crucial role for prion protein membrane anchoring in the neuroinvasion and neural spread of prion...

JOURNAL OF VIROLOGY, Feb. 2011, p. 1484–1494 Vol. 85, No. 40022-538X/11/$12.00 doi:10.1128/JVI.02167-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Crucial Role for Prion Protein Membrane Anchoring in theNeuroinvasion and Neural Spread of Prion Infection�

Mikael Klingeborn,1 Brent Race,1 Kimberly D. Meade-White,1 Rebecca Rosenke,2James F. Striebel,1 and Bruce Chesebro1*

Laboratory of Persistent Viral Diseases1 and Rocky Mountain Veterinary Branch,2 Rocky Mountain Laboratories,National Institute of Allergy and Infectious Diseases, Hamilton, Montana

Received 14 October 2010/Accepted 19 November 2010

In nature prion diseases are usually transmitted by extracerebral prion infection, but clinical disease resultsonly after invasion of the central nervous system (CNS). Prion protein (PrP), a host-encoded glycosylphos-phatidylinositol (GPI)-anchored membrane glycoprotein, is necessary for prion infection and disease. Here, weinvestigated the role of the anchoring of PrP on prion neuroinvasion by studying various inoculation routes inmice expressing either anchored or anchorless PrP. In control mice with anchored PrP, intracerebral or sciaticnerve inoculation resulted in rapid CNS neuroinvasion and clinical disease (154 to 156 days), and after tongue,ocular, intravenous, or intraperitoneal inoculation, CNS neuroinvasion was only slightly slower (193 to 231days). In contrast, in anchorless PrP mice, these routes resulted in slow and infrequent CNS neuroinvasion.Only intracerebral inoculation caused brain PrPres, a protease-resistant isoform of PrP, and disease in bothtypes of mice. Thus, anchored PrP was an essential component for the rapid neural spread and CNSneuroinvasion of prion infection.

Prion diseases, also known as transmissible spongiform en-cephalopathies (TSE diseases), are fatal neurodegenerativediseases of humans and animals. TSE diseases include scrapiein sheep, chronic wasting disease (CWD) in cervids, and bo-vine spongiform encephalopathy (BSE) in cattle as well askuru, Gerstmann-Straussler-Scheinker syndrome (GSS), andfamilial, sporadic, iatrogenic, and variant forms of Creutzfeldt-Jakob disease (CJD) in humans. Within a species, TSE dis-eases are easily transmissible by intracerebral inoculation ofinfected tissue homogenates, whereas transmission to a newspecies is usually inefficient (16). A hallmark of prion diseasesis the accumulation in infected tissues of a partially pro-tease-resistant isoform of the prion protein ([PrP] PrPres).The detection of PrPres by immunoblotting or immunohis-tochemistry is often used as an important diagnostic featureof prion disease. PrPres is generated by misfolding andaggregation of host-encoded protease-sensitive prion pro-tein, PrPsen, which is attached to the outer leaflet of theplasma membrane via a glycosylphosphatidylinositol (GPI)moiety (59). PrPsen is required for susceptibility to prioninfection and for pathogenesis and transmission of priondiseases (10, 14).

The fastest route of TSE infection is via direct entry to thecentral nervous system (CNS) by intracerebral (i.c.) or intraspi-nal inoculation (39, 42). However, natural and experimentalprion infection models include a number of different routes ofexposure outside the CNS where disease onset is usuallyslower. Most notably these include intraperitoneal (i.p.), intra-venous (i.v), intraneural (i.n.), intratongue (i.t.), subcutaneous,

intranasal, and oral routes of exposure (7, 9, 13, 26, 27, 40, 43,51). Peripheral infection is often accelerated by local amplifi-cation of agent in follicular dendritic cells (FDC) of lymphoidorgans, followed by spread via local nerves to the CNS (12, 30,43, 47, 49, 53). Alternatively, neuroinvasion via peripheralnerves after i.p., i.v., i.n., i.t., or ocular inoculation may occurwithout agent amplification in lymphoid tissues (5, 40, 42,43, 55). In most cases neurons are the final route of spreadto the CNS, but these neurons must express PrP to befunctional in this respect (8, 55). The mechanism by whichscrapie infectivity is transported along peripheral nerves tothe CNS is not well understood, and some studies suggestthat conventional axonal transport is not the main mecha-nism (28, 40, 44, 45).

To study if PrPres spread to the CNS in peripheral nerveswas dependent on membrane anchoring of PrP, we comparedwild-type mice expressing anchored PrP with transgenic mice(tg44�/�) expressing only anchorless PrP, i.e., lacking the usualGPI membrane anchor (18). Intracerebral scrapie inoculationof tg44�/� mice leads to high levels of infectivity and amyloidPrPres in CNS and results in a fatal amyloid brain disease (17).In these mice the PrP amyloid-associated neuropathology andcerebral amyloid angiopathy were similar to observations inseveral human GSS patients expressing PrP molecules lackingthe GPI anchor (25, 34, 56). Thus, anchorless PrP transgenicmice appeared to be an excellent model for these unusualforms of familial prion disease.

In the present experiments we employed five differentroutes of inoculation outside the CNS. For all extracerebralroutes of inoculation, GPI anchoring of PrP was requiredfor efficient spread of infectivity to the brain. Therefore,anchored PrP appeared to be an essential part of the mech-anism of rapid neural spread of PrPres and prion infectivityto the CNS.

* Corresponding author. Mailing address: Laboratory of PersistentViral Diseases, Rocky Mountain Laboratories, National Institute ofAllergy and Infectious Diseases, Hamilton, MT 59840. Phone: (406)363-9354. Fax: (406) 363-9286. E-mail: [email protected].

� Published ahead of print on 1 December 2010.

1484

MATERIALS AND METHODS

Experimental mice and tissue collection for histochemical and biochemicalanalysis. All mice were housed at the Rocky Mountain Laboratories (RML) inan AAALAC-accredited facility, and research protocols and experimentationwere approved by the NIH RML Animal Care and Use Committee (protocolnumber 2007-50). This study was carried out in strict accordance with the rec-ommendations in the Guide for the Care and Use of Laboratory Animals of theNational Institutes of Health.

Transgenic GPI anchorless PrP mice (tg44) expressed the transgene in ho-mozygous form (�/�) and did not express normal GPI-anchored mouse PrP(17). Mice were bred and genotyped at RML. Weanling C57BL/10 mice wereobtained from Harlan Laboratories, Indianapolis, IN. Animals were observeddaily for onset and progression of scrapie and euthanized when late-stage scrapiesigns were consistently present. One C57BL/10 mouse from each inoculationroute was euthanized at 100 days postinfection (dpi) for histochemical andbiochemical analyses. Tissues were also collected from C57BL/10 mice when theywere in an advanced stage of scrapie. In a similar fashion, some tg44�/� micefrom each group were euthanized at various days postinfection and at a terminalstage of disease or no later than 600 dpi for histochemical and biochemicalanalyses. A portion of each tissue was flash frozen in liquid nitrogen and kept at�80°C for future use in biochemical analyses. A second portion was immersed in10% neutral buffered formalin (3.7% formaldehyde) for histochemistry studies.At necropsy of mice, brain was collected first to avoid contamination with PrPresfrom other tissues.

