JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.7.3453–3461.2001

Apr. 2001, p. 3453–3461 Vol. 75, No. 7

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Branched Polyamines Cure Prion-Infected Neuroblastoma CellsSURACHAI SUPATTAPONE,1,2 HOLGER WILLE,1,2 LISA UYECHI,3 JIRI SAFAR,1,2

PATRICK TREMBLAY,1,2 FRANCIS C. SZOKA,3 FRED E. COHEN,1,4,5,6

STANLEY B. PRUSINER,1,2,6* AND MICHAEL R. SCOTT1,2

Institute for Neurodegenerative Diseases,1 and Departments of Neurology,2 Biopharmaceutical Sciences,3 Cellular andMolecular Pharmacology,4 Medicine,5 and Biochemistry and Biophysics,6 University of California at San Francisco,

San Francisco, California 94143

Received 30 October 2000/Accepted 14 December 2000

Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able topurge PrPSc, the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a)cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl.Acad. Sci. USA 96:14529–14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 mg of PPI gen-eration 4.0/ml for 4 weeks not only reduced PrPSc to a level undetectable by Western blot but also eradicatedprion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyaminesin vitro disaggregated the prion rods, reduced the b-sheet content of PrP 27-30, and rendered PrP 27-30susceptible to proteolysis. The susceptibility of PrPSc to proteolytic digestion induced by branched polyaminesin vitro was strain dependent. Notably, PrPSc from bovine spongiform encephalopathy-infected brain wassusceptible to PPI-mediated denaturation in vitro, whereas PrPSc from natural sheep scrapie-infected brainwas resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched poly-amines act within this acidic compartment to mediate PrPSc clearance. Branched polyamines are the first classof compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting,and strain-typing reagents for prion diseases.

Prion diseases are caused by an infectious protein (20, 25).These invariably fatal illnesses cannot be cured using routineantimicrobial agents, and materials contaminated with prionscannot be disinfected by conventional methods. Therefore, it isimportant to identify compounds that can be used either astherapeutic or disinfecting reagents for prion diseases. Ongo-ing epidemics of new variant Creutzfeldt-Jakob disease andbovine spongiform encephalopathy (BSE) in the United King-dom highlight the urgency of this task.

We recently reported that branched polyamines could purgescrapie-infected neuroblastoma (ScN2a) cells of PrPSc, the dis-ease-causing isoform of the prion protein (33). The ability ofthese compounds to eliminate PrPSc from ScN2a cells de-pended upon a highly branched structure and a high surfacedensity of primary amino groups. The most potent compoundsidentified were generation 4.0 polyamidoamine (PAMAM)and polypropyleneimine (PPI) dendrimers. Dendrimers arebranched polyamines manufactured by a repetitive divergentgrowth technique, allowing the synthesis of successive, well-defined “generations” of homodisperse structures. In the cur-rent study, we demonstrate that branched polyamines cureprion-infected cells and identify the site and mechanism ofpolyamine-mediated prion clearance. We also demonstratethat these compounds can be employed in a rapid and sim-ple assay to discriminate between different prion strains invitro.

MATERIALS AND METHODS

Chemical compounds. High-molecular-weight polyethyleneimine (PEI) waspurchased from Fluka. SuperFect transfection reagent was purchased from Qia-gen. All other polyamines were purchased from Sigma-Aldrich. Fluorescein-labeled PPI was synthesized by mixing 30 mg of fluorescein isothiocyanate(FITC) with 1 mg of PPI generation 4.0 in 2 ml of ethanol overnight at 4°C.Labeled PPI was separated from residual, unreacted FITC using a Sephadex P-2column.

Cultured cells. Cultures of ScN2a cells were maintained as described previ-ously (33). Cytotoxicity after treatment with polyamines was assessed in ScN2acells by the following four methods: (i) examination of morphology under phasecontrast microscopy, (ii) observation of growth curves and cell counts for 3 weeksafter treatment, (iii) vital staining of living cells with 0.4% trypan blue (Sigma-Aldrich), and (iv) assay of dehydrogenase enzymes with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich). For ScN2a cellstreated with either PAMAM or PPI generation 4.0 continuously for 1 week, the50% toxic dose was ;50 mg/ml.

