Institute of Neuropathology, University Hospital Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland1
Author for correspondence: Adriano Aguzzi. Fax +41 1 2554402. e-mail Adriano{at}pathol.unizh.ch
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Abstract |
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Introduction |
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The LRS clearly plays an important role in the transport of the scrapie agent. In several animal models, including hamsters and mice, lymphoid organs such as the spleen are early sites of accumulation and replication of the agent following intraperitoneal (i.p.) inoculation (Eklund et al., 1967 ; Kimberlin & Walker, 1986
, 1989
). Although B-lymphocytes are required for efficient neuroinvasion of the agent, they do not need to express PrPC. It appears that their role in neuroinvasion consists at least in part of lymphotoxin
-mediated induction of follicular dendritic cell maturation (Montrasio et al., 2000
; Klein et al., 1998
). In order to replicate prions within lymphatic tissues follicular dendritic cells may need to express PrPC (Brown et al., 1999
).
Although various components of the immune system play a pivotal role in scrapie neuroinvasion, there is substantial evidence that the PNS may be important for neuroinvasion of prions as well (Lasmezas et al., 1996 ). Adoptive bone marrow transfer of PrPC-expressing cells into PrPC knockout mice restored accumulation and replication of prions in the lymphatic tissue, yet not transport of the agent to the brain (Blättler et al., 1997
). These results indicated that a non-haematopoetic PrPC-expressing tissue is required for efficient neuroinvasion. Further experiments using Prnp knockout mice expressing transgenic PrPC under a neuron-specific promoter provided evidence that this tissue may be the PNS (Race et al., 2000
).
Here we show that transgenic mice overexpressing PrPC under the control of its own regulatory sequences (Fischer et al., 1996 ) support rapid neuroinvasion upon i.n. and footpad (f.p.) inoculation of the infectious agent. The route of neuroinvasion was consistent with direct intranerval spread in all transgenic mice, and in only one subset of i.n. inoculated wild-type mice. The use of two different routes of inoculation in transgenic and wild-type mice enabled us to calculate the actual rate of spread of the infectious agent in the PNS of tga20 mice as a function of PrPC expression.
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Methods |
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Western blot analysis.
Homogenates (10%, w/v) of sciatic nerve, spinal cord or brain were prepared as described (Büeler et al., 1993 ) and, where indicated, digested with 20 µg/ml proteinase K for 30 min at 37 °C. Unless otherwise stated in the figure legends, 50 µg of total protein was then electrophoresed through 12%SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were probed with monoclonal antibody 6H4 to mouse PrPC (Korth et al., 1997
), and developed by enhanced chemiluminescence (Amersham). Quantification of protein was accomplished by scanning membranes with a Kodak image station 440. The signal intensities produced by serial dilutions of tga20 sciatic nerve homogenates were measured and compared to the signal intensity of a specified amount of wild-type sciatic nerve homogenate using the 1D image analysis software (Kodak). All three bands corresponding to the different glycosylation states of PrPC (un-, mono- and diglycosylated) were included in the measurement.
Histoblots.
The histoblot technique was performed according to protocols of Taraboulos et al. (1992 ). Frozen sections of 8 µm thickness were mounted on uncoated glass slides and immediately pressed 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 using proteinase K concentrations of 50 and 100 µg/ml 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 serum. Incubation with primary antibody 6H4 (Korth et al., 1997
) was carried out at a dilution of 1:2000 in 1% non-fat milk serum at room temperature for 1 h. Detection was accomplished with an alkaline phosphatase-conjugated goat anti-mouse antibody at a concentration of 1:2000. Visualization was achieved with nitro blue tetrazolium and bromo-chloro-indolyl phosphate according to the protocols of the supplier.
Histological studies.
Brain, spinal cord, sciatic nerves and muscles from selected mice were fixed with 4% buffered formalin, inactivated by 1 h with 98% formic acid and embedded in paraffin. Sections were cut to a thickness of 5 µm. For cryosectioning tissues were snap-frozen in cryoprotectant compound and cut with a cryostat to a thickness of 6 µm. Sections were subjected to routine stainings with haematoxylineosin and to immunostaining for glial fibrillary acidic protein (GFAP) according to standard procedures. Gliosis (a non-specific but early indicator of brain damage) was visualized by the presence of large immunostained reactive astrocytes. The clinical diagnosis of scrapie was confirmed by histological analysis of brains or spinal cords.
