246 Research Article

IntroductionThe causative event in prion disease is thought to be a change inconformation of the predominantly -helical C-terminal domainof cellular prion protein (PrPC) that increases its -pleated sheetcontent as the protein converts to the infectious PrPSc isoform(Prusiner, 1998). This conversion rarely occurs de novo as a singlemolecule reaction. Pre-existing PrPSc oligomers powerfullystimulate the conversion (Silveira et al., 2005), not only recruitingadjacent PrPC to their amyloidogenic conformation and therebypropagating infection, but also acting as template to specify thenew conformation. For there is not a single conformation thatcharacterises PrPSc fibrils, nor a single set of pathologicalcharacteristics that define prion diseases, but rather multipledifferent strains of disease that differ in their molecular andpathogenic characteristics (Aguzzi, 1998; Bruce et al., 1994).These different strains are stably propagated through manygenerations in animals or cell lines, and require intimate contactbetween PrPSc as template, and PrPC as substrate, to dictate theprecise conformation adopted on the growing amyloid seed.Interaction between PrPC and PrPSc is also required forneurotoxicity, since PrPSc in the brain kills only PrPC-expressingneurons by a mechanism that remains to be elucidated (Brandneret al., 1998; Chesebro et al., 2005; Mallucci et al., 2003; Mallucciet al., 2007).

Where do the infectious and cellular forms of PrP meet? A rangeof evidence indicates the infectious interaction occurs either at thecell surface or in some post-surface organelle to which the proteinsare trafficked (Caughey and Baron, 2006; Krammer et al., 2009;

Marijanovic et al., 2009; Morris et al., 2006). PrPC, beingglycosylphosphatidylinositol (GPI)-anchored to the surfacemembrane, occupies lipid ‘rafts’ (Brügger et al., 2004; Tarabouloset al., 1995). On neurons and N2a cells, PrPC rapidly leaves theseordered lipid domains to enter disordered membrane and then coatedpits, from where it is recycled back to the surface, the whole processtaking only a few minutes (Morris et al., 2006; Shyng et al., 1994;Shyng et al., 1993; Sunyach et al., 2003). The fate of infectiousPrPSc on the cell surface has not been followed, although it appearsto be corralled in heavier rafts than PrPC (Naslavsky et al., 1997;Vey et al., 1996).

We have recently shown that the endocytic partner of PrPC onsensory neurons is LRP1 (low-density lipoprotein receptor-relatedprotein 1) (Parkyn et al., 2008). This enormous (600 kDa) receptoris derived from four linear repeats of the LDL receptor (Fig. 1),and retains in two repeats (clusters 2 and 4) high affinity bindingsites for a range of ligands. These include the proteins that mediatelipid and sterol uptake in neurons (apo-lipoprotein E and2macroglobulin), regulate the extracellular adhesive and proteolyticenvironments, and remove A amyloid fibrils from the surroundingcerebrospinal fluid (Deane et al., 2004; May and Herz, 2003).Expression of ‘minireceptors’ composed of individual ligand bindingclusters 2 or 4 (Fig. 1) has facilitated the analysis of ligand bindingto LRP1 and shown that most ligands bind to both clusters(Obermoeller-McCormick et al., 2001). LRP1 (often shortened toLRP in the literature) should not be confused with the lamininreceptor precursor (also abbreviated as LRP), a multifunctional dualcytoplasmic-surface protein of 37/67 kDa that has been identified

Neuronal low-density lipoprotein receptor-relatedprotein 1 binds and endocytoses prion fibrils viareceptor cluster 4Angela Jen1,*, Celia J. Parkyn1,*, Roy C. Mootoosamy1,*, Melanie J. Ford1, Alice Warley2, Qiang Liu3, Guojun Bu3, Ilia V. Baskakov4, Søren Moestrup5, Lindsay McGuinness6, Nigel Emptage6 and Roger J. Morris1,‡

1Wolfson Centre for Age Related Disease, and 2Centre for Ultrastructural Imaging, King’s College London, SE1 1UL, UK3Department Pediatrics, Washington University School of Medicine, St Louis Children’s Hospital, St Louis, MO 63110, USA4Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore MD 21201, USA5Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark6Department Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK*These authors contributed equally to this work‡Author for correspondence (roger.morris@kcl.ac.uk)

Accepted 19 October 2009Journal of Cell Science 123, 246-255 Published by The Company of Biologists 2010doi:10.1242/jcs.058099

SummaryFor infectious prion protein (designated PrPSc) to act as a template to convert normal cellular protein (PrPC) to its distinctive pathogenicconformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrPC is thelow-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that bindsand rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds twomolecules of most ligands independently to its receptor clusters 2 and 4, PrPC and PrPSc fibrils bind only to receptor cluster 4. PrPSc

fibrils out-compete PrPC for internalization. When endocytosed, PrPSc fibrils are routed to lysosomes, rather than recycled to the cellsurface with PrPC. Thus, although LRP1 binds both forms of PrP, it traffics them to separate fates within sensory neurons. The bindingof both to ligand cluster 4 should enable genetic modification of PrP binding without disrupting other roles of LRP1 essential toneuronal viability and function, thereby enabling in vivo analysis of the role of this interaction in controlling both prion and LRP1biology.

Key words: Endocytosis, LRP1, Lysosome, Neuron, Prion protein

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as a receptor for normal and infectious PrP (Gauczynski et al., 2006;Gauczynski et al., 2001; Pflanz et al., 2009).

The N-terminal domain of PrPC is necessary and sufficient forendocytosis of this protein, and in particular the basic motif(KKRPKP) at its immediate N-terminus is essential (Sunyach etal., 2003). This N-terminal domain binds to human LRP1 with aKd of approximately 20 nM (Parkyn et al., 2008). The assembly ofamyloid fibrils of both native and recombinant PrP by interactionsbetween their C-terminal domains leaves their N-terminal domainsaccessible for antibody binding (Jeffrey et al., 1997; Novitskaya etal., 2006; Safar et al., 1998). Should these N-terminal domains bindLRP1, each receptor molecule could bring together the template(PrPSc) and substrate (PrPC) for the conformational conversion thatproduces prion disease (Morris et al., 2006).

The binding of infectious PrPSc to cells has been studied usingfluorescently tagged fibrils proteolytically digested with proteinaseK (Horonchik et al., 2005; Magalhaes et al., 2005). This step degradesnon-PrP protein within the fibrils to ensure that only the proteaseresistant PrPRes is labelled, thereby validating the use of the fluorescenttag to follow PrPSc uptake by the cells (Magalhaes et al., 2005); but

it also removes much of the N-terminal domain of PrP that is presentin freshly isolated infectious fibrils (Hope et al., 1986).

