was provided from basic Cu2Cl (Micronutrients, Indianapolis, IN), Mn from MnSO4·H2O (Sulfamex, Veracruz, Mexico), and Mo from NaMoO4 (Eastern Minerals Inc., Henderson, NC). Molybdenum, a strong Cu antagonist, was used in conjunction with a low-Cu diet to induce Cu deficiency (Suttle, 1991).
The 12 multiparous cows began the study in September, approximately 60 to 90 d prepartum, and remained on their respective treatments for 360 d. Calves were weaned at approximately 180 d of age. Cows were grazed in treatment groups on tall fescue pastures and were systematically rotated through the pastures to minimize any pasture effects. During the winter months, the cows were grazed on stockpiled tall fescue and supplemented with corn silage. The tall fescue pastures averaged 7.6 mg of Cu/kg of DM and 77.7 mg of Mn/kg of DM, and the corn silage averaged 5.9 mg of Cu/kg of DM and 44.4 mg of Mn/kg of DM.
The Cu-deficient treatments received 75 mg of Mo/ head per d in a corn supplement for the first 14 d to begin the depletion of Cu stores. For the first 120 d, the cows received their respective treatments through a free-choice mineral. The Cu-adequate treatment (control) contained 1,000 mg of Cu/kg of DM and 2,000 mg of Mn/kg of DM in the mineral. Based on free-choice mineral consumption, control cows consumed approximately 130 mg of supplemental Cu/head per d and 260 mg of supplemental Mn/head per d, which equates to 11.1 mg of Cu/kg of DM and 22.2 mg of Mn/kg of DM, assuming an average daily DMI of 2% of BW. These daily intakes of Cu and Mn are adequate to meet the recommended requirements (NRC, 1996).
The –Cu treatment contained 500 mg of Mo/kg of DM and 2,000 mg of Mn/kg of DM. Daily consumption by this treatment group averaged 60 mg of supplemental Mo/head per d or 4.7 mg of Mo/kg of DM, and 240 mg of supplemental Mn/head per d or 18.9 mg of Mn/kg of DM. The –Cu+Mn treatment contained 500 mg of Mo/ kg of DM and 50,000 mg of Mn/kg of DM. Consumption by this treatment group averaged 60 mg of supplemental Mo/head per d or 4.8 mg of Mo/kg of DM, and 6,000 mg of supplemental Mn/head per d or 483 mg of Mn/ kg of DM. From d 120 to 360, the cows received a daily corn gluten feed supplement (1 kg/head per d) that provided Mo (30 mg/head per d) to treatments –Cu and –Cu+Mn. Daily Mo supplementation more effectively depleted liver Cu stores. During this time, the cows continued to receive supplemental Cu (control) and Mn via the free-choice mineral. Supplemental Cu was provided from Cu2(OH)3Cl (Micronutrients), Mn from MnSO4·H2O (Sulfamex), and Mo from NaMoO4 (Eastern Minerals Inc.).
Liver biopsies were obtained, as described by Tiffany et al. (2003), on d 30, 120, 240, and 300 for the determination of liver Cu and Mn concentrations. On d 0, 30, 120, 240, and 300, jugular blood samples were collected into heparinized tubes (Vacutainer 9735, Becton Dickinson, Franklin Lakes, NJ) specifically designed for trace mineral analysis, for plasma Cu determination.
After receiving the treatments for 360 d, the cows were euthanized to acquire brain tissue for prion analysis. A liver sample (approximately 100 g) was collected, transported on dry ice, and frozen (–20°C) until analysis. The obex portion of the brain stem between the cerebellum and spinal cord, which contains the motor nucleus of the vagus nerve, was removed through the occipital foramen by using the spoon technique (USDA, 2004). The obex was transported on dry ice and stored (–80°C) until analysis. Because the obex region of the bovine brain is optimal for BSE detection and prion analysis (USDA, 2004), all data pertaining to brain prions are from obex samples.
