Decreased brain copper due to copper deficiency has no effect on bovine prion proteins

Biochemical and Biophysical Research Communications 352 (2007) 884–888 www.elsevier.com/locate/ybbrc

Decreased brain copper due to copper deficiency has no effect on bovine prion proteins q Leon R. Legleiter a, Jason K. Ahola a

b,1

, Terry E. Engle b, Jerry W. Spears

a,*

Department of Animal Science and Interdepartmental Nutrition Program, North Carolina State University, Raleigh, NC, USA b Department of Animal Science, Colorado State University, Fort Collins, CO, USA Received 15 November 2006 Available online 1 December 2006

Abstract Copper (Cu) is believed to be integral in prion biology and the lack of Cu or replacement by other metal ions on prions may be involved in prion diseases. This theory has not been evaluated in the bovine. Thus, mature cows were used to determine the effects of Cu deficiency on brain Cu concentrations and prion functional characteristics. Two Cu states were induced, Cu-adequate (n = 4) and Cu-deficient (n = 4). Copper deficiency resulted in decreased (44%) brain Cu concentrations but had no effect on prion concentrations. Based on Western blot analysis, the molecular weights, glycoform distributions, and elution profiles of brain prions were not affected by Cu status. Importantly, Cu status did not affect prion proteinase degradability as all prions were completely degraded by proteinase K. In conclusion, Cu status affected bovine brain Cu concentrations but had no detectable effects on brain prion protein characteristics. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Bovine; Brain; Copper; Prion protein; Copper deficiency; Bovine spongiform encephalopathy

Prions, proteinaceous infectious particles, are the infectious agents in transmissible spongiform encephalopathies (TSEs) [1]. The family of TSEs includes Creutzfeldt–Jakob disease, scrapie, chronic wasting disease, and bovine spongiform encephalopathy (BSE), among others [2]. These fatal neurodegenerative diseases manifest as genetic, infectious, or sporadic disorders, all of which result from the misfolding of the cellular prion protein (PrPc) isoform to the infective and disease causing isoform (PrPsc) [3] that is partially resistant to proteinase K (PK) degradation [4]. Metal ions, particularly copper (Cu), may be involved in TSEs [5–7]. It has been established that PrPc cooperatively

q Use of trade names in this publication does not imply endorsement by the North Carolina Agric. Research Serv. or criticism of similar products not mentioned. * Corresponding author. Fax: +1 919 515 4463. E-mail address: [email protected] (J.W. Spears). 1 Present address: Department of Animal and Veterinary Sciences, Caldwell Research and Extension Center, University of Idaho, Caldwell, ID, USA.

0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.11.108

binds Cu ions with an affinity in the micromolar range [8– 10]. The binding of Cu ions by PrPc may serve to stabilize the protein [11,12] and allow for acquisition of function [13]. Although the function(s) of PrPc has yet to be elucidated, several proposed functions that involve Cu have been described [14,15]. Most notably, PrPc may play a key role in anti-oxidant defense via its Cu-dependent superoxide dismutase (SOD)-like activity [14,16,17]. Prion proteins may also bind manganese (Mn) ions [5,6]. However, an imbalance in Cu and Mn that allows for Mn ions to replace Cu on PrPc results in dramatic changes in PrPc biochemical characteristics [5,7,18]. Interestingly, large decreases in brain Cu and increases in brain Mn have been associated with TSEs [19–21]. Thus, it appears that Cu is critical for the normal function of PrPc, and its absence may lead to biochemical changes in PrPc that could have implications in TSEs, particularly BSE. However, the relevancy of these findings has not been tested in the bovine. Testing this theory in the bovine is imperative since bovine spongiform encephalopathy is of major concern to animal and human health.

