Advertisement
Research Article

Genesis of Mammalian Prions: From Non-infectious Amyloid Fibrils to a Transmissible Prion Disease

  • Natallia Makarava,

    Affiