1 Laboratory of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
2 Laboratory of Veterinary Radiology, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
Correspondence
Motohiro Horiuchi
horiuchi{at}vetmed.hokudai.ac.jp
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ABSTRACT |
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Present address: Prion Disease Research Center, National Institute of Animal Health, Kannondai, Tsukuba, Ibaragi, 305-0856, Japan.
Present address: Laboratory of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818, Japan.
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INTRODUCTION |
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Conversion of PrPC to PrPSc is believed to involve direct interaction of the two PrP isoforms. Although the molecular mechanism of conversion is not yet fully understood, it is known that mature PrPC expressed on the cell surface is a substrate for PrPSc formation, and a process that involves a conformational transformation takes place in subcellular compartments associated with the degradation pathway of PrPC, including a sphingolipid-rich membrane microdomain, called a lipid raft (Caughey & Raymond, 1991; Naslavsky et al., 1997
; Vey et al., 1996
).
Because of the emergence of variant CJD and iatrogenic CJD by dura matter transplantation, especially in Japan, the establishment of therapeutics for prion disease is urgently needed. Therapeutics have been directed at the binding of the two PrP isoforms, as well as the process of conformational transformation, since the conversion of PrPC to PrPSc is associated with neuronal pathogenicity. To date, many substances have been reported to inhibit PrPSc formation in cell culture and/or cell-free systems, including amyloid-binding dyes (Caughey & Race, 1992), sulfated glycosaminoglycans (Caughey & Raymond, 1993
), tetrapyrrole compounds (Caughey et al., 1998
), cysteine protease inhibitors (Doh-Ura et al., 2000
), substituted tricyclic derivatives such as chlorpromazine and quinacrine (Doh-Ura et al., 2000
; Korth et al., 2001
), branched polyamines (Supattapone et al., 1999
, 2001
), peptides (Chabry et al., 1998
; Soto et al., 2000
) and conversion-incompetent PrP (Holscher et al., 1998
; Horiuchi et al., 2000
; Kaneko et al., 1997
). Some of these have already been examined in vivo. For instance, sulfated glycosaminoglycans and tetrapyrrole compounds were effective when administered at early stages of infection or simultaneously with the scrapie-affected brain inoculum (Ehlers & Diringer, 1984
; Ladogana et al., 1992
; Priola et al., 2000
). Polyene antibiotics prolonged the incubation period, even when administered at the middle-late stage of infection (Demaimay et al., 1997
), but the effects appeared to depend on the prion strains and host animals studied (Demaimay et al., 1999
; Xi et al., 1992
). Recently, Doh-Ura and colleagues (2004)
showed that intraventricular administration of pentosan polysulfate and quinine prolonged the incubation periods in a prion-infected transgenic mouse model, even at a late stage of infection (Doh-Ura et al., 2004
; Murakami-Kubo et al., 2004
). Further in vivo studies are expected to lead to the establishment of effective therapeutics for prion diseases. However, to achieve more efficient therapeutics, it is essential to elucidate the mechanisms of action and to investigate proper delivery of drugs based on pharmacokinetics.
Anti-PrP antibodies have also been reported to inhibit the formation of PrPSc in cultured cells and/or cell-free systems (Enari et al., 2001; Horiuchi & Caughey, 1999
; Kaneko et al., 1995
; Peretz et al., 2001
). Transgenic mice expressing an anti-PrP mAb on B cells (Heppner et al., 2001
), immunization with recombinant PrP (Sigurdsson et al., 2002
) and passive immunization with an anti-PrP mAb (White et al., 2003
) antagonized the peripheral inoculation of scrapie-affected brain inoculum. These in vivo experiments suggested the possible use of anti-PrP antibodies as a therapy for prion diseases. However, it remains unclear how anti-PrP antibodies can antagonize PrPSc formation in cells. To address this point, in the current study, we evaluated a panel of anti-PrP mAbs against diverse epitopes for inhibition of PrPSc formation. We found that a mAb recognizing the octapeptide repeat sequence, a region that is not essential for PrPSc formation, reduced PrPSc accumulation in cells persistently infected with prions. Furthermore, our data suggest a possible link between cell-surface retention of PrPC by anti-PrP antibodies and inhibition of PrPSc formation in cells.
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METHODS |
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Cell culture.
The mouse neuroblastoma cell line Neuro2a (CCL-131; ATCC) was cultured in Dulbecco's modified Eagle's medium (ICN Biomedicals) with 10 % fetal bovine serum (FBS) and non-essential amino acids. Mouse neuroblastoma cells persistently infected with prions, originally established by Race et al. (1987), were cloned by limiting dilution. Subclone I3/I5-9, which possessed a high level of PrPSc, was used in this study. I3/I5-9 cells were maintained in Opti-MEM (Invitrogen) containing 10 % FBS and cells passaged fewer than 20 times were used for experiments.
Treatment of cells persistently infected with prions and sample preparation.
Almost-confluent I3/I5-9 cells in 25 cm2 flasks were split 1 : 20 into 35 mm tissue culture dishes. On day 2, the medium was replaced with 3 ml Opti-MEM containing 4 % FBS and each test compound or mAb, and the cells were cultured for a further 3 days. For PrPC detection, the cells were washed with PBS and lysed with 300 µl lysis buffer A (1 % Zwittergent 3-14, 150 mM NaCl, 50 mM Tris/HCl, pH 7·5) supplemented with protease inhibitors (2 mM EDTA, 1 µg pepstatin ml1, 2 µg leupeptin ml1, 2 µM bestatin and 1 µg aprotinin ml1). After the removal of cell debris by low-speed centrifugation, samples were centrifuged at 45 000 r.p.m. for 30 min at 4 °C using the TLA 100.3 rotor of a Beckman Optima TLX and the resulting supernatants were used as a source of PrPC. For the detection of PrPSc, cells were lysed with 300 µl lysis buffer B (5 mM EDTA, 0·5 % Triton X-100, 0·5 % sodium deoxycholate, 150 mM NaCl, 10 mM Tris/HCl, pH 7·5) and kept on ice for 30 min. Cell debris was removed by centrifugation for 5 min at 1000 r.p.m. A portion of the sample (10 %) was removed for determination of protein concentration using the DC protein assay (Bio-Rad) and the remaining portions were treated with 20 µg proteinase K ml1 for 20 min at 37 °C. Proteolysis was terminated by the addition of 1 mM Pefabloc (Roche). The samples were then treated with DNase I (100 µg ml1) and RNase A (5 µg ml1) for 15 min at room temperature and centrifuged at 70 000 r.p.m. for 2 h at 4 °C using the TLA 100.3 rotor of a Beckman Optima TLX. The resulting pellets were dissolved in SDS-PAGE sample buffer.
SDS-PAGE and immunoblotting.
SDS-PAGE was carried out using NuPAGE 12 % Bis-tris gels and MOPS-SDS running buffer according to the manufacturer's instructions (Invitrogen). After SDS-PAGE, proteins were transferred on to Immobilon-P PVDF membranes (Millipore) using a Transblot Mini Cell wet-type blotting apparatus (Bio-Rad) and NuPAGE transfer buffer (Invitrogen) at 60 V for 2 h. Immunoreactive proteins were detected using X-ray film as described elsewhere (Kim et al., 2004). For quantitative analysis, immunoreactive proteins were visualized using the Western-Star Protein detection kit (TROPIX) according to the supplier's instructions and processed with an LAS-1000 lumino image analyser (Fujifilm). The intensity of the bands was quantified using Science Lab 98 Image Gauge software (Fujifilm).
Flow cytometric analysis.
Adherent cells were treated with ice-cold PBS containing 0·1 % collagenase (Wako) and dispersed by pipetting. Cells were washed with 0·5 % FBS in PBS (FBS/PBS) and incubated with anti-PrP mAbs diluted with 0·5 % FBS/PBS for 30 min on ice. Cells were washed three times with 0·5 % FBS/PBS and incubated with 1 : 2000-diluted Alexa 488-labelled Fab fragment of goat anti-mouse IgG (Molecular Probes) for 30 min. After washing, cells were stained with 5 µg propidium iodide ml1 in 0·5 % FBS/PBS for 5 min and analysed using an EPICS XL-ADC flow cytometer (Beckman Coulter). All procedures were carefully carried out under chilled conditions.
Indirect immunofluorescence assay.