Scrapie inoculation of mice. Young adult mice were inoculated with an RML/Chandler scrapie brain homogenate from C57BL/10 mice. For intracerebral,intraperitoneal, intravenous, and intravenous injection followed with a mockintracerebral needle stab, 50 �l of a 1% brain suspension containing 1.0 � 106

50% infective doses (ID50) was inoculated. One ID50 is the dose causing infec-tion in 50% of C57BL/10 mice. Some routes of inoculation did not allow injectionof large volumes, so we reduced the volume and increased the percentage ofbrain suspension to more closely match the ID50 inoculated. Mice inoculated byintrasciatic, perisciatic, intravitreal, and supraciliary routes received 2 �l of a10% brain suspension containing 4 � 105 ID50, and mice inoculated in thetongue received 5 �l of a 10% suspension containing 1 � 106 ID50.

Route-specific techniques are described below.For intracerebral inoculation, mice were anesthetized with isoflurane and

injected in the left parietal lobe with a 27-gauge, 0.5-in. needle.For intravenous injections mice were restrained in a chute and inoculated in

the tail vein. Groups of mice receiving an intracerebral needle stab were anes-thetized with isoflurane immediately following their i.v. scrapie inoculation, anda stab wound was made in the left parietal lobe using a sterile 27-gauge needlewith no inoculum (23, 39). The needle was inserted through the skull anddirected into the left parietal lobe; following a slight retraction of the needle, itwas redirected into a second region of the parietal lobe.

For intraneural inoculation mice were anesthetized with an intraperitonealinjection of a combination of ketamine (80 mg/kg), xylazine (16 mg/kg), andacepromazine (2 mg/kg). The lateral surface of the left rear leg was clipped andprepared for surgery. Once mice had reached a medium-to-deep plane of anes-thesia, a 1-cm incision was made just caudal to the midfemoral region. Bluntdissection was used to retract muscles and expose the sciatic nerve. After visu-alization of the nerve, a curved forceps was placed under the nerve and used toelevate the nerve for easier inoculation. A 30-gauge needle attached to a Ham-ilton syringe was inserted through the nerve sheath and threaded into the nerve.The needle was then moved back and forth 1 to 2 mm within the nerve sheathapproximately 10 times (6), and 2 �l of 10% brain suspension was then injectedwithin the nerve sheath. The skin incision was closed with 7-mm surgical skinstaples.

For perineural inoculation, the same methods described for the intraneuralinoculation were used to expose the nerve, but the nerve sheath was not pene-trated by the needle, and the inoculum was placed outside the nerve rather thandirectly into the nerve sheath.

For tongue inoculation mice were anesthetized as described in the intraneuralsection. A 30-gauge needle was directed at an acute angle into the right side ofthe tongue to the level of the subepithelium where 5 �l of inoculum was injected.

For intraocular inoculations mice were anesthetized as described for the in-traneural nerve inoculations. Inoculum was deposited either in the vitreouschamber (36) or in the supraciliary space of the eye (1) using a 32-gauge needleattached to a Hamilton syringe. Results were similar for both ocular routes.

Immunoblotting analysis of PrPres. To detect PrPres by immunoblotting,tissue homogenates (20%) were prepared in 10 mM Tris-HCl (pH 7.4) using aMini-Beadbeater (Biospec products, Bartlesville, OK). All homogenates were

sonicated for 1 min using a Vibracell cup-horn sonicator (Sonics, Newtown, NJ)and briefly vortexed. PrPres preparation was done as previously described (48).Briefly, an aliquot of a 20% tissue homogenate was adjusted to 100 mM Tris-HCl(pH 8.3), 1% Triton X-100, and 1% sodium deoxycholate and treated for 45 minat 37°C with proteinase K at a final concentration of 50 �g/ml. The reaction wasstopped by the addition of Pefabloc SC (Roche) to a final concentration of 4 mM,and the sample was placed on ice for 5 min. An equal volume of 2� Laemmlisample buffer (Bio-Rad, Hercules, CA) containing 10% �-mercaptoethanol wasadded; samples were boiled for 5 min and then frozen at �80°C until needed.Freshly boiled samples were electrophoresed on 16% SDS-PAGE gels (Invitro-gen, CA). Immunoblots were probed with D13 anti-PrP antibody (InPro Bio-technology, S. San Francisco, CA) followed by a peroxidase-conjugated anti-human IgG secondary antibody (Sigma, St. Louis, MO). Bands were detectedusing enhanced chemiluminescence substrate (ECL) as directed by the manu-facturer (GE Healthcare Life Sciences, Piscataway, NJ).

The amount of brain PrPres detected by immunoblotting was defined relativeto the signal detected in i.c. inoculated tg44�/� mice at terminal stage, andscoring was as follows: 3, 25 to 100% of terminal mouse; 2, 5 to 24% of terminalmouse; 1, 1 to 4% of terminal mouse; 0, no detectable signal (�1% of signal interminal mouse).

Neuropathology and IHC. Tissues were removed and placed in 10% neutralbuffered formalin (3.7% formaldehyde) for 3 to 5 days before dehydration andembedding in paraffin. Serial 5-�m sections were cut using a standard Leicamicrotome, placed on positively charged glass slides, and dried overnight at 56°C.Slides were then deparaffinized using standard procedures, rehydrated to aque-ous conditions, and processed for standard hematoxylin and eosin (H&E) stain-ing or immunohistochemistry (IHC) analysis. For immunohistochemical detec-tion of PrPres using Fast Red chromogen (Ventana, Tucson, AZ), tissue sectionswere pretreated in citrate buffer, pH 6.0, and heated to 120°C at 20 lb/in2 for 20min in a Decloaking Chamber (Biocare, Walnut Creek, CA) for antigen re-trieval, followed by staining in the Ventana automated NexES stainer. Stainingfor PrP was done using human anti-mouse PrP antibody D13 at a dilution of1:500 (InPro Biotechnology) at 4°C for 16 h, followed by a biotinylated anti-human IgG at 1:500 (Jackson ImmunoResearch, West Grove, PA) and strepta-vidin-alkaline phosphatase with Fast Red chromogen.

Immunohistochemical detection of PrPres using diaminobenzidine (DAB)chromogen (DAB Map kit; Ventana) was done on selected slides at later timesin the course of the experiments as follows. Antigen retrieval and staining wereperformed using a Ventana automated Discovery XT stainer. PrPres antigenswere exposed by incubation in CC1 buffer (Ventana) containing Tris-borate-EDTA, pH 8.0, for 188 min at 95°C. Staining for PrP was done using humananti-mouse PrP antibody D13 at a dilution of 1:500 (InPro Biotechnology) at37°C for 2 h, followed by a biotinylated anti-human IgG at 1:500 (JacksonImmunoResearch) and avidin-horseradish peroxidase with DAB as chromogen.The staining for PrPres was observed using an Olympus BX51 microscope usingMicrosuite FIVE software.

Immunofluorescence microscopy. For immunofluorescence staining for PrPresand astroglia or PrPres and microglia, tissue sections were pretreated in citratebuffer, pH 6.0, and heated to 120°C at 20 lb/in2 for 20 min for PrPres antigenretrieval. Staining was performed using D13 human anti-mouse PrP (1:500),rabbit anti-glial fibrillary acidic protein ([GFAP] 1:1,000; Dako, Carpinteria,CA), and rabbit anti-Iba1 (1:1,000; Wako, Richmond, VA). D13 plus eitheranti-GFAP or anti-Iba1 antibodies was coincubated for 2 h at room temperature,followed by coincubation with Alexa-Fluor 568-labeled anti-human IgG plusAlexa-Fluor 488-labeled anti-rabbit IgG (Invitrogen, Carlsbad, CA) for 30 min atroom temperature in darkness. Both secondary antibodies were used at a 1:200dilution. After samples were rinsed with double-distilled H2O (ddH2O), cover-slips were applied to tissue sections with ProLong Gold antifade reagent with4�,6�-diamidino-2-phenylindole (DAPI; Invitrogen) to stain nuclei and protectfluorescence from fading. The staining for PrPres, astroglia, and microglia wasobserved using an Olympus BX51 microscope using Microsuite FIVE software.

