PrPC expression in the peripheral nervous system is a determinant of prion neuroinvasion

Markus Glatzel1 and Adriano Aguzzi1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Transmissible spongiform encephalopathies are often propagated by extracerebral inoculation. The mechanism of spread from peripheral portals of entry to the central nervous system (neuroinvasion) is complex: while lymphatic organs typically show early accumulation of prions, and B-cells and follicular dendritic cells are required for efficient neuroinvasion, actual entry into the central nervous system occurs probably via peripheral nerves and may utilize a PrPC-dependent mechanism. This study shows that transgenic mice overexpressing PrPC undergo rapid and efficient neuroinvasion upon intranerval and footpad inoculation of prions. These mice exhibited deposition of the pathological isoform of the prion protein (PrPSc) and infectivity in specific portions of the central and peripheral sensory pathways, but almost no splenic PrPSc accumulation. In contrast, wild-type mice always accumulated splenic PrPSc, and had widespread deposition of PrPSc throughout the central nervous system even when prions were injected directly into the sciatic nerve. These results indicate that a lympho-neural sequence of spread occurs in wild-type mice even upon intranerval inoculation, while overexpression of PrPC leads to substantial predilection of intranerval over lymphoreticular spread. The rate of transport of infectivity in peripheral nerves was ca. 0·7 mm per day, and prion infectivity titres of sciatic nerves were much higher in tga20 than in wild-type mice, suggesting that overexpression of PrPC modulates the capacity for intranerval transport.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Prion diseases are transmissible fatal neurodegenerative diseases. Although the infectious agent is most efficiently propagated through intracerebral inoculation, peripheral infection is the natural route of transmission in most prion diseases. Oral administration is most probably involved in kuru, BSE and the new variant of Creutzfeldt–Jakob disease, while parenteral administration of growth hormone and gonadotropins has resulted in iatrogenic Creutzfeldt–Jakob disease. In all these instances transport of the infectious agent from the site of entry into the body to the central nervous system (CNS) is of crucial importance (Hill et al., 1997 ; Scott et al., 1999 ). A large body of evidence indicates that both the lymphoreticular system (LRS) and the peripheral nervous system (PNS) are involved in scrapie neuroinvasion (Baldauf et al., 1997 ; Kimberlin & Walker, 1988 ; Klein et al., 1997 ; Lasmezas et al., 1996 ). Transport along the PNS was suggested by experiments showing that intranerval (i.n.) injection of infectivity could bypass the need for extraneural replication of the agent (Kimberlin et al., 1983b ). Also, the first pathological lesions and replication of infectivity after peripheral administration of the scrapie agent occur in spinal cord and medulla oblongata: these sites are consistent with entry via peripheral nerves like the vagal or splanchnic nerves (Beekes et al., 1998 ; Cole & Kimberlin, 1985 ).

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 {beta}-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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Scrapie inoculation.
Mice were inoculated with a 1% homogenate of heat- and sarcosyl-treated brain prepared from mice infected with the Rocky Mountain Laboratory (RML) scrapie strain. Twenty µl was used for f.p. injections while 1 µl was administered i.n. For f.p. and i.p. injections mice were anaesthetized with Rompun (Bayer). For i.n. injection, mice were anaesthetized with Xylazin/Ketamin and the right sciatic nerve was surgically exposed by dislodging the M. gluteus superficialis and the M. biceps femoris. The nerve was gently placed onto a metal plate (20x5x0·5 mm) and 1 µl of RML inoculum was injected with a 34-gauge Hamilton syringe over a period of 5 min (Bassant et al., 1986 ). The nerve was anatomically repositioned and the skin was closed with USP 4·0 nylon sutures. The i.p. injected mice received 100 µl of a 0·1% (high dose) or 100 µl of a 0·01% (low dose) homogenate of heat-and sarcosyl-treated RML inoculum. Mice of the respective control groups received the same amounts of mock inoculum, consisting of similarly prepared brain homogenate from uninfected mice. Mice were monitored every third day, and scrapie was diagnosed according to standard clinical signs.

{blacksquare} 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.

{blacksquare} 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.

