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Peripheral Circulation of the Prion Infectious Agent in Transgenic Mice Expressing the Ovine Prion Protein Gene in Neurons Only

  1. Carole Crozet1,a,
  2. Stéphane Lezmi1,
  3. Frédéric Flamant2,
  4. Jacques Samarut2,
  5. Thierry Baron1 and
  6. Anna Bencsik1,1
  1. 1 Agence Française de Sécurité Sanitaire des Aliments, Unité Agents Transmissibles Non Conventionnels, Institut Fédératif de Recherche 128, Biosciences Lyon-Gerland, Lyon, France
  2. 2 Ecole Normale Supérieure de Lyon, Laboratoire de Biologie Moléculaire et Cellulaire, Institut Fédératif de Recherche 128, Biosciences Lyon-Gerland, Lyon, France
  1. Reprints or correspondence: Dr. Anna Bencsik, AFSSA, Unité ATNC, 31 ave. Tony Garnier, 69364 Lyon, France (a.bencsik{at}lyon.afssa.fr).

  1. Presented in part: Keystone Symposia: Molecular Aspects of Transmissible Spongiform Encephalopathies (Prion Diseases), Breckenridge, Colorado, 2–6 April 2003 (abstract 123).

  • ↵a Present affiliation: Institut de Génétique Humaine, UPR1142 Centre National de la Recherche Scientifique, Montpellier, France.

 
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Abstract

Background. For prion diseases, even if a large body of evidence indicates that both the lymphoreticular system (LRS) and peripheral nerves are involved in scrapie neuroinvasion, the processes by which prions invade the central nervous system are only partially understood.

Methods. Transgenic Tg(OvPrP4) mice, which express the ovine prion protein (PrP) gene under the rat neuron—specific enolase promoter on a knockout background, were used to study prion extracerebral circulation after scrapie prions were inoculated via the intracerebral (ic) and the intraperitoneal (ip) route.

Results. Surprisingly, PrPSc was detected in the spleens of mice inoculated ic with prions. Moreover, the absence of the ovine PrPC in nonneural tissue at the periphery did not stop neuroinvasion after ip challenge. Additionally, pilot studies performed in Tg(OvPrP4) mice that had undergone splenectomy before ic prion inoculation showed that the time course of the disease is delayed.

Conclusions. Given that these mice express the ovine PrP gene in neuronal cells but not in nonnervous tissue, our results suggest that PrPC expressed by cells of the LRS are not necessary for neuroinvasion or for their ability to accumulate PrPSc and emphasize the importance of extracerebral circulation of PrPC or PrPSc for the development of the disease.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that affect humans as well as sheep, goats, and cattle. One of the main characteristics of these encephalopathies is the accumulation of a pathological protein, prion protein (PrP)Sc that is insoluble and partially protease resistant [1]. This isoform results from the conversion of the normal host prion protein, PrPC, which is soluble and protease sensitive [1–3]. PrPC is an ubiquitously expressed glycosylphos-phatidylinositol (GPI)—anchored glycoprotein that is predominantly expressed by neurons but is also expressed by astrocytes, oligodendrocytes, and cells of lymphoreticular system (LRS) as well as follicular dendritic cells (FDCs) [4–6]. According to the prion hypothesis, the abnormal isoform PrPSc itself constitutes infectivity and acts as a seed or as a template in the conversion of host PrPC into PrPSc [1, 7, 8]. It has also been proposed that PrPC may act as a receptor for the infectious agent, with the conversion considered to be an epiphenomenon of the infectious process [9].

Although prions are most efficiently propagated in animal models through intracerebral (ic) inoculation, peripheral infection is the usual route of transmission in most of acquired prion diseases. Nevertheless, the neuroinvasion process by which prions invade the central nervous system (CNS) is only partially understood. However, a large body of evidence indicates that both the host lymphoid system and peripheral nerves are involved in prion replication and neuroinvasion [10–16]. Indeed, lymphoid organs, including the spleen, are early sites of PrPSc accumulation and replication after intraperitoneal (ip) inoculation [17]. As suggested by PrPScdeposit analysis and bone marrow graft experiments, replication at the periphery mainly requires FDCs [18]. The identity of PrPSc-replicating cells, however, remains controversial, because several types of cells, including DCs, may also carry infectivity [19]. With regard to the mechanism of PrPSc invasion into the CNS, it has been shown that orally administrated prions can apparently reach the brain via the parasympathetic vagal nerves [20], indicating that the autonomous nervous system may be involved. Implication of the peripheral nervous system is also confirmed by experiments showing that, after ip delivery of prions, disease can be delayed by sympathectomy or can be accelerated by sympathetic hyperinnervation of lymphoreticular organs [21].