RESULTS

Slow neural spread after intraneural scrapie inoculation. Inmice and hamsters, i.n. inoculation in the sciatic nerve isknown to be an efficient extracerebral route of scrapie infec-tion, which results in rapid neuroinvasion and clinical scrapie(2, 6, 40). In agreement with these results, we found similarrapid development of clinical brain disease after both i.c. andi.n. inoculation in C57BL/10 mice expressing anchored PrP;

VOL. 85, 2011 PRION NEUROINVASION 1485

however, after perineural inoculation clinical disease was sig-nificantly delayed (Table 1). To test whether PrP anchoringhad a role in neural spread and neuroinvasion, we comparedi.c. and i.n. routes in homozygous anchorless PrP mice, linetg44�/� (17). In these transgenic mice the two routes werequite different. After i.c. inoculation PrPres amyloid depositionwas detected in the brain starting at 146 dpi (Fig. 1A), andclinical neurological disease was seen at 298 to 321 dpi (Fig.1A). In contrast, after i.n. inoculation, mice had no PrPresamyloid in brain or clinical brain disease even after 600 dpi(Fig. 1A). However, i.n. inoculation resulted in some neuralspread and accumulation as PrPres was detected by immuno-blotting (Fig. 1B) and by IHC (Fig. 1C to F) in spinal cord andsciatic nerve and nerve roots on the ipsilateral (inoculated)side at late times postinfection. Furthermore, a higher fre-quency of PrPres detection was found in nerve and lumbarspinal cord than in thoracic and cervical spinal cord regions,which suggested slow progression of PrPres up the nerve andthe spinal cord (Table 2). In addition, between 500 and 600dpi, clinical signs of rear leg weakness and/or paralysis werenoted on the ipsilateral side, indicating that significant damagemight have been caused by the local PrPres deposition in thesenerves or in lumbar spinal cord (Fig. 1C to F). However, thelack of PrPres in brain and the small amount in upper spinalcord following i.n. inoculation, in spite of extensive PrPresdeposition in the sciatic nerve, indicated that neural spread ofPrPres was slow in tg44�/� mice. Therefore, GPI-anchoredPrP was associated with rapid neural spread and brain infec-tion following scrapie inoculation via sciatic nerve, whereasanchorless PrP was not.

PrPres deposition in nerves and muscles of the tongue. Wenext tested tongue inoculation as this extraneural route is knownto result in CNS neuroinvasion via the hypoglossal nerve in ani-mals expressing anchored PrP (4, 5, 7, 50). In C57BL/10 mice wealso found 100% incidence of clinical scrapie after inoculationvia the i.t. route (Table 1). In contrast, in tg44�/� mice, i.t.inoculation resulted in deposition of PrPres in tongue muscleand nerves (Fig. 2A and B). However, no PrPres was found inbrain (Fig. 2C), and no clinical signs were observed in any ofthe six mice analyzed from 354 to 600 dpi. The absence of CNSneuroinvasion, together with the presence of PrPres in nervebranches in the tongue, suggested that spread of infectivity by

cranial nerves was very inefficient in tg44�/� mice compared tospread in C57BL/10 mice. This conclusion was in agreementwith results from sciatic nerve inoculation.

Infection of retina and brain after ocular scrapie inocula-tion. Ocular (i.o.) scrapie inoculation was also tested as thisroute is known to produce significant local infection as well asCNS neuroinvasion via the optic nerve and optic tract in miceexpressing anchored PrP (22, 24, 42, 57). In C57BL/10 mice wefound 100% incidence of clinical scrapie and abundant PrPresin brain at 231 � 32 days after inoculation via the i.o. route(Table 1). In contrast, in tg44�/� mice, after i.o. inoculationPrPres was not detected in brain until 357 dpi. PrPres amyloidplaques were found in the brains of 8 of 11 tg44�/� micebetween 357 and 604 dpi (Fig. 3A and B). Two of the eightpositive mice also had clinical signs of CNS disease (Fig. 3A).Interestingly, the majority of PrPres plaques in brain were notlocated in visual areas but were in cerebellum and cerebralcortex, often associated with the pia mater of the meninges andadjacent leptomeningeal blood vessels entering the brain (Fig.3C and D).

After i.o. inoculation of tg44�/� mice, large perivascularPrPres amyloid deposits were found in the inoculated eye byimmunohistochemistry at 232 dpi (Fig. 4A and C). The mostextensive PrPres accumulation was found surrounding bloodvessels in the retinal ganglion layer (Fig. 4A, B, F, and G). Atthe site where the optic nerve enters the retina, PrPres depo-sition was readily detected in optic nerve at 232 dpi (Fig. 4C toE) and was extracellular but closely associated with GFAP-expressing astrocytes (Fig. 4E and F) and Iba1-positive acti-vated microglia and/or macrophages with large plump cell bod-ies and thickened processes (Fig. 4G). PrPres was also found inclose association with the pia mater in optic nerve (Fig. 4D andE). From this location PrPres might spread from the pia to thecerebrospinal fluid (CSF) in the adjacent subarachnoid spaceand subsequently via CSF to cortical and cerebellar meninges(Fig. 3C and D). The relatively high incidence of neuroinvasionin i.o. inoculated tg44�/� mice indicated that spread of infec-tivity from retina to brain was more efficient than spread fromsciatic nerve or tongue inoculation sites, but the progressionwas still very slow compared to that in mice with anchored PrP(Table 1).

Limited brain infection after intravenous or intraperitonealscrapie inoculation. In a previous study, i.c. and i.p. inoculatedheterozygous tg44�/� mice had readily detectable levels ofinfectivity in blood plasma (54). This finding together with theprominent perivascular distribution of PrPres in brain after i.c.inoculation (17, 18) prompted us to investigate the possibilityof hematogenous neuroinvasion in tg44�/� mice. In C57BL/10mice, i.v. and i.p. scrapie inoculation caused 100% incidence ofbrain disease at 203 � 6 and 193 � 5 dpi, respectively (Table1), and accumulation of high levels of PrPres was detectedin brain at similar times (Fig. 5B, lanes 2 and 3). However,perivascular PrPres accumulation was not a prominent featurein C57BL/10 mice. Therefore, there was no obvious morpho-logical evidence for hematogenous neuroinvasion in C57BL/10mice. Furthermore, previous experiments by several laborato-ries using mice expressing anchored PrP suggested that neu-roinvasion after either i.v. or i.p. inoculation proceeds by am-plification in lymphoid tissues, followed by spread to the CNS

TABLE 1. Influence of inoculation route on incidence andincubation period of scrapie disease in C57BL/10 mice

Inoculationroutea

Incubationperiod (dpi)b

Incidence ofdiseasec

i.c. 154 � 6 5/5i.n. 156 � 2 9/9p.n. 233 � 7 4/4i.t. 198 � 12 7/7i.o. 231 � 32 6/6i.p. 193 � 5 3/3i.v. 203 � 6 6/6i.v. � i.c. stab 190 � 6 9/9

a The ID50 dose of RML scrapie inoculated via the different routes is de-scribed in Materials and Methods. i.c., intracerebral; i.n., intraneural; p.n., per-ineural; i.t., intratongue; i.o., intraocular; i.p., intraperitoneal; i.v., intravenous.

b Number of days postinoculation when mice were euthanized due to clinicalscrapie. Values are means � 1 standard deviation.

c Number of mice positive for clinical scrapie/total number of mice in group.