{blacksquare} 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 haematoxylin–eosin 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.

{blacksquare} 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.

{blacksquare} 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 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
tga20 mice overexpress PrPC in the PNS
Previous studies have shown that tga20 mice overexpress PrPC in the CNS about tenfold (Fischer et al., 1996 ). We investigated the level of PrPC expression in the PNS in this mouse line. Western blot analysis of sciatic nerves of tga20 mice compared to C57Bl/6 mice show that these mice also overexpress PrPC in the PNS (Fig. 1A). Quantification of the amount of PrPC expressed on the sciatic nerve was performed by measuring the signal intensity of serial dilutions of tga20 and wild-type sciatic nerve extracts using a Kodak image station and appropriate software. The expression of PrPC in the PNS of tga20 mice was determined to be five to seven times higher than in wild-type mice (Fig. 1B). In addition to the different expression level there is a marked difference between the various PrPC glycotypes as assessed by the electrophoretic pattern of PrPC between wild-type and tga20 nerves. In tga20 mice the strongest band is the high molecular mass band (diglycosylated PrPC), whereas in wild-type nerves a lower molecular mass band (monoglycosylated PrPC) gives the strongest signal (Fig. 1A).



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Fig. 1. (A) Western blot analysis of PrPC in the spinal cords (sc) and sciatic nerves (scn) of C57Bl/6 (Bl/6) and tga20 mice. The amount of total protein loaded is indicated for each lane. (B) PrPC content in sciatic nerves was quantified by direct recording of Western blot chemiluminescence using the Kodak 1D image analysis system. A standard curve was produced by plotting the signal intensity produced by 5–40 µg of tga20 sciatic nerve homogenate. The arrow indicates that the PrPC content of 40 µg wild-type sciatic nerve corresponds to 8 µg tga20 sciatic nerve. (C) Western blot analysis of PrPC of spleens and lymph nodes (LN) of C57Bl/6 (Bl/6) and tga20 mice. The amount of total protein loaded in each lane was 120 µg.

 
Overexpression of PrPC in the PNS leads to efficient neuroinvasion following intranerval and footpad inoculation
Studies aimed to investigate the transport of prions in the PNS often rely on inoculation routes that target specifically the PNS. In this study we used two different routes of administration of the infectious agent: the i.n. and f.p. routes. To study the effect of overexpression of PrPC on the PNS we inoculated tga20 mice and wild-type mice using these inoculation routes. All of the tga20 mice inoculated with RML scrapie prions (n=14 for i.n.; n=9 for f.p.) developed scrapie (Fig. 2). The incubation time until terminal scrapie was 89±16 days for i.n. injected mice. F.p. injected tga20 mice developed scrapie 33 days later (123±17 days). Wild-type mice exposed to prions according to the same protocol (n=5 for i.n.; n=4 for f.p.) developed terminal disease 195±29 days after i.n. inoculation and 178±3 days after f.p. injection (Fig. 2).



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Fig. 2. Incubation time (days) from inoculation until terminal scrapie disease in i.n. and f.p. injected tga20 mice compared to i.n., f.p. and i.p. injected C57Bl/6 (Bl/6) mice. tga20 mice developed clinical scrapie at early time-points with a difference of about 30 days between the i.n. and f.p. injected group. Wild-type mice did not show such a clear difference between i.n. and f.p. routes of inoculation.

 
Among wild-type mice inoculated i.n., segregation into two subgroups was observed: a ‘fast-onset’ group came down with scrapie very rapidly, while the remaining group of mice developed the disease later, with an incubation time similar to that of i.p. injected mice. A similar phenomenon was observed earlier (Kimberlin et al., 1983b ).

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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Fig. 3. Western blot detection of PrPC and PrPSc. Upper blot, extracts of spinal cords; lower blot, extracts of sciatic nerves. Both the side of i.n. prion injection (ipsi) and the contralateral (contra) side were examined. Genotypes of mice, route of inoculation and proteinase K digest are indicated above each lane. As a control, we analysed terminally sick intracerebrally inoculated CD1 mice (CD1), mock injected wild-type mice (Bl/6) and PrP knockout mice (PrPo/o). The amount of total protein loaded in each lane was 50 µg except for the Bl/6 sciatic nerve, where it was 25 µg. While PrPSc was readily detectable in the spinal cords of all examined mice, no proteinase K-resistant PrP could be detected in any of the investigated sciatic nerve samples.