To prepare samples for infectivity assays, 100-mm-diameter plates (Falcon) ofconfluent cells were washed three times with 5 ml of phosphate-buffered saline,scraped into 2 ml of phosphate-buffered saline, and homogenized by repeatedextrusion through a 26-gauge needle. Prion infectivity was determined by intra-cerebral inoculation of 30 ml of cell homogenate into Tg(MoPrP)4053 mice. Micewere observed for clinical signs of scrapie, and a subset of diagnoses wereconfirmed by neuropathological examination. Samples were prepared for sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as described previously(33).

Mixture of brain homogenates and purified prions with polyamines in vitro.Brain homogenates were prepared as described previously (33). Except for Tg(BoPrP) samples, 50 ml of 1-mg/ml brain homogenate was mixed with 450 ml of1% NP-40–50 mM sodium acetate (pH 3.0) (final measured pH 5 3.6) plus orminus 60 mg of PPI generation 4.0/ml and shaken constantly for various periodsat 37°C.

Purified prions were prepared as described previously (21), utilizing bothproteinase K digestion and sucrose gradient sedimentation, and resuspended in1% NP-40–1-mg/ml bovine serum albumin (BSA). For pH studies, 475 ml of0.5-mg/ml purified RML PrP 27-30 in 1% NP-40–1-mg/ml BSA was mixed with25 ml of 1 M buffers from pH 3 to 8 (sodium acetate for pHs 3 to 6 and Trisacetate for pHs 7 and 8) plus or minus 60 mg of PPI generation 4.0/ml for 2 h at37°C with constant shaking. The final pH value of each sample was measured

* Corresponding author. Mailing address: Institute for Neurodegen-erative Diseases, Box 0518, University of California, San Francisco,CA 94143-0518. Phone: (415) 476-4482. Fax: (415) 476-8386. E-mail:hang@itsa.ucsf.edu.

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directly with a calibrated pH electrode (Radiometer Copenhagen). For com-pound screening, 475 ml of 0.5-mg/ml purified RML PrP 27-30 in 1% NP-40–1-mg/ml BSA was mixed with 25 ml of 1 M sodium acetate (pH 3.0) plus 60 mg ofpolyamine/ml for 2 h at 37°C with constant shaking.

Following incubations, each sample was neutralized with an equal volume of0.2 M HEPES (pH 7.5) containing 0.3 M NaCl and 4% Sarkosyl. Samples nottreated with proteinase K were mixed with an equal volume of 23 SDS samplebuffer. For proteinase K digestion, samples were incubated with 20 mg of pro-teinase K (Boehringer Mannheim)/ml for 1 h at 37°C. Proteolytic digestion wasterminated by the addition of 8 ml of 0.5 M phenylmethylsulfonyl fluoride.Digested samples were then mixed with equal volumes of 23 SDS sample buffer.All samples were boiled for 5 min prior to SDS-polyacrylamide gel electrophore-sis. Western blotting was performed as previously described (27), using human-mouse chimeric Fab D13.

The mixture protocol was modified for Tg(BoPrP) samples. Incubations werecarried out at room temperature, and deoxycholate was substituted for Sarkosylin the neutralization buffer. Protease-treated samples were concentrated 10-foldby centrifugation for 1 h at 100,000 3 g. Immunoblotting was performed with Fabclone P as the primary antibody to recognize bovine PrP.

FTIR. Samples were lyophilized and resuspended in D2O. Prior to the spec-troscopic measurements, the samples were centrifuged briefly (14,000 3 g for 2min), and 1.5-ml samples from the bottom of the tube were enclosed between2 AgCl windows (International Crystal Laboratories, Garfield, N.J.), creating apath length of 50 mm. Spectra were recorded with a Perkin-Elmer (Norwalk,Conn.) System 2000 Fourier transform infrared resonance (FTIR) spectropho-tometer. Blank controls identical in buffer conditions and PPI content were usedto subtract any nonprotein contributions from the spectra. Spectral analysis andself-deconvolution were carried out as previously described (6) and modified(15).

