1 Institute of Neuropathology, University Hospital of Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland
2 Division of Psychiatry Research, University of Zürich, August Forel Str. 1, CH-8008 Zürich, Switzerland
3 Pulmonary Division, Children's Hospital, Harvard Medical School, Boston, USA
Correspondence
Markus Glatzel
markus.glatzel{at}usz.ch
Adriano Aguzzi
adriano{at}pathol.unizh.ch
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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Formally, the accumulation of PrPSc in brain and extraneural organs is the result of the differential between its de novo generation and its clearance. Surprisingly, clearance of PrPSc is exceedingly efficient in vivo. Very large amounts of PrPSc injected into brain or peritoneum of Prnp-deficient mice (Büeler et al., 1992), which are incapable of replicating prions, resulted in a very fast decrease to concentrations below detectability (Büeler et al., 1993
).
Although the relative resistance of PrPSc to protease digestion is the basis of laboratory assays for prion infections, it is obvious that PrPSc undergoes proteolytic processing in cell-culture models of prion diseases and in vivo (Büeler et al., 1993; Enari et al., 2001
; Glatzel et al., 2003
). Proteolytic processing of PrPC and PrPSc differs and cleavage of PrPSc may lead to the occurrence of a 20 kDa PrPSc fragment referred to as C2, whereas cleavage of PrPC leads to the formation of an 18 kDa fragment termed C1 (Chen et al., 1995
; Jiménez-Huete et al., 1998
). Although protein kinase C-dependent cleavage of PrPC by ADAM10 and TACE has been reported, the protease responsible for PrPSc cleavage remains enigmatic (Checler & Vincent, 2002
; Vincent et al., 2001
). It was suggested that the alleged PrPSc-processing protease could belong to the metalloprotease family of enzymes, yet no studies have been undertaken until now to address the exact nature of this activity (Jiménez-Huete et al., 1998
).
Neprilysin, a zinc metalloprotease, has been shown to catabolize neurotoxic amyloid- protein that accumulates in Alzheimer's disease (Iwata et al., 2001
; Mohajeri et al., 2004
). The localization of neprilysin, a type II integral membrane protein, the relatively broad substrate specificity of this peptidase and the amyloid-
-degrading properties suggest a possible involvement of neprilysin in PrPC/PrPSc catabolism (Turner, 2003
). Involvement of neprilysin in degradation of aggregated PrPSc is further supported by similarities between PrPSc and amyloid-
. Both proteins are deposited in the extracellular space in the form of amyloid and both proteins may act as neurotoxins, either as aggregates or in the form of soluble oligomers (Aguzzi & Haass, 2003
).
Here, we investigated whether PrPC or PrPSc represents a substrate for the enzymic activity of neprilysin by exposing mice (i) entirely lacking neprilysin, (ii) expressing reduced amounts of neprilysin or (iii) overexpressing neprilysin, to infectious prions.
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METHODS |
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Measurement of the Nep peptidase activity.
Neprilysin enzyme activity was measured in brain homogenates of Nep-tg (n=4) and wild-type (wt) littermates (n=4) as described previously, with minor modifications (Shirotani et al., 2001). In brief, 34 µg protein homogenized in 150 mM NaCl, 100 mM Tris/HCl, 1 % Triton X-100 (pH 7·8) was incubated for 1 h at 37 °C with 100 µM Z-Ala-Ala-Leu-p-nitroanilide (ZAAL-pNA; Bachem) in 50 mM HEPES buffer (pH 7·2). Thereafter, 0·8 mU leucine aminopeptidase (Sigma) was added to the reaction mixtures, incubated for an additional 20 min at 37 °C (Mohajeri et al., 2004
) and OD405 was measured. To inhibit neprilysin activity, 40 µM thiorphan (Sigma) was added for 5 min at room temperature before the addition of ZAAL-pNA. The results were confirmed by measuring several amounts of the Nep-tg samples diluted in similarly prepared brain homogenate of a Nep-KO mouse that exhibits no neprilysin enzyme activity (data not shown).
Scrapie infections.