We have followed Baron et al. (Baron et al., 2006) and Greil etal. (Greil et al., 2008) in studying a native (non-proteolysed) fibrilpreparation with the N-terminal domain of PrP intact. We haveisolated PrPSc fibrils from mouse brain infected with ME7 strainof scrapie and tagged them with Alexa Fluor 594 fluorochrome.Although PrP is the dominant labelled protein in this preparation,it is difficult to exclude the possibility that it is some fluorescentcontaminant, unrelated to prion infection, that is bound andendocytosed by the neurons. We have therefore tested our mainconclusions with fluorescently labelled pure recombinant PrPfibrils, and by detection of PrPSc fibrils by their protease resistance(PrPRes) in immunoblotting.

ResultsProduction of PrPSc fibrils that bind to sensory neuronsPrPSc fibrils, isolated from mouse brain terminally infected withME7 strain of scrapie, showed the appropriate PrP bands onimmunoblots, before and after digestion with proteinase K(supplementary material Fig. S1). These same bands were thoseprimarily labelled in the fibrils by either biotin or Alexa Fluor 700using N-hydroxysuccinimide linkage to couple the label to thefibrils. The same linkage was used to couple Alexa Fluor 488 and594 to fibrils to follow their cellular trafficking.

When isolated, the fibrils were large (~10 m) aggregates (Fig.2A,B) that did not bind to the sensory neurons. Sonication releasedmany smaller oligomers of ~20 nm (Fig. 2C-E) and increased by100-fold the proportion of Alexa-Fluor-594-labelled fibrils thatbound to the neurons (supplementary material Table S1). The smalleroligomers remained in the supernatant after the short centrifugationused to pellet the large unsonicated aggregates (Fig. 2E) andaccounted for most of the binding to neurons (supplementarymaterial Table S1). Binding of the sonicated fibrils to the neuronswas saturable (supplementary material Fig. S2). The whole sonicatedfraction (without size separation) was used in these studies and isreferred to as PrPSc fibrils. Stored aliquots of labelled fibrils werere-sonicated for 90 minutes immediately before addition to cells.

Binding and uptake of PrPSc by sensory neuronsAdult sensory neurons in culture spread their axons over thesubstrate with their large (15-50 m diameter) cell bodies protrudinginto the medium above. Their uptake of surface-labelled proteinswas analysed in optical sections of ~3 m taken through 5-12 cells,reconstructed from deconvolved serial sections taken every 100 nmin the Z-axis towards the middle of the cell body [5-20 m abovethe substrate (Parkyn et al., 2008)]. Sonicated Alexa-Fluor-594-labelled fibrils were pre-incubated with neurons for 30 minutes at12-15°C to allow binding to the cell surface; unbound fibrils wereremoved by washing, and the cells transferred to 37°C to allow thetrafficking of this pulse of surface-bound PrPSc fibrils to befollowed. Other surface proteins simultaneously labelled withAlexa-Fluor-488-coupled ligands were Thy-1 (which is notendocytosed and so delineates the cell surface at 37°C) and PrPC,both labelled with Fab antibody fragments (Sunyach et al., 2003);transferrin (Tf; binding to the Tf receptor) and activated 2-macroglobulin (2M*, binding to LRP1), both endocytosed rapidlyvia coated pits.

Alexa-Fluor-594-PrPSc fibrils were rapidly endocytosed, with96.2±2.6% internalized by 2 minutes (Fig. 3A,B), significantly more(P0.02) than the 85.6±13.5% of surface-labelled PrPC internalized

Fig. 1. Schematic comparison of the modular structure of LRP1, itsminireceptors and PrPC. The domains of LRP1 [adapted from Li et al. andSpringer (Li et al., 2000; Springer, 1998)], the minireceptors mLRP2 andmLRP4 containing, respectively, ligand-binding clusters 2 and 4(Obermoeller-McCormick et al., 2001), and PrPC, for which the flexible N-and structured C-terminal domains, and GPI anchor, are indicated; grey linesrepresent the surface membrane. LRP1 domains and the site of furin cleavageare indicated (see legend); Roman numerals indicate the four repeats of theligand-binding complement-like domains; endocytic motifs (Li et al., 2000)are indicated in the cytoplasmic domain. EGF, epidermal growth factorreceptor; HA, ’flu haemagglutinin.

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by a separate population of neurons (supplementary material TableS2). Endocytosed PrPSc accumulated in perinuclear tubularstructures, where it colocalized at 2 minutes with Tf (Fig. 3C; ofinternalized protein, 28.4±20.2% of PrPSc colocalized with Tf, and72.2±28.3% Tf colocalized with PrPSc) and particularly with theLRP1 ligand, 2M* (Fig. 3D; 66.7±18.1% of PrPSc colocalized with2M*, and 69.6±14.6% of 2M* colocalized with PrPSc;supplementary material Table S3). PrPSc uptake in the presence of1 M 2M* was identical (95.8±3.4%; P0.8) to its uptake in theabsence of this LRP1 ligand.

When imaged at substrate level, virtually all Alexa-Fluor-594-PrPSc

fibrils were on neurons, including their processes (Fig. 3E) with thesubstrate cells themselves not binding labelled fibrils (Fig. 3F).

Trafficking of endogenous PrPC (prelabelled with Alexa-Fluor-488-Fab) and exogenous Alexa-Fluor-594-PrPSc on the same neuronswas examined. After 2 minutes at 37°C, 91.6±7.7% of PrPSc (red),but only 38.4±9.3% of PrPC (green) was internalized (Fig. 3G),compared with 82.4±4.9% of PrPC in control cells not exposed toPrPSc (P<0.001 for PrPC endocytosed). Even after 4 minutes at 37°C(Fig. 3H), only 60.9±27.0% of PrPC had been internalized (comparedwith >98% by control cells without additional PrPSc fibrils). Thisdisplacement of PrPC endocytosis by PrPSc suggests that the two

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forms of PrP compete for the same binding site, in contrast to thelack of competitive inhibition by PrPSc of 2M* endocytosis.