Cu and Mn Analysis
Liver, brain, and feed samples used for the analysis of Cu and Mn were prepared by using a microwave digestion (Mars 5, CEM Corp., Matthews, NC) procedure described by Gengelbach et al. (1994). Before microwave digestion, approximately 0.3 g of dried tissue or 0.5 g of dried feed was allowed to digest overnight in trace mineral grade nitric acid (Fisher Scientific, Fair Lawn, NJ). Tissue and feed Cu and Mn were determined by acetylene flame atomic absorption spectrophotometry (AA-6701F, Shimadzu Scientific Instruments, Kyoto, Japan).
Plasma Cu was determined as described by Legleiter et al. (2007). Briefly, plasma was diluted 1:3 (vol/vol) in 5% trace mineral grade nitric acid (Fisher Scientific), centrifuged at 1,200 x g for 20 min, and analyzed for Cu by using acetylene flame atomic absorption spectrophotometry (Shimadzu Scientific Instruments).
Protein Extraction and Western Blot Analysis
Total protein was extracted from brain tissue based on the procedure of Wong et al. (2000), as previously reported (Legleiter et al., 2007). Briefly, 1 g of chilled obex tissue was homogenized on ice in 9 mL of chilled extraction buffer, immediately centrifuged at 5,000 x g for 20 min at 4°C, and the clarified supernatant was equilibrated based on protein concentration. The protein-equilibrated supernatants were aliquoted into microcentrifuge tubes and stored at –80°C until analysis.
Electrophoresis and Western blot (WB) procedures have previously been described in detail (Legleiter et al., 2007). All electrophoresis and WB supplies were purchased from Invitrogen Corp. (Carlsbad, CA), unless otherwise stated. Polyacrylamide gel electrophoresis was performed by using precast NuPAGE Novex 10% Bis-Tris gels and the Novex X-Cell Surelock Mini-Cell electrophoresis system. Magic Mark XP Western Protein Standard molecular weight (MW) marker was used for MW determination. To serve as positive and negative controls, recombinant PrPc ab753 (Abcam Inc., Cambridge, MA) and water, respectively, were treated exactly as the samples. Proteins were separated on gels under denaturing conditions and were subsequently transferred onto a polyvinylidene difluoride membrane. Prion proteins were probed by using anti-PrPc mAb 6H4 (Prionics AG, Schlieren-Zurich, Switzerland) diluted 1:10,000 (vol/vol) in Tris-buffered saline with Tween, and the WB was visualized by using the Western Breeze Chemiluminescent Kit. Western blot images were captured on autoradiography film (Kodak X-OMAT LS, Eastman Kodak Co., Rochester, NY) and analyzed by using Image Quant TL (Amersham Biosciences, Piscataway, NJ). Analysis included band identification, MW determination based on standardized MW markers, and relative optical densitometry for each glycoform of PrPc. All WB analyses were based on 2 to 3 WB replicates.
PK Digestion
To determine the effects of treatment on prion proteinase degradability, samples were first exposed to PK (Bio-Rad Laboratories, Inc., Hercules, CA), as described by Thackray et al. (2002), before PAGE and WB. Brain extracts were digested with 250 µg of PK/mL for 1 h at 37°C (Legleiter et al., 2007). Both unexposed and PK-exposed samples for each animal were run parallel to one another on the same gel. The PK protocol described by Brown et al. (2000), in which samples were exposed to 0, 2, 10, and 25 µg of PK/mL of 10% brain tissue homogenate for 30 and 60 min, followed by PAGE and WB, was used to more sensitively test the effects of treatment on the proteinase degradability of prion proteins. For both PK tests, the reaction was stopped with the addition of loading buffer and reducing agent and by heating to 70°C. Proteinase degradability was determined by comparing the WB elution profiles of PrPc exposed to PK with those not exposed to PK. Complete PK degradation resulted in no detectable immunoreactive prion proteins in PK-treated lanes on the WB.
ELISA
The PrPc concentrations in brain tissue homogenate were determined by using a double-antibody sandwich ELISA kit (Cayman Chemical Co., Ann Arbor, MI), as previously reported (Legleiter et al., 2007). Absorbances were read at 405 nm by using a plate reader (Synergy HT, BioTek Instruments Inc., Winooski, VT). Ellman’s reagent was used as the blank, and absorbances were corrected accordingly.