1486 KLINGEBORN ET AL. J. VIROL.

by peripheral nerves rather than by hematogenous neuroinva-sion (8, 12, 29, 30, 43, 47, 49, 53).

In contrast to mice with anchored PrP, tg44�/� mice inoc-ulated by i.v. or i.p. routes had no brain PrPres until after 384dpi (Fig. 5A). However, by 600 dpi, 3 of 12 i.p. inoculated mice

and 3 of 11 i.v. inoculated mice had PrPres detectable byimmunoblotting in brain (Fig. 5A). Two of these i.v. inoculatedmice had clinical signs at late times (Fig. 5A). Representativeimmunoblots are shown in Fig. 5B. By immunohistochemistry,PrPres was found primarily adjacent to meninges of cerebel-

FIG. 1. PrPres deposition in sciatic nerve and CNS following intracerebral and sciatic nerve scrapie inoculation of tg44�/� mice. (A) Relativelevels of PrPres deposition detected by immunoblotting in brain of tg44�/� mice after scrapie inoculation via intracerebral (i.c.), intraneural (i.n.),and perineural (p.n.) routes. Solid symbols represent mice with clinical neurological signs as previously described (17). Relative amount of brainPrPres detected by immunoblotting is defined in Materials and Methods. (B) Immunoblot of PrPres in sciatic nerve and CNS following i.c. (lanes1 to 4) and i.n. (lanes 5 to 10) scrapie inoculation. Brain samples from clinical C57BL/10 mice (WT) inoculated via indicated routes are in lanes1 and 5. Tissues from an i.c. inoculated tg44�/� mouse are in lanes 2 to 4 (308 dpi). Brain and/or lumbar spinal cord samples from two i.n.inoculated tg44�/� mice are in lanes 6 and 7 (600 dpi) and lane 8 (600 dpi). Ipsilateral sciatic nerves are shown for two i.n. inoculated tg44�/� micein lanes 9 and 10 (both 600 dpi). Lane 8 was overexposed to show PrPres in lumbar spinal cord. Lanes 1, 2, and 5 were loaded with 0.25 mg, andall other lanes were loaded with a 1-mg tissue equivalent. Approximate molecular sizes of 18 and 22 kDa are indicated. (C to F) Immunohisto-chemical detection of PrPres deposits in sciatic nerve and lumbar spinal cord following sciatic nerve scrapie inoculation in tg44�/� mice.(C) Extensive PrPres deposition was found in the ipsilateral sciatic nerve at 454 dpi. (D) Numerous amyloid plaques (arrows) seen by hematoxylinand eosin staining of the sciatic nerve seen in panel C. (E) PrPres deposition was found in nerve roots (N) and white matter tracts (WM) but onlyrarely in gray matter tracts (GM) on the ventral aspect of the lumbar spinal cord at 600 dpi. (F) The boxed area in panel E magnified. Arrows showblood vessels surrounded by PrPres. Bars, 100 �m (C, D, and E) and 50 �m (F).


lum and cerebrum (Fig. 5C and D) and in rare cases was alsoseen in the choroid plexus (Fig. 5C). This was similar to themeningeal PrPres seen after i.o. inoculation (Fig. 3C and D).The low incidence and slow tempo of CNS infection after i.v.and i.p. scrapie inoculation in mice expressing anchorless PrPcompared to mice expressing anchored PrP supported ourprevious conclusions that anchored PrP had an important in-fluence on neuroinvasion after peripheral prion inoculation.

To investigate the role of the blood-brain barrier (BBB) inhematogenous spread of scrapie to the brain, we mechanicallydisturbed the BBB at the time of i.v. inoculation. Mice wereinoculated by the i.v. route with scrapie, and within 2 minthereafter a needle stab to the brain was made with a cleanneedle (23, 39). In C57BL/10 mice the mean incubation periodwas 190 dpi after i.v. inoculation with the i.c. stab compared to203 dpi without the i.c. stab, which was a small but statisticallysignificant difference (Mann-Whitney; 95% confidence interval[CI], P 0.0024). In tg44�/� mice, the combined i.v. and stab(i.v.�stab) method of infection compared to i.v. inoculationalone increased the incidence of neuroinvasion (Fig. 6A). Fur-thermore, PrPres deposition was clearly evident in the brainalong the i.c. stab needle track at 225 dpi (Fig. 6B and C),suggesting that scrapie invasion of the brain occurred via leak-age from blood into the stab wound. Thus, these experimentssupported the conclusion that the BBB was an obstacle to earlyneuroinvasion by blood infectivity in both C57BL/10 mice andtg44�/� mice.

Spread of PrPres to extraneural tissues after peripheralinfection routes in tg44�/� mice. In several previous studiesafter either i.c. or i.p. scrapie infection of heterozygous tg44�/�

mice, extensive prion infectivity and/or PrPres deposition wasdetectable by immunoblotting or IHC at extraneural sites suchas lymphoid tissues, brown and white fat, heart, tongue, skel-etal muscle, and plasma (18, 54, 60). In the present studiesafter inoculation of tg44�/� mice by five peripheral routesdescribed above, brown fat had prominent PrPres detectablestarting at 146 to 230 dpi (Table 3). PrPres was also found inheart, spleen, white fat, and colon (lamina propria) at timesprior to detection in brain. Thus, in the absence of PrP an-choring, spread of PrPres to extraneural sites was rapid, inmarked contrast to the slow and inefficient spread to the CNSfound in these same mice. Therefore, spread to these differentlocations appeared to be mediated by distinct mechanisms.

DISCUSSION

Most cases of natural transmission of prion infection arebelieved to occur at peripheral sites, followed by spread to theCNS. In the present study we investigated the role of PrPmembrane anchoring in scrapie neuroinvasion using periph-eral sites of infection which mimic various types of natural oriatrogenic infection. Five different routes of scrapie inoculationoutside the brain, i.n., i.t., i.o., i.p., and i.v., were used inC57BL/10 mice expressing anchored PrP and in tg44�/� trans-genic mice that express only PrP lacking the GPI anchor. In all

FIG. 2. (A) Immunohistochemical detection of PrPres deposits intongue nerves at 600 days following scrapie inoculation in the tongueof a tg44�/� mouse. Arrows indicate PrPres deposits in close associa-tion with neurolemma. (B) Widespread PrPres accumulation mostlyalong capillaries between muscle cells in tongue of mouse from panelA. (C) Sagittal section of medullar area of brain stem of the samemouse showing no detectable PrPres. Cerebellum and fourth ventricleare visible in the upper left corner. Inset shows higher magnification ofboxed area. Light brown diffuse staining is PrPsen, which is also seenin uninoculated animals (data not shown), and is easily distinguishedfrom PrPres (panels A and B) in tg44�/� mice. Bars, 50 �m (A, B, andinset in C) and 500 �m (C).