Inhibition of LRP1 lowers binding and uptake of PrPSc

Receptor associated protein (RAP), the specific inhibitor of LRPreceptors (Iadonato et al., 1993; Moestrup et al., 1993) strongly

Fig. 2. Electron micrographs of prion fibrils. Negatively stained samples ofPrPSc fibrils before (A,B) and after (C-E) sonication are shown. Those in Cand D have not been further centrifuged (and are typical of the fibrils added tothe neuronal cultures). (E) The supernatant left after a sonicated fraction wascentrifuged at 14,000 g for 2 minutes. Arrows in C-E point to smalleroligomers of about 20 nm released by sonication.

Fig. 3. Endocytosis of sonicated prion fibrils by neurons. Binding anduptake of Alexa-Fluor-594-labelled PrPSc fibrils (red) by sensory neurons iscompared with other ligands labelled with Alexa Fluor 488 (green). Thechromatin was labelled with DAPI (blue) throughout this and followingfigures, which show cells with typical (average) levels of labelling. Scale bars:5m. (A,B)PrPSc fibrils, and Alexa Fluor 488-Fab for Thy-1, on neuronsplaced at 37°C for 30 (A) and 120 (B) seconds. (C,D)PrPSc fibrils, and Alexa-Fluor-488-labelled transferrin (C) or 2M* (D), on neurons placed at 37°C for2 minutes. In C, Tf but not PrPSc fibrils was re-added to the medium at 37°C toenable Tf binding to delineate the surface. (E,F)PrPSc fibril binding anduptake at the level of substrate cells. E shows Alexa-Fluor-594-PrPSc fibrils(arrows) on an axon emerging from a neuronal cell body. F shows a cluster ofAlexa-Fluor-594-PrPSc below the lowest extremity of the neuronal nucleus(arrow; the majority of this cell and its nucleus lies above this plane); adjacentsubstrate cells (nuclei visible) have not bound the fibrils. These are the lowestimages (normally not used) collected in the Z-axis series for neurons allowedto endocytose PrPSc fibrils for 2 minutes. (G,H)PrPSc fibrils out-compete PrPC

for internalization. Neurons were prelabelled at 12-15°C with Alexa-Fluor-488-conjugated Fab to label PrPC, and with Alexa-Fluor-594-labelled PrPSc

fibrils; the cells were washed and transferred to 37°C and fixed after 2 (G) or 4(H) minutes.

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inhibited the binding and uptake of PrPSc by wild-type (WT) neurons(PrP+/+; Fig. 4A,B and Table 1). This binding was independent ofPrPC expression since knockout (KO; PrP–/–) neurons bound andinternalized PrPSc, and this was inhibited by 80 nM RAP (Fig. 4C,Dand Table 1).

Amyloid fibrils composed purely of recombinant PrP (PrPRec)(Bocharova et al., 2005) bound to sensory neurons and were >95%internalized within 2 minutes at 37°C; this binding was inhibitedby 80 nM RAP (Fig. 4E,F and Table 1).

LRP1 protein levels were transiently (for several hours) reducedto 10-25% of normal levels by delivering three independent siRNAs

specific for LRP1 [siRNALRP1.1-3 (Parkyn et al., 2008)] using themembrane-crossing peptide penetratin-1, and assayed 2 hours afterapplying the siRNAs to avoid the multiple effects of longer termsuppression of neuronal LRP1 (Parkyn et al., 2008). All threesiRNALRP1 inhibited the binding and uptake of PrPSc (Fig. 4G-J,Table 1 and supplementary material Table S4).

PrPC and PrPSc are internalized by binding to ligandbinding cluster 4 but not 2The LRP1-binding activity of stably transfected N2a cells expressinglow levels of endogenous LPR1 (control cells transfected with emptyvector), or expressing the minireceptors mLRP2 (M2 cells) ormLRP4 (M4 cells), was assessed by incubating them with trace(1 nM) levels of Alexa-Fluor-488-labelled RAP, and Alexa-Fluor-594-PrPSc fibrils (Table 2). The cells expressing mLRP2 had six,and the cells expressing mLRP4 had thirty, times the level of RAPbinding compared with the parental control cells. Only the M4 cellsbound Alexa Fluor 594-PrPSc fibrils significantly above controls,and ten times more than the M2 cells (Table 2).

To assess the interaction between the endogenous PrPC expressedby these cells and the LRP1 minireceptors, the surface levels ofPrPC, and the amount internalized within 5 minutes at 37°C, were

Fig. 4. Inhibition of LRP1 inhibits the uptake of PrPSc fibrils by neurons.(A,B)Neurons were pre-incubated at 37°C for 2 hours with vehicle only (A) or80 nM RAP (B), and then for 30 minutes at 12-15°C with Alexa-Fluor-594-laelled PrPSc (red) before being returned to 37°C for 2 minutes. Cells werefixed and surface immunolabelled for LRP1 (green). (C,D)KO (PrP–/–)neurons were incubated with vehicle (C) or 80 nM RAP (D) and then withPrPSc fibrils as above, with Tf (green) added as an endocytic control; cellswere placed for 2 minutes at 37°C before fixing and processing. (E,F)WT(PrP+/+) neurons were preincubated for 2 hours at 37°C with vehicle (E) or80 nM RAP (F), and then at 12-15°C with recombinant PrP fibrils labelledwith Alexa Fluor 594 (red) and Alexa-Fluor-488-Tf (green), then placed at37°C for 2 minutes. (G-J)WT neurons were pre-incubated for 2 hours withsiRNAControl (G) or siRNALRP1.1-3 (H-J) and then PrPSc bound for 30 minutes at12-15°C before being returned to 37°C for 2 minutes. Alexa Fluor-488-Tflabelling, done as a positive control, is not shown so as not to obscure thereduction of PrPSc binding. Scale bars: 5m.

Table 1. Inhibition of LRP1 ligand binding (RAP) orexpression level (siRNA) reduces binding of PrPSc, or

recombinant PrPRec, fibrils to sensory neurons

% of Neuronal control

Inhibitor genotype Fibrils binding P-value

80 nM RAP PrP+/+ PrPSc 8.3±3.0 0.001PrP–/– PrPSc 25.0±5.6 0.05PrP+/+ PrPRec 19.1±13.2 0.05

siRNALRP1.1 (LRP<15%) PrP+/+ PrPSc 19.5±6.3 0.01siRNALRP1.1 (LRP<15%) PrP–/– PrPSc 17.2±3.0 0.01 siRNALRP1.2 (LRP<15%) PrP+/+ PrPSc 16.2±13.2 0.01siRNALRP1.3 (LRP<25%) PrP+/+ PrPSc 30.4±22.6 0.05

Inhibition of binding of fluorescent fibrils expressed as a percentage ofbinding to neurons (8-12 cells analysed for each condition) compared withcontrols treated with either vehicle (RAP) or 250 nM siRNAControl.Transferrin binding, included as a positive control in each experiment, did notdiffer from control values (P>0.5). The extent of inhibition of LRP1expression by the siRNAs (shown in brackets in the first column) wasassessed by immunolabelling cells with fluorochrome-labelled anti-LRP1antibody.

