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No propagation of prions in mice devoid of PrP
Cell, Vol. 77, 967-966,
July I, 1994, Copyright
0 1994 by Cell Press
Matters Arising
No Propagation of Prions in Mice Devoid of PrP The “protein only” hypothesis proposes that the prion, the infectious agent of transmissible spongiform encephalopathies such as scrapie, mad cow disease, and CreutzfeldJakob disease, is PrPSC, a modified form of the normal host protein PrPC. Propagation of the prion is ascribed to the conversion of PrPC into PrPS” under the influence of PrPS” (Prusiner, 1991). PrP knockout mice (Pm-p”), when inoculated with mouse scrapie prions, remained healthy and free of scrapie symptoms for at least 2 years while wild-type controls all died within 6 months (Biieler et al., 1993). Infectivity in brain was determined in two independent titration experiments. Scrapie-inoculated wild-type animals revealed a prion titer of 105.‘-1 O’.’ LDEUU/g brain tissue by 8 weeks and 107.7-10B.B LDso U/g by 20 weeks. In the case of scrapie-inoculated animals devoid of PrP, brain homogenates taken at 2,8, 12, and 25 weeks after inoculation showed no infectivity. One single sample, a pool of four brain homogenates prepared 20 weeks after infection, had a low titer of infectivity, about 103 LDm U/g. The most likely explanation was persistence of inoculum or contamination; however, low level propagation of scrapie
Table
1. Titration
Experiment 1
of Scrapie
Infectivity
Time after Inoculation
in Brain Homogenates
agent had to be considered (Biieler et al., 1993; Cheseboro and Caughey, 1993). We have now reassayed the 20-week-old samples from this experiment and confirmed the titer found previously (Table 1, experiment 1). In addition, we assayed brains of PrP knockout mice from the same experiment 25,33, and 48 weeks after inoculation and found no infectivity (Table 1, experiment 1). We repeated the entire experiment, inoculating a different group of PrP knockout mice with mouse scrapie prions and titrating brain homogenates at 16, 18, 20, and 22 weeks after inoculation (Table 1, experiment 2). The 16-, 18-, and 22-week-old samples showed no scrapie infectivity. For the 20 week timepoint, four single brains were titrated on eight indicator mice each. Of 32 animals, 1 showed scrapie symptoms at 261 days; however, all others stayed healthy for more than 300 days, at which time the experiment was ended. Under the unlikely assumption that the single diseased indicator mouse was not the result of contamination, this would represent an average content of about 20 LDso units per brain. Because each animal was inoculated with about 10’ LDw units, it is clear that prions were not propagated in the absence of PrP. Prusiner et al. (1993), using the same strain of PrP knockout mice, also failed to find propagation of infectious agent. We conclude that PrP is not only essential for the devel-
of Scrapie-Inoculated
Prp”b Mice
Number of Brains Pooled
Concentration Homogenate
20 20 25 25 33 33 33 48 48
4 4 4 4 Single Single Single Single Single
5 1 5 1 5 5 5 5 5
6 5 0 0 0 0 0 0 0
of of of of of of of of of
16 18 20 20 20 20 22
4 4 Single Single Single Single 4
1 1 1 1 1 1 1
0 0 0 1 0 0 0
of 6 of a of 6 of6 of 8 of 0 of 9
(weeks)
of (%)
CD1 Mice with Scrapie Symptoms 6’ 6b 5 6 4 5 6 5 6
Inoculation of Pm-p” mice, titration of infectivity, and monitoring for scrapie symptoms were described earlier (Biieler et at., 1993). In brief, Pm-p mice were inoculated intracerebrally with 30 pl of a IO-fold diluted (phosphate-buffered saline, 5% bovine serum albumin) Chandler-derived mouse prion preparation (ML; obtained from S. B. Prusiner). Brains were recovered at the times indicated. In experiment 1 the hemisphere ipsilateral and in experiment 2 the hemisphere contralateral to the one inoculated were processed to a 10% (w/v) homogenate. After centrifuging for 10 min at 1500 rpm (about 600 x g), the supernatant was stored at -20°C. For titration, homogenates were diluted 19fold and 30 ul samples (not heated) were injected intracerebrally into Swiss CD1 mice. Mice were examined every 2 days for scrapie symptoms. a Mean time to death, 216 f 26 days. b Mean time to death, 213 f 16 days. c Killed after 274 days in moribund state; histopathology of the brain revealed astrocytosis and vacuolation. Calculation of the average titer of the 30 ul inoculum was carried out as follows. Of 32 mice inoculated with 26week-old brain homogenate, 1 contracted disease; therefore, on the basis of a Poisson distribution P@, = 31/32 = eern and m = 3.2 x 10mz infectious units per sample or 33 x 102 x 3.2 x IO-* = 106 infectious units per gram of brain. Because one infectious unit = 0.69 LOW units and a brain weighs about 0.3 g, there are approximately 20 LDw units Of infectivity per brain.