In the CNS, the cell type expressing the PrP gene also appears to be important for the successful development of the disease. This is outlined by infectious studies in PrP knockout mice with ectopic and cell-restricted expression of PrPC that have shown the ability of neurons and/or astrocytes to propagate prions [22, 23].

We have developed a transgenic mouse line, Tg(OvPrP4) mice, that express the ovine PrP (OvPrP) gene under the rat neuron—specific enolase (NSE) promoter [22, 24] on a murine Prnp—/— genetic background. These mice express the transgene solely in the neuronal compartment [25]. In addition, these mice develop a prion disease after inoculation with sheep-scrapie isolates of different origins [25] with greater efficiency than do similarly challenged C57Bl/6 mice. This transgenic mouse model is, thus, an efficient tool for the study of scrapie strains from natural disease. In the present work, we use Tg(OvPrP4) mice as a model to study PrPSc extracerebral circulation. Because the OvPrP gene is expressed only in neuronal cells, we logically supposed that, after ic challenge, no PrPSc should be observed in the spleen given that the PrP gene is not expressed there, as has been previously found in transgenic mice carrying the hamster PrP (haPrP) gene [12]. Nonetheless, we did detect PrPSc accumulation in the follicules of the spleens of ic inoculated transgenic Tg(OvPrP4) mice. This unexpected observation raised the question of how the prion agent circulates in extracerebral tissue. The present article focuses on this question and on understanding how the spleen is involved in disease in Tg(OvPrP4) mice. First, we show that PrPSc can circulate from the brain to the periphery (and inversely) after both ip and ic inoculation. Second, we report a pilot study conducted in transgenic Tg(OvPrP4) mice that had undergone splenectomy before ic prion infection. Preliminary results show that the time course of the disease is delayed in splenectomized Tg(OvPrP4) mice, compared with that in nonsplenectomized mice.

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Materials and Methods

Animals. The transgenic Tg(OvPrP4) mice used in the present study express the OvPrP gene (AA136RR154QQ171 genotype) solely in neurons and do not express the endogenous murine PrP gene. The generation of these mice and their susceptibility to scrapie have been described elsewhere [25]. In these mice, the murine PrP gene has been inactivated by successive crossing with Prnp—/— knockout mice [26]. The transgenic mice were housed in an autonomous, filtered, pressurized enclosure, in accordance with the guidelines of the French Ethical Committee (decree 87–848) and the European Community Directive 86/609/EEC.

Inoculation and monitoring of mice. The transgenic mice (5 weeks old) were inoculated ic (n = 25) or ip (n = 16) with 20 μL of a 10% (wt/vol) brain homogenate in 5% glucose. The brains came from naturally scrapie-affected sheep that originated from 2 different regions of France and that were detected by the French Epidemiological Surveillance Network (D. Calavas, Agence Française de Sécurité Sanitaire des Aliments, Lyon, France). The mice were then checked weekly for the presence of such clinical signs as leanness, hunched posture, hind-limb paralysis, equilibrium trouble, plastic tail, prostration, tremors, ruffled fur, abnormal gait, and clasping feet [25].

As soon as a mouse showed one of these clinical signs, it was isolated and monitored daily. Mice were killed when the intensity of clinical symptoms appeared to be life threatening; in some cases, mice were found dead. The whole brain was quickly removed and was either fixed (with 2% paraformaldehyde in 0.1 mol/L PBS [pH 7.4]) for immunohistochemical (IHC) analysis or frozen and stored at −80°C for Western blot (WB) analysis, as described elsewhere [25].

Production of splenectomized mice. Before inoculation, 3-week-old mice (n = 8) were anesthetized (4.25% chloral hydrate, 13.2% nembutal, 39.6% propylene glycol, and 2.12% MgSO4) and splenectomized.

Reverse-transcription polymerase chain reaction (RT-PCR) analysis. RT-PCR analysis of OvPrP mRNA was performed with 1 μg of total RNA and 30 cycles of amplification, as described elsewhere [25].