SOD Analysis
Total SOD activity of the brain tissue homogenates was measured by using an SOD kit (Cayman Chemical Co.). Purified SOD was used to construct a standard curve for quantification of the sample SOD activity. Ten microliters of protein-equilibrated brain tissue homogenate was added to duplicate wells in addition to 200 µL of the radical detector (tetrazolium salt). Addition of 20 µL of xanthine oxidase to each well and incubation for 20 min allowed the formation of superoxide radicals and subsequent color formation. Absorbances were read at 450 nm with a plate reader (BioTek Instruments Inc.). One unit (U) of SOD was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Assays were performed in duplicate, and SOD activities were expressed as units per milligram of protein.
In addition to total SOD activity, Cu:Zn SOD and Mn SOD activities of the brain tissue homogenates were determined by using the same assay. Potassium cyanide (2 mM) was used to inhibit Cu:Zn SOD in the brain tissue homogenates, which subsequently allowed for the detection of Mn SOD activity. Copper:zinc SOD activity was then calculated by subtracting Mn SOD activity from total SOD activity (Brown and Besinger, 1998).
Immunopurified Prion Protein Analyses
Prion proteins from all brain tissue homogenates were purified by immunoprecipitation, similar to that described by Brown et al. (1999). The mAb 6H4 was coupled to protein G-agarose (Sigma-Aldrich Inc., St. Louis, MO) and subsequently mixed with brain tissue homogenates overnight at 4°C in microtube spin columns (SigmaPrep Spin column kit SC1000, Sigma-Aldrich Co., St. Louis, MO). The beads were extensively washed, and the proteins were subsequently eluted from the beads with the addition of 50 mM Gly (pH 4.0) and were neutralized with 100 mM Tris-HCl (pH 8.0). The protein concentrations of the purified PrPc eluates were determined by using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories). The purity of the immunopurified prion eluates was confirmed (>90%) by electrophoresis (Invitrogen) on polyacrylamide gels followed by Coomassie Blue staining, and the presence of PrPc was confirmed via dot blotting. The SOD-like activity of immunopurified PrPc was determined as described for brain tissue homogenates. Immunopurified PrPc eluates were also analyzed for Cu and Mn concentrations by using flameless atomic absorption spectrophotometry (Shimadzu Scientific Instruments).
Statistical Analysis
All data were analyzed as a completely randomized design with PROC MIXED (SAS Inst. Inc., Cary, NC). In the model, treatment served as the fixed effect and animal was used as a random variable. Treatment means were separated by using 2 preplanned, single df, orthogonal contrasts: 1) control vs. –Cu and –Cu+Mn; and 2) –Cu vs. –Cu+Mn. Effects were considered significant at P < 0.05.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Both treatments designed to induce Cu deficiency (–Cu and –Cu+Mn) resulted in liver Cu stores of less than 20 mg/kg of DM and would be considered Cu deficient (Underwood, 1981). Plasma Cu on d 300 averaged 0.49 µg/mL for the Cu-deficient cows, which was less (P = 0.001) than that of control cows, at 0.92 µg/mL.
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). Further, Cu-deficient cows (–Cu) tended (P = 0.06) to have less obex Cu than Cu-deficient animals receiving high dietary Mn (–Cu+Mn; Table 1).
Liver Mn concentrations at the end of the study were similar across treatments (Table 1). Obex Mn concentrations in –Cu+Mn cattle averaged 1.6 mg/kg of DM, which tended to be greater (P = 0.09) than the 1.4 mg/ kg of DM in –Cu cattle (Table 1).
Brain prion protein concentrations were evaluated by using both densitometric analysis of WB for relative comparisons and an ELISA for quantification. Densitometric analysis of WB indicated that total prion protein band relative optical densities were similar across all treatments (Figure 1A). Likewise, brain prion protein concentrations, as determined by ELISA, were similar across treatments (Figure 1B). Thus, based on both ELISA-quantified and WB relative optical densities, PrPc concentrations were not affected by Cu deficiency or Cu deficiency in combination with high dietary Mn.