Cells grown in eight-well slides (Nunc) were fixed with 100 % methanol for 20 min at 20 °C. Fixed cells were blocked with 5 % FBS/PBS for 30 min at room temperature, after which they were incubated with hybridoma supernatants or mAbs diluted in 1 % FBS/PBS for 30 min at room temperature. After washing with PBS, cells were incubated with 1 : 1000-diluted Alexa 488-labelled Fab fragment of goat anti-mouse IgG for 30 min. Finally, the slides were mounted with PBS containing 50 % glycerol and 1 % n-propyl gallate (Wako) and examined using a fluorescence microscope equipped with a cooled CCD unit (CoolSNAP HQ; Roper).
Cell growth and cytotoxicity.
The effect of mAbs on cell growth was analysed using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay (Ishiyama et al., 1996) and cytotoxicity was analysed by lactate dehydrogenase (LDH) release assay using the LDH-Cytotoxic Test (Wako).
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RESULTS |
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Fig. 3 shows the long-term effect of mAbs on PrPSc formation. Treatment for 6 days with mAb 110, 44B1, 31C6 (Fig. 3
) or 72 (data not shown) reduced PrPSc to an almost undetectable level, and no re-emergence of PrPSc was observed in the following 6 and 12 days of incubation in the absence of mAbs. On the contrary, mAbs that did not bind to cell-surface PrPC showed little effect on PrPSc accumulation even after long-term treatment.
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Effect of anti-PrP mAbs on total amount of PrPC
Fig. 1(a, lower panel) shows total PrPC in the I3/I5-9 cells treated with mAbs for 3 days. The intensities of PrPC bands were normalized with
-sarcomeric actin on the same blot and PrPC levels relative to cells treated with negative control mAb (P1-284) are indicated at the bottom. Although there was a certain degree of variation, no marked difference was observed in the total amount of PrPC. In contrast, after long-term treatment (6 days), the total amount of PrPC in I3/I5-9 cells treated with mAb 110 or 44B1 appeared to be higher than that with the negative-control mAb or other anti-PrP mAbs (Fig. 3
, top right panel). To confirm this further, we repeated the same experiment at least three times for the four inhibitory mAbs, 110, 31C6, 44B1 and 72. Relative PrPC levels in cells treated with these four mAbs were 168±38, 88±23, 183±54 and 103±33 %, respectively. These results suggested that the effect of mAbs on PrPC level varied depending on the mAb: mAbs 110 and 44B1 increased total PrPC levels following long-term treatment, while mAbs 31C6 and 72 did not affect the total PrPC level.
Cell-surface localization of the mAbPrPC complex
The N-terminal portion of PrP, including the octapeptide repeat, is not essential for PrPSc formation and/or prion propagation (Flechsig et al., 2000; Rogers et al., 1993
). The finding that not only the mAbs recognizing the C-terminal part of PrP, such as 31C6 and 44B1, but also mAb 110 inhibited PrPSc accumulation in the neuroblastoma cells, together with the fact that only the mAbs that bound to cell-surface PrPC showed an inhibitory effect, implied that the mAbPrPC interaction on the cell surface is essential for inhibition of PrPSc accumulation. To investigate this further, we analysed the dynamics of anti-PrP mAbs after their binding to the cell surface (Fig. 4
). Neuro2a cells were treated with 10 µg mAb 31C6 ml1 for 1 h, after which the cells were cultured for an additional 4 h without mAb. Cells were then harvested and stained with an Alexa 488-conjugated secondary antibody. As a control, cells cultured with mAb 31C6 for 1 h were immediately stained with the secondary antibody. Flow cytometric analysis showed no difference in fluorescence intensity between the two preparations, suggesting that the mAbPrPC complex remained on the cell surface, even after the additional 4 h culture in the absence of mAb. As I3/I5-9 cells are established by repeated limiting dilution, Neuro2a cells may not be a suitable uninfected control for I3/I5-9 cells. Hence, we carried out the same experiment using I3/I5-9 cells. It is known that elimination of PrPSc parallels the reduction of prion infectivity. Considering biosafety issues, we used I3/I5-9 cells cured of PrPSc by long-term treatment with mAb 44B1 for flow cytometric analysis. mAb 31C6 (Fig. 4
) and the three other inhibitory mAbs, 110, 44B1 and 72 (data not shown), showed the same retention of mAbPrPC complexes as observed with Neuro2a cells.