Cell 968
opment of clinical scrapie disease and neuropathology, but also for the propagation of the prion. Andreas Sailer,‘t Hansruedi Biieler, ‘t Marek Fischer,t Adrian0 Aguui,s and Charles Weissmannt tlnstitut ftir Molekularbiologie I Universitiit Zurich CH-8093 Zurich Switzerland Slnstitut fiir Neuropathologie Universitiitsspital Ziirich CH-8091 Ziirich Switzerland *These
authors
contributed
equally
to this work.
Biieter, H., Aguzzi, A., Sailer, A., Greiner, R. A., Autenried, M., and Weissmann, C. (1993). Cell 73, 1339-1347. Chesebro, Prusiner,
B., and Caughey, S. B. (1991).
Science
B. (1993).
P., Aguet,
Curr. Viol. 3, 696-698.
252, 1515-1522.
Prusiner, S. 3, Groth. D., Serban, A., Koehler, R., Foster, D., Torchia, M., Burton, D., Yang, S-L., and DeArmond, S. J. (1993). Proc. Natl. Acad. Sci. USA 90, 10608-19612.
July I, 1994, Copyright
0 1994 by Cell Press
Matters Arising
No Propagation of Prions in Mice Devoid of PrP The “protein only” hypothesis proposes that the prion, the infectious agent of transmissible spongiform encephalopathies such as scrapie, mad cow disease, and CreutzfeldJakob disease, is PrPSC, a modified form of the normal host protein PrPC. Propagation of the prion is ascribed to the conversion of PrPC into PrPS” under the influence of PrPS” (Prusiner, 1991). PrP knockout mice (Pm-p”), when inoculated with mouse scrapie prions, remained healthy and free of scrapie symptoms for at least 2 years while wild-type controls all died within 6 months (Biieler et al., 1993). Infectivity in brain was determined in two independent titration experiments. Scrapie-inoculated wild-type animals revealed a prion titer of 105.‘-1 O’.’ LDEUU/g brain tissue by 8 weeks and 107.7-10B.B LDso U/g by 20 weeks. In the case of scrapie-inoculated animals devoid of PrP, brain homogenates taken at 2,8, 12, and 25 weeks after inoculation showed no infectivity. One single sample, a pool of four brain homogenates prepared 20 weeks after infection, had a low titer of infectivity, about 103 LDm U/g. The most likely explanation was persistence of inoculum or contamination; however, low level propagation of scrapie
Table
1. Titration
Experiment 1
of Scrapie
Infectivity
Time after Inoculation
in Brain Homogenates
agent had to be considered (Biieler et al., 1993; Cheseboro and Caughey, 1993). We have now reassayed the 20-week-old samples from this experiment and confirmed the titer found previously (Table 1, experiment 1). In addition, we assayed brains of PrP knockout mice from the same experiment 25,33, and 48 weeks after inoculation and found no infectivity (Table 1, experiment 1). We repeated the entire experiment, inoculating a different group of PrP knockout mice with mouse scrapie prions and titrating brain homogenates at 16, 18, 20, and 22 weeks after inoculation (Table 1, experiment 2). The 16-, 18-, and 22-week-old samples showed no scrapie infectivity. For the 20 week timepoint, four single brains were titrated on eight indicator mice each. Of 32 animals, 1 showed scrapie symptoms at 261 days; however, all others stayed healthy for more than 300 days, at which time the experiment was ended. Under the unlikely assumption that the single diseased indicator mouse was not the result of contamination, this would represent an average content of about 20 LDso units per brain. Because each animal was inoculated with about 10’ LDw units, it is clear that prions were not propagated in the absence of PrP. Prusiner et al. (1993), using the same strain of PrP knockout mice, also failed to find propagation of infectious agent. We conclude that PrP is not only essential for the devel-