L.R. Legleiter et al. / Biochemical and Biophysical Research Communications 352 (2007) 884–888

Further, the relationship between Cu deficiency and prion protein biology has received little attention. To our knowledge no research has been conducted to evaluate the relationship between Cu deficiency, brain Cu concentrations, and the biology of prion proteins in the bovine. Thus, the objective of this study was to determine the effects of Cu deficiency in the bovine on brain Cu concentrations and prion protein biochemical characteristics. Materials and methods Animals. To achieve two contrasting Cu states (Cu-deficient and Cuadequate), the study consisted of a depletion phase (216 d), designed to induce Cu deficiency, followed by a repletion phase (159 d). Feeding supplemental molybdenum (Mo) and sulfur (S) to ruminants allows for the formation of ruminal thiomolybdates, which tightly bind Cu and thereby greatly reduce absorption. Continual exposure to a low Cu diet coupled with high levels of Mo and S will induce Cu deficiency in cattle [22]. Eight mature Angus cows (8.2 ± 1.8 yr) were fed a low Cu (5.03 mg Cu/kg DM) diet consisting of chopped alfalfa hay and corn stalks supplemented with 5 mg Mo/kg of diet DM and 0.3% sulfur for 216 d to induce Cu deficiency. Copper status was monitored throughout the study via liver Cu concentrations. Liver biopsies were performed using the truecut technique described by Pearson and Craig [23], as modified by Engle and Spears [24]. Cattle were determined to be Cu-deficient when liver Cu concentrations fell below 30 mg/kg DM [25]. Following Cu depletion, cows were randomly assigned to one of two treatments: (1) Cu-deficient ( Cu; no supplemental Cu), (2) Cu-adequate (+Cu; 10 mg supplemental Cu/kg DM from CuSO45H2O). Cattle were individually fed a ground alfalfa hay-based diet (7.9 mg Cu/kg DM) formulated to meet NRC [26] requirements with the exception of Cu. After receiving the treatments for 159 d the cows were humanely euthanized and cerebral and liver tissue was collected and stored at 80 °C until analysis. All animal care, handling, and sampling procedures were approved by the Colorado State University Animal Care and Use Committee before the initiation of the experiment. Copper and manganese analysis. Liver samples were analyzed for Cu concentration as described by Engle et al. [27]. Brain samples for Cu and Mn analysis were prepared using a microwave digestion (Mars 5, CEM) procedure described by Gengelbach et al. [28]. Approximately 0.5 g of dried tissue was added to 5 mL of trace mineral grade nitric acid (Fisher Scientific) and allowed to digest overnight prior to microwave digestion. Copper and Mn concentrations in the ashed brain samples were determined using flame atomic absorption spectrophotometry (GFA-6500, Shimadzu). Protein extraction and Western blot analysis. Total protein was extracted from brain tissue as described by Wong et al. [17]. Approximately 1 g of chilled cerebral tissue was homogenized on ice in 9 mL of chilled extraction buffer (0.01 M PBS, 1% Nonidet P40, 10% w/v complete EDTA-free protease inhibitor cocktail tablets; Roche) with a Polytron homogenizer in a 50 mL polycarbonate tube. Homogenates were immediately centrifuged at 5000g for 20 min at 4 °C and the supernatant was analyzed for total protein using the Bio-Rad DC protein assay kit (BioRad) so that samples could be equilibrated based on protein concentration. The protein equilibrated 10% brain tissue homogenates were aliquotted into microcentrifuge tubes and stored at 80 °C until analysis. Using the Novex X-Cell Surelock Mini-Cell electrophoresis system (Invitrogen), proteins were separated on 10% Bis–Tris NuPAGE gels in MOPS electrophoresis buffer under denaturing conditions (Invitrogen). Magic Mark XP Western Protein Standard molecular weight marker (Invitrogen) was used for molecular weight (MW) determination. Recombinant PrPc (ab753, Abcam) was used as a positive control. The eluted proteins were transferred to a PVDF membrane using the XCell II Blot Module and NuPAGE Transfer Buffer with 10% methanol and 0.1% anti-oxidant. Immunoreactive PrPc were probed with 1:10,000 diluted anti-PrPc mAb 6H4 (Prionics) for 1 h. The Western blot (WB) was visu-