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). Additionally, the banding patterns of PrPc from all brain tissue homogenates appeared to be similar when visually inspected (Figure 2).
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). Alternatively, if the samples had been positive for BSE, fragments of PK would have been clearly visible on the WB because of their resistance to PK digestion, as has been demonstrated previously (Wong et al., 2001b; Deloncle et al., 2006).
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). However, this visible band was present in all samples, likely because of a very low and less than saturable PK enzyme concentration. Both PK tests indicated that dietary Cu and Mn levels did not affect PrPc proteinase degradability and that animals were BSE negative.
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). Manganese SOD in brain tissue was further increased (P = 0.04) in Cu-deficient cows receiving high dietary Mn (–Cu+Mn) compared with those that were only Cu deficient (–Cu). However, total and Cu:Zn SOD activities of brain tissue homogenates were not affected by treatment. The immunopurified PrPc eluates from the brain tissue homogenates had similar SOD-like activities, averaging 55.7 U/mg (9.7 SEM, P > 0.35), and did not differ in Cu concentration, averaging 0.64 mg of Cu/g of protein (0.24 SEM, P > 0.29). Manganese was detectable at low concentrations (<1 ng/g) in some immunopurified PrPc eluates, but most samples had undetectable concentrations of Mn.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), brain Cu was decreased by approximately 44% in Cu-deficient cows.
Liver Mn concentrations were not affected by the –Cu+Mn treatment, likely because of the low absorption of dietary Mn (Hurley and Keen, 1987) and effective homeostatic control mechanisms for Mn. Homeostatic regulation of Mn in the body is primarily a function of increased hepatobiliary excretion, which can increase up to 200-fold in cattle in response to Mn loading (Hall and Symonds, 1981). Further, feeding high levels of Mn may also decrease intestinal absorption (Abrams et al., 1977). A recent study did demonstrate linear increases in liver and longissimus muscle Mn concentrations in cattle fed supplemental Mn ranging from 0 to 240 mg/ kg of DM (Legleiter et al., 2005); however, these were young, growing cattle and the absolute increases in liver and muscle Mn appeared to have minimal biological significance. Even though liver Mn concentrations did not change in the current study, brain Mn concentrations did tend to increase in cattle receiving high dietary Mn.
Although brain Cu and Mn did tend to be affected by the diet, the magnitude of these brain metal changes, particularly Mn, was less than those seen in TSE-affected tissues. For example, Thackray et al. (2002) detected an approximate 1.5- to 2-fold increase in brain Mn in TSE-infected mice, whereas Wong et al. (2001b) reported up to 10-fold increases in brain Mn in patients having Creutzfeldt-Jakob disease. Thus, it may not be possible via dietary means to induce changes in brain Cu and Mn of the magnitude seen in TSE-affected tissues.
There is substantial evidence linking Cu to prion biology (Brown et al., 1997; Wong et al., 2000; Kramer et al., 2001) and compelling data supporting a relationship between brain metal ion perturbations and TSE. The observational data (Purdey, 2000; Wong et al., 2001b; Thackray et al., 2002) and results from controlled experiments with rodent models and in vitro techniques (Brown et al., 2000; Tsenkova et al., 2004; Deloncle et al., 2006) implicate a Mn for Cu replacement on PrPc in the pathogenesis of TSE. This theory may be particularly relevant to sporadic TSE. However, until now, the relevance of these findings has not been tested in the bovine, which is particularly important given the substantial differences in trace mineral metabolism between rodents and ruminants. The current study using mature cows indicates that Cu deficiency and Cu deficiency coupled with high dietary Mn had minimal effects on the biochemical properties of PrPc. Most notably, all prion proteins were equally and fully degradable by PK, indicating that the treatments did not induce a PrPc to infective isoform conversion, the hallmark change in all TSE.