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measured (Table 2). Surface levels of PrPC on M4 cells were nearlythree times the level on control cells, and four times that on M2cells. Over 50% of the high level of PrPC on the M4 cells wasendocytosed within 5 minutes, compared with only a third of thePrPC on control cells (i.e. M4 cells endocytosed five times morePrPC than the control cells). The low amount of surface PrPC onM2 cells was barely endocytosed, ten times less than the M4 cells.Together, these results indicate that the fourth but not second ligand-binding cluster of LRP1 binds both PrPSc and PrPC.

As an independent test of the interaction of PrPSc and PrPC withthe minireceptors, the later were immunoprecipitated from detergentlysates of the N2a cell lines after allowing PrPSc to bind.Immunoprecipitates of the minireceptors were digested withproteinase K (PK) to remove PrPC from the sample, enabling PrPSc

to be identified by antibody binding to the residual protease-resistantPrPRes, which was detected only in the mLRP4 cells (Fig. 5A,B).

To determine the interaction of PrPC with the minireceptors,the control and minireceptor cells were found to expresscomparable levels of total PrPC, as seen in immunoblots (Fig. 5C,actin control in G), despite their different levels of the surfaceprotein (Table 2). Full-length LRP1 is proteolytically cleaved intrans Golgi by furin to yield an 85 kDa cytoplasmic plustransmembrane fragment and a 550 kDa extracellular fragmentthat contained all ligand binding clusters (Fig. 1). The 550 kDachain of native LRP1 could only be detected on these cells usingmore sensitive detection, confirming their very low level ofendogenous LRP1 expression. Furin cleavage of full-lengthminireceptors (190 kDa) produces two fragments, both with amolecular mass of 85 kDa (Obermoeller-McCormick et al., 2001).In the cell lysate, the uncleaved 190 kDa form of mLRP2predominates, whereas furin-cleaved mLRP4 is more prominent(Fig. 5D), showing that more mLRP4 than mLRP2 progressesfrom the ER through the Golgi to the cell surface, in keeping withthe fivefold higher surface level of mLRP4 (Table 2).Immunoprecipitation of the minireceptors via their HA tag

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preferentially selected for the furin-cleaved 85 kDa forms (Fig.5F). mLPR4 but not mLRP2 co-immunoprecipitated PrPC (Fig.5E).

Endocytosed PrPSc is routed to lysosomes, not recycledback via endosomesNot only do PrPSc and PrPC bind to LRP1 on the neuronal surface,their initial endocytic trafficking is to the same perinuclearendosomal compartments (Fig. 3H). However, PrPC is recycled backto the neuronal surface (Morris et al., 2006; Sunyach et al., 2003),whereas labelled PrPSc fibrils were not similarly recycled, at leastwithin the time scale of these studies. An extended experiment wasset up in which sensory neurons were pre-labelled with PrPSc fibrils(30 minutes at 12-15°C), which for this experiment were notremoved by washing but remained in the medium for 0.5-30 minutesincubation at 37°C before fixation and immunolabelling forLAMP2, a marker of late endosomes and lysosomes (Cuervo andDice, 1996).

LAMP2 marked tubular compartments located initially in thecortical regions of the cell past which the endocytosed PrPSc fibrilsrapidly moved to concentrate in deeper, perinuclear compartments(Fig. 6A,B). By 10-30 minutes, however, the LAMP2-labelledlysosomes had moved into the cell interior and the proportion oflabelled PrPSc fibrils that colocalized with LAMP2 steadily rose(Fig. 6C,D, Fig. 7). The total amount of Alexa Fluor 594fluorescence associated with PrPSc fibrils bound to or internalizedby the neurons increased over the first 10 minutes of incubation at37°C, then decreased by 30 minutes (Figs 6 and 7).

To gain a better view of the fate of labelled PrPSc fibrilsfollowing endocytosis, we examined differentiated hippocampalorgan cultures in which the pyramidal cell neurites, flattened on

Table 2. Binding of PrPSc and RAP (A), and endocytosis ofsurface PrPC (B), by control N2a cells and stable

transfectants expressing LRP1 minireceptors

Control mLRP2 mLRP4(%) (%) (%)

A. BindingBinding of added RAP 100±125 576±492* 2,934±2,067***Binding of added PrPSc 100±128 57±81 594±775***

B. Traffic of endogenous PrPc

PrPc on surface at 0 minutes 100±66 71±46 282±175*PrPc endocytosed at 5 minutes 34±29 5±7** 54±48(**)

For A, cells were pre-incubated (20 minutes, 12-15°C) with Alexa-Fluor-594-PrPSc fibrils and 1 nM Alexa-Fluor-488-RAP, washed, fixed and totalfluorescence binding to individual cells, normalised to 1 m3 of cell volume,determined using Volocity software. Results are expressed as a percentage ofbinding to the empty-vector-transfected control cells.

For B, endogenous PrPC on the cells was labelled with Alexa-Fluor-594-Fab of 2S antibody (20 minutes, 12-15°C, 5 g/ml Fab), and either fixed andexamined immediately for total surface Fab bound, or placed at 37°C for 5minutes, residual surface Fab stripped by two washes in 100 mM acetatebuffer pH 3.0, then fixed and internal Fab determined as above. For eachcondition, 10 individual cells were analysed: values are mean ± s.d. *P<0.05;**P<0.01; ***P<0.001, each compared with the control cells except for (**)

for endocytosis of PrPC by mLRP4 cells, which is compared with mLRP2cells.