WB analysis of PrPC. The presence of PrPC was analyzed by WB analysis using a mixture of SAF32 (recognizing the 61—67-aa region of OvPrP), SAF61 (recognizing the 164—157-aa region of OvPrP), and SAF84 (raised against the 142—160-aa human PrP peptide) mouse monoclonal antibodies, as described elsewhere [27].

Detection of abnormal PrP in transgenic mice inoculated with scrapie prions. PrP IHC was performed as described elsewhere [25]. SAF84 monoclonal antibody recognizing the human 142—160-aa PrP sequence (0.5 μg/mL; provided by J. Grassi, Commissariat à l'Energie Atomique/Service de Pharmacologie et d'Immunologie, Saclay, France) was used to detect PrPSc. The double-immunostaining procedure used to allow detection of PrPSc and noradrenergic endings has been described elsewhere [28]. The presence of protease-resistant protein was investigated by WB analysis using the RB1 polyclonal antibody raised against synthetic bovine 105—120-aa PrP peptide, as described elsewhere [25, 29].

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Results

OvPrP expression in Tg(OvPrP4) mice. In the present study, we used Tg(OvPrP4) mice that express the OvPrP gene under the control of the neuron-specific enolase promoter. RT-PCR analysis of OvPrP mRNA showed that the transgene is selectively expressed in the brains of the Tg(OvPrP4) mice but not in any nonnervous tissues (figure 1A) [25]. This was confirmed by ELISA (data not shown). In particular, these mice did not express the OvPrP gene in the spleen (figure 1). Still, WB analysis showed that the OvPrP gene—encoded protein was present in the brain as well as in vagual (data not shown) and sciatic (figure 1B) nerves, indicating that transgenic PrPC can be present in peripheral tissues.

Figure 1.
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Figure 1.

A, Reverse-transcription polymerase chain reaction analysis performed on RNA extracts from Tg(OvPrP4) mice brain and spleen. Although ovine prion protein (OvPrP) transcripts are present in the brains of Tg(OvPrP4) mice, no OvPrP mRNA is detectable in the spleens of Tg(OvPrP4) mice or in the brains of Prnp—/— knockout mice (right lane). B, Western blot analysis of Tg(OvPrP4) sciatic nerves, using a mixture of SAF32 (recognizing the 61—67-aa region of OvPrP), SAF61 (recognizing the 164—157-aa region of OvPrP), and SAF84 (raised against the 142—160-aa human PrP peptide) mouse monoclonal antibodies. The positive control (C°+) and the negative control (C°−) consist, respectively, of murine wild-type PrP-expressing tissue and PrP knockout tissue. The 2 analyzed samples consist of 2 pools of sciatic nerves from 3 Tg(OvPrP4) mice. Ovine PrPC is present both in the positive control lane and in each pooled sciatic nerve sample of Tg(OvPrP4) origin.

Tg(OvPrP4) mouse susceptibility: ic versus ip route. The susceptibility of Tg(OvPrP4) mice to the ic route of scrapie prion infection has been established [25] by use of 2 sheepscrapie isolates (table 1). Mice have also been inoculated with additional sheep-scrapie isolates, which similarly led to the development of a spongiform encephalopathy [25, 30]. Because Tg(OvPrP4) mice do not express the OvPrP gene in the LRS, we did not expect these mice to be susceptible to scrapie after ip inoculation. Surprisingly, Tg(OvPrP4) mice developed the disease after ip inoculation of sheep-scrapie isolates (isolate 1, n = 9; isolate 2, n = 4), as shown by the presence of PrPSc in the brains of the inoculated mice (figure 2). Compared with the development of disease observed for the ic route (death at a mean ± SE of 201 ± 91 and 238 ± 7 days after inoculation for isolates 1 and 2, respectively), the time course of the disease for the ip route was delayed, leading to death at a mean ± SE of 413 ± 90 and 349 ± 111 days after inoculation for isolates 1 and 2, respectively (P = .0005, for both; t test) (table 1). The distribution of PrPSc within the brain in Tg(OvPrP4) mice inoculated by the ip route was not different from that in Tg(OvPrP4) mice challenged ic, showing a predominant accumulation in the septal, thalamic, hypothalamic, and substantia nigra areas (figure 3). Protease-resistant PrP (PrPres) accumulation was also detectable in the spleens of ip inoculated mice (figure 3). These results demonstrate that the scrapie agent can invade the brain in our model even after ip inoculation.

Figure 2.
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Figure 2.