Confocal microscopy. Confocal images were obtained using a Bio-Rad (Her-cules, Calif.) laser scanning confocal microscope (MRC-1024) outfitted with aNikon Diaphot 200 microscope and a helium-neon laser. Laser power was set at10% and scanned with a slow speed across the sample. Individual laser linesconfirmed the lack of “bleed-through” between detection channels. The imageswere averaged with a Kalman filter (n 5 4).

RESULTS

Branched polyamines cure prion-infected neuroblastomacells. ScN2a cells were incubated with 3 mg of PPI/ml in sup-plemented Dulbecco’s modified Eagle’s medium for 4 weeksand then cultured for an additional 2 weeks in polyamine-freemedium. This transient exposure to PPI was not cytotoxic (seeMaterials and Methods) and completely purged the cells ofprotease-resistant PrPSc (Fig. 1A, lanes 2 and 4). In contrast,protease-sensitive PrPC bands migrating between 32 and 38kDa appear to be present at similar levels in cells treated withPPI (lane 2) and in uninfected N2a cells (data not shown).Elimination of PrPSc was measured by the disappearance ofthe protease-resistant core of PrPSc, denoted PrP 27-30. Theelimination of PrP 27-30 appeared to be relatively specific,since the steady-state levels of proteins in PPI-treated cellswere similar to those in control ScN2a cells (Fig. 1B). To assessthe effect of PPI treatment on prion infectivity, homogenatesprepared from polyamine-treated and control ScN2a cells wereinoculated into Tg(MoPrP)4053 mice. The average scrapie in-cubation time was 61 6 2 days for mice inoculated with controlScN2a cells and .200 days for mice inoculated with ScN2acells treated with PPI (n/n0 5 0/13) (Fig. 1C). These incubationtimes indicate that the titer of infectious prions in ScN2a cellswas reduced from ;106 50% infective dose (ID50) units/100-mm plate to ,102 ID50 units/plate by PPI treatment (Fig. 1D).Thus, exposure to PPI eliminates measurable prion infectivityfrom ScN2a cells.

Branched polyamines act directly on purified RML prions.Having established that branched polyamines reduce prion

infectivity, we sought to identify the mechanism by which thesecompounds eliminate PrPSc. Our first objective was to deter-mine the molecular target of branched polyamines. Previously,we developed an in vitro assay which was used to show thatthese compounds could render PrPSc protease susceptiblewhen mixed directly with crude brain homogenates (33). Weperformed a similar assay with PrP 27-30 purified from mousebrains infected with RML prions to determine whether or notthe molecular target of branched polyamines was present inthis highly purified preparation. PrP 27-30 in purified prepa-rations of RML prions was rendered protease sensitive bybranched polyamines with a similar acidic pH optimum (Fig.2A) and structure-activity profile (Fig. 2B) as previously ob-tained in crude brain homogenates (33). Treatment of purifiedprions with branched polyamines in vitro also diminished in-fectivity. We incubated 15 mg of RML prion rods per ml in50 mM sodium acetate (pH 3.0)–1% NP-40–1-mg/ml BSA for2 h at 37°C, with or without 60 mg of PPI generation 4.0/ml, andmeasured prion infectivity using a scrapie prion incubationtime assay in Tg(MoPrP)4053 mice. PPI treatment reducedprion infectivity from 107 ID50 units/ml to 105 ID50 units/ml(data not shown).

PrPSc susceptibility to PPI-induced conformational changeis sequence and strain specific. Although the PrP sequence iswell conserved among mammals, a small number of amino acidsubstitutions retard prion transmission across species (27).Furthermore, prions can exist as different phenotypic strainsthat yield distinct incubation times, neuropathology, anddistribution of PrPSc upon infection of susceptible hosts. Incertain cases, these phenotypic differences can be correlatedwith differences in the conformation of PrPSc (3, 26, 29, 37).We sought to determine whether different species and strainsof rodent prions, which presumably contain different confor-mations of PrPSc, vary in their susceptibility to branched poly-amines. Homogenates were prepared from the brains ofrodents infected with one of several Syrian hamster (SHadesignations), mouse (Mo designations), or artificial prionstrains. Individual samples were mixed with 60 mg of PPI gen-eration 4.0/ml in vitro for 2 h at 37°C, neutralized, and sub-jected to limited proteolysis. The results indicate that suscep-tibility to the PPI dendrimer is dependent on both the prionstrain and PrP sequence (Fig. 3A).