Fig. 5. Co-immunoprecipitation of PrPSc and PrPC with mLRP4 (M4) butnot mLRP2 (M2) minireceptor of LRP1. (A,B)Cells were incubated with(A) or without (B) PrPSc fibrils, the cell lysates were sonicated thenminireceptors immunoprecipitated by their HA epitope. PrP was detected afterproteinase K digestion (500g/ml, 30 minutes, 37°C) by immunoblotting withSAF83 antibody (Demart et al., 1999). PrPRes is seen in A; proteinase Kremoves all trace of PrPC expressed by the cells (B). (C)Total PrPC indetergent lysates of the cells detected by immunoblotting using SAF 83 (actinlevels shown in G). (D)Immunoblot of LRP1 (MMMM antibody specific forthe cytoplasmic domain shared by LRP1 and minireceptors) in the cell lysates.(E)Immunoblot of PrPC co-immunoprecipitated with the minireceptors via theHA epitope. (F)Immunoprecipitate of minireceptors (via their HA epitope),detected by immunoblotting for the LRP cytoplasmic domain. (G)Actinimmunoblots from cell lysates used in C-F.

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the substrate, could be viewed continuously in a single plane offocus.

Hippocampal neurons rapidly took up labelled PrPSc fibrilssimilarly to the sensory neurons and transported them to lysosomeswithin their cell bodies and neurites (Fig. 6E; 50.9±10.2% of labelledPrPSc fibrils colocalized with Lysotracker, and 69.7±5.7% ofLysotracker colocalized with labelled PrPSc fibrils). Lysosomes inneurites that had taken up PrPSc fibrils did not move perceptiblyduring 60 minutes of observation, allowing the fate of their ingestedAlexa Fluor 594-fibrils to be followed. Within 5-15 minutes afterfocusing on neurites, lysosomes that fluoresced green with PrPSc

fibrils rapidly (with 15 seconds) lost their fluorescence (Fig. 6Fshows two examples), and by 60 minutes very little greenfluorescence remained in the culture.

Since the endocytosis of PrPC on hippocampal neurons has notpreviously been described, we note here that endogenous surfacePrPC labelled with Alexa Fluor 488-Fab (Parkyn et al., 2008) wasrapidly endocytosed and transported retrogradely along processeswithout going into lysosomes. This movement was significantlyfaster, and over longer distances, than the transport of PrPSc toproximal lysosomes (supplementary material Table S5).

Binding of PrPSc fibrils to LRP1, detected by proteaseresistance (PrPRes)The protease resistance of PrPSc fibrils was used as an independentcheck on their binding by sensory neurons. We refer to the fibrilsso detected as PrPRes, although this includes any endogenous PrPC

that has been converted by the initial exogenous fibrils.PrPRes was bound by both WT and KO neurons, and was inhibited

by RAP and siRNALRP1.1 (Fig. 8A), confirming LRP1 as the receptorinvolved.

To test the long-term fate of bound fibrils, cultures initiallyincubated with PrPSc fibrils were maintained for up to 12 (Fig. 8B)and 15 weeks (supplementary material Fig. S3A). In WT cultures,PrPRes persisted at reduced levels (30-50% of the initial level) for6 weeks, and then rose steeply to be more than seven times theinitial level by 12 weeks (determined by scanning the gels; Fig.

8C; the increase was tenfold when estimated by comparison of a1:10 dilution with the 0 week sample; supplementary material TableS6). KO levels of PrPRes remained below that of the initial inoculum,whereas cultures from Tg20 mice, whose sensory neurons express14-fold higher levels of surface PrPC than WT neurons (Parkyn etal., 2008), showed a slightly faster increase in PrPRes than WTneurons by 8 weeks (Fig. 8B,C; 2.7-fold over the initial value byscanning of gels, or fourfold by comparing a 1:10 dilution with the0 week sample; supplementary material Fig. S3B and Table S6).

The effect of transient inhibition of LRP1 during the initialincubation with PrPSc fibrils, upon longer term development ofPrPRes, was analysed after 4 weeks (Fig. 8D). Inhibition of initialfibril binding in these cultures by siRNALRP1.1was indicated by thehigher level of PrPRes remaining in the medium after that incubation(Fig. 8D). Four weeks later, the level of PrPRes in the control andLRP1-suppressed cultures was very similar. The major differencewas in total PrP, and so of PrPC, which was distinctly higher in thesiRNALRP1.1-treated cultures.

Fig. 6. PrPSc fibrils are routed to lysosomes in sensory and hippocampal neurons. (A-D)Adult sensory neurons prelabelled at 12-15°C with Alexa-Fluor-594-PrPSc fibrils (red) and held at 37°C for 0.5, 2, 10 and 30 minutes, respectively, before being fixed, permeabilized and immunolabelled (green) for LAMP2. Imagescollected in a Z-axis series (34 sections at 100 nm intervals) have been deconvolved and reassembled in 3D; chromatin was labelled with DAPI (blue). (E)Culturedhippocampal neurons at 37°C were incubated for 15 minutes with Lysotracker (red) and Alexa-Fluor-488-PrPSc fibrils (green; note the change in fluorochrome),then washed extensively with artificial cerebrospinal fluid and photographed in the same buffer. Arrowhead indicates a pyramidal neuron cell body withcolocalized Lysotracker and PrPSc fibrils; small arrows indicate similar colocalization in ‘spots’ along neurites. (F)Stills from a film of neurites in the hippocampalneuron culture shown in E, focused on four spots of Alexa-Fluor-488-PrPSc fibrils within lysosomes (red Lysotracker channel not shown for clarity), two of which(arrows) loose their fluorescence within the 15 second period shown. Scale bars: 5m.

Fig. 7. Fate of endocytosed Alexa-Fluor-594-PrPSc fibrils. Variation inintensity of fluorescence of PrPSc fibrils (black bars) and of its percentageoverlap with LAMP2 immunolabelling (grey bars), as a function of time ofincubation at 37°C with sensory neurons.

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DiscussionIdentification of a neuronal surface receptor for PrPSc is of interestprimarily because it must play a role in determining whether andhow PrPSc interacts with PrPC on neurons. Since this study, and aprevious investigation of the neuronal endocytic partner for PrPC,both led to the well characterized, rapidly endocytosed neuronalsurface receptor LRP1, we focus here on the interaction betweenPrP and LRP1. Recent reviews (e.g. Caughey and Baron, 2006;Krammer et al., 2009; Linden et al., 2008) provide a widerperspective on interactions proposed for PrP with other receptors,and their implication for prion infection.