Susceptibility of Tg(OvPrP4) mice after intracerebral (ic) or intraperitoneal (ip) inoculation of 2 sheep-scrapie isolates. Prion protein (PrP)Sc accumulates in the brain of the Tg(OvPrP4) mice regardless of the route of inoculation, demonstrating that the scrapie agent can invade the brain from the periphery even in the absence of PrPC expression at the periphery. Lane 1, Sheep-scrapie isolate 2 (sh2); lane 2, Tg(OvPrP4) mice inoculated ic with isolate 2 (Tg ic2); lane 3, Tg(OvPrP4) mice inoculated ip with isolate 2 (Tg ip2); lane 4, Tg(OvPrP4) mice inoculated ic with isolate 1 (Tg ic1); lane 5, Tg(OvPrP4) mice inoculated ip with isolate 1 (Tg ip1).

Figure 3.
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Figure 3.

Pattern of pathological prion protein (PrP)Sc accumulation in the brains of Tg(OvPrP4) mice after intracerebral (ic) or intraperitoneal (ip) inoculation of sheep-scrapie isolate 2. The inoculation route does not influence the distribution of PrPSc, which predominantly accumulates in the septum area and accumbens septi nucleus (1), hypothalamus (2), substantia nigra (3), and thalamus (4). The Western blot analysis shown at bottom illustrates the detection of protease-resistant PrP (PrPres) in the spleens of 2 Tg(OvPrP4) mice after ip inoculation of sheep-scrapie isolate 2.

PrPSc circulation fromthe brain to the periphery. A second question is whether infectivity can be transported from the brain to the periphery. To test this possibility, we analyzed infectivity indirectly by checking the presence of PrPSc in the spleen after ic inoculation. In our experiments, in every available spleen from Tg(OvPrP4) mice inoculated with the 2 sheepscrapie isolates, there was PrPSc accumulation (table 1). Moreover, the mice that died 87 days after inoculation with isolate 1, which were negative for PrPSc in the brain by WB and weakly positive by IHC analysis, showed the presence of PrPres in the spleen (table 1 and figure 4A, 4C, 4E, and 4G). This demonstrates that, after ic inoculation, PrPres was detectable by WB first in the spleen. The mice that died after 231 days were positive for PrPSc in the brain as well as in the spleen, both by WB and IHC analysis (table 1 and figure 4B, 4D, 4F, and 4H).

Figure 4.
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Figure 4.

Abnormal prion protein (PrP)Sc accumulation in the brains and spleens of Tg(OvPrP4) mice after intracerebral inoculation of sheep-scrapie isolate 1. The mice dying 87 days after inoculation are negative for PrPSc accumulation by Western blot (A) and weakly positive by immunohistochemical analysis (C; see arrow and E) in the brain but show the presence of a low level of protease-resistant PrP (PrPres) in the spleen (A), whereas the mice dying after 231 days are highly positive in the brain (B, D, and F; black deposits) and in the spleen (H).

IHC analysis of the spleens from ic inoculated mice showed PrPSc accumulation within germinal centers in dense areas, reminiscent of FDC-like clusters in germinal centers (figure 5). In addition, double labeling of PrPSc and tyrosine hydroxylase showed that neuronal processes invade the follicular areas (figure 5); some of them were even in close vicinity to the PrPSc deposits (figure 5C and 5D). These results indicate that PrPSc can travel from the brain to the spleen.

Figure 5.
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Figure 5.

Abnormal prion protein (PrP)Sc immunohistochemistry in the spleens of Tg(OvPrP4) mice after intracerebral inoculation of sheep-scrapie isolates 1 and 2. PrPSc accumulates in dense areas, reminiscent of follicular dendritic cell—like clusters in germinal centers (black deposits). Double labeling of PrPSc (black deposits) and tyrosine hydroxylase (red) shows that neuronal processes invade the follicular areas (A and B); some of them are even in close vicinity to PrPSc labeling (C and D). A similar pattern is observed for intraperitoneally inoculated mice (data not shown).

