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From the Department of Pathology,*
University of
Melbourne, Melbourne, Australia; the Mental Health Research
Institute,
Parkville, Victoria, Australia; and
the Center for Molecular Biology,
University
of Heidelberg, Heidelberg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the normal function of PrPc is unknown, a possible role for PrP in modulating neuronal oxidative stress has been recently identified. Studies using PrP-/- neuronal cultures have shown that a lack of PrP expression increases susceptibility to superoxide anions (O2-)14 or copper-mediated toxicity.19 This appears to be related to decreased copper/zinc superoxide dismutase (Cu/ZnSOD) activity in PrP-/- cultured neurons and brain.20 The exposure of wild-type neurons to PrP106–126 also reduced Cu/ZnSOD activity and predisposed cells to increased O2- toxicity. If PrPc is associated with antioxidant activity, then the interaction between PrP106–126/PrPres and PrPc could impair these antioxidative defenses (through loss of function) and predispose neurons to increased free radical toxicity.
Another important antioxidant pathway in neurons involves glutathione reduction (GSH) and oxidation. The reduced form of GSH is critical for detoxification of cellular hydrogen peroxide (H2O2), prevention of lipid and protein oxidation,21 and the transport, detoxification, and sequestration of copper.22,23 GSH metabolism and peroxidation appear to play a central role in a number of neurodegenerative disorders.24,25 We therefore studied the relationship between PrPc expression on H2O2 toxicity and the glutathione pathway. As H2O2 is a major metabolic and excitotoxic byproduct, we wished to determine whether PrP-/- neurons were more vulnerable to peroxide toxicity than WT neurons. Our studies revealed a significant increase in H2O2 toxicity in PrP-/- neurons. This may be related to the decreased glutathione reductase (GR) activity observed in PrP-/- neurons in vitro and in vivo. The toxic PrP106–126 peptide also induced a significant reduction in GR activity in neuronal cultures, suggesting that altered GR activity may be relevent to PrPres toxicity in vivo. These studies support the important extensive role PrPc has in antioxidant activity in neurons and that perturbations to PrP function in CJD may predispose neurons to peroxidative damage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PolyL-lysine and 3,[4,5 dimethylthiazol-2yl]-2,5 diphenyltetrazolium bromide (MTT) were from Sigma Chemical Co. (Australia). Glutamine, glucose and gentamicin sulfate were obtained from Gibco BRL (Australia). Fetal calf serum (FCS) and horse serum were from the Commonwealth Serum Laboratories (Sydney, Australia). 1,3-bis(2-chloroethyl)-1-nitrosurea (BCNU) was from Bristol Myers Squibb (UK).
Primary Cultures
PrP-/- mice were obtained from Dr C. Weissmann, Institut fur Molekularbiologie I (Zurich, Switzerland). The absence of PrP expression in the PrP-/- mice has been previously reported26 and was confirmed by immunoblot analysis in our laboratory (unpublished observations). Wild-type (WT) control mice (C57BL6J x 129/Sv) correspond to genetically matched mice from which the PrP-/- mice were derived26 and are of the same background as those used in previous studies of PrP-/- mice.14,19,20,27 Primary cultures of cerebellar granule neurons (CGN) were established from PrP-/- and WT mice using previously described methods.28 Cerebellar granule neurons were plated at 350,000 cells/cm2 in Basal Medium Eagle (BME) (Gibco BRL) with 10% (v/v) FCS. Cultures were maintained at 37°C in 5% CO2. Cytosine arabinofuranoside Ara C (10 µmol/L) was added at day 2.
Measurement of Neuronal Cell Viability and Cell Death
Cell viability was determined by measuring the redox potential of the cells using the MTT assay as previously described.28,29 Culture medium was replaced with 0.6 mg/ml MTT in control salt solution (CSS) for 2 hours. The supernatant was removed and cells solubilized with dimethyl sulfoxide. Aliquots of 100 µl were measured with a spectrophotometer at 570 nm. The MTT assay compared well to other cell viability assays such as Alamar Blue (Serotec, Sydney, Australia) and provided an accurate indication of neuronal viability under the conditions used.