We found it necessary to sonicate PrPSc fibrils isolated frominfected brain to reduce them to smaller (~20 nm) oligomers beforethey would bind and be endocytosed by sensory neurons. The initiallarge aggregates presumably results from the isolation procedure,since ME7 strain of scrapie has diffuse, sub-light microscopic (<1m) fibrils deposited in infected brain (Jeffrey et al., 1997).Previous studies with unsonicated PrPSc fibrils observed they werenot taken up by neurons until they were slowly broken down intosmaller units by biological processes at the cell surface (Baron etal., 2006; Magalhaes et al., 2005); with fibrils of varied size, smalleroligomers are taken up faster (Greil et al., 2008). Sonication hasbeen used to break PrPSc fibrils of the RML strain into their mostinfectious units, which are oligomers of 17-27 nm correspondingto 14-28 PrP molecules (Silveira et al., 2005; Caughey et al., 2009).

Sonicated PrPSc fibrils were internalized even faster than PrPC,presumably because they were pre-bound to their receptor at 12-15°C, allowing endocytosis to commence immediately when thetemperature was raised. By contrast, only a portion of surface PrPC

is bound to LRP1 at any time, the remainder being contained withinlipid ‘rafts’ which exclude LRP1 (Chen et al., 2009). PrPSc bindingwas inhibited by RAP, indicating the receptor to be an LRP-familymember, where LRP1 is the only member detected on sensoryneurons (Parkyn et al., 2008), and the only member endocytosedthis rapidly (Li et al., 2001). Three different siRNAs that lower theexpression of LPR1 proportionately inhibited the binding of PrPSc

fibrils. Together, these results strongly implicate LRP1 as theneuronal receptor that binds and endocytoses the smaller oligomericfibrils of PrPSc.

In fact, PrPSc fibrils competed with endogenous PrPC forendocytosis and so presumably for binding to LRP1. The ligandbinding sites on LRP1 have been analysed by the use ofminireceptors (Mikhailenko et al., 2001; Obermoeller-McCormicket al., 2001) or recombinant proteins (Neels et al., 1999) that expressthe four ligand-binding clusters singly and in combination. Mostendogenous ligands of LRP1 bind separately to both clusters 2 and4, so the receptor is divalent for these ligands (Neels et al., 1999;Obermoeller-McCormick et al., 2001). Clusters 1 and 3 retain onlyvestigial binding domains. RAP binds with high affinity to clusters2 and 4, and weakly to cluster 3, the only LRP ligand known to doso (Neels et al., 1999). Also unique is the binding of 2M* to asite spanning clusters 1 and 2, and not to cluster 4 (Mikhailenko etal., 2001), which explains its failure to compete with PrPSc forbinding and internalization. Pseudomonas exotoxin A binds onlyto cluster 4 (Obermoeller-McCormick et al., 2001). The binding ofboth PrPC and PrPSc fibrils only to cluster 4, and not 2, is thereforeunparalleled for an endogenous ligand of LPR1. This propertyshould allow the selective inactivation of the PrP binding site incluster 4 to enable an otherwise functional LRP1 to be made. Thisshould allow stable inhibition of PrP-LRP1 interactions in otherwisehealthy neurons.

Fig. 8. Fate of ingested PrPSc fibrils, determined as PrPRes, in long-termcultures of sensory neurons. All samples were sonicated for 90 minutesbefore splitting into samples, one-tenth for non-proteolysed analysis and nine-tenths for PK treatment with PrPRes, detected by SAF83. (A)PrPRes, with actinas a loading control, in wild-type (WT) and knockout (KO) cultures, treatedfor 1.5 hours with vehicle or 80 nM RAP, or siRNAControl or siRNALRP1.1

before PrPSc fibrils were incubated with the cells for 30 minutes at 37°C.(B)PrPSc fibrils, incubated for 30 minutes with sensory neurons from WT, KOor Tg20 mice, were maintained in long-term culture for the weeks shown. Tokeep the signal within a linear range of intensity, ECL Plus development of theperoxidase signal was used rather than the more sensitive ECL Advanced usedin A and D. WT and KO PrPRes samples were run on the same gel to enabledirect comparison (week 12* for WT is a one-tenth dilution of the 12-weekPrPRes sample). Tg20 samples were run separately. Both monomer and dimerbands of PrPRes are shown. (C)PrPRes recovered in cultures after the timesshown were measured by scanning the bands and normalized against actinlevels (mean ± s.d., n≥ 3; supplementary material Table S6). Asterisks indicatethat the value differs from the KO value at that time point with *P<0.05 or*P<0.01. (D)Cultures were pre-treated with siRNAControl or siRNALRP1.1 for1.5 hours before incubating for 0.5 hours with PrPSc fibrils, then washed andmaintained in culture for 4 weeks. The left panel shows that less PrPRes wasbound initially by the cells incubated with siRNALRP1.1, whereas by 4 weeksthe level of PrPRes in both control and LRP1-inhibited cells was comparable(middle panel). PrPC levels (right panel) were, however, strongly stimulated inthe cultures in which LRP1 levels were originally suppressed. Actin levels areshown in lower inset on the right.

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253LRP1 is the neuronal receptor for prion fibrils

Currently, the standard approach of stably inhibiting theexpression of native LRP1 to study its role in prion infection is notpossible because of its multiple roles essential for neuronal viability(May and Herz, 2003; May et al., 2004). The limitations of eventransient inhibition of full-length LRP1 for studying long-termeffects upon prion infectivity were evident in this study. Transientknockdown of LRP1 during infection lowered the uptake of fibrils,but even at 4 weeks stimulated PrPC expression and thus possiblythe rate of conversion of PrPC to PrPRes to compensate for the lowerlevel of PrPSc bound initially. The interdependence of levels of PrPC

and LRP1 on the cell surface (and not the total amount expressedby neurons) has been noted previously [inhibition of LRP1 led toaccumulation of PrPC in biosynthetic compartments at the expenseof cell surface levels; PrPC-over-expressing Tg20 mice had 1.8-foldhigher levels of surface LRP1 (Parkyn et al., 2008)]. The sameinterdependent biosynthetic trafficking is presumably responsiblefor the elevated levels of surface (not total) PrPC and minireceptormLRP4 on transfected N2a cells, compared with the levels onmLRP2-expressing cells, since only the former minireceptor bindsPrPC. A stable experimental system in which neurons respond onlyto prion infection, rather than to interdependent changes in surfaceexpression of PrPC and LRP1, is needed to elucidate the role ofLRP1 in prion infection.