The varying susceptibility of different strains is most clearlyillustrated by the six mouse strains analyzed (paired lanes 7 to12). Mo(RML), Mo(22a), and Mo(139A) were susceptible toPPI-induced conformational change (paired lanes 7, 9, and 11,respectively). In contrast, Mo(Me7) and Mo(87V) were resis-tant (paired lanes 8 and 10, respectively) and Mo(C506) wasmarginally susceptible to PPI-induced conformational change(paired lanes 12).

The effect of PrP sequence can be seen by comparing therelative susceptibilities of SHa(RML), MH2M(RML), andMo(RML). Whereas Mo(RML) was susceptible to PPI-in-duced conformational change (paired lanes 7), SHa(RML) wasresistant (paired lanes 4). MH2M(RML) displayed an inter-mediate level of susceptibility to PPI (paired lanes 5); MH2Mis a chimeric PrP molecule in which amino acids 94 to 188 ofthe mouse sequence have been replaced by the correspondingSyrian hamster residues (28). Thus, SHaPrPSc appears to be

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more resistant to PPI-induced conformational change thanMoPrPSc.

We investigated whether the varying susceptibilities to PPIdisplayed by different strains and species of prions might becaused by kinetic differences. To test this possibility, we incu-bated samples of each prion isolate with PPI generation 4.0 forvarious periods of time. Even after incubation with PPI for 3days, PrPSc in samples of resistant isolates did not becomemore susceptible to protease digestion (Fig. 3B). Thus, thedifferences in susceptibilities of different prion strains and se-

quences are not caused simply by differences in the kinetics ofPrPSc unfolding.

Recently, it was demonstrated that Tg(BoPrP)Prnp0/0 micewere susceptible to both BSE and natural sheep scrapie (30).Furthermore, these two prion strains remain distinct duringpassage through Tg(BoPrP)Prnp0/0 mice (30). These trans-genic mice therefore provided an opportunity to compare thesusceptibility of BSE and scrapie prions to branched poly-amines. We incubated brain homogenates from Tg(BoPrP)Prnp0/0 (BSE) and Tg(BoPrP)Prnp0/0 (sheep scrapie) mice

FIG. 1. Treatment of scrapie-infected neuroblastoma cells with PPI dendrimer. ScN2a cells were treated with 3 mg of PPI generation 4.0/ml insupplemented Dulbecco’s modified Eagle’s medium or control medium for 4 weeks. After two additional weeks of culture in compound-freemedium, cells were harvested for analysis. (A) PrP immunostain. Apparent molecular masses based on migration of protein standards are 30 and27 kDa. (B) Silver stain was performed as previously described (23). Apparent molecular masses based on migration of protein standards are 49,36, 25, and 19 kDa. For panels A and B, samples were assigned lanes as follows: 1, undigested control; 2, undigested, PPI treated; 3, proteinaseK-digested control; 4, proteinase K digested, PPI treated. All samples possessed equivalent protein concentrations prior to proteinase K digestion.(C) Infectivity bioassay of cell homogenates in Tg(MoPrP)4053 indicator mice. Filled circles, control cells; open squares, PPI-treated cells. (D) Thecalibration of Tg(MoPrP)4053 mice (36) with RML prions was performed as described previously (22). The brain homogenate used for calibrationwas prepared from a large pool of CD1 Swiss mice inoculated intracerebrally with RML prions. Each data point is an average 6 the standard errorof the mean obtained from three end-point titrations. Fewer than 100% of mice developed scrapie when the infectivity of the inocula was ,102

ID50 units/ml. The data correlating the end-point titer to the time intervals from inoculation to onset of clinical illness were best fitted using theleast squares method. ID50, 50% infectious dose.

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with PPI generation 4.0 in vitro. The PrPSc in scrapie-infectedTg mice was not susceptible to PPI-induced conformationalchange (Fig. 3C, lanes 1 and 2). In contrast, .90% of the PrPSc

in BSE-infected Tg mice was rendered protease sensitive bytreatment with PPI (Fig. 3C, lanes 3 and 4).