Semi-thin sections.
After fixation with 0·5% glutaraldehyde, samples were fixed with osmic tetroxide and embedded in epoxy resin. Sections 3 µm thick were stained with toluidine blue.
Infectivity bioassays.
Spinal cord and sciatic nerve homogenates (10% in 0·32 M sucrose) were prepared from infected animals by homogenizing the tissues using a pellet mixer and by sonicating the samples for 5 min with a sonifier (Branson 450) at a constant output power of 400 W. Thirty µl (diluted 1:10 in PBS and 1% BSA) was administered intracerebrally to groups of four (in one sample three) tga20 mice for each sample. The incubation time until development of terminal scrapie sickness was determined and infectivity titres were calculated (Prusiner et al., 1982 ) using the relationship y=11·45-0·088x, where y is LD50 and x is incubation time (days) to terminal disease (Fischer et al., 1996
).
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Results |
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Control groups consisting of wild-type mice inoculated i.p. with different amounts of RML inoculum (high and low dose) developed terminal scrapie at 190±5 days (high dose of inoculum, n=4) and at 214±2 days (low dose of inoculum, n=4).
None of the mock injected tga20 or wild-type mice developed scrapie (n=4 for tga20 i.n.; n=3 for tga20 f.p.; and n=2 for C57Bl/6 i.n. or f.p.). These mice were sacrificed at day 205 (tga20) or 333 (C57Bl/6) after injection. Finally, we did not observe any clinical or histopathological signs of disease in Prnpo/o mice following i.n. or f.p. injection of RML inoculum (n=2 for i.n., n=2 for f.p.): these mice were sacrificed 333 days after inoculation.
Predominantly intranerval spread of prions in tga20 mice
In order to elucidate the predominant route of transport to the CNS in tga20 mice and in wild-type mice, we analysed the content and localization of PrPSc and of scrapie infectivity in sciatic nerves, spinal cords and brains of inoculated mice. In the sciatic nerves of both tga20 and wild-type mice we were not able to detect any proteinase K-resistant PrP by Western blot analysis (Fig. 3).
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Selected spinal cords of i.n. injected wild-type mice were assayed for the presence of infectivity by bioassay with tga20 indicator mice. Prion titres were calculated to be 6·1 and 6·4 logLD50xg-1 (Table 1).
No accumulation of PrPSc in spleens of tga20 mice following i.n. and f.p. injection
In wild-type mice accumulation of PrPSc in the LRS occurs very early following i.p. injection (Eklund et al., 1967 ; Kimberlin & Walker, 1989
). To examine the role of the spleen in neuroinvasion following i.n. and f.p. injection of tga20 and wild-type mice, we performed Western blots of selected spleens from i.n. and f.p. injected mice. In all of the tested wild-type mice we could detect sizeable PrPSc accumulation in spleens, whereas PrPSc could not be detected in spleens of tga20 mice, or was present in very low amounts (Fig. 6
). This unexpected finding may, in principle, point to lower expression levels of PrPC in lymphoreticular organs of tga20 transgenic mice: we therefore determined the expression levels of PrPC in spleens and in inguinal lymph nodes of tga20 mice. However, similarly to what was observed in other tissues, PrPC was massively overexpressed in these tissues (Fig. 1
). We conclude that i.n. and f.p. injection of peripheral nerves overexpressing PrPC facilitates intranerval spread so extensively that lymphoinvasion of prions becomes marginal or absent.
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Discussion |
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The pattern of PrPSc distribution in histoblots of tga20 mice showed selective accumulation of PrPSc in areas representing projections of the sensory pathway. It is conceivable that the selective accumulation of PrPSc in tga20 mice is due to an altered proteinase K sensitivity of cerebral versus spinal PrPSc. To control for this possibility we performed Western blot analysis of various brain regions. Proteinase K-resistant PrPSc could be demonstrated in cortical areas and in the brain stem of wild-type mice, whereas tga20 mice were practically devoid of proteinase K-resistant PrPSc in cortical areas. The fact that we found proteinase K-resistant PrPSc in the brain stem of tga20 mice (data not shown) demonstrates that this is not due to an altered proteinase K sensitivity of PrPSc.