 
This was further investigated by inoculating tga20 indicator mice with homogenates of selected sciatic nerves: all transmitted sciatic nerves showed infectivity. When titres were calculated by the incubation time method, sciatic nerves of tga20 mice were shown to contain significantly more infectivity than those of wild-type mice. The calculated titres were 5·4 and 5·6 logLD50xg-1 for tga20 sciatic nerves, as opposed to 3·8 and 4·2 logLD50xg-1 for wild-type nerves (Table 1).


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Table 1. Incubation times and titres of infectivity of selected samples transmitted to indicator mice

 
The sciatic nerves of seven wild-type and seven tga20 mice were then examined histologically. In semi-thin sections we could see signs of axonal degeneration in the sciatic nerves of both i.n. and f.p. injected tga20 mice. In contrast, no signs of axonal degeneration were seen in the sciatic nerves of i.n. and f.p. injected wild-type mice or of mock injected tga20 mice (Fig. 4). Because degeneration was observed in tga20 mice but not in wild-type mice, it is likely to represent a consequence of retrograde prion spread within the PNS of tga20 mice rather than Wallerian degeneration secondary to CNS affection.



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Fig. 4. Histology of thoracic spinal cords (A–D) and sciatic nerves (E, F) from a terminally sick tga20 mouse compared to a mock injected tga20 mouse. In both mice the injection was i.n. Strong gliosis (D) and spongiosis (B) are visible in the spinal cord of the terminally sick mouse. (A)–(B) Haematoxylin–eosin stain; (C)–(D) GFAP immunohistochemistry. Original magnifications, x100. (E)–(F) Semi-thin sections of injected sciatic nerves from a mock injected tga20 mouse (E) and terminally sick tga20 mouse (F). Axonal degeneration is obvious in (F): single axons undergoing Wallerian degeneration are indicated by an arrow. Original magnification, x260.

 
tga20 mice injected i.n. and in the f.p. show selective accumulation of PrPSc in specific areas of the CNS
To further investigate the spread of infectivity from the PNS to the CNS we performed Western blot analyses of selected thoracic spinal cords of i.n. and f.p. injected tga20 and wild-type mice. In all of the examined mice (n=12) we detected proteinase K-resistant PrPSc in the spinal cord (Fig. 3). The large amount of PrPSc detectable in the spinal cords of tga20 stands in contrast to the very low amount of PrPSc that was detected in the brains of these mice (Fischer et al., 1996 ), whereas in wild-type mice the amount of PrPSc in spinal cords and in brains was similar (Fig. 5).



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Fig. 5. (A) Accumulation of PrPSc in brains. Histoblots showing immunoreactive PrPC in brain sections natively (upper row) and after digestion with increasing levels of proteinase K (second and third rows). Prnpo/o mice (first column) show no immunoreactivity, while mock inoculated wild-type mice show proteinase K-sensitive PrPC (second column), but no proteinase K-resistant PrPSc. Terminally sick i.n. or f.p. injected wild-type mice contained large amounts of both PrPC and PrPSc (fourth and sixth columns). Terminally sick i.n. and f.p. injected tga20 mice accumulated PrPSc selectively in specific brain areas belonging to the sensory nervous system (thalamus) (third and fifth columns). The boxes indicate areas where material for Western blotting was obtained. (B) Western blot analysis of cortical areas of terminally sick i.n. or f.p injected wild-type and transgenic mice. The treatment and genotype of the mice correspond to (A). No PrPSc was detected in the cortex of transgenic mice, while wild-type mice showed abundant cortical PrPSc. (C) Left side, histoblot [close-up from (A), second row third column] showing accumulation of PrPSc in the thalamus of an i.n. inoculated tga20 mouse. Right side, coronal section through a mouse brain (modified after Sidman et al., 1971 ). Asterisks indicate the posterior nucleus of the thalamus.