Brain Cu and Mn concentrations also had no effect on immunopurified PrPc SOD-like activity. However, the purported SOD-like activity of PrPc has been questioned, because the protein was found to have minimal, if any, SOD activity both in vitro (Jones et al., 2005) and in vivo (Hutter et al., 2003). In fact, Jones et al. (2005) found PrPc to have SOD-like activities of <20 U/ mg, which is less than 2% of the level of authentic SOD. This is similar to the PrPc SOD-like activities reported herein, indicating that the activities we detected would have minimal biological significance.
The concentrations of PrPc were similar across all treatments. Waggoner et al. (2000) demonstrated that mice expressing 0, 1, and 10 times the normal concentrations of PrPc had similar brain Cu concentrations and cuproenzyme activities. Taken together, these findings question the importance of PrPc in brain Cu metabolism. There were also no detectable changes in immunopurified PrPc Cu and Mn concentrations. The Cu concentration in the immunopurified PrPc precipitates equates to a molar ratio of 0.25, or 0.25 atoms of Cu per molecule of PrPc. Further, Mn was undetectable in the immunopurified PrPc precipitates, which agrees with a recent study demonstrating that PrPc binds Cu but not Mn (Garnett and Viles, 2003). Although there is intense disagreement regarding PrPc and its ability to bind metal ions, if PrPc does lack the ability to bind Mn, then the hypothesis tested here would indeed be rejected. Overall, these data do not support the hypothesis that an imbalance in dietary Cu and Mn results in perturbed brain Cu and Mn concentrations, leading to altered prion protein biochemical characteristics.
Future research investigating the relationship between Cu, Mn, and brain prions in the bovine should consider the effects of the length and severity of brain metal imbalances. Although brain Cu and Mn appeared to be altered in cows exposed to low-Cu diets and low-Cu diets coupled with high dietary Mn, the magnitude of the perturbations in brain Cu and Mn may not have been great enough to effectively alter PrPc biology. Although the liver Cu concentrations of cows in the Cu-deficient treatments were well below 20 mg/kg of DM, which was indicative of severe Cu deficiency, their average plasma Cu concentration of 0.49 µg/mL on d 300 indicates that they may have been only marginally deficient. Thus, a more severe Cu deficiency may depress brain Cu even further than seen in this study. Likewise, a more significant increase in brain Mn than seen in this study may be required to affect prion biology. Although the respiratory and gastrointestinal tracts are the primary means of Mn entry into the body, most Mn toxicities in humans are associated with inhalation (Oberdoerster and Cherian, 1988), primarily because inhaled Mn is more likely to reach the central nervous system before hepatic clearance (Heilig et al., 2005). In rats, administering MnCl2 via oral gavage and intra-peritoneal injection had no effect on cerebellum and striatum Mn concentrations, but intratracheal instillation significantly increased Mn in both tissues (Roels et al., 1997). Although little is known about the effects of Mn inhalation in the bovine, this route of exposure may produce much greater brain Mn concentrations that could have deleterious effects on PrPc. Finally, the cows in this study were exposed to Cu deficiency and high dietary Mn for approximately 1 yr, with minimal effects on brain prion proteins. Prolonged exposure to these imbalances in Cu and Mn may bring forth different results.
We have previously demonstrated in the bovine that Cu status appears to have minimal effects on brain PrPc (Legleiter et al., 2007); however, to our knowledge, this is the first study investigating the relationships between dietary Cu and Mn and brain prion protein characteristics using the bovine as a model. We conclude that exposing mature cows to low-Cu diets, or low-Cu diets plus high dietary Mn, for 1 yr had minimal effects on prion protein biology.
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Footnotes |
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2 Appreciation is extended to D. Askew, L. Cole, D. Jackson, G. Shaeffer, J. Dickerson, and J. Woodlief for their assistance in sampling and animal care.
3 Corresponding author: Jerry_Spears{at}ncsu.edu
Received for publication April 15, 2007. Accepted for publication June 29, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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  L. R. Legleiter, J. W. Spears, and H. C. Liu Copper deficiency in the young bovine results in dramatic decreases in brain copper concentration but does not alter brain prion protein biology J Anim Sci, November 1, 2008; 86(11): 3069 - 3078. [Abstract] [Full Text] [PDF]
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