The selective accumulation of PrPSc speaks in favour of neuroinvasion via the PNS and strengthens the hypothesis that tga20 mice transport the scrapie agent mainly in the PNS. In wild-type mice we could not see such a targeted distribution of PrPSc. This prompted us to assess the involvement of the LRS in i.n. and f.p. injected tga20 mice. As expected, wild-type mice showed typical accumulation of PrPSc in the spleen, indicating colonization of the immune system (Lasmezas et al., 1996 ; Mabbott et al., 1998
). In contrast, tga20 mice injected i.n. and in the f.p. did not show significant PrPSc accumulation in spleens. These results indicate that wild-type mice respect a lympho-neural sequence of pathogenesis even after direct administration of prions into nerves, while tga20 mice transport prions predominantly in the PNS. Finally, a subset of i.n. injected wild-type mice may use direct PNS neuroinvasion, and develop disease significantly earlier.
The fact that we did not find any PrPSc by Western blot analysis of the sciatic nerves of wild-type and transgenic mice may surprise, especially in view of the infectivity readily detectable by bioassay of the same samples. However, considerable amounts of infectivity that are not associated with detectable PrPSc deposits have been observed before (Manson et al., 1999 ), and are probably due to the limited sensitivity of the Western blot technique.
Using the difference in incubation times of the f.p. and the i.n. inoculated tga20 mice we attempted to estimate the velocity of transport of infectivity in the PNS. The distance between the footpad and the mid sciatic nerve, where the i.n. injection is performed, is 2·1 cm on average, and the difference in incubation times of the i.n. and f.p. injected mice is about 30 days. Because tga20 mice transport primarily in the PNS after inoculation at either of these two sites, we calculated the speed of transport in the PNS by dividing the distance between the different sites of inoculation by the difference in incubation time. The calculated rate of spread of infectivity is 0·7 mm per day. This velocity is similar to that reported for wild-type mice where the rate of spread was calculated to be around 1 to 2 mm per day (Kimberlin et al., 1983b ). Neither of these values correspond to fast axonal transport or to slow axonal transport (McEwen & Grafstein, 1968
), whereas PrPC was reported to be transported with fast axonal transport with a velocity of about 1 cm/h (Borchelt et al., 1994
). The possibility that the transport of PrPSc in the PNS may not occur through axonal transport mechanisms was raised recently (Groschup et al., 1999
; Hainfellner & Budka, 1999
) and is compatible with our data.
The bioassay data gathered in this study provide intriguing insights into the kinetics of intranerval spread. Prion infectivity titres of tga20 sciatic nerves were up to 1·8 log higher than those observed in wild-type mice, yet the velocity of transport was similar in wild-type and in transgenic mice. Therefore, PrPC availability in the nerve modulates the capacity of intranerval spread, but does not affect its velocity. Perhaps the significantly higher titres in the sciatic nerves of tga20 mice are indicative of a mode of transport in which PrPC localized on the PNS is converted into PrPSc in a domino fashion centripetally towards the CNS. A similar phenomenon may occur in the CNS (Brandner et al., 1996 ). Another possibility to explain the difference between wild-type and tga20 mice is the difference in the glycotype ratio of PrPC expressed by the electrophoretic pattern of PrPC between wild-type and tga20 nerves. In sciatic nerves of tga20 mice the diglycosylated form of PrPC seems to be abundant, whereas in wild-type mice monoglycosylated PrPC is predominant. It is conceivable that different glycosylation states of PrPC may influence the transport of PrPSc.
Besides confirming a central role of PrPC in the PNS in prion neuroinvasion, the present study provides surprising evidence that mice which overexpress PrPC can effect strictly intranerval neuroinvasion and bypass LRS pathogenesis. One may wonder whether similar phenomena may underlie neuroinvasion of BSE prions in cows, which also appear to bypass the LRS.
Because overexpression of PrPC leads to increased intranerval prion titres, PrPC may well be rate-limiting for prion spread. In order to test this hypothesis (which bears some relevance to the prospect of post-exposure prophylaxis for prion diseases) we are currently attempting to express PrPC conditionally in the PNS using virus vector-mediated gene transfer (Glatzel et al., 2000 ).
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Acknowledgments |
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References |
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Received 16 May 2000;
accepted 3 August 2000.