Reduced scrapie disease after ic inoculation in splenectomized mice. To address the possibility that the circulation of PrPSc between the brain and spleen is involved in the development of the disease after ic inoculation of Tg(OvPrP4) mice, we inoculated by the ic route Tg(OvPrP4) mice that had undergone splenectomy. The results reported in table 1 and figure 6A showed that, for the splenectomized mice inoculated with isolate 1, the incubation period was delayed (mean ± SE, 395 ± 72 days after inoculation), compared with that for nonsplenectomized mice (mean ± SE, 201 ± 91 days after inoculation). In addition, no PrPSc accumulation was detected by either WB or IHC analysis for isolate 1. For isolate 2, of the 4 splenectomized mice, the 2 that died 258 and 448 days after inoculation did not show any PrPSc accumulation. Slight deposits of PrPSc were detected in the brains of the mice that died 569 and 638 days after inoculation. The pattern of PrPSc accumulation in the mice that died at 569 and 638 days showed slight PrPSc deposits in areas similar to those in the mice ic or ip inoculated with isolate 2 (figure 6A).

Figure 6.
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Figure 6.

Schematic pattern of abnormal prion protein (PrP)Sc accumulation in the brains of the splenectomized Tg(OvPrP4) mouse dying 569 days after intracerebral inoculation of sheep-scrapie isolate 2. A, SAF84 immunohistochemical (IHC) analysis of PrPSc (black deposits) in the septum area and accumbens septi nucleus (1), hypothalamus (2), substantia nigra (3), and thalamus (4). B and C, PrPSc IHC analysis showing slight brown deposits in the germinal centers of the Peyer patches, compared with control without primary antibody (P). Similarly, PrPSc was detectable in the Peyer patches of Tg(OvPrP4) mice after intraperitoneal inoculation with sheep-scrapie isolate 1 (E and F).

Table 1.
Table 1.

Incubation periods and prion protein (PrP)Sc status in the brain and spleen of each transgenic mouse inoculated either intracerebrally (ic), intraperitoneally (ip), or ic after splenectomy with ovine brain material from 2 different cases of scrapie (isolate 1 and isolate 2).

Extracerebral accumulation of PrPSc. Because disease was delayed in splenectomized Tg(OvPrP4) mice, it was of interest to identify other possible areas of PrPSc accumulation, such as organs of the LRS. IHC analysis showed slight deposits in the germinal centers of the Peyer patches in the intestine analyzed (figure 6B–6D), as well as in nonsplenectomized mice (data not shown) and in ip inoculated mice (figure 6E and 6F). This indicates that Peyer patches were also able to accumulate or retain PrPSc in Tg(OvPrP4) mice.

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Discussion

We have produced OvPrP-transgenic mice on a murine Prnp—/— background suitable not only for the detection of the sheep-scrapie infectious agent but also for the study of the traffic of the infectious agent between cerebral and extracerebral compartments. Indeed, we show here that the use of the NSE promoter resulting in the absence of the OvPrPC protein in nonneural tissue at the periphery allowed the detection of PrPSc deposits not only in the brain but also at least in the spleen and in Peyer patches of the intestine after ic or ip challenge. These observations have at least 2 important implications: (1) PrPSc can be transported backward and forward from the brain to the periphery and (2) this transport might play a key role in the development of the disease.

We have shown that, after ip challenge of Tg(OvPrP4) mice with natural sheep-scrapie isolates, the neuron-specific expression of the OvPrP gene was sufficient to allow the transport of infectivity from the peritoneal cavity to the brain, even if PrPC is not supposed to be expressed by cells of the LRS according to the NSE promoter. PrPC can be present at the periphery at the surface of the nervous fibers. This may thus promote the transfer of infectivity from the peritoneal cavity to the brain, probably allowed by a possible capture of the infectious agent at the level of these nerve endings. This is consistent with the findings of several studies showing that, after peripheral challenge of mice, peripheral nerves, which express PrPC, may be the final common pathway for neuroinvasion in vivo [12, 31–34]. In addition, given that PrPC has been shown to be retrogradely transported from the axon ending along peripheral nerves [35], PrPSc could also be subjected to the same mechanism of transport. Otherwise, it could be consistent with the proposed “domino mechanism” hypothesis [21], by which incoming PrPSc converts resident PrPC on the cell surface, thus propagating the infection. Moreover, it has been reported that transgenic mice overexpressing PrPC under the control of its own regulatory sequences [32, 36] support rapid neuroinvasion on intranervous and footpad inoculation of the infectious agent, indicating a direct neuronal spread in these transgenic mice [32]. In addition, after ip delivery of prions, disease can be delayed by sympathectomy or can be accelerated by sympathetic hyperinnervation of lymphoreticular organs [32]. Some other approaches addressing the dynamics of scrapie pathogenesis by tracing the spread of the agent have shown that the infectious agent enters the CNS at the thoracic spinal cord and then spreads rostrally to the brain [17, 37–39]. It is also noteworthy that experiments with transgenic mice (NSE HaPrP/MoPrP—/—) inoculated with the 263K hamster-scrapie strain [12] have also demonstrated that HaPrP expression in neurons was also sufficient for infection after oral or ip infection, but no accumulation of PrPSc was detected in the spleen. This indicates that the TSE agent may be capable of bypassing the LRS and may proceed directly to the brain via peripheral nerves.