Mice were infected intraperitoneally (i.p.) with 100 µl brain homogenate diluted in PBS and containing 6 or 3 logLD50 intracerebral (i.c.) units of the Rocky Mountain laboratory (RML) scrapie strain (passage 5). For i.c. inoculations, 30 µl inoculum with 3x105 or 3x102 LD50 i.c. units was administered. Mice were monitored every second day and scrapie was diagnosed according to standard clinical criteria. Mice were sacrificed on the day of onset of terminal clinical signs of scrapie. The data for terminal disease are given as means±SD.
Infectivity bioassay with tga20 indicator mice.
Assays were performed on 1 % (w/v) brain homogenates. Tissues were homogenized in 0·32 M sucrose with a microhomogenizer (TreffLab) diluted in PBS/5 % BSA. When the solution appeared homogeneous, it was spun for 5 min at 500 g. Supernatants (30 µl) were inoculated i.c. into groups of five or six tga20 mice (Fischer et al., 1996). Incubation time until development of terminal scrapie sickness was determined and infectivity titres were calculated for 1 g inoculated tissue by using the relationship y=11·450·088x, where y is the number of i.c. LD50 units and x is the incubation time (days) to terminal disease (Prusiner et al., 1982
).
Western blot analysis.
Brain homogenates were adjusted to 8 mg protein ml1 and treated with proteinase K where indicated (50 µg ml1, 30 min, 37 °C). Total protein (50 µg) was electrophoresed through an SDS-PAGE gel (12 %). Proteins were transferred to nitrocellulose by wet blotting. Membranes were blocked with Tris/HCl-buffered saline/Tween 20 (TBST) containing 5 % Top Block, pH 7·4 (Sigma), incubated with mAb POM 1 to PrP (M. Polymenidou, M. Vey & A. Aguzzi, unpublished data) or with mAb 56C6 to neprilysin (Novocastra) and visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) (Glatzel et al., 2001).
Histology and immunohistochemistry.
Paraffin-embedded sections from brain were stained with haematoxylin and eosin. Immunostaining to glial fibrillary acidic protein (GFAP) was performed with a rabbit antiserum against GFAP (1 : 300 dilution; DAKO) and detected with biotinylated swine anti-rabbit serum (1 : 250 dilution; DAKO) and diaminobenzidine (Sigma).
Histoblots.
Histoblots were performed according to Taraboulos et al. (1992). Briefly, frozen sections were mounted on uncoated glass slides and pressed immediately on a nitrocellulose membrane wetted in lysis buffer. Membranes were air-dried for at least 24 h. For detection, they were rehydrated in TBST and limited proteolysis was performed by using proteinase K concentrations of 50 and 100 µg ml1 at 37 °C for 4 h. Blots were then denatured in 3 M guanidinium thiocyanate for 10 min and blocked for 1 h in 5 % non-fat milk. Incubation with primary antibody XN to PrP was carried out at a dilution of 1 : 2000 in 1 % non-fat milk at room temperature for 1 h (Glatzel & Aguzzi, 2000
). Detection was accomplished with an alkaline phosphatase-conjugated goat anti-mouse antibody (1 : 2000). Visualization was achieved with nitro blue tetrazolium and BCIP according to the protocols of the supplier (Sigma).
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RESULTS |
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PrPC is not a substrate for the enzymic activity of neprilysin
Because of the relatively broad substrate specificity of neprilysin, we investigated whether PrPC might represent a substrate for degradation by neprilysin (Turner, 2003). If this were correct, one would expect decreased protein levels of PrPC in mice overexpressing neprilysin. Conversely, the complete lack of neprilysin expression in neprilysin-knockout mice could lead to increased protein levels of PrPC in these mice.
In order to test these possibilities, we assessed the relative quantities of PrPC in mice overexpressing and lacking neprilysin by Western blot analysis (Fig. 1d). There were no significant alterations of PrPC protein levels between these transgenic lines of mice and appropriate wt controls, as evidenced by the ratios of the signal intensities of PrPC to corresponding
-actin bands (0·21, 0·27 for Nep/; 0·27, 0·24 for Nep-tg; 0·19, 0·23 for wt; Fig. 1d
). The fact that neither depletion nor overexpression of neprilysin seems to influence the turnover of PrPC showed convincingly that PrPC did not represent a substrate for the enzymic activity of neprilysin.