of Scrapie-Inoculated
Prp”b Mice
Number of Brains Pooled
Concentration Homogenate
20 20 25 25 33 33 33 48 48
4 4 4 4 Single Single Single Single Single
5 1 5 1 5 5 5 5 5
6 5 0 0 0 0 0 0 0
of of of of of of of of of
16 18 20 20 20 20 22
4 4 Single Single Single Single 4
1 1 1 1 1 1 1
0 0 0 1 0 0 0
of 6 of a of 6 of6 of 8 of 0 of 9
(weeks)
of (%)
CD1 Mice with Scrapie Symptoms 6’ 6b 5 6 4 5 6 5 6
Inoculation of Pm-p” mice, titration of infectivity, and monitoring for scrapie symptoms were described earlier (Biieler et at., 1993). In brief, Pm-p mice were inoculated intracerebrally with 30 pl of a IO-fold diluted (phosphate-buffered saline, 5% bovine serum albumin) Chandler-derived mouse prion preparation (ML; obtained from S. B. Prusiner). Brains were recovered at the times indicated. In experiment 1 the hemisphere ipsilateral and in experiment 2 the hemisphere contralateral to the one inoculated were processed to a 10% (w/v) homogenate. After centrifuging for 10 min at 1500 rpm (about 600 x g), the supernatant was stored at -20°C. For titration, homogenates were diluted 19fold and 30 ul samples (not heated) were injected intracerebrally into Swiss CD1 mice. Mice were examined every 2 days for scrapie symptoms. a Mean time to death, 216 f 26 days. b Mean time to death, 213 f 16 days. c Killed after 274 days in moribund state; histopathology of the brain revealed astrocytosis and vacuolation. Calculation of the average titer of the 30 ul inoculum was carried out as follows. Of 32 mice inoculated with 26week-old brain homogenate, 1 contracted disease; therefore, on the basis of a Poisson distribution P@, = 31/32 = eern and m = 3.2 x 10mz infectious units per sample or 33 x 102 x 3.2 x IO-* = 106 infectious units per gram of brain. Because one infectious unit = 0.69 LOW units and a brain weighs about 0.3 g, there are approximately 20 LDw units Of infectivity per brain.
Cell 968
opment of clinical scrapie disease and neuropathology, but also for the propagation of the prion. Andreas Sailer,‘t Hansruedi Biieler, ‘t Marek Fischer,t Adrian0 Aguui,s and Charles Weissmannt tlnstitut ftir Molekularbiologie I Universitiit Zurich CH-8093 Zurich Switzerland Slnstitut fiir Neuropathologie Universitiitsspital Ziirich CH-8091 Ziirich Switzerland *These
authors
contributed
equally
to this work.
Biieter, H., Aguzzi, A., Sailer, A., Greiner, R. A., Autenried, M., and Weissmann, C. (1993). Cell 73, 1339-1347. Chesebro, Prusiner,
B., and Caughey, S. B. (1991).
Science
B. (1993).
P., Aguet,
Curr. Viol. 3, 696-698.
252, 1515-1522.
Prusiner, S. 3, Groth. D., Serban, A., Koehler, R., Foster, D., Torchia, M., Burton, D., Yang, S-L., and DeArmond, S. J. (1993). Proc. Natl. Acad. Sci. USA 90, 10608-19612.