The adult sensory neuron cultures did serve to demonstrate thepersistence of PrPRes in the neurons for weeks after the fluorescenttag was hydrolysed in the lysosomes. Importantly, the markedaccumulation of PrPRes after a lag phase of 6 weeks demonstratedthat prion infection, as indicated by conversion of PrPC to PrPRes,had occurred. Furthermore, Tg20 neurons, despite their very highlevels of PrPC expression, increased their PrPRes levels only slightlyfaster than the WT neurons. Although the course of prion disease ismore than twice as fast in Tg20 compared to WT mice, in the endstages of the disease the Tg20 mice have half the levels of PrPRes

compared to WT animals (Fischer et al., 1996). It will be interestingin this system to identify when prion-dependent neuronal death occurs;in preliminary experiments to date we have failed to maintain infectedTg20 neurons for 12-15 weeks. These sensory neuron cultures contain,in addition to neurons, fibroblasts, Schwann cells and some satellitecells that together we refer to as substrate cells. Their failure to bindPrPSc fibrils, or to express PrPC (Parkyn et al., 2008) suggests theyare unlikely to play a direct role in the conversion process, a viewthat is borne out by the lack of PrPRes in cultures in which the neuronshad died (supplementary material Fig. S3A).

Why is PrPC recycled back to the cell surface whereas PrPSc fibrilsare routed to lysosomes? A possible factor is size – PrPC is clustered(Brügger et al., 2004; Sunyach et al., 2003) but not known to bephysically aggregated at the cell surface, whereas the size of PrPSc

oligomers could signal their transfer to lysosomes. Infectivity ofPrPSc is critically linked to size, with oligomers up to hexomersbeing non-infectious, those of 14-28 subunits being maximallyinfectious, and then diminishing in proportion to increasing oligomersize (Riesner et al., 1996; Silveira et al., 2005). It will be of interestto determine the lower size limit that triggers routing of PrPSc tolysosomes. There is also an upper limit of 120 nm on the size ofcargo that can be endocytosed via coated pits (Conner and Schmid,2003), so that only oligomers of intermediate size are likely to beendocytosed by LRP1 and trafficked to lysosomes.

Despite the rapid quenching of fluorescence in the lysosomes,the fibrils detected as PrPRes persist for weeks in the cells. Possiblyit is only the PrPSc fibrils that are conveyed to the lysosome thatbecome infectious. Although in every endocytic cycle most PrPC

is returned to the surface, a small proportion is routed to lysosomesfor degradation, ensuring overall that PrPC has a short half-life(Shyng et al., 1993). Given the long persistence of the fibrils, thelysosome may trap PrPSc as in a filter through which all PrPC isfunnelled for degradation, thus maximizing over several weeks theopportunity for interaction between the two forms of PrP.

This proposal is at variance with the suggestion, arising fromstudies on stably infected GT1 and N2a cell lines, that neither earlynor late endosomes are the site of prion conversion, but rather it isthe recycling endosome (Marijanovic et al., 2009). For stableinfection of proliferating cell lines, the rate of PrPSc production mustmatch that of cell division (doubling every day or so) and not becytotoxic, neither of which are characteristics of prion infectioneither in vivo or in the cultured sensory neurons. Differentiated adultneurons never divide; their endocytic trafficking of both forms ofPrP occurs considerably more rapidly, and their production of PrPRes

is very much slower, than the cell lines. We see no recycling offibrils that would give them access, in sensory neurons, to therecycling endosomes identified as the likely site of conversion inrapidly dividing cell lines.

While the primary role of LRP1 is lipid uptake, it also plays asignificant role as a scavenger receptor, removing protease andmatrix complexes from extracellar space (May and Herz, 2003).PrPC is emerging as an active partner for LRP1 in the scavengerrole. PrPC through its His-containing octapeptide repeats can bindand endocytose pathological levels of Cu2+ (Wells et al., 2006) andthe toxic metabolite hemin (Lee et al., 2007). PrPC has recentlybeen shown to be a high-affinity ligand for A oligomers (Laurenet al., 2009), suggesting that the endocytotic trafficking of PrPC

mediated by LRP1 may be important in removing these neurotoxicoligomers in the brain. LRP1 further plays a complex role incontrolling the metabolism of Alzheimer precursor protein (APP)(Bu, 2009; Marzolo and Bu, 2009), so this receptor may be the linkbetween PrPC expression and APP metabolism (Parkin et al., 2007).

That a single receptor should mediate such a diverse range ofroles is unusual, but LRP1 is most unusual, being 5-10 times largerthan most receptors, with 31 repeats of its ligand-binding domainextracellularly, and with intracellular motifs that bind a range ofadaptor proteins linking LRP1 to neuronal proteins including APP,and neurotransmitter and growth factor receptors (May and Herz,2003). Heparan sulphate and lactoferrin are key regulators of LRP1activity (e.g. Mahley and Ji, 1999; Nathan et al., 2002; Wang et al.,2004), which may explain their roles in modulating PrPC traffickingand PrPSc fibril binding to cells (Ben-Zaken et al., 2003; Hijazi etal., 2005; Horonchik et al., 2005; Iwamaru et al., 2008; Paquet etal., 2007). Deciphering the individual roles of this massive, multi-faceted receptor is complex. PrP, despite its small size, is provingto have its own complex biology and pathology that includes aunique relationship to LRP1 during biosynthesis and endocytictrafficking of the normal, and pathogenic, forms of PrP.Understanding the functional consequences of this interaction willnot be straightforward, but promises to be interesting.

Materials and MethodsGeneral methodsFluorochrome labelling of reagents and their use to follow endocytosis; siRNAinhibition; immunoprecipitation; immunoblotting and protein determination have beendescribed previously (Parkyn et al., 2008; Sunyach et al., 2003). The 2S anti-PrP Fab[against amino acids 142-160 in the C-terminal domain of mouse PrP (Ford et al.,2002)] used to label PrPC failed to detect intact PrPSc fibrils, shown by the failure ofthe Fab to react at all with PrP0/0 neurons to which PrPSc fibrils were bound (notshown). Alexa-Fluor-700-labelled PrPSc fibrils were visualized using an ODYSSEYInfrared Imaging system (LI-COR Biosciences).