Branched polyamines mediate PrPSc denaturation. The ex-istence of prion strains resistant to branched polyamines sug-gests that PrPSc molecules in these strains might exist in con-formations which are more resistant to denaturation thanPrPSc molecules in polyamine-susceptible strains. To test thishypothesis, we examined the effect of adding urea to SHa(Sc237) brain homogenate treated with and without PPI gen-eration 4.0. In the presence of urea, PrPSc was more suscepti-ble to protease digestion in samples treated with PPI, whereasno difference in protease resistance could be detected in theabsence of urea (Fig. 4A). Thus, additional denaturation en-ables PrPSc molecules in a resistant strain to become suscep-tible to branched polyamines. This result suggests that thegeneral mechanism of action of branched polyamines mightbe to assist PrPSc denaturation. Consistent with this concept,branched polyamines render PrPSc protease sensitive moreefficiently at lower pH values (Fig. 2A). Furthermore, poly-amine-treated PrPSc did not regain protease resistance afterprolonged neutralization (Fig. 4B) or dialysis (data not shown).

Finally, we excluded the possibility that acidification might berequired only to activate the dendrimer by demonstrating thatpreacidified PPI generation 4.0 could not render PrPSc pro-tease sensitive at a neutral pH (Fig. 4C).

To visualize the effect of branched polyamines on prions, weexamined the ultrastructure of purified prion rods treated invitro with PPI generation 4.0. By electron microscopy, RMLprion rods were disaggregated after incubation for 2 h at 37°Cwith PPI (Fig. 5B). In contrast, SHa(Sc237) PrP 27-30 rodsremained intact after treatment with PPI (Fig. 5D). Disaggre-gation of purified RML prion rods by treatment with PPIwas accompanied by a loss of b-sheet secondary structure, asjudged by FTIR spectroscopy. Whereas control rods were 57%b-sheet, 25% a-helix, 7% b-turn, and 11% random coil, PPI-dissociated PrPSc was 47% b-sheet, 25% a-helix, 15% b-turn,and 13% random coil (Fig. 5E).

To investigate further the mechanism of polyamine-induceddisaggregation of PrP 27-30, we performed a kinetic study invitro using purified mouse RML prion rods and various con-centrations of PPI. The results indicate that polyamine-in-duced PrPSc disaggregation is not a catalytic process and re-quires a stoichiometry of approximately one PPI molecule perfive PrP 27-30 molecules in purified RML prion preparations(data not shown).

FIG. 2. Mixture of purified prions with branched polyamines in vitro. (A) Purified mouse RML prion rods were incubated with 60 mg of PPIgeneration 4.0/ml or control buffer at different pH values, as indicated. (B) Samples containing purified mouse RML PrP 27-30 were incubatedwith various polyamines, shown in lanes as follows: 1 and 2, control; 3, poly-L-lysine; 4, PAMAM 0.0; 5, PAMAM 1.0; 6, PAMAM 2.0; 7, PAMAM3.0; 8, PAMAM 4.0; 9, PAMAM-OH 4.0; 10, PPI 2.0; 11, PPI 4.0; 12, linear PEI; 13, high-molecular-weight PEI; 14, low-molecular-weight PEI;15, average-molecular-weight PEI; 16, Qiagen SuperFect. For panels A and B, all samples possessed equivalent protein concentrations and weresubjected to limited proteolysis. Apparent molecular masses based on migration of protein standards are 30 and 27 kDa. PK, proteinase K.

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PPI accumulates in lysosomes. Branched polyamines appar-ently require acidic conditions to render PrPSc protease sensi-tive when mixed with brain homogenates or purified prions invitro (Fig. 2A). However, these compounds successfully cure

living cells of prion infection when added to culture mediabuffered at pH 7.4 (Fig. 1). One possible explanation for thisdiscrepancy is that branched polyamines might localize withprions within an acidic intracellular compartment. PrPSc has

