Cellular Prion Protein Mediates Toxic Signaling of Amyloid Beta

Prion diseases in humans and animals comprise a group of invariably fatal neurodegenerative diseases characterized by the formation of a pathogenic protein conformer designated PrPSc and infectious particles denoted prions. The cellular prion protein (PrPC) has a central role in the pathogenesis of prion disease. First, it is the precursor of PrPSc and infectious prions and second, its expression on neuronal cells is required to mediate toxic effects of prions. To specifically study the role of PrPC as a mediator of toxic signaling, we have developed novel cell culture models, including primary neurons prepared from PrP-deficient mice. Using these approaches we have been able to show that PrPC can interact with and mediate toxic signaling of various beta-sheet-rich conformers of different origins, including amyloid beta, suggesting a pathophysiological role of the prion protein beyond prion diseases. Copyright (C) 2011 S. Karger AG, Base

Anti-prion drug mPPIg5 inhibits PrP(C) conversion to PrP(Sc).

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future

The heat shock response is modulated by and interferes with toxic effects of scrapie prion protein and amyloid β

The heat shock response (HSR) is an evolutionarily conserved pathway designed to maintain proteostasis and to ameliorate toxic effects of aberrant protein folding. We have studied the modulation of the HSR by the scrapie prion protein (PrPSc) and amyloid β peptide (Aβ) and investigated whether an activated HSR or the ectopic expression of individual chaperones can interfere with PrPSc- or Aβ-induced toxicity. First, we observed different effects on the HSR under acute or chronic exposure of cells to PrPSc or Aβ. In chronically exposed cells the threshold to mount a stress response was significantly increased, evidenced by a decreased expression of Hsp72 after stress, whereas an acute exposure lowered the threshold for stress-induced expression of Hsp72. Next, we employed models of PrPSc- and Aβ-induced toxicity to demonstrate that the induction of the HSR ameliorates the toxic effects of both PrPSc and Aβ. Similarly, the ectopic expression of cytosolic Hsp72 or the extracellular chaperone clusterin protected against PrPSc- or Aβ-induced toxicity. However, toxic signaling induced by a pathogenic PrP mutant located at the plasma membrane was prevented by an activated HSR or Hsp72 but not by clusterin, indicating a distinct mode of action of this extracellular chaperone. Our study supports the notion that different pathological protein conformers mediate toxic effects via similar cellular pathways and emphasizes the possibility to exploit the heat shock response therapeutically

mPPIg5 treatment of ScN2a cells and treatment of lysates from RML and 22L infected N2a cells.

<p><b>A</b>) RML infected N2a cells and 22L infected N2a#58 cells were lysed in 4% sarkosyl (in PBS pH 7.4). 100 µg of lysate was then left untreated or treated with 2.4 mg/ml of mPPIg5 dendrimers for 3 hours before analysis for protease resistant PrP^Sc content by PK digestion and immunoblot. <b>B</b>) RML infected N2a cells were treated with increasing concentrations of mPPIg5 for 4 days before analysis by PK digest and immunoblot for PrP^Sc. Apparent molecular mass based on migration of protein standards is indicated for 17, 25, and 30 kDa in both panel A and B. n = 3.</p

Validation of nSCA.

<p><b>A</b>) N2a cells and <b>B</b>) ScN2a cells were inoculated for 1.5 hours with increasing dilutions of PrP^27–30. The infective media was removed and replaced with regular DMEM for an additional 3 hours before cells were repeatedly washed with PBS and 20,000 cells attached per well of an ELISPOT plate. Cells were examined via the nSCA and mSCA. Positive result is defined as >30 spots/20,000 cells. The software used to count spots (infected cells) cannot count accurately above 1000 spots so a dose response curve is not observed with the mSCA for the ScN2a cells inoculated with PrP^27–30. Error bars represent SD; n = 2.</p

mPPIg5 effect on subcellular distribution of PrP^C.

<p><b>A</b>) Confocal microscopy was used to examine the cellular localization of PrP^C. N2a#58 cells grown on coverslips were mock treated, treated with 20 µg/ml mPPIg5 or treated with 200 µg/ml suramin for 48 hours. Cells were then permeabilized or left intact before immuno-staining with SAF32 and Alexa488 secondary antibody for PrP^C. Hoechst 33342 was used to stain DNA. All images were acquired using equal settings. n = 2. <b>B</b>) Phosphatidylinositol-specific phospholipase C (PIPLC) was used to validate the confocal microscopy result. N2a#58 cells were treated with 20 µg/ml mPPIg5 (lanes 2, 4, 6 and 8) or mock treated (lanes 1, 3, 5 and 7) for 48 hours. Cells were washed with PBS then treated for two hours with 0.5U/ml PIPLC in serum free DMEM (lanes 3, 4, 7 and 8) or mock treated with serum free DMEM (lanes 1, 2, 5 and 6). The media was collected, ethanol precipitated and examined for PrP^C by immunoblotting with SAF83 antibody (lanes 5–8). The remaining cells were lysed and a portion examined for PrP^C by immunoblot (lanes 1–4). Apparent molecular mass based on migration of protein standards is indicated for 17, 25, and 30 kDa. <b>C</b>) Biosynthesis of PrP^C was examined by metabolic labelling. N2a cells were transfected with PrP^C and labelled with [³⁵S] methionine for 1 hour. After labelling cells were either analysed directly (Pulse), or incubated in fresh medium for additional 4 h (Chase). When indicated, mPPIg5 was present during the starving, labelling and chase periods (final concentration 20 µg/ml). PrP was immunoprecipitated by using the monoclonal anti-PrP antibody 3F4 and analysed by SDS-PAGE and autoradiography. Apparent molecular mass based on migration of protein standards is indicated for 16, 22, and 36 kDa.</p

Schematic description of dendrimers PPIg4, PPIg5 and mPPIg5 used in this study.

<p><b>A</b>) mPPIg5 is obtained by reductive amination of PPIg5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055282#pone.0055282-Klajnert1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055282#pone.0055282-Fischer1" target="_blank">[14]</a>. <b>B</b>) Molecular structure of cationic 4th generation poly(propylene imine) (PPIg4) with the smallest size used in biological experiments. <b>C</b>) Molecular weights for the dendrimers utilised in this study.</p

Effect of NH₄Cl on FL PrP^Sc levels.

<p>22LN2a#58 cells were mock treated or treated with mPPIg5 (20 µg/ml), STI571 (10 µM) or suramin (200 µg/ml) in the presence or the absence of 2 mM NH₄Cl for 48 hours before analysis for FL PrP^Sc content via nSCA. The number of FL PrP^Sc containing cells in 20,000 mock treated control cells (- NH₄Cl) is taken as the 100% value on the Y axis. All subsequent results are calculated from this. Results marked a, b, c and d are all statically significantly different from one another (p<0.001); Analysed using a Two-way ANOVA for PrP^res, Dendrimer × NH₄Cl (F_4,88  = 7.99; P<0.001). Post-hoc analysis by Tukeys test; Error bars represent SD; n = 3.</p

Summary of the standard scrapie cell assay and variants developed in this study.

<p>Summary of the standard scrapie cell assay and variants developed in this study.</p