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First published online 20 May 2003
doi: 10.1242/jcs.00494

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Specific inhibition of pathological prion protein accumulation by small interfering RNAs

Nathalie Daude*, Mathieu Marella* and Joëlle Chabry{ddagger}

Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 6097, Centre National de la Recherche Scientifique. 660, route des lucioles, 06560 Valbonne, France



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  		Fig. 1. Dose-response effect of siRNA duplexes on PrP-sen expression and PrP-res accumulation in infected N2aS12sc+. (A) Immunoblot of a representative experiment performed with transfected N2aS12sc+ cells in the absence (0) or in the presence of increasing concentrations of Prnp gene-specific siRNA or of scrambled siRNA duplexes. One-tenth of the post-nuclear cell lysates were directly mixed with the same volume of 2x denaturing loading buffer. PrP-sen was first detected with the SAF83 mouse monoclonal antibody. The same blot was then incubated in the presence of rabbit polyclonal antibodies raised against ERK proteins (40-42 kDa) as a control of protein loading. (B) 90% of the lysates of untransfected or siRNAs-transfected N2aS12sc+ cells were PK-digested as described under Materials and Methods and loaded onto a 12% polyacrylamide gel. Partially PK-digested PrP-res was assayed with SAF83 monoclonal antibody. Molecular mass markers are indicated on the left in kilodaltons (kDa). (C) Densitometry analysis performed on blots developed and exposed as described above were done with `National Institutes of Health' IMAGE software. The results of four independent experiments are shown and expressed as a percentage of control levels ± s.e.m. (bars).

 


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  		Fig. 2. Effect of moPrnp-specific siRNA on the accumulation of PrP-res in GT-1 cells infected with 22L mouse scrapie strain. Infected GT-1 cells were transfected in the absence (0) or in the presence of specific or scrambled siRNA duplexes. Four days post-transfection, cells were lysed and the post-nuclear supernatants were PK-treated as described in Materials and Methods. PrP-res was detected with the SAF83 monoclonal antibody. The blot was developed by using an enhanced chemiluminescence system and exposed on x-ray film. Brackets on the right side indicate the immuno-detected bands corresponding to the un-, mono- and diglycoforms of PrP-res. Molecular mass markers in kilodaltons (kDa) are indicated on the left.

 


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  		Fig. 3. Transfection efficiency and sub-cellular localization of fluorescent siRNA duplex. (A) Confocal microscopy images of neuroblastoma cells transfected with either 400 nM of fluorescein-conjugated siRNA duplex or single strand fluorescein-conjugated siRNA as control. At the indicated times after transfection, N2aS12 cells were fixed with paraformaldehyde and stained with propidium iodide (red) for 10 minutes at room temperature under permeabilization conditions. Images were obtained with a laser scanning confocal microscope. Magnification, x40. (B) A representative fluorescein-siRNA-positive cell was observed as described above. The yellow color indicates red (propidium iodide) and green (fluorescein) dyes superimposed. The dashed line between arrowheads represents the Y-slice optical section. Arrows indicate a single fluorescent dot. N, nucleus.

 


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  		Fig. 4. Long-term effect of Prnp-specific siRNA on the PrP-res accumulation. N2aS12sc+ cells were transfected without (0) or with 400 nM Prnp-specific siRNA duplex. At confluence, cells were lysed and one-tenth of the samples were analysed in the absence of PK-treatment (–PK), whereas the remaining samples were PK-digested (+PK). These samples are labeled as 0 passage. Alternatively, cells were cultured for 1 and 2 weeks with splitting 1:4 every 4 days, i.e. 2 and 4 passages, respectively. PrP-sen and PrP-res levels were then detected by immunoblotting with SAF83 monoclonal antibodies as described in Materials and Methods. Molecular mass markers in kilodaltons (kDa) are indicated on the left.

 

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© The Company of Biologists Ltd 2003