Depletion of neprilysin does not alter incubation times to terminal prion disease
Mice deficient for neprilysin (Nep/) and mice with decreased levels of neprilysin expression (Nep+/) were challenged with various amounts of infectious prions and the incubation time to terminal prion disease was measured. After high-dose (3x105 LD50) or low-dose (3x102 LD50) i.c. prion injection, there was no significant difference in incubation times compared with wt mice (high dose: 164±4 days, n=6 for wt; 161±4 days, n=7 for Nep+/; 165±7 days, n=7 for Nep/ mice; low dose: 192, 197 days, n=2 for wt; 200±17 days, n=5 for Nep+/; 188±10 days, n=8 for Nep/ mice; Fig. 2).
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The lack of an effect of neprilysin on prion pathogenesis is further corroborated by biochemical and histological analysis of terminally sick mice. Immunoblot analysis confirmed that there were comparable amounts of PrPSc in the brains of terminally sick wt, Nep+/ and Nep/ mice (Fig. 3). We investigated the distribution of protease-resistant PrPSc in wt, Nep+/ and Nep/ mice by histoblot analysis of CNS tissue (Fig. 4
). This analysis shows an even, cortically accentuated distribution of PrPSc throughout the entire cerebral hemispheres. Accordingly, histological analysis of mice with complete or partial absence of neprilysin showed unaltered patterns of vacuolization and reactive astrogliosis within the CNS tissue when assayed 100 days following i.c. prion challenge (Fig. 5
).
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DISCUSSION |
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As a candidate, we have investigated whether neprilysin is involved in degradation of PrPC or PrPSc. This was achieved by subjecting genetically modified mice expressing either reduced levels of neprilysin or no neprilysin at all and mice overexpressing neprilysin to infectious prion preparations. We did not find any evidence that PrPC or PrPSc represents a target for neprilysin. On the contrary, the evidence that neprilysin plays no role at all in prion/PrPSc is overwhelming: all investigated parameters of prion pathogenesis, including the amount and the distribution of PrPSc in infected brains at various time points, the amount of infectivity in the CNS and neuropathological changes of mid- and end-stage brains, were identical in mice lacking or overexpressing neprilysin and wt mice.
Proteins belonging to the metalloprotease family, most of which are expressed in CNS tissue, seem to be involved in degradation of extracellular deposited amyloid in vivo. It has been shown that neprilysin is the most potent amyloid-degrading metalloprotease (Iwata et al., 2001). As neprilysin displays a broad substrate specificity and is localized subcellularly in the vicinity of PrP (Iwata et al., 2001
; Turner, 2003
), it is surprising that it has no impact on prion pathogenesis.
The findings described here do not negate the involvement of other metalloproteases in PrPSc catabolism. Indeed, studies focusing on possible substrates of metalloproteases provided evidence that matrix metalloprotease MMP-9 is able to degrade amyloid- and might possess PrPSc-degrading properties (Backstrom et al., 1996
). The availability of reliable cell-culture models for prion diseases, in combination with high-throughput tools to assess prion infectivity, should significantly facilitate the investigation of PrPSc proteolysis (Enari et al., 2001
; Klöhn et al., 2003
).
Given that our understanding of PrPSc metabolism is far from complete, this field of prion research continues to represent an important target for the future. In view of the fascinating developments in Alzheimer's disease research, where insights into amyloid- precursor protein processing have led to the identification of the mechanisms of amyloid-
generation and, based on this knowledge, to the development of therapeutic strategies, the investigation of the proteolytic cascade leading to the removal of aggregated PrPSc might hold the key to the discovery of novel prophylactic or therapeutic strategies against prion diseases (Aguzzi & Haass, 2003
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 December 2004;
accepted 10 March 2005.
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