Induction of Oxidative Stress
Peroxide toxicity was induced in day 4 CGN cultures by adding H2O2 diluted from 30% stock solution to culture media for 3, 6, and 24 hours, followed immediately by MTT assay of cell viability. Data represents the mean and standard error of the mean (SE) of experiments performed in at least 3 to 4 cultures measured in triplicate. To determine the effect of BCNU on H2O2 toxicity, BCNU was added 1 hour before addition of H2O2. A BCNU stock solution (1 mmol/L) was prepared in absolute ethanol and stored at -70°C before use.
Exposure of Neuronal Cultures to Amyloidogenic Peptides
PrP106–126 and a scrambled version of this peptide were synthesized as previously described.30 Peptides were dissolved in fresh, serum-free culture medium at a concentration of 2 mg/ml before use. PrP106–26 displayed fibrillogenic and neurotoxic properties consistent with previous reports.18,28 CGN cultures were exposed to 80 µmol/L PrP106–126 or 80 µmol/L scrambled PrP106–126. These concentrations did not alter cell viability after 24 hours’ exposure.
Measurement of Glutathione Peroxidase and Glutathione Reductase Levels in Primary Cultures
Determination of glutathione peroxidase (GPx) and GR levels in CGN cultures and brain tissue were performed using the respective assay kits per the manufacturer’s instructions (Oxis International Inc., R&D Systems, Minneapolis, MN). Protein estimation was performed with a BCA protein assay kit (Pierce, Rockford, IL) on aliquots of homogenized cultures before centrifugation. For GPx activity, control and H2O2-treated CGN cultures and 2-week-old brain tissue were homogenized for 10 seconds in a polytron homogenizer in ice-cold 50 mmol/L Tris-HCl containing 1 mmol/L 2ß-mercaptoethanol, pH 7.5, centrifuged at 8500 x g for 10 minutes at 4°C, and the supernatants were stored at -70°C. For GR activity, control, H2O2- and peptide-treated CGN cultures and 2-week-old brain tissue were homogenized as above in ice-cold 60 mmol/L KPO4 buffer, pH 7.5, centrifuged, and stored at -70°C. Determination of GR activity was based on the equation: (A340 minutes/6220 mol/L-1 cm-1) x 5 = mU/ml, where A340 = change in absorbance per minutes at 340 nm, 6220 mol/L-1 cm-1 = the molar extinction coefficient of NADPH, and 5 = assay dilution. Determination of GPx activity was achieved by the equation: (A340 minutes/6220 mol/L-1 cm-1) x 16 = mU/ml, where 16 = assay dilution. Results for both GR and GPx assays were then adjusted to give data as mU/mg protein. One GR unit reduces 1 µmol of oxidized glutathione (GSSG) per minute at 25°C and pH 7.6. One GPx unit consumes 1 µmol NADPH per minute at 25°C and pH 7.6.
Statistical Analysis
Data represent the mean and SE of experiments performed in at least 3 to 4 cultures measured in triplicate. In all cases, comparisons of data were performed with analysis of variance and Newman-Keuls tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether PrPc expression affects neuronal survival under our basal culture conditions, CGN cultures were grown from PrP-/- and WT mice for up to 8 days and cell viability was determined with the MTT assay. PrP-/- and WT cultures grown with the astrocyte growth inhibitor, Ara C, added at day 2 showed no significant difference in cell viability between day 1 and day 8 in vitro. (Cell viability in PrP-/- cultures was 97 ± 4.0% of the WT value after 8 days in vitro). These data indicate that under the conditions used, PrPc expression did not affect basal cell survival in neuronal cultures.