FIG. 3. Treatment of different prion strains with PPI in vitro. (A) Samples containing 1% (wt/vol) brain homogenates were incubated for 2 hat 37°C with 60 mg of PPI generation 4.0/ml. Paired lanes are designated as follows: 1, SHa(Sc237); 2, SHa(139H); 3, SHa(DY); 4, SHa(RML);5, Tg(MH2M)Prnp0/0(RML); 6, Tg(PrP106)Prnp0/0(RML); 7, Mo(RML); 8, Mo(Me7); 9, Mo(22a); 10, Mo(87V); 11, Mo(139A); 12, Mo(C506).Minus symbols denote untreated, control samples and plus symbols designate samples exposed to 60 mg of PPI generation 4.0/ml for 2 h at 37°C.(B) Samples containing 1% (wt/vol) Mo(RML) or SHa(Sc237) were incubated at 37°C with 60 mg of PPI generation 4.0/ml or control buffer forthe time periods indicated. For panels A and B, all samples possessed equivalent protein concentrations and were subjected to limited proteolysis.Apparent molecular masses based on migration of protein standards are 30 and 27 kDa. (C) Brain homogenates from Tg(BoPrP)Prnp0/0 mice wereincubated with 60 mg of PPI generation 4.0/ml or control buffer. Lanes: 1, Tg(BoPrP)Prnp0/0 (sheep scrapie); 2, Tg(BoPrP)Prnp0/0 (sheep scrapie)plus PPI; 3, Tg(BoPrP)Prnp0/0 (BSE); 4, Tg(BoPrP)Prnp0/0 (BSE) plus PPI. Minus symbols denote undigested, control samples and plus symbols des-ignate samples subjected to limited proteolysis. All samples possessed equivalent protein concentrations prior to proteolysis. Apparent molecularmasses based on migration of protein standards are 30 and 27 kDa.

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previously been shown to accumulate in lysosomes (35). There-fore, we sought to determine whether branched polyamineslocalize to this same compartment. We incubated N2a cellswith fluorescein-labeled PPI and LysoTracker Red and per-

formed dual channel confocal microscopy to compare the lo-calization of the two compounds. Our results indicate thatfluorescein-labeled PPI accumulates in the lysosomes of livingcells (Fig. 6).

DISCUSSION

Branched polyamines as therapeutic agents. A major find-ing of this study is that branched polyamines eliminate prioninfectivity from living cells that were chronically infected. Toour knowledge, this is the first class of compounds shown tocure an established prion infection. Polyene antibiotics, ani-onic dyes, sulfated dextrans, anthracylines, porphyrins, phthalo-cyanines, dapsone, and a synthetic b-breaker peptide all pro-long scrapie incubation times in vivo but only if administeredprophylactically (1, 11, 12, 16–19, 31, 34).

The unique ability of branched polyamines to cure an estab-lished prion infection in cells suggests that these compoundsmight also reverse disease progession in animals. However, twofactors could potentially limit the use of these compounds astherapeutic reagents against prion diseases. One potential lim-itation is that branched polyamines might not act on all strainsof prions in vivo. This possibility is shown by in vitro studies inwhich some strains and species of prions were more resistantthan others to branched polyamine-induced disaggregation(Fig. 3). It remains to be determined whether prion strains re-sistant to branched polyamine-induced disaggregation in vitrowould also be resistant to treatment by these compounds invivo. Treatment of more resistant strains might require therapywith branched polyamines in combination with another classof prion-directed compounds. Significantly, PPI demonstratessubstantial in vitro activity against BSE (Fig. 3C).

The second potential limitation of branched polyamines isthat these highly charged compounds might not cross theblood-brain barrier. If this proves to be the case, it may bepossible to deliver branched polyamines directly to the cere-brospinal fluid through an intraventricular reservoir or perhapsto synthesize them as prodrugs capable of crossing the blood-brain barrier. Preliminary studies indicate that continuous in-traventricular infusion of PPI generation 4.0 is tolerated byFVB mice up to a total dose of approximately 0.5 mg/animal(data not shown). Further studies are required to characterizethe biodistribution of dendrimers and to optimize their deliv-ery to prion-infected neurons in vivo.