TABLE 2. PrPres deposition in brain, three levels of spinal cord,and ipsilateral sciatic nerve following sciatic nerve scrapie

inoculation of tg44�/� micea

Site

Frequency of PrPres deposition (no. of positivesamples/total no. of samples tested) at:

155–230 dpi 349–454 dpi 544–600 dpi

Brain 0/2 0/2 0/7Cervical cord 0/2 0/2 2/7Thoracic cord 0/2 0/2 1/7Lumbar cord 0/2 1/2 4/7Sciatic nerveb 1/2 2/2 7/7

a Mice were inoculated in sciatic nerve with RML scrapie at 4 � 105 ID50.b At 15 dpi, sciatic nerve near the inoculation site was negative for PrPres by

immunoblotting and IHC (data not shown), suggesting that PrPres detected atlater times was newly generated.


of these routes, neuroinvasion was severely affected by the lackof anchored PrP. Strikingly, in tg44�/� mice, three routes (i.n.,i.t., and i.o.) resulted in no or slow neuroinvasion of the brain(Fig. 1A, 2C, and 3A), whereas these same routes gave efficientneuroinvasion via neural spread in mice with anchored PrP(Table 1) (5, 22, 40). However, the poor neuroinvasion of brainafter i.n., i.t., and i.o. inoculation in tg44�/� mice was not dueto a lack of PrPres generation in nerves as PrPres could bedetected in sciatic nerve, branches of the hypoglossal nerve intongue, and optic nerve at later times after inoculation. Thus,a lack of anchored PrP appeared to affect the speed and effi-ciency of neuroinvasion via nerves in tg44�/� mice but did notblock accumulation of PrPres in peripheral nerves.

Another possible interpretation of these findings is thatpresence of anchorless PrP, rather than absence of anchoredPrP, might alter neuroinvasion. However, in our previous ex-periments using mice coexpressing anchored and anchorlessPrP, i.p. scrapie inoculation resulted in CNS neuroinvasion in100% of mice between 275 and 357 dpi (18; also and B. Raceand B. Chesebro, unpublished data). Therefore, lack of neu-

roinvasion after peripheral scrapie inoculation in tg44�/� miceappears to depend more on the absence of anchored PrPrather than on the presence of anchorless PrP.

Two other caveats might also influence the interpretationof our results. First, peripheral nerves in mice expressing noPrPsen (Prnp�/� mice) or mice expressing only anchorlessPrPsen (heterozygous tg44�/� mice) were recently found todevelop abnormal axonal morphology at around 60 weeks ofage (11), which might influence spread of PrPres. However,this axonal problem was not detected in younger mice such asthose used for our infections and thus would not be expectedto influence the tempo of neuroinvasion in our experiments.Second, PrPsen expression levels in tg44�/� mice are 8-foldlower than in C57BL/10 mice (17), and this lower expressionlevel might also influence the rate of neuroinvasion by neuraland other peripheral routes tested here. However, in othertransgenic mice expressing anchored PrP at 10-fold lower lev-els than C57BL/10, we previously found that i.p. scrapie infec-tion gave 90 to 100% incidence of neuroinvasion and clinicalscrapie with incubation periods somewhat longer than the i.c.

FIG. 3. PrPres deposition in brain and eyes following ocular scrapie inoculation in tg44�/� mice. (A) Relative amount of PrPres depositiondetected in brain by immunoblotting at various times after inoculation. Solid symbols represent mice with clinical neurological signs. (B) Immu-noblot of PrPres in brain and eyes following ocular scrapie inoculation. High levels of PrPres can be seen at terminal stage of disease in brains (Br)of an i.o. inoculated C57BL/10 (WT) mouse (lane 1), an i.c. inoculated tg44�/� mouse at 308 dpi (lane 2), and an i.o. inoculated tg44�/� mouseat 469 dpi (lane 3), whereas no PrPres was detected in a 520-day-old uninoculated (un) tg44�/� mouse (lane 4). In lanes 5 to 10, brains and eyesfrom two other i.o. inoculated tg44�/� mice are shown (animal 286, 441 dpi; animal 291, 435 dpi). Ipsilateral (I) eyes (lanes 7 and 10) have highlevels of PrPres, while the contralateral (C) eyes (lanes 6 and 9) have undetectable levels of PrPres. Brains had either moderate (lane 8) or low(lane 5) levels of PrPres. Lanes 1 to 3 were loaded with 0.25 mg, and all other lanes were loaded with a 1-mg tissue equivalent. Approximatemolecular sizes of 18 and 22 kDa are indicated by the two bars on the right hand side. (C) Immunohistochemical staining using monoclonalantibody D13 shows PrPres amyloid plaque deposition in a sagittal section of the cerebellum of a tg44�/� mouse at 357 days after ocular scrapieinoculation. (D) Higher magnification of panel C shows perivascular PrPres plaques in the molecular layers (M), as well as in meninges betweentwo lobules, around blood vessels (arrows) and in the granular layer (G) of the third cerebellar lobule. Bars, 500 �m (C) and 100 �m (D).


FIG. 4. Immunohistochemical and immunofluorescence detection of PrPres deposition in eye and optic nerve at 232 dpi after ocular scrapieinoculation in several tg44�/� mice. (A) Extensive PrPres deposition (red, Fast Red staining) in retinal ganglion layer at 232 dpi. The eye was cutat an angle tangential to the ganglion layer, resulting in the appearance of a flap of ganglion layer extending into the vitreal space in the centerof the section. Fewer PrPres deposits are present in the inner nuclear layer (INL) and only rarely were deposits detected in the outer nuclear layer(ONL) or other retinal cell layers outside INL. (B) At higher magnification of the boxed area in panel A, PrPres deposits are evident in the ganglionlayer, and many PrPres plaques have a distinct perivascular appearance (arrows). (C) In a different mouse at 232 dpi the section shows widespreadPrPres accumulation (brown, DAB staining) in the optic nerve entering the retina as well as in the ganglion layer of the retina and in the vitreousfluid. (D) At higher magnification of the boxed area in panel C, PrPres can be seen in optic nerve (ON), vitreous fluid (VF), ganglion layer (G),and inner nuclear layer (INL) and around two retinal blood vessels (arrows). PrPres plaques adjacent to the pia mater of optic nerve are indicated


route (median of 360 days versus 260 days) (35). Therefore,lack of PrP anchoring, rather than low PrPsen levels, appearsto account for the low incidence of neuroinvasion seen afteri.p. infection in tg44�/� mice.

Slow neural spread of PrPres in tg44�/� mice was particu-larly apparent after i.n. inoculation, where the earliest detec-tion of PrPres in lumbar spinal cord was at 349 dpi (Table 2).With an average distance of 20 mm from the inoculation site inthe sciatic nerve to lumbar spinal cord, the rate of neural

PrPres spread was calculated to be roughly 0.06 mm per day.Previous studies have reported rates of spread of scrapie in-fectivity and/or PrPres ranging from 0.5 to 3.3 mm per day inmice and hamsters with anchored PrP (6, 28, 40, 41, 46). Thus,the rate of neural PrPres spread in tg44�/� mice was approx-imately 10 to 50 times lower than in mice and hamsters withanchored PrP, demonstrating that neural PrPres spread wasseverely affected in the absence of the PrP GPI anchor. In bothtg44�/� mice and mice with anchored PrP, neural spread of

by arrowheads. (E) Double immunofluorescence staining of the optic nerve of mouse shown in panels C and D at the level of entry into the retina.PrPres deposits (red) and an extensive network of GFAP-expressing astrocytes (green) can be seen in the optic nerve. This astrocyte network wasalso seen in optic nerve from uninfected tg44�/� mice (data not shown). Arrows indicate PrPres plaques in close proximity to the pia mater onthe edge of the nerve. (F) Double immunofluorescence staining of retina at high magnification shows amyloid PrPres deposits (red), mostlyperivascular and in close proximity to perivascular GFAP-expressing astrocytes (green). Close to the lower edge of the picture, the outer nuclearlayer of the retina stains strongly with the nuclear stain DAPI (blue). (G) Iba1-positive microglia or macrophages (green) were detected in closeproximity to PrPres deposits (red) in the retinal ganglion layer. Most of the Iba1-positive cells have large plump cell bodies (arrows) and thickenedprocesses, suggesting an activated phenotype. Bars, 500 �m (A and C), 100 �m (D and E), and 50 �m (B, F, and G).