In addition, in our transgenic mice, PrPSc can be transported not only from the periphery to the brain but also from the brain to the periphery. The anterograde transport of PrPSc toward the spleen is not, at first glance, surprising, given that, after ic inoculation of wild-type mice, levels of infectivity in the spleen increase marginally earlier than they do after ip inoculation [15, 40]. Nevertheless, the presence of PrPSc in the spleen remained unexpected. One explanation to this spleen accumulation may be attributed to a capture of PrPSc in the spleen at the nerve-ending surface. Moreover, the FDC-like pattern of PrPSc accumulation observed is particularly important because FDCs have been identified as one of the cell types of the lymphoid system that sustain replication of the agent in TSE [18, 41–43]. Given that FDCs are antigen-presenting cells, the presence of PrPSc in the LRS might result from the uptake of PrPSc present in nervous fibers by FDCs. Moreover, PrPC is attached to the cell surface by a GPI anchor, and such GPIanchored proteins are known to be transferable from cell to cell [44]. Given the intimate association of the extensively interdigitate FDCs with autonomic fibers that innervate lymphoid organs, it would not be impossible that PrPC-negative FDCs would become transiently PrPC- or PrPSc-positive cells after contact with PrPC- or PrPSc-producing nervous fibers. This suggests that FDCs might therefore indirectly participate in the amplification process in our model, or at least play a role in storage [16, 28, 45, 46]. This is also supported in our model by the double labeling that suggested close vicinities of PrPSc-accumulating cells with tyrosine hydroxylase—positive noradrenergic fibers. In addition, it has been proposed that the anatomic location of FDCs relative to central arteries and nerves might also play a role in the efficiency of neuroinvasion after injection [47]. In mice deficient in the CXCR5 chemokine receptor, FDCs and B cells are located closer to the central artery and nerves than in wild-type mice. These knockout mice have shorter incubation periods after peripheral infection with the scrapie agent, possibly as a result of an increased efficiency of neuroinvasion, given the proximity of FDCs to splenic nerves [47].

That we find detectable PrPSc levels in the spleen and in the Peyer patches indicate first a retargeting of the infectious agent in the lymphoid system and second that the involvement of the LRS in the development of the disease seems to be more important than we first thought. Reasons for this possible implication of the spleen in our model are not easy to identify. Preliminary results with splenectomized mice also indicated that the infectious agent may require spleen partners in the LRS, although it is difficult to speculate on that because the pilot study was performed in a limited number of animals; in particular, the prion infection could be attested by PrPSc detection in the brain for only 1 sheep isolate, homozygous for the ARQ allele. Because this may be due to the nature of the strain present in the isolate, as has been proposed for SCID mice infected at variable infectious titers [48], or to a possible transmission barrier resulting from genotype discrepancies between PrPs of the host and infectious donor [25] for isolate 1, we must perform many more experiments in splenectomized mice with a larger number of animals and with different strains and titers.

To conclude, our results suggest that PrPC expressed by cells of the LRS are not necessary for neuroinvasion or for their ability to accumulate abnormal PrP. In addition, nervous fibers may facilitate prion propagation first by increasing prion uptake by the autonomic nerve terminals that extensively innervate lymphoid organs and second by promoting the circulation of PrPSc in the host in a centripetal and/or centrifugal manner between the CNS and the periphery. Taken together, our results show that the extracerebral circulation of PrPC or PrPSc present in our model seems to be relevant for the development of the disease.

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Acknowledgments

We are grateful to Cedric Lambert, for the surgical manipulations; Dominique Canal and Jérémy Verchère, for Western blot analyses; Emilie Antier and Clément Lavigne, for sciatic nerve sampling; and Sandrine Philippe, for statistical advice.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: La Fondation pour la Recherche Médicale (grant to C.C.).

  • Received August 16, 2006.
  • Accepted November 2, 2006.
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