Molecular target, mechanism, and site of action. The abilityof branched polyamines to render PrPSc sensitive to proteolyticdigestion in purified prion preparations (Fig. 2) suggests thatthe molecular target of these compounds must be either (i)PrPSc itself, (ii) an acid-induced unfolding intermediate ofPrPSc, or (iii) a very tightly bound, cryptic molecule whichcopurifies with PrPSc. Cross-linking experiments indicate thatphotoaffinity-labeled PPI generation 4.0 binds PrP 27-30 avidly(data not shown), but unfortunately these results cannot proveconclusively that PrP is the molecular target of branched poly-amines. If the molecular target is PrP, at least one of thepolyamine binding sites must be contained within the aminoacid sequence of the PrP106 deletion mutant (32), since PPIrenders PrPSc106 protease sensitive (Fig. 3A, lane 6). The 106amino acids present in PrP106 are residues 89 to 140 and 177to 231. PPI also renders a spontaneously protease-resistant,

FIG. 4. Denaturation of PrPSc is enhanced by PPI. (A) Samplescontaining 1% (wt/vol) SHa(Sc237) brain homogenates were incu-bated for 2 h at 37°C with 60 mg of PPI generation 4.0/ml or controlbuffer, plus various concentrations of urea as indicated. All sampleswere subjected to limited proteolysis. (B) Samples containing 1% Mo(RML) brain homogenate in 1% NP-40–50 mM sodium acetate (pH3.6) were incubated at 37°C for 2 h with either no addition (odd lanes)or 60 mg of PPI/ml (even lanes). All samples were neutralized with anequal volume of 0.2 M HEPES (pH 7.5) containing 0.3 M NaCl and4% Sarkosyl. Lanes: 1 and 2, samples not subjected to protease diges-tion; 3 and 4, samples immediately subjected to limited proteinase Kdigestion; 5 and 6, samples incubated at pH 7.5 for an additional 16 hat 37°C before proteinase K digestion. (C) Samples containing 1% Mo(RML) brain homogenate were treated in the following manner forlanes: 1, control sample at pH 3.6; 2, mixed with 60 mg of PPI/ml at pH3.6 for 2 h; 3, mixed with 60 mg of PPI/ml at pH 7.0 for 2 h; 4, incubatedalone at pH 3.6 for 2 h and then mixed with 60 mg of PPI/ml (preti-trated to pH 7.0) for 10 min; 5, incubated alone at pH 7.0 for 2 h andthen mixed with 60 mg of PPI/ml (pretitrated to pH 3.0) for 10 min. Allincubations were carried out at 37°C. Minus symbols denote undi-gested, control samples and plus symbols designate samples subjectedto limited proteolysis by proteinase K. All samples possessed equiva-lent protein concentrations prior to proteolysis. Apparent molecularmasses based on migration of protein standards are 30 and 27 kDa.

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61-amino-acid-long PrP deletion mutant, PrP(D23–88,D141–221), susceptible to protease digestion (33a), further delimitingthe boundaries of at least one putative binding site to residues89 to 140 and 222 to 231.

Several lines of evidence suggest that branched polyaminesrender PrPSc molecules protease sensitive by dissociating PrPSc

aggregates. (i) RML prion rods treated in vitro with PPI be-come disaggregated, as judged by electron microscopy (Fig. 5).(ii) Prion strains resistant to branched polyamines in vitroappear to be more amyloidogenic than polyamine-susceptiblestrains, as judged by neuropathology (4, 7, 13, 14). (iii) Theability of branched polyamines to render PrPSc protease sen-sitive in vitro is enhanced by conditions which favor PrPSc

disaggregation. These conditions include acidic pH (Fig. 2A)and the presence of urea (Fig. 4A).

Theoretically, it is possible that the mechanism by whichbranched polyamines remove PrPSc and prion infectivity from

ScN2a cells does not relate to the ability of these compoundsto disaggregate prions in vitro. However, this is unlikely be-cause the relative potency of 14 different polyamines in elim-inating PrPSc from ScN2a cells correlates with the relativeability of these same compounds to render PrPSc sensitive toproteolysis in crude brain homogenates and purified prepara-tions of RML PrP 27-30 in vitro (33) (Table 1). The structure-activity profile obtained from these studies indicates that poly-amines become more potent at eliminating PrPSc as theybecome more branched and possess more surface primaryamines. With PPI dendrimers, this effect reaches a plateau atthe fourth generation; PPI generation 5.0 is no more potentthan PPI generation 4.0 at either removing PrPSc from cells orrendering PrPSc protease sensitive in vitro. Homodisperse, uni-form PPI and PAMAM dendrimers were more potent than theheterogeneous preparations of PEI or SuperFect, a heat-frac-tured dendrimer.