FIG. 5. (A) Relative amount of PrPres deposition detected by immunoblotting in brain of tg44�/� mice after intravenous (i.v.) and intraper-itoneal (i.p.) scrapie inoculation. Solid symbols represent mice with clinical neurological signs. (B) Immunoblot of PrPres in brain and after i.v.or i.p. scrapie inoculation. Brains from three C57BL/10 (WT) mice (lane 1, i.c. at 144 dpi; lane 2, i.v. at 197 dpi; lane 3, i.p. at 188 dpi) and a tg44�/�

mouse (lane 4, i.c. at 308 dpi) at terminal stage of disease are shown. Brain from i.v. inoculated tg44�/� mice at 603 dpi (lane 5), 560 dpi (lane6), and 483 dpi (lane 7) showed various levels of PrPres. Brains of i.p. inoculated tg44�/� mice showed undetectable PrPres (lane 8, 601 dpi) orvery low PrPres (lanes 9 and 10, 601 and 580 dpi, respectively). Lanes 5 and 6 and 8 to 10 were loaded with 1.0 mg, and all other lanes were loadedwith 0.25-mg tissue equivalents. Approximate molecular masses of 18 and 22 kDa are indicated on the right-hand side. (C and D) Immunohis-tochemical staining using monoclonal antibody D13 shows PrPres amyloid plaque deposition in brain after intraperitoneal scrapie inoculation oftg44�/� mice. (C) PrPres plaques (red, Fast Red) were detected in the choroid plexus (arrow) and radiating out from blood vessels (arrowheads)in the meninges of the 10th cerebellar lobule and the fissure between the 9th and 10th cerebellar lobules at 740 days after i.p. inoculation. (D) Inanother mouse at 601 dpi after i.p. inoculation, PrPres deposits (brown, DAB stain) were found only along the meninges in the posterior portionof the cerebral cortex. Bar, 200 �m.


PrPres was much slower than conventional retrograde axonaltransport (i.e., 85 mm to 430 mm per day [31]). The very slowneural spread in tg44�/� mice might be due to limited and slowdiffusion of both inoculated and newly formed PrPres in inter-stitial spaces of the nerve. In contrast, in the presence ofanchored PrP, neural spread is likely to be a faster cell-asso-ciated process. Our data show that anchored PrP is a vital partof this process of neural transmission after infection at periph-eral sites. The exact details of this process remain unclear (33),but they appear to differ from the mechanisms involved inneuroinvasion by A�-amyloid as this material showed no evi-dence for neuroinvasion after inoculation by i.v., i.o., oral, andintranasal routes (20). However, in a more recent report, neu-roinvasion after i.p. inoculation of A�-amyloid was observed(21).

Our results show that both i.v. and i.p. routes of scrapieinoculation in tg44�/� mice resulted in slow and inefficientneuroinvasion compared to mice expressing anchored PrP(Fig. 5A). Previous studies have shown that PrP expression in

nerves is necessary for neuroinvasion of the CNS after both ofthese routes of scrapie inoculation in mice (8, 28, 42, 55). Afterboth i.v. and i.p. scrapie inoculation in mice with anchored PrP,CNS neuroinvasion is thought to occur via splanchnic nervesinnervating lymphoid tissues, where scrapie infectivity is am-plified (3, 19, 29). Thus, the poor neuroinvasion of the CNS intg44�/� mice after i.v. and i.p. inoculation might be due topoor spread of PrPres in splanchnic nerves, similar to sciaticand cranial nerves innervating the tongue. Similar results wereseen in recent studies of G3 transgenic mice which produceonly unglycosylated PrP (15). In G3 mice PrP is GPI anchoredto membranes in the Golgi apparatus inside the cell ratherthan on the plasma membrane. G3 mice are susceptible to i.c.infection by 79A scrapie, but after i.p. inoculation they do notdevelop clinical disease or PrPres in brain. These results sug-gest that PrP must be anchored to the cell plasma membranefor effective CNS neuroinvasion.

As has been discussed in a preceding paper, lack of PrPanchoring in tg44�/� mice leads to extensive formation ofPrPres in an amyloid form (17). Possibly, large aggregates ofamyloid PrPres might spread from peripheral inoculation sitesto CNS less efficiently than the usual PrPres aggregates foundin typical scrapie disease in mice, sheep, and other models.However, this conclusion seems unlikely for several reasons.First, tg44�/� mice were inoculated with brain homogenatesfrom C57BL/10 mice expressing anchored PrP, and the inoc-ulum contained little, if any, amyloid PrPres. Second, intg44�/� mice only CNS neuroinvasion was slow, whereasPrPres and infectivity appeared to spread efficiently in extran-eural tissues such as brown fat, heart, colon, and spleen (Table3). Since plasma of infected tg44�/� mice also contained sig-nificant amounts of infectivity (54), it is possible that extran-eural spread in tg44�/� mice is hematogenous or lymphatic.

Interestingly, in mice with anchored PrP, there is also evi-dence against the hematogenous route of neuroinvasion. Afteri.p. and i.v scrapie inoculation, CNS neuroinvasion was notobserved in mice lacking PrP expression in nerves (8) or afteri.p. inoculation of sympathectomized mice (29). In contrast,studies in sheep and goats (32, 58) found that initial sites ofPrPres deposition in CNS were near the circumventricularorgans (CVO) of the brain, which might indicate their role asa possible site of entry of prions from blood. However, thefenestrated capillaries of the CVO, which allow passage ofsmall peptides (up to 50 amino acids), would not be likely toallow penetration by full-length PrPsen or larger multimers of

TABLE 3. Spread of PrPres to brain and brown fat afterinoculation of tg44�/� mice with scrapie brain

homogenate by various routes

TissueEarliest PrPres detection (dpi) by inoculation routea

i.c. i.n. i.t. i.o. i.p. i.v. i.v. � stab

Brain 146 600 600 357 559 385 149Brown fatb 146 230 155c 148 155 149 149

a As detected by immunoblotting or IHC. Inoculation details are presented inthe Materials and Methods section. See Table 1 for abbreviations of the inocu-lation routes.

b PrPres was usually seen first in brown fat and subsequently in white fat, heart,spleen, and colon.

c After i.t. inoculation, earliest PrPres was found in white fat.

FIG. 6. The effect of blood-brain barrier manipulation on PrPresdeposition in brain of tg44�/� mice after intravenous scrapie inocula-tion. (A) Relative amount of PrPres detected by immunoblotting inbrains of tg44�/� mice after intravenous scrapie inoculation followedby a mock intracerebral needle stab (i.v. � stab) was higher anddetected earlier than after intravenous inoculation alone. Solid sym-bols represent mice with clinical neurological signs, and open symbolsrepresent mice without clinical signs. (B) Immunohistochemical stain-ing with monoclonal antibody D13 at the site of the mock needle stabin the cerebral cortex at 225 dpi after i.v.�stab inoculation in a tg44�/�

mouse. A large PrPres plaque is evident at the dorsal surface adjacentto the pia mater. (C) H&E staining shows disruption of several neu-ronal cell layers along the needle track corresponding to locations ofPrPres amyloid plaques seen in panel B. Bar, 200 �m.


