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North Carolina State
University Return to main menu Copper Deficiency Alone and in the Presence of high Dietary Manganese
does not greatly affect Brain Prion Protein Characteristics in the Mature
Bovine: Implications for Trace Minerals Cu and Mn and Bovine Spongiform
Encephalopathy L. R. Legleiter, J. W. Spears,
K. E. Lloyd, S. L. Hansen, and R. S. Fry
Summary Brain Cu concentrations
were decreased in copper (Cu) deficient cows and brain manganese (Mn) tended
to be higher in Cu deficient cows receiving high levels of dietary Mn. Bovine brain prion concentrations,
proteinase degradability, glycoform distributions, and molecular weight were
not affected by Cu deficiency alone or coupled with high dietary Mn in mature
Angus cows. Brain and prion protein
superoxide dismutase (SOD) activities were altered due to treatment. Current ongoing research is being
conducted to better describe the relationship between Cu, Mn, and prion
protein biology in the bovine. Introduction Bovine spongiform
encephalopathy (BSE) and similar neurodegenerative diseases such as scrapie
and Cruetzfeldt-Jakob disease are caused by abnormal prion proteins,
apparently mutated from the naturally occurring cellular prion (Prusiner,
1991). While
it is known that abberrent 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 that Cu plays important roles both in the structure and function of
the prion protein (Lehmann, 2002).
Brown et al. (1997) showed that the cellular prion protein binds up to
six, depending on species, Cu ions.
Further, there may be a relationship between Cu and prion antioxidant
activity, as the prion protein has superoxide dismutase activity when Cu is
bound (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 in rodent models and cell cultures, limited work
has been done in the bovine. We feel
it is imperative that similar research be conducted utilizing the bovine as a
model because Cu absorption and metabolism in the ruminant is significantly
different than that of the rodent.
Further, the bovine serves as a relevant model for BSE. The
research described here and current ongoing studies examine the effects of Cu
deficiency alone and in the presence of high dietary Mn on prion protein
concentrations, prion proteinase degradability, prion protein superoxide
dismutase-like activity, and other biochemical properties indicative of
abnormal prion biology that may have significance with respect to BSE. Materials
and methods Twelve mature Angus cows
were used to determine the effects of Cu deficiency alone and in the presence
of high dietary Mn on brain Cu and Mn concentrations and brain prion protein
characteristics. The cows were
randomly assigned to one of the following three treatments: 1) control, 2) Cu deficient (-Cu), and 3)
Cu deficient plus high dietary Mn (-Cu+Mn; Table 1). The Cu antagonist molybdenum (Mo) was used
to induce Cu deficiency. The cows
were grazed on pasture and received treatments via a daily corn gluten
feed-based supplement. The cows were
on study for 240 days and were Cu deficient, based on liver Cu concentrations
(< 20 mg Cu/kg DM), at approximately day 140. Liver and brain samples
were taken immediately after euthanasia.
Tissue Cu and Mn concentrations were measured via flame and flameless
atomic absorption spectrophotometry.
Prions were purified using immunoprecipitation and were
electrophoretically separated and transferred to polyvinylidene diflouride
membranes. After Western blotting,
prions were probed with primary and secondary antibodies and visualized using
chemiluminescence. Superoxide
dismutase (SOD) activity was measured in both brain tissue homogenates and
purified prion proteins. Proteinase K
degradability of prion proteins was determined by incubation with proteinase
K followed by Western blotting. Brain
prion protein concentrations were determined using an enzyme-linked
immunosorbent assay. Results and
Discussion All brain tissue analysis
reported here was conducted on the obex region of the brain which is where
prion proteins are highly concentrated and where BSE testing is
conducted. Liver Cu concentrations
were lower (P = 0.001) in animals
receiving the Cu deficient treatments (-Cu and -Cu+Mn) compared to control
cows (Table 2).
Most importantly, the liver Cu concentrations were less than 20 mg/kg
DM which indicates induced Cu deficiency was achieved. Brain Cu concentrations tended (P = 0.06) to be lower in Cu deficient
animals compared to the controls.