885

alized using the Western Breeze Chemiluminescent Kit (Invitrogen). After extensive rinsing the membrane was incubated with the secondary antibody, anti-mouse conjugated to alkaline phosphatase, for 30 min followed by exposure to 2.5 mL of chemiluminescent substrate (CDP-Star) for 5 min. To capture the WB image, Kodak X-OMAT LS film (Eastman Kodak) was exposed to the membrane and developed using an auto-developer (Kodak X-OMAT Clinic 1 Processor, Eastman Kodak). The WB images were analyzed using Image Quant TL (Amersham Biosciences). Analysis included MW determination, glycoform distribution, and relative optical densitometry. Proteinase K digestion. To determine the effects of Cu status on prion proteinase degradability, samples were exposed to proteinase K (PK; BioRad) as described by Thackray et al. [21] prior to electrophoresis and WB. Briefly, 250 lL PK/mL 10% brain tissue homogenate was allowed to digest for 1 h at 37 °C. Proteinase degradability was determined by comparing WB elution profiles of PrPc exposed to PK with those not exposed to PK. The inability to detect immunoreactive PrPc bands from samples exposed to PK indicated complete degradation by PK. Enzyme linked immunosorbent assay (ELISA). PrPc was quantitated using a double antibody sandwich enzyme-linked immunosorbent assay (Cayman). Brain homogenates were incubated in duplicate wells on a 96well plate pre-coated with anti-PrPc mouse monoclonal antibody for 2 h at room temperature. A standard curve was constructed using known quantities of rPrPc ab753. Wells were rinsed with wash buffer five times followed by addition of the second anti-PrPc antibody conjugated to acetylcholinesterase and incubated for 2 h. Ellman’s Reagent was allowed to incubate for 30 min and absorbances were read at 405 nm using a plate reader (Synergy HT, Bio-Tek). Superoxide dismutase (SOD) analysis. Total SOD activity of the brain tissue homogenates was measured using a SOD assay kit (Cayman). Purified SOD was used as a positive control and to construct a standard curve for sample SOD activity quantitation. Brain tissue homogenates were mixed with 200 lL of the radical detector (tetrazolium salt) in duplicate wells. The addition of 20 lL of xanthine oxidase to each well and incubation for 20 min allowed for the formation of superoxide radicals and subsequent color formation. Absorbances were read at 450 nm using a plate reader (Synergy HT, Bio-Tek). One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radicals. Statistical analysis. Means were compared using one-way analysis of variance (ANOVA) using Proc Mixed in SAS (SAS Institute, Inc.). Effects were considered significant at P < 0.05. Values reported are means with standard errors of the means (SEM).

Results Liver and brain copper The objective of inducing two distinct Cu states (Cu-deficient and Cu-adequate) in the mature bovine was successful. After receiving treatments for 159 d cows receiving +Cu were Cu-adequate while the Cu cows had dramatically less (P = 0.01) liver Cu and were all Cu-deficient (Table 1). The induced Cu deficiency resulted in decreased (P = 0.007) brain Cu concentrations in the Cu-deficient cows compared to Cu-adequate. Brain Mn was not different between the two treatment groups. Prion protein characteristics Based on visual inspection, the electrophoretic profiles of the PrPc after polyacrylamide gel electrophoresis and WB were similar across treatments (Fig. 1). Further, relative optical densities of total PrPc were not different among

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Table 1 Effect of dietary Cu level on liver Cu and brain Cu and Mn concentrationsa,b

2

Treatmentsc Liver Cu, mg/kgd Brain Cu, mg/kg Brain Mn, mg/kg a b c d

23.5 ± 9.9 5.9 ± 0.9 0.7 ± 0.1

+Cu

P values

90.6 ± 9.9 13.4 ± 1.1 0.9 ± 0.1

0.003 0.003 0.47

PrPc, ng/g

1.5

Cu

Samples collected after 159 d on treatment. Values are expressed as means ± SEM. Cu-deficient ( Cu); Cu-adequate (+Cu). Less than 30 mg liver Cu/kg DM is considered Cu-deficient.