PrPres (52). After i.v. and i.p. inoculation of tg44�/� mice, noPrPres deposition was found in CVO regions (data not shown).The PrPres found in brain of the few positive i.v. or i.p. inoc-ulated mice was mostly in close proximity to the meninges ofthe cerebral cortex (Fig. 5D) and occasionally also in the cho-roid plexus (Fig. 5C), suggesting that spread of infectivity fromblood to CSF was a possible route of neuroinvasion at very latetimes after i.p. or i.v. inoculation.

Ocular inoculation of tg44�/� mice resulted in more fre-quent CNS invasion than with the other peripheral routestested. Extensive PrPres was found in the retina and opticnerve of i.o. inoculated tg44�/� mice (Fig. 4). In mice andhamsters with anchored PrP, the optic tract is thought to be themain route for cerebral neuroinvasion of scrapie after i.o.inoculation (22, 24, 42, 57). However, in tg44�/� mice inocu-lated by the i.o. route, the majority of PrPres plaques detectedat early stages of neuroinvasion were associated with meningesand submeningeal brain parenchyma and were not associatedwith the optic tract in the brain (Fig. 3C and D). This suggestedthat neural spread via the optic tract was not the main route ofspread to the CNS in these mice. Alternatively, spread fromretina to brain might be via CSF, which is in the subarachnoidspace of the optic nerve and can flow between the optic nerveand the brain (37, 38). If PrPres could cross the pial membraneto the subarachnoid space (Fig. 4D and E, arrows), it couldenter the CSF and spread to other pial surfaces in brain.This appeared to occur after i.o. inoculation of tg44�/� miceas most PrPres plaques in the brain were found in closeproximity to the meninges in the cerebellum and cerebralcortex (Fig. 3D).

In summary, the present data appeared to support the con-clusion that the presence of GPI-anchored PrP is an importantfactor in facilitating efficient CNS scrapie infection after inoc-ulation by several different peripheral routes. Anchored PrPmight be part of the transport process of PrPres by peripheralnerves to the CNS. However, the precise mechanism of thisprocess remains to be elucidated. The current experiments alsosuggested that CNS neuroinvasion by the hematogenous routeis unlikely to occur at high incidence in tg44�/� mice becauseafter i.p. inoculation these mice have significant blood infec-tivity (54) but only rare late neuroinvasion. Thus, the BBBseems to be a barrier to prion infectivity in tg44�/� mice, andthis is similar to previous conclusions from studies in nontrans-genic mice with anchored PrP (8, 29).

ACKNOWLEDGMENTS

We thank Dan Long and Lori Lubke for histological technical sup-port and Lisa Kercher for technical assistance with intraocular scrapieinoculations. We also thank Byron Caughey, Karin Peterson, AndrewTimmes, and John Portis for critical evaluation of the manuscript,Lynne Raymond for assistance in breeding the transgenic mice, and EdSchreckendgust for animal husbandry.

This research was supported by the intramural research program ofthe National Institute of Allergy and Infectious Diseases, NationalInstitutes of Health.

REFERENCES

1. Atherton, S. S., C. K. Newell, M. Y. Kanter, and S. W. Cousins. 1991.Retinitis in euthymic mice following inoculation of murine cytomegalovirus(MCMV) via the supraciliary route. Curr. Eye Res. 10:667–677.

2. Ayers, J. I., A. E. Kincaid, and J. C. Bartz. 2009. Prion strain targetingindependent of strain-specific neuronal tropism. J. Virol. 83:81–87.

3. Baldauf, E., M. Beekes, and H. Diringer. 1997. Evidence for an alternative

direct route of access for the scrapie agent to the brain bypassing the spinalcord. J. Gen. Virol. 78:1187–1197.

4. Bartz, J. C., C. Dejoia, T. Tucker, A. E. Kincaid, and R. A. Bessen. 2005.Extraneural prion neuroinvasion without lymphoreticular system infection.J. Virol. 79:11858–11863.

5. Bartz, J. C., A. E. Kincaid, and R. A. Bessen. 2003. Rapid prion neuroinva-sion following tongue infection. J. Virol. 77:583–591.

6. Bartz, J. C., A. E. Kincaid, and R. A. Bessen. 2002. Retrograde transport oftransmissible mink encephalopathy within descending motor tracts. J. Virol.76:5759–5768.

7. Bessen, R. A., S. Martinka, J. Kelly, and D. Gonzalez. 2009. Role of thelymphoreticular system in prion neuroinvasion from the oral and nasal mu-cosa. J. Virol. 83:6435–6445.

8. Blattler, T., et al. 1997. PrP-expressing tissue required for transfer of scrapieinfectivity from spleen to brain. Nature 389:69–73.

9. Bons, N., et al. 1999. Natural and experimental oral infection of nonhumanprimates by bovine spongiform encephalopathy agents. Proc. Natl. Acad. Sci.U. S. A. 96:4046–4051.

10. Brandner, S., et al. 1996. Normal host prion protein (PrPC) is required forscrapie spread within the central nervous system. Proc. Natl. Acad. Sci.U. S. A. 93:13148–13151.

11. Bremer, J., et al. 2010. Axonal prion protein is required for peripheralmyelin maintenance. Nat. Neurosci. 13:310–318.

12. Brown, K. L., et al. 1999. Scrapie replication in lymphoid tissues depends onprion protein-expressing follicular dendritic cells. Nat. Med. 5:1308–1312.

13. Brown, P., D. C. Gajdusek, C. J. Gibbs, Jr., and D. M. Asher. 1985. Potentialepidemic of Creutzfeldt-Jakob disease from human growth hormone ther-apy. N. Engl. J. Med. 313:728–731.

14. Bueler, H., et al. 1993. Mice devoid of PrP are resistant to scrapie. Cell73:1339–1347.

15. Cancellotti, E., et al. 2010. Glycosylation of PrPC determines timing ofneuroinvasion and targeting in the brain following transmissible spongiformencephalopathy infection by a peripheral route. J. Virol. 84:3464–3475.

16. Chesebro, B. 2003. Introduction to the transmissible spongiform encepha-lopathies or prion diseases. Br. Med. Bull. 66:1–20.

17. Chesebro, B., et al. 2010. Fatal transmissible amyloid encephalopathy: a newtype of prion disease associated with lack of prion protein membrane an-choring. PLoS Pathog. 6:e1000800.

18. Chesebro, B., et al. 2005. Anchorless prion protein results in infectiousamyloid disease without clinical scrapie. Science 308:1435–1439.

19. Cole, S., and R. H. Kimberlin. 1985. Pathogenesis of mouse scrapie: dynam-ics of vacuolation in brain and spinal cord after intraperitoneal infection.Neuropathol. Appl. Neurobiol. 11:213–227.

20. Eisele, Y. S., et al. 2009. Induction of cerebral beta-amyloidosis: intracerebralversus systemic Abeta inoculation. Proc. Natl. Acad. Sci. U. S. A. 106:12926–12931.

21. Eisele, Y. S., et al. 2010. Peripherally applied A�-containing inoculatesinduce cerebral �-amyloidosis. Science 330:980–982.

22. Fraser, H. 1982. Neuronal spread of scrapie agent and targeting of lesionswithin the retino-tectal pathway. Nature 295:149–150.

23. Fraser, H. 1979. Scrapie: a transmissible degenerative CNS disease, p. 194–210. In P. O. Behan and F. C. Rose (ed.), Progress in neurological research,with particular reference to motor neurone disease. Pitman Medical Pub-lishing Co. Ltd., Tunbridge Wells, England.

24. Fraser, H., and A. G. Dickinson. 1985. Targeting of scrapie lesions andspread of agent via the retino-tectal projection. Brain Res. 346:32–41.