 
The results above suggested selective targeting of specific CNS areas with PrPSc in tga20 mice following i.n. and f.p. injection of prions. This was studied in more detail by histoblot analyses of brains. Indeed, the topography of PrPSc accumulation was strikingly different in tga20 and wild-type mice. Wild-type mice exhibited abundant accumulation of PrPSc in cortex and hippocampus, while in tga20 transgenic mice these regions did not contain detectable PrPSc (Fig. 5A, B, C). The sites of accumulations in tga20 mice were superimposable on the main targets of the projections of the sensory pathway, whereas wild-type mice accumulated PrPSc much more diffusely in the telencephalon.

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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Fig. 6. Western blot analysis of spleens. Immunodetection with antibody 1B3 (Farquhar et al., 1996 ) revealed the presence of proteinase K-resistant PrPSc in spleens of terminally sick wild-type (n=2) and tga20 (n=2) mice after i.n. and f.p. injection of scrapie prions. No or very little proteinase K-resistant PrPSc could be detected in the i.p. or f.p. injected tga20 mice. Indicated above each lane is the genotype of the mouse, the route of inoculation of infectivity, and proteinase K digest. Control experiments were performed with terminally sick intracerebrally inoculated CD1 mice (CD1); mock injected wild-type mice (Bl/6) and PrP knockout mice (PrP0/0). The amount of total protein loaded in each lane was 80 µg.

 
Determination of the rate of spread in the PNS of tga20 mice
The results documented above clearly indicate that, in tga20 mice, prions bypass the lymphoid tissue and spread intranervally. By comparing the incubation times of the f.p. and the i.n. injected mice we attempted to gain some insight into the rate of spread in the PNS in these mice. The average distance between the f.p. and the i.n. sites of inoculation was determined to be 2·1 cm. The difference in the incubation times of these two groups of animals is approximately 30 days. Therefore, the actual rate of spread in the PNS of the tga20 mice is around 0·7 mm per day.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
A wealth of studies points to the importance of the PNS for neuroinvasion (Beekes et al., 1998 ; Kimberlin et al., 1983a ; Kimberlin & Walker, 1988 ). We have previously shown that expression of PrPC in a sessile compartment that cannot be reconstituted by adoptive bone marrow transfer is necessary for neuroinvasion of prions (Blättler et al., 1997 ). Several lines of evidence suggest that at least a part of this compartment is the PNS (Race et al., 2000 ). To clarify the role of neural PrPC expression in scrapie neuroinvasion via the PNS, we took advantage of transgenic mice that overexpress PrPC (Fischer et al., 1996 ). These mice were inoculated both i.n. and via the f.p. route. All of the inoculated tga20 mice came down with clinical scrapie, with a difference in incubation times between the i.n. and f.p. routes of about 30 days. In contrast, the incubation times of i.n. and f.p. injected wild-type mice were not significantly different from those of mice inoculated i.p. A detailed breakdown of the incubation times of i.n. inoculated wild-type mice (Fig. 2) suggested that two groups can be differentiated: ‘fast transporting’ and ‘slow transporting’ mice. Kimberlin et al. (1983b ) had observed this phenomenon in a previous study and remarked that the fast transporting group may possibly accomplish neuroinvasion directly via the injected nerve, whereas the slower transporting group may use other routes of neuroinvasion. Accordingly, the efficiency could be increased by damaging the injected nerve. In contrast to wild-type mice, in i.n. injected tga20 mice there was no segregation in two groups.

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 ).


   Acknowledgments
 
We thank M. Peltola and M. König for technical help, and Dr Farquhar for the gift of antibody 1B3. This work was supported by the Kanton of Zürich, the Bundesämter für Gesundheit, Veterinärwesen, Bildung und Wissenschaft, and by grants from the Swiss National Research Program NFP38/NFP38+ and of the companies Baxter and Migros.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Baldauf, E., Beekes, M. & Diringer, H.(1997). Evidence for an alternative direct route of access for the scrapie agent to the brain bypassing the spinal cord.Journal of General Virology78, 1187-1197.[Abstract]

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Received 16 May 2000; accepted 3 August 2000.