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PrPSc FibrilsPrPSc fibrils were prepared as described previously (Hope et al., 1986). Briefly, anhomogenate of five ME7-infected mouse brains was solubilized at 12-15°C in 10%Sarkosyl, a low speed (30 minutes at 10,000 g, 15°C) pellet was removed and thesupernatant pelleted at 205,000 g for 160 minutes. This pellet, resuspended in 0.6 MKI plus 6 mM Na2S2O3, was centrifuged through a 20% sucrose cushion at 255,000 gfor 90 minutes. The pellet was dispersed in water by short (15 minutes) sonicationin a cuphorn sonicator (Sonics and Materials VCX500 ultrasonic processor with a53 mm cuphorn, pulsing for 5 seconds with 1-second intervals to deliver 10,000joules/minute, water cooled to 23°C), with subsequent steps done aseptically. Proteinconcentration, determined by the Bio-Rad Protein Assay on an aliquot of fibrilssolubilized in 1 M NaOH then neutralized with 1 M HCl, was adjusted to 2 mg/mland 200 l aliquots stored at 4°C. For labelling with Alexa fluorochromes or biotin,thawed samples were pelleted (14,000 g, 2 minutes), resuspended in 0.1 M Na2HCO3buffer pH 8.0, and labelled with N-hydroxysuccinimide ester of fluorochromes(Molecular Probes) or biotin (Pierce) at half the manufacturer’s recommended levelto limit reaction with lysine residues important for the endocytosis of PrPC (Sunyachet al., 2003). Fibrils were dialysed for 2 days against sterile PBS; immediately beforeuse they were sonicated as above for 90 minutes and added at 5 g/ml to tissueculture supernatant for labelling cells. For these experiments, four differentpreparations of fibrils were made, three from archived stocks and one from miceinfected with aliquots from the first batch of sonicated fibrils. The fibrils used herehave also been used in a companion study of fibril uptake by mouse colon in vivo,in which the appearance, over the space of months, of PrPRes in lymphoreticular tissueand then brain is followed by the onset of terminal scrapie in the mice (R.C.M.,unpublished data).

Mouse full-length recombinant PrP encompassing residues 23-230 was expressedand purified as described previously (Bocharova et al., 2005) with modifications(Breydo et al., 2005) and converted to amyloid fibrils according to the procedure ofBocharova et al. (Bocharova et al., 2005). It was fluorochrome labelled as above,and sonicated for 1 minute before use.

Electron microscopy (EM)Cell suspension (2 l) was dropped into Pioloform-coated EM grids and allowed tosettle for 2 minutes. Excess solution was drawn off carefully by blotting the edge ofthe grid with the cut edge of a piece of hardened filter paper. The preparations werenegative stained by dropping 2 l 1% aqueous uranyl acetate onto the grids, and thiswas then removed after 90 seconds by blotting as described above. The preparationwas allowed to dry before examination in a FEI Tecnai12 electron microscope; imageswere captured with a Gatan BioScan camera.

Cell culturePrimary sensory neurons were routinely cultured from the dorsal root ganglia of 4-to 5-week-old C57Bl6 mice. Cells from each mouse, dispersed evenly on ten poly-D-lysine- and laminin-coated 13 mm glass coverslips were cultured in a 24-well plateat 37°C with 5.0% CO2 in phenol-red-free neurobasal medium (Gibco) with B-27serum-free supplement, plus 7.5 pg/ml nerve growth factor (NGF), 200 MGlutaMAX-1, and 1 g/ml cholesterol as low-density lipoproteins (Sigma Aldrich)for 3-7 days prior to commencing experiments (Parkyn et al., 2008; Sunyach et al.,2003). For detection of PrPRes after long-term culture, mice of 129/0la (PrP+/+), NPUPrP0/0 KO strain (Manson et al., 1994), and Tg20 mice over-expressing PrPC (Fischeret al., 1996) were used (99 mice in total). Dissociated ganglia of each single mousewere applied to a 16 mm coverslip to provide sufficient material for assay. Mice wereprocessed in two groups (differing in genotype) of three animals, on consecutivedays. Three days after the second group was introduced to culture, all wells wereincubated with sonicated fibrils at 37°C, washed thoroughly three times, then returnedto culture with one-third of the medium changed twice a week. On harvesting, cellswere solubilized in 0.5% Brij 96-PBS with 400 Kunitz unit/ml of DNase, sonicatedfor 30 minutes to ensure PrPRes was dispersed evenly, and one-tenth volume removedto determine (and equalize) protein concentration and immunoblot for PrPC and actin,with the remainder used to determine PrPRes. PK digestion of PrPSc fibrils was for 1hour at 37°C with 50 g PK/ml for PrPSc fibrils, and 500 g/ml protein of cell lysate.Peroxidase reaction was developed with ECL Advanced or (for more accuratequantification) ECL Plus (both GE Healthcare), and bands measured by scanninginto ImageJ software at a resolution of 1200 dpi using a Canon Canoscan 3200F.

Postnatal day 4 or 5 rat organotypic hippocampal cultures were maintained for aweek in DMEM with 20% horse serum before use; lysosomes were labelled andimaged as described previously (McGuinness et al., 2007). N2a cells, stablytransfected with empty vector or the LRP1 minigene containing cluster 2 or 4 ligandbinding sites were produced as described for CHO-LRP null cells (Obermoeller-McCormick et al., 2001). We previously noted that N2a cells and sensory neuronsshould not be chilled below 12-15°C during the pre-incubation to bind ligands priorto endocytosis. Here it was found that if hippocampal pyramidal neurons were chilledeven to 20°C they were slow to recover vesicle trafficking; they were incubated at37°C with 5 g/ml PrPSc fibrils or anti-PrP Fab, and 75 nM Lysotracker Red DND99(Molecular Probes, Invitrogen).

N2a control and minireceptor lines were fragmented by N2 bomb cavitation (Chenet al., 2009); the post-nuclear membrane fraction was solubilized in lysis buffer(Obermoeller-McCormick et al., 2001) and cleared by ultracentrifugation (100,000 g,

1 hour) and minireceptors immunoprecipitated using the Dynal Protein GImmunoprecipitation Kit (Invitrogen); the appropriate antibody was pre-bound to thebeads before addition to the lysates [anti-HA was monoclonal 12CA5 from Babco;affinity purified sheep 2S antibody for PrP (Ford et al., 2002) and rabbit MMMMantiserum for LRP1 cytoplasmic domain (Zerbinatti et al., 2004)].

We thank Chris Birkett and Andy Gill (Institute for Animal Health,Compton), and Oduolo Abiolo and Steve Whatley (Institute ofPsychiatry, King’s College London) for donating three different batchesof mouse brains terminally infected with ME7 scrapie strain. This workwas supported by BBSRC grants 18/BS516350 and BB/C506680/1,and an MRC doctoral training studentship to C.J.P. Deposited in PMCfor release after 6 months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/123/2/246/DC1

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