25. Ghetti, B., et al. 1996. Vascular variant of prion protein cerebral amyloidosiswith tau-positive neurofibrillary tangles: the phenotype of the stop codon 145mutation in PRNP. Proc. Natl. Acad. Sci. U. S. A. 93:744–748.

26. Gibbs, C. J., Jr., H. L. Amyx, A. Bacote, C. L. Masters, and D. C. Gajdusek.1980. Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie tononhuman primates. J. Infect. Dis. 142:205–208.

27. Gibbs, C. J., Jr., et al. 1985. Clinical and pathological features and laboratoryconfirmation of Creutzfeldt-Jakob disease in a recipient of pituitary-derivedhuman growth hormone. N. Engl. J. Med. 313:734–738.

28. Glatzel, M., and A. Aguzzi. 2000. PrP(C) expression in the peripheral ner-vous system is a determinant of prion neuroinvasion. J. Gen. Virol. 81:2813–2821.

29. Glatzel, M., F. L. Heppner, K. M. Albers, and A. Aguzzi. 2001. Sympatheticinnervation of lymphoreticular organs is rate limiting for prion neuroinva-sion. Neuron 31:25–34.

30. Glaysher, B. R., and N. A. Mabbott. 2007. Role of the GALT in scrapie agentneuroinvasion from the intestine. J. Immunol. 178:3757–3766.

31. Goldstein, L. S., and Z. Yang. 2000. Microtubule-based transport systems inneurons: the roles of kinesins and dyneins. Annu. Rev. Neurosci. 23:39–71.

32. Gonzalez, L., et al. 2010. Pathogenesis of natural goat scrapie: modulation byhost PRNP genotype and effect of co-existent conditions. Vet. Res. 41:48.

33. Heikenwalder, M., C. Julius, and A. Aguzzi. 2007. Prions and peripheralnerves: a deadly rendezvous. J. Neurosci. Res. 85:2714–2725.

34. Jansen, C., et al. 2010. Prion protein amyloidosis with divergent phenotypeassociated with two novel nonsense mutations in PRNP. Acta Neuropathol.119:189–197.


35. Kercher, L., C. Favara, C. C. Chan, R. Race, and B. Chesebro. 2004. Dif-ferences in scrapie-induced pathology of the retina and brain in transgenicmice that express hamster prion protein in neurons, astrocytes, or multiplecell types. Am. J. Pathol. 165:2055–2067.

36. Kercher, L., C. Favara, J. F. Striebel, R. LaCasse, and B. Chesebro. 2007.Prion protein expression differences in microglia and astroglia influencescrapie-induced neurodegeneration in the retina and brain of transgenicmice. J. Virol. 81:10340–10351.

37. Killer, H. E., G. P. Jaggi, J. Flammer, N. R. Miller, and A. R. Huber. 2006.The optic nerve: a new window into cerebrospinal fluid composition? Brain129:1027–1030.

38. Killer, H. E., et al. 2007. Cerebrospinal fluid dynamics between the intra-cranial and the subarachnoid space of the optic nerve. Is it always bidirec-tional? Brain 130:514–520.

39. Kimberlin, R. H., S. Cole, and C. A. Walker. 1987. Pathogenesis of scrapie isfaster when infection is intraspinal instead of intracerebral. Microb. Pathog.2:405–415.

40. Kimberlin, R. H., S. M. Hall, and C. A. Walker. 1983. Pathogenesis of mousescrapie. Evidence for direct neural spread of infection to the CNS afterinjection of sciatic nerve. J. Neurol. Sci. 61:315–325.

41. Kimberlin, R. H., and C. A. Walker. 1982. Pathogenesis of mouse scrapie:patterns of agent replication in different parts of the CNS following intra-peritoneal infection. J. R. Soc. Med. 75:618–624.

42. Kimberlin, R. H., and C. A. Walker. 1986. Pathogenesis of scrapie (strain263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly.J. Gen. Virol. 67:255–263.

43. Kimberlin, R. H., and C. A. Walker. 1989. The role of the spleen in theneuroinvasion of scrapie in mice. Virus Res. 12:201–211.

44. Kratzel, C., D. Kruger, and M. Beekes. 2007. Prion propagation in a nerveconduit model containing segments devoid of axons. J. Gen. Virol. 88:3479–3485.

45. Kratzel, C., J. Mai, K. Madela, M. Beekes, and D. Kruger. 2007. Propagationof scrapie in peripheral nerves after footpad infection in normal and neuro-toxin exposed hamsters. Vet. Res. 38:127–139.

46. Kunzi, V., et al. 2002. Unhampered prion neuroinvasion despite impairedfast axonal transport in transgenic mice overexpressing four-repeat tau.J. Neurosci. 22:7471–7477.

47. Mabbott, N. A., J. Young, I. McConnell, and M. E. Bruce. 2003. Follicular

dendritic cell dedifferentiation by treatment with an inhibitor of the lympho-toxin pathway dramatically reduces scrapie susceptibility. J. Virol. 77:6845–6854.

48. Meade-White, K., et al. 2007. Resistance to chronic wasting disease in trans-genic mice expressing a naturally occurring allelic variant of deer prionprotein. J. Virol. 81:4533–4539.

49. Montrasio, F., et al. 2000. Impaired prion replication in spleens of micelacking functional follicular dendritic cells. Science 288:1257–1259.

50. Mulcahy, E. R., J. C. Bartz, A. E. Kincaid, and R. A. Bessen. 2004. Prioninfection of skeletal muscle cells and papillae in the tongue. J. Virol. 78:6792–6798.

51. Powell-Jackson, J., et al. 1985. Creutzfeldt-Jakob disease after administra-tion of human growth hormone. Lancet 2:244–246.

52. Price, C. J., T. D. Hoyda, and A. V. Ferguson. 2008. The area postrema: abrain monitor and integrator of systemic autonomic state. Neuroscientist14:182–194.

53. Prinz, M., et al. 2003. Positioning of follicular dendritic cells within thespleen controls prion neuroinvasion. Nature 425:957–962.

54. Race, B., K. Meade-White, M. B. Oldstone, R. Race, and B. Chesebro. 2008.Detection of prion infectivity in fat tissues of scrapie-infected mice. PLoSPathog. 4:e1000232.

55. Race, R., M. Oldstone, and B. Chesebro. 2000. Entry versus blockade ofbrain infection following oral or intraperitoneal scrapie administration: roleof prion protein expression in peripheral nerves and spleen. J. Virol. 74:828–833.

56. Revesz, T., et al. 2009. Genetics and molecular pathogenesis of sporadic andhereditary cerebral amyloid angiopathies. Acta Neuropathol. 118:115–130.

57. Scott, J. R., and H. Fraser. 1989. Transport and targeting of scrapie infec-tivity and pathology in the optic nerve projections following intraocularinfection. Prog. Clin. Biol. Res. 317:645–652.

58. Siso, S., M. Jeffrey, and L. Gonzalez. 2009. Neuroinvasion in sheep trans-missible spongiform encephalopathies: the role of the haematogenous route.Neuropathol. Appl. Neurobiol 35:232–246.

59. Stahl, N., D. R. Borchelt, K. Hsiao, and S. B. Prusiner. 1987. Scrapie prionprotein contains a phosphatidylinositol glycolipid. Cell 51:229–240.

60. Trifilo, M. J., et al. 2006. Prion-induced amyloid heart disease with highblood infectivity in transgenic mice. Science 313:94–97.


crucial role for prion protein membrane anchoring in the neuroinvasion and neural spread of prion...

Documents