Treatment of scrapie-infected neuroblastoma cells with PPI dendrimer. ScN2a cells were treated with 3 μg of PPI generation 4.0/ml in supplemented Dulbecco's modified Eagle's medium or control medium for 4 weeks. After two additional weeks of culture in compound-free medium, cells were harvested for analysis. (A) PrP immunostain. Apparent molecular masses based on migration of protein standards are 30 and 27 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, proteinase K-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) The calibration of Tg(MoPrP)4053 mice (36) with RML prions was performed as described previously (22). The brain homogenate used for calibration was prepared from a large pool of CD1 Swiss mice inoculated intracerebrally with RML prions. Each data point is an average ± the standard error of 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 the least squares method. ID₅₀, 50% infectious dose.

Treatment of different prion strains with PPI in vitro. (A) Samples containing 1% (wt/vol) brain homogenates were incubated for 2 h at 37°C with 60 μg 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)Prnp^0/0(RML); 6, Tg(PrP106)Prnp^0/0(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 μg 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 μg of PPI generation 4.0/ml or control buffer for the 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)Prnp^0/0 mice were incubated with 60 μg of PPI generation 4.0/ml or control buffer. Lanes: 1, Tg(BoPrP)Prnp^0/0 (sheep scrapie); 2, Tg(BoPrP)Prnp^0/0 (sheep scrapie) plus PPI; 3, Tg(BoPrP)Prnp^0/0 (BSE); 4, Tg(BoPrP)Prnp^0/0 (BSE) plus PPI. Minus symbols denote undigested, control samples and plus symbols designate samples subjected to limited proteolysis. All samples possessed equivalent protein concentrations prior to proteolysis. Apparent molecular masses based on migration of protein standards are 30 and 27 kDa.

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

Mixture of purified prions with branched polyamines in vitro. (A) Purified mouse RML prion rods were incubated with 60 μg of PPI generation 4.0/ml or control buffer at different pH values, as indicated. (B) Samples containing purified mouse RML PrP 27-30 were incubated with 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, PAMAM 3.0; 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 were subjected to limited proteolysis. Apparent molecular masses based on migration of protein standards are 30 and 27 kDa. PK, proteinase K.

Denaturation of PrP^Sc is enhanced by PPI. (A) Samples containing 1% (wt/vol) SHa(Sc237) brain homogenates were incubated for 2 h at 37°C with 60 μg of PPI generation 4.0/ml or control buffer, plus various concentrations of urea as indicated. All samples were subjected to limited proteolysis. (B) Samples containing 1% Mo(RML) brain homogenate in 1% NP-40–50 mM sodium acetate (pH 3.6) were incubated at 37°C for 2 h with either no addition (odd lanes) or 60 μg of PPI/ml (even lanes). All samples were neutralized with an equal volume of 0.2 M HEPES (pH 7.5) containing 0.3 M NaCl and 4% Sarkosyl. Lanes: 1 and 2, samples not subjected to protease digestion; 3 and 4, samples immediately subjected to limited proteinase K digestion; 5 and 6, samples incubated at pH 7.5 for an additional 16 h at 37°C before proteinase K digestion. (C) Samples containing 1% Mo(RML) brain homogenate were treated in the following manner for lanes: 1, control sample at pH 3.6; 2, mixed with 60 μg of PPI/ml at pH 3.6 for 2 h; 3, mixed with 60 μg of PPI/ml at pH 7.0 for 2 h; 4, incubated alone at pH 3.6 for 2 h and then mixed with 60 μg of PPI/ml (pretitrated to pH 7.0) for 10 min; 5, incubated alone at pH 7.0 for 2 h and then mixed with 60 μg of PPI/ml (pretitrated to pH 3.0) for 10 min. All incubations were carried out at 37°C. Minus symbols denote undigested, control samples and plus symbols designate samples subjected to limited proteolysis by proteinase K. All samples possessed equivalent protein concentrations prior to proteolysis. Apparent molecular masses based on migration of protein standards are 30 and 27 kDa.

PPI localizes to lysosomes. N2a cells grown to 50% confluence on coverslips were incubated for 4 h with 3 mM FITC-PPI and 1 h with 75 nM LysoTracker Red (Molecular Probes). Fluorescence confocal microscopy in green and red channels was performed as described in Materials and Methods. Bar = 5 μm.