Previous studies have shown that PrP-/- neurons
are more susceptible to superoxide toxicity.14,20
To
determine whether PrP-/- neurons are more
susceptible to other forms of oxidative stress, we tested
PrP-/- neurons against peroxidative toxicity.
Four-day-old cultures of PrP-/- and WT CGN
(3.5 x 105
cells/cm2)
were exposed to different concentrations of
H2O2 for 3, 6, and 24 hours
and cell viability was measured using the MTT assay. The cell density
used is consistent with previous studies on CGN oxidative
toxicity.31-33
The MTT assay measures cellular redox
potential and is a sensitive indicator of neuronal cell
viability.29
The MTT assay was used in preference to cell
death assays (such as the lactate dehydrogenase assay) as nonlethal
changes to neuronal viability are an important indicator of neuronal
dysfunction and increased susceptibility to oxidative
insults.28,34,35
H2O2 induced a significant
decrease in neuronal viability after 3 hours’ exposure (Figure 1A)
. At this time point there was no
difference in cell viability between PrP-/- and
WT cultures. However, after a 6- or 24-hour treatment with
H2O2, cell viability was
significantly lower (*P < 0.05, **P <
0.01) in the PrP-/- cultures as compared to WT
treated with 
40 µmol/L
H2O2 (Figure 1, B and C)
. At 100 µmol/L
H2O2,
PrP-/- neurons revealed approximately 40 and
60% lower viability than WT neurons after 6 and 24 hours’ treatment,
respectively (**P < 0.01, Figure 1, B and C
). These
findings clearly demonstrate that, in vitro, neurons lacking
PrPc are significantly more susceptible to
peroxide toxicity than WT neurons.
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Perturbations to the activity of the GSH-metabolizing enzymes GPx or GR can mediate increased sensitivity to H2O2 in neurons.25 To determine whether increased susceptibility to H2O2 toxicity in PrP-/- neurons was related to perturbed GSH metabolism, we measured the activity of these two main GSH-associated enzymes, GPx and GR, in CGN cultures and cerebellum.
Assessment of GR levels in cultures under basal conditions revealed
that PrP-/- neurons had approximately 17%
lower GR activity than WT neurons at day 4 in vitro
(*P < 0.05, Figure 2
,
Control). To determine whether
H2O2 exposure affected the
GR activity in PrP-/- or WT neurons, 60
µmol/L H2O2 was added to
cultures for 6 or 24 hours. In both WT and
PrP-/- neurons, 60 µmol/L
H2O2 did not significantly
alter GR activity after 6 hours (Figure 2)
. However, a 24-hour exposure
to 60 µmol/L H2O2 caused
a 31% lower GR activity in PrP-/- neurons
compared to WT neurons (**P < 0.01, Figure 2
). This
treatment regimen (60 µmol/L
H2O2 for 24 hours) resulted
in 35% lower cell viability in PrP-/- compared
to WT neurons (Figure 1C)
. To determine whether these in
vitro GR levels are consistent with in vivo GR levels,
2-week-old WT and PrP-/- mouse cerebella were
assayed and GR activity was found to be approximately 30% lower in
PrP-/- mice (**P < 0.01,
Figure 2
). These findings indicate that the difference in GR activity
between WT and PrP-/- neurons in
vitro is not a culture-derived artifact.
