Christina Orru recently pointed me to a paper published in Chinese a couple of years ago by Gao Chen (高晨), a scientist in China’s CJD surveillance unit who spent some time at RML learning RT-QuIC [Gao 2012]. No one here has been able to read the article; I knew those years I spent studying Chinese would come in handy at some point during my new scientific career!

Chinese CJD surveillance is relatively new, but in the data collected so far, D178N-129M has been the most frequently seen mutant haplotype [Gao 2011], so Dr. Gao was interested in the biochemical properties of HuPrP with the D178N mutation. The paper presents the results of unseeded RT-QuIC reactions on three substrates:

• HuPrP 23-231 D178N
• HuPrP 90-231 D178N
• HuPrP 90-231 wild-type

The paper has basically three results:

1. Figure 2 shows HuPrP 90-231 D178N spontaneously forming amyloid starting at about 10 or 12 hours at physiological salt concentrations (130 mM NaCl) while HuPrP 23-231 D178N does not do so at anywhere from 130 to 300 mM NaCl. These plates were run at 42°C with no SDS, 10μM EDTA, 10μM ThT, 1 mg/mL rPrP.
2. Figure 3 shows that the ThT signal from unseeded HuPrP 90-231 D178N reactions begins to rise after about 8h when only ~9 μM SDS is added, and can be shortened to <5h when ~70 μM SDS is present. Even at that high SDS concentration, HuPrP 90-231 without the mutation does not spontaneously form amyloid.
3. Figure 4 shows that the spontaneous amyloids derived of HuPrP 90-231 D178N are PK resistant.

Based on these data it would be tempting to conclude that the D178N mutation has a destabilizing or amyloidogenic effect in vitro and that this effect requires or at least is enhanced by N terminal truncation of the protein. But I can see a few reasons why these results should be considered as pretty preliminary:

• Properties of recombinant PrP can vary from batch to batch. The different conversion properties of HuPrP 90-231 D178N compared to the other substrates tested are confounded with batch effects.
• The proteins were all tested at a concentration of 0.1 mg/mL as is standard in RT-QuIC. HuPrP D178N 90-231 weighs 16.15 kDa, while HuPrP D178N 23-231 weighs 22.84 kDa, so the molar concentration of HuPrP D178N 90-231 in Figure 2 is actually 41% higher:
(1/16.15)/(1/22.84)
# 1.414241
• It is not clear how many technical replicates are being presented. Probably four replicates per curve since that’s standard in the Caughey lab but I do not see it stated anywhere. The total number of experiments presented here is small so it would be good to have a bit more data just to know that, for instance, the positive signal from HuPrP D178N 90-231 in Figure 2 is consistent and not just a one-time fluke.

In spite of these limitations, this paper is to my knowledge the first time someone has done RT-QuIC using a substrate with a genetic prion disease mutation, and the suggestion that this appears to accelerate spontaneous conversion is interesting. RT-QuIC products appear to be a different conformation than authentic infectious PrPSc, so does the D178N mutation destabilize PrPC or does it predispose to both the QuIC amyloid conformation and the FFI PrPSc conformation?