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DISCUSSION |
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Although the cell-surface binding of mAb 110 was lower than that of the other mAbs (Fig. 1b, left panel), it inhibited PrPSc formation as efficiently. This may be explained by the presence of an 18 kDa N-terminally truncated PrPC. This truncated PrPC fragment is produced by cleavage of PrPC around residue 112 during the recycling process (Chen et al., 1995
) so that it is not recognized by mAb 110. Recently, Mishra et al. (2002)
reported that the N-terminally truncated form comprised as much as 4050 % of PrPC on the cell surface. This could account for the lower signals obtained using mAb 110. Because N-terminally truncated PrPC is unlikely to act as a substrate for prion propagation and/or PrPSc formation (Lawson et al., 2001
; Weissmann, 1999
), the binding of mAb 110 to PrPC possessing the N-terminal portion is apparently sufficient for the inhibition of PrPSc formation.<~?tpb=7pt>
In this work, we have demonstrated both quantitatively and qualitatively that mAbs that bind to cell-surface PrPC remain attached to the membrane, even after withdrawal of the mAbs from the culture medium. This suggests that the mAbPrPC complex on the cell surface is not preferentially internalized into the cell. Mature PrPC expressed on the cell surface is thought to be internalized via either clathrin-coated or -uncoated vesicles from which it enters the degradation pathway (Peters et al., 2003; Shyng et al., 1994
; Sunyach et al., 2003
). Because PrPSc formation is believed to take place in the subcellular compartments that include cell membrane during the degradation pathway (Borchelt et al., 1992
; Caughey & Raymond, 1991
), it is possible that mAb treatment could interfere with the regular PrPC metabolism simply by retaining it on the cell surface. We suspected that the cell-surface retention of PrPC would result in an increase in total PrPC. Actually, two mAbs, 110 and 44B1, obviously increased the total amount of PrPC, while two other mAbs 31C6 and 72 did not influence the total amount of PrPC. It is conceivable that binding of mAbs to specific epitopes of cell-surface PrPC might result in downregulation of PrPC synthesis; however, further experiments are required to resolve this.
It was recently reported that polyclonal antibodies against dimeric recombinant PrP inhibited PrPSc formation in the cell, while the corresponding Fab fragments had little effect on PrPSc formation (Gilch et al., 2003). This suggests that antibody-mediated cross-linking of PrPC on the cell surface is important for inhibition of PrPSc formation. Whether cross-linking of PrPC by IgG is required for the retention of the mAbPrPC complex under our experimental conditions remains to be determined. Treatment of cells persistently infected with prions using antibodies against the laminin receptor precursor/laminin receptor (LRP/LR) reduced PrPSc accumulation (Leucht et al., 2003
). Because binding of LRP/LR to PrPC could be involved in PrP metabolism (Gauczynski et al., 2001
), it is conceivable that antibodies interfere with the interaction between PrPC and a molecule(s) that participates in PrPC internalization.
Many reagents, including small molecules, recombinant PrP and anti-PrP antibodies, have been identified as potential inhibitors of prion propagation. It is important to elucidate their mechanisms of action, not only for the establishment of therapeutics but also for an understanding of prion replication. In the present study, we have demonstrated that blocking of the internalization of PrPC with anti-PrP mAbs prevents PrPSc accumulation. Although anti-PrP mAbs recognizing specific epitopes have recently been reported to induce neuronal death in the hippocampus and cerebellum (Solforosi et al., 2004), we have not found an apparent adverse effect on the cell growth and clinical manifestation by intraventricular inoculation of the anti-PrP mAbs used in this study (data not shown). Further analyses using prion-infected animals are necessary for evaluation of anti-PrP antibodies as therapeutics for treating prion diseases.
After the submission of this paper, a paper was published by Perrier et al. (2004) in which it was described that recognition by mAb SAF34 of the octapeptide repeat region on the N-terminal part of human PrP inhibited PrPSc formation in prion-infected neuroblastoma cells.
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ACKNOWLEDGEMENTS |
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Received 16 March 2004;
accepted 26 July 2004.