We determined that the process by which PPI renders PrPSc

protease sensitive in vitro was not catalytic. Instead, this pro-cess appeared to require a fixed stoichiometric ratio of PPI toPrPSc of approximately 1:5. How could PPI denature prion

FIG. 5. Ultrastructure and secondary structure of purified prionrods treated with PPI in vitro. (A to D) Samples of purified 100-mg/mlPrP 27-30 in 0.1% NP-40–50 mM sodium acetate (pH 3.0) buffer wereincubated overnight at 37°C. Negative-stain electron microscopy wasperformed as described previously (32). Panels: A, Mo(RML) prionrods; B, Mo(RML) prion rods plus 60 mg of PPI generation 4.0/ml; C,SHa(Sc237) prion rods; D, SHa(Sc237) prion rods plus 60 mg of PPIgeneration 4.0/ml. The negative stain used was 2% uranyl acetate;scale bar 5 100 nm. (E) FTIR spectra of 0.1 mg of purified Mo(RML)prion rods/ml incubated overnight at 37°C with (dotted line) or without(solid line) 60 mg of PPI generation 4.0/ml.

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rods stoichiometrically? One possible explanation is that indi-vidual amino groups on the surface of PPI might bind to PrPSc

monomers or oligomers that exist in equilibrium with a largeaggregate under acidic conditions. The dendrimer might thenpry bound PrPSc molecules apart from the aggregate and/orprevent such molecules from reaggregating.

Another possible mechanism of polyamine-induced prionclearance from ScN2a cells is that branched polyamines facil-itate PrPSc transport from the plasma membrane through theendocytic pathway into secondary lysosomes. Several lines ofevidence indicate that the cellular site of action of branchedpolyamines is secondary lysosomes. (i) Fluorescein-tagged PPIand PrPSc both localize to lysosomes (8, 35) (Fig. 6). (ii) ThepH optimum of PrPSc denaturation in vitro is ,5.0. When cul-tured cells were studied with fluorescent acidotropic pH mea-surement dyes, secondary lysosomes were the most acidic cel-lular compartment detected, with pH values of ;4.4 to 4.5 (2,10). (iii) The lysosomotropic agent chloroquine attenuates the

ability of branched polyamines to eliminate PrPSc from ScN2acells (33). Our studies raise the possibility that lysosomal pro-teases normally degrade PrPSc in prion-infected cells at a slowrate and that polyamines accelerate this process by denaturingPrPSc.

Other applications of branched polyamines. Beyond theirpotential use as therapeutic agents and research tools, branchedpolyamines might also be useful as prion strain-typing reagentsand/or prion decontaminants. Presently, typing of prion strainsis time-consuming and requires the inoculation of samples intoseveral strains of inbred animals to obtain incubation time andneuropathology profiles (4, 9). Recently, antibody-based PrPSc

conformational stability assays able to distinguish prion strainshave been developed (26) (D. Peretz, unpublished data). Inthis study, we observed that different species and strains ofprions displayed varying susceptibilities to branched poly-amine-induced denaturation in vitro (Fig. 3). These resultssuggest that a polyamine-based in vitro protease digestion as-say could, in principle, be used as a simple and rapid diagnosticmethod for prion strain typing. One practical application whicharises from our results is that a polyamine-based assay could beused to distinguish between BSE and natural scrapie in flocksof domestic sheep.

Currently, it is very difficult to remove prions from skin,clothes, surgical instruments, foodstuffs, and surfaces (5). Stan-dard prion decontamination requires either prolonged auto-claving or exposure to harsh protein denaturants such as 1 NNaOH or 6 M guanidine thiocyanate (24). Branched dendrim-ers are nontoxic and relatively inexpensive. These compoundsmay therefore be suitable for use as disinfecting reagents tolimit the commercial and iatrogenic spread of prion diseases.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes ofHealth (NS14069, AG02132, and AG10770) and by a gift from theLeila and Harold Mathers Foundation. S.S. was supported by theBurroughs Wellcome Fund Career Development Award and by anNIH Clinical Investigator Development Award (K08 NS02048-02).

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