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,
Control). Similarly, a 6- and 24-hour
H2O2 exposure (60 µmol/L)
caused no significant change in GPx activity in
PrP-/- or WT cultures (Figure 3)
. The activity
of GPx was also measured in tissue extracts obtained from 2-week-old
mouse cerebellum and was the same in both WT and
PrP-/- mice (Figure 3)
. These data indicate
that PrPc expression is probably not an important
modulator of neuronal GPx and is consistent with a recent
report.36
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To determine whether the reduced GR activity in
PrP-/- neurons could account for the
differences in cell viability in response to
H2O2 toxicity, the
GR-specific inhibitor BCNU was used.37
WT and
PrP-/- CGN were treated with different
concentrations of BCNU and measured after 6 hours for GR
activity. BCNU induced a dose-dependent reduction in enzyme
activity in both PrP-/- and WT cultures
(*P < 0.05, **P < 0.01, Figure 4A
). The susceptibility of the
BCNU-treated cultures to
H2O2 was tested by
cotreating with 60 µmol/L
H2O2. There was a
significant decrease in cell viability with increasing BCNU
concentration in both PrP-/- and WT cultures
(*P < 0.01, Figure 4B
). BCNU alone had no effect on
cell viability at concentrations up to 100 µmol/L. To examine whether
a direct correlation exists between GR activity and
H2O2 toxicity, we compared
WT neurons exposed to 0.1 µmol/L BCNU with untreated
PrP-/- neurons. Both cultures revealed a GR
activity of approximately 27 mU/mg protein (Figure 4, A and C)
and after exposure to 60 µmol/L
H2O2 for 6 hours there was
a similar decrease in cell viability (Figure 4C)
. These data
demonstrate a direct link between GR activity and peroxide toxicity and
confirm that the lower GR activity in PrP-/-
neurons can result in increased sensitivity to
H2O2 toxicity.
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It has been demonstrated that the toxic PrP fragment PrP106–126
can reduce SOD activity and GSH levels in primary
neurons.20,38
We therefore tested the effect of
PrP106–126 on neuronal GR activity. Exposure of WT and
PrP-/- CGN to a nontoxic concentration of
PrP106–126 (80 µmol/L) for 24 hours resulted in significantly lower
GR activity compared to untreated controls (approximately 50 and 44%
decrease in GR activity, respectively, *P < 0.01,
Figure 5A
). Exposure of CGN to a
nonfibrillogenic scrambled version of PrP106–126 failed to alter GR
activity. Exposure of PrP106–126-treated cultures to 60 µmol/L
H2O2 for 6 hours resulted
in significantly increased toxicity compared to
H2O2 alone
(*P < 0.01, Figure 5B
). These results provide evidence
that decreased GR activity may be relevent to the mechanism of PrP
toxicity in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We believe the increased vulnerability of PrP-/- neurons to H2O2 is relevant to the pathophysiology of prion disease, because H2O2 toxicity is an important mediator of oxidative stress in neurons.24 Peroxides play an important role in the pathology of several neurodegenerative disorders, as indicated by perturbations to GPx and GR activities and increased lipid peroxidation in Alzheimer’s disease, CJD, and amyotrophic lateral sclerosis.25 Peroxides may also be involved in Aß and PrP106–126 toxicity in vitro14,21 and scrapie in vivo.40 H2O2 is generated from many intracellular metabolic processes or through excitotoxicity41 and is released by activated microglia.42 H2O2 can also interact with copper to form the hydroxyl radical (OH·), one of the most destructive biological molecules generated in cells.43 Because PrPc is involved in neuronal copper binding44 and modulation of copper neurotoxicity,19 interactions between H2O2 and copper via PrPc could promote neuronal degeneration in prion disease.
To counter H2O2 toxicity,
neurons contain the GSH pathway and catalase.41
The
finding that PrP-/- neurons had lower GR, but
not GPx, activity may be related to the increased sensitivity of
PrP-/- neurons to
H2O2. This finding is
consistent with the lower SOD activity observed in
PrP-/- neurons, which resulted in increased
sensitivity to O2-
toxicity.20
The lack of significant differences in GPx
levels between PrP-/- and WT neurons under
basal conditions or after
H2O2 exposure is also
consistent with recent studies in PrP-/- and WT
myoblasts, myotubes, and mouse brain.36,45
This indicates
that increased peroxide toxicity in PrP-/-
neurons is not due to impaired GPx activity. However, the analogously
lower GR activity in PrP-/- neurons in
vitro and PrP-/- mouse cerebellum in
vivo indicates that lower GR levels are unlikely to be irrelevant
culture artifacts. Moreover, inhibition of GR activity in WT neurons
with BCNU, a GR-specific inhibitor,46-48
increased
susceptibility to peroxide toxicity and correlated well with the GR
level and toxicity in PrP-/- neurons (Figure 4C)
. Furthermore, the PrP106–126 peptide reduced GR levels (this
study) and SOD activity20
in neurons and increased
susceptibility to H2O2
toxicity, supporting the relevance of these findings to prion diseases.