1

0.5

0 Cu deficient

1

2

3

4

5

6

7

8

9

10

40 kD →

Treatment Fig. 2. Effects of dietary Cu level on brain prion protein concentration. Brain prion proteins (PrPc) were quantitated using a double-sandwich antibody enzyme-linked immunsorbent assay. Known concentrations of recombinant PrPc (ab753, Abcam) were used to construct a standard curve. Values are expressed as means ± SEM and are based on duplicate assays. (ANOVA, P = 0.95).

30 kD →

Treatment:

-Cu

+Cu

+Cu

-Cu

-Cu

+Cu

-Cu

+Cu

β -actin:

Table 2 Effect of dietary Cu level on prion protein glycoform molecular weights and relative distributionsa Treatmentsb

125 Relative optical density, %

Cu adequate

Cu

+Cu

P values

36.6 ± 0.2 32.3 ± 0.1 27.1 ± 0.2

36.6 ± 0.2 32.1 ± 0.1 27.2 ± 0.2

0.91 0.58 0.91

33.8 ± 0.7 23.7 ± 1.1 42.5 ± 1.0

34.6 ± 0.7 24.7 ± 1.1 40.7 ± 1.0

0.42 0.56 0.24

c

Molecular weight (kDa) Diglycosylated Monoglycosylated Unglycosylated Glycoform distributionc (%) Diglycosylated Monoglycosylated Unglycosylated

100 75 50 25

a

Values are expressed as means ± SEM. Cu-deficient ( Cu); Cu-adequate (+Cu). c Estimated based on Western blot analysis using Image Quant TL (Amersham). b

0 Cu deficient

Cu adequate Treatment

Fig. 1. A Western blot of immunoreactive PrPc from bovine brain tissue homogenates from all eight cows using mAb 6H4 (Prionics). (A) Elution profiles of PrPc from Cu-adequate (+Cu) and Cu-deficient ( Cu) cows. Lane 1, molecular weight markers (Magic Mark XP, Invitrogen). Lane 2, positive control (rPrPc ab753, Abcam). (B) Relative optical densities of prion protein bands, normalized for b-actin, were determined by densitometric analysis of Western blots (n = 3). The mean relative optical density of prion protein bands from Cu-adequate cows is expressed as a percent of the mean optical density of prion protein bands from Cudeficient cows. (ANOVA, P = 0.13).

was unaffected by treatment (Table 2). Copper status also did not affect PrPc proteinase degradability as all prions were completely degraded by PK and no longer visible on the WB (Fig. 3). This indicates that no resistant isoforms (PrPsc) were present in any of the cows and all animals were BSE negative. Total SOD activity of the brain tissue homogenates was not affected by dietary Cu (Fig. 4). Discussion

treatments, indicating brain Cu did not influence PrPc concentrations (Fig. 1). Likewise, using an ELISA to quantitate PrPc, no differences were seen in prion concentrations between the two treatments with an average of 1.2 ng of PrPc/g cerebral tissue (Fig. 2). Molecular weights of the three PrPc glycoforms were not affected by Cu status and ranged from 36.6 to 27.1 kDa for the diglycosylated and unglycosylated, respectively (Table 2). Additional WB analysis showed that the glycoform distribution

This study demonstrated that brain Cu content in the mature bovine is affected by Cu status. Specifically, Cu deficiency resulted in decreased brain Cu. This is a key finding because any effects of brain Cu concentration on prion protein biology are dependent upon the potential for changes in brain Cu concentration to occur. Suttle and Angus [29] noticed an 18% decrease in brain Cu of young calves that were Cu-depleted. Lambs born to unsupplemented ewes, compared to Cu-supplemented ewes,

L.R. Legleiter et al. / Biochemical and Biophysical Research Communications 352 (2007) 884–888

1

2

3

PK digestion: -

+

-

Treatment:

+Cu

4

+

-Cu

5

6

7

8

9 10 11 12 13 14 15

-

+

-

+

-

-Cu

+Cu

-Cu

+

-

+

+Cu

-

+ -Cu

-

887

16

+

+Cu

Fig. 3. Western blots of immunoreactive PrPc from Cu-adequate (+Cu) and Cu-deficient ( Cu) cows used to test proteinase K (PK) degradability. Samples in even numbered lanes (PK+) were digested with 250 lg PK/mL 10% brain tissue homogenate for 1 h at 37 °C prior to loading on the gel.