Further, animals receiving -Cu tended (P = 0.06) to have lower brain Cu than those animals on the -Cu+Mn
treatment. Liver Mn was not affected
by treatment, but brain Mn tended (P =
0.09) to be higher in -Cu+Mn animals compared to -Cu animals. These data indicate that brain Cu and Mn
are affected by Cu deficiency and dietary Mn, respectively. Decreasing brain Cu and increasing brain
Mn allowed us to test the hypothesis that Mn can replace Cu on the prion
protein and cause biochemical changes relevant to BSE. 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 on the Western blot, was not affected by treatment. Further,
using Western blots, the prion protein glycoform distribution was not altered
due to treatment. Prion protein
concentrations were similar across treatments and averaged 2.1 ug/g of brain
tissue. While Cu deficiency and
high dietary Mn did not affect prion concentrations, molecular weights, and
proteinase degradability, there were effects on SOD activity (Figures 1 and 2). There are two primary isoforms of the SOD
enzyme. The Cu/Zn SOD isoform uses Cu
in its active site while Mn SOD utilizes Mn at the catalytic site. Brain tissue total SOD activity (Cu/Zn SOD
and Mn SOD combined) and Cu/Zn SOD activity were not affected by treatment
(Figure 1). However, Mn SOD activity
of brain tissue homogenates was greater (P
= 0.05) in Cu deficient animals compared to the controls. Mn SOD was further increased (P = 0.04) in the brain of animals
receiving the -Cu+Mn treatment compared to those receiving the -Cu treatment. Similarly, Mn SOD of purified prion
proteins was increased (P = 0.02)
in Cu deficient animals compared to the controls (Figure 2). This increase in prion Mn SOD activity
tended (P = 0.09) to increase total
SOD activity of prion proteins. Altering the brain Cu and
Mn concentrations appears to have affected brain and prion protein SOD
activities. While brain Cu was
decreased in the Cu deficient animals, this did not decrease brain and prion
Cu/Zn SOD activities. However, brain
and prion Mn SOD activities were increased in animals that were Cu deficient
alone (-Cu) and in the presence of high dietary Mn (-Cu+Mn). It is possible that a Cu deficiency that
leads to depressed brain Cu results in increased oxidative stress that is
countered by increased Mn SOD activities.
However, the biological significance of these changes in Mn SOD
activities is unclear. Ongoing
Research Research in our
laboratory is continuing to elucidate the relationships between Cu, Mn, and
prion proteins in the bovine brain. Specifically, we hope to quantify the bound Cu and Mn ions on
the purified prion proteins from this and other studies. Further, we have conducted several studies
to test the effects of age of animal on the relationship between Cu, Mn, and
prion proteins in the bovine. Implications Except for the changes in
SOD activity, Cu deficiency alone and in the presence of high dietary Mn had
minimal effects on bovine prion protein biochemical characteristics. Most notably, the proteinase degradability
of the prion proteins, one of the underlying changes associated with prion
diseases, was not affected by treatment.
Thus, it would appear that a relatively short-term Cu deficiency in
the bovine, alone or coupled with high Mn intake, does not significantly
affect prion protein biology in a manner that would implicate it in the
physiopathology of BSE. References Brown, D.
R., K. Qin, J. W. Herms, A. Madlung, J. Manson, R. Strome, P. E. Fraser, T.
Kruck, A. Bohlen, W. Schulz-Schaeffer, A. Giese, D. Westway, and H.
Kretzschmar. 1997. The cellular prion protein binds copper in
vivo. Nature 390:684-687. 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. Lehmann,
S. 2002. Metal ions and prion diseases.
Curr. Opinion Chem. Biol.
6:187-192. 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. Table 1. Treatments designed to alter Cu and Mn status. Table 2. Treatment effects on tissue Cu and Mn
concentrations. Figure
1. Brain tissue SOD activity. Figure
2. Purified prion protein SOD
activity. |