A consequence of lower GR activity may be increased copper toxicity. Copper is a highly reactive metal and Cu(I) can combine with H2O2 to form OH·.49 GSH is an important chelator of intracellular copper49 and may contribute to transport of copper to Cu/ZnSOD and metalloproteins.50 Lower GR activity would reduce the cellular capacity to regenerate glutathione, resulting in the destabilization of Cu homeostasis and increased OH· formation. In support of this, we have shown that GSH depletion dramatically increases Cu toxicity with no effect on Fe toxicity in cultured neurons.35 Future studies will need to establish that the level of reduced GSH is lower in PrP-/- neurons due to the impaired GR activity. Perovic et al38 have shown that PrP106–126 depletes neuronal GSH levels in vitro, a phenomenon that may be related to the reduced GR activity we observed in neurons treated with PrP106–126. Significantly, treatment of cultures with flupirtine to prevent GSH depletion also inhibited PrP106–126 neurotoxicity.38 These studies strongly suggest that perturbations to GSH metabolism could be involved in neurodegeneration in prion disease. PrPres may induce a similar loss of GR and GSH function in neurons and therefore increase susceptibility to both O2- and H2O2. The fact that PrP106–126 inhibits Cu/ZnSOD20 and GR activity in both WT and PrP-/- neurons indicates that the peptide can induce oxidative stress without direct interaction with PrPc. Only WT neurons are susceptible to PrP106–126-induced cell death,12 suggesting that resistance to PrP106–126 toxicity by PrP-/- neurons may result in part from lower ROS generation in these cultures. This is consistent with the findings that microglia (which can release O2-, H2O2, and glutamate) are required for PrP106–126 toxicity in vitro and that WT microglia are more toxic than PrP-/- microglia when incubated with PrP106–126.12
The evidence presented here and in previous studies44,45
indicates that PrPc expression modulates neuronal
antioxidant activity. This is supported by the reduced antioxidant
activity (GR and Cu/ZnSOD) in neurons devoid of
PrPc in vitro and in vivo
and by the correlation between PrPc expression
levels and Cu/ZnSOD activity in neurons overexpressing
PrPc.45,51
Whether
PrPc directly interacts with GR and Cu/ZnSOD or
acts upstream of antioxidant enzyme activation is still to be
determined. The ability of PrP106–126 to inhibit GR and Cu/ZnSOD
activity in PrP-/- neurons indicates that these
antioxidant enzymes are affected by regulatory molecules downstream
from PrPc (Figure 6)
. Moreover, lower SOD and/or GR
activity could also result from oxidative damage to the antioxidant
enzymes. Interestingly, Barker et al52
demonstrated that
oxidative stress in rat brain significantly and specifically inhibited
GR activity, an enzyme particularly susceptible to oxidative damage
from peroxynitrite (ONOO-), a by-product of
O2- and NO. Other studies have
similarly shown that GR is particularly susceptible to oxidative damage
from excessive free radical generation in vitro and in
vivo.53,54
However, the pathways linking
PrPc expression to Cu/ZnSOD and GR activity are
not understood and their study will be important in understanding how
PrP modulates these key antioxidant enzymes.
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Acknowledgements |
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Footnotes |
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Supported by grants from the National Health and Medical Research Council of Australia (to R. C. and C. L. M.) and The National Pituitary Hormones Advisory Council (to R. C. and S. J. C.).
Accepted for publication July 7, 1999.
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