300

SOD, U/mL

250 200 150 100 50 0 Cu deficient

Cu adequate Treatment

Fig. 4. Superoxide dismutase (SOD) activity of brain tissue homogenates from Cu-deficient and Cu-adequate cows. The SOD activity is expressed as U/mL of protein equilibrated brain tissue homogenate. One unit (U) of SOD is defined as the amount of enzyme required to exhibit 50% dismutation of the superoxide radicals. Values are expressed as means ± SEM and are based on triplicate assays. (ANOVA, P = 0.67).

were born with markedly reduced brain Cu concentrations (60% decreased brain Cu, [30]; 74% decreased brain Cu, [31]). Mature ewes that received no supplemental Cu also had decreased (64%) brain Cu concentrations [30]. Few studies have given attention to the effects of Cu status on brain tissue Cu concentrations in adult animals, particularly mature cows. We believe this is the first report of decreased brain Cu in mature cows due to Cu deficiency. Further, the magnitude of the decrease in brain Cu, 44% less brain Cu in Cu cows versus +Cu cows, is noteworthy. The decreased brain Cu due to Cu deficiency had no apparent effects on the biochemical properties of PrPc. The demonstrated ability of PrPc to bind Cu ions both in vitro [11,12] and in vivo [8], coupled with the observation that PrPc-null mice have significantly lower brain Cu concentrations, has suggested that PrPc and brain Cu are intimately linked. However, in the present study the concentrations of PrPc, similar to those reported in sheep [32], were not affected by brain Cu content. This is in agreement with Waggoner et al. [33], who demonstrated that mice expressing 0, 1, and 10 times the normal level of PrPc had similar brain Cu concentrations and cuproenzyme activities, causing them to question the importance of PrPc in brain Cu metabolism.

Prions have also been demonstrated to have a Cu-dependent SOD-like activity that contributes to the total SOD activity of brain tissue [14,16]. We were unable to detect any changes in total SOD activity in the brain tissue samples, indicating that the decreased brain Cu concentrations in Cu cows had no effect on total SOD enzyme activity or the purported SOD-like activity of PrPc. More recent research has questioned the role of PrPc as an anti-oxidant molecule, as the protein was found to have minimal, if any, SOD activity both in vitro [34] and in vivo [35]. Importantly, the proteinase degradability of PrPc was unaffected as all animals across both treatments had readily degradable PrPc when exposed to PK. The hallmark change in the conversion of PrPc to PrPsc is incurred partial protease resistance. Thus, the decreased brain Cu did not induce a PrPc to PrPsc conversion. Further, it has been demonstrated that a Mn for Cu substitution on PrPc allows for increased protease resistance [5,7]. Although we did not measure PrPc associated Cu and Mn, the lack of biochemical changes in PrPc, particularly PK degradability, may indicate that the Cu deficiency did not result in decreased PrPc bound Cu or a replacement of Cu with Mn. All other biochemical characteristics of PrPc measured were unaffected by cow Cu status and subsequent brain Cu content. We analyzed WB so that any changes in glycoform MW or relative distribution would be detected. Significant changes in these properties would have strengthened the data supporting a functional relationship between Cu and PrPc. However, finding no differences in PrPc between cows with large differences in brain Cu concentrations questions the relevancy of previously reported findings regarding PrPc and Cu. There are compelling data supporting a relationship between brain metal ion perturbations and TSE. Several studies have implicated a Mn for Cu replacement on PrPc in the pathogenesis of TSE [5,7,18,19,21]. However, the relevancy of these findings has not been tested in the bovine. It would be interesting to examine the effects of a Cu deficiency coupled with exposure to high dietary Mn on bovine brain PrPc. In the present study we noticed no differences in brain Mn concentrations between the Cu-adequate and Cu-deficient cows. To our knowledge this is the first study designed to examine the relationship between Cu deficiency, brain Cu concentration, and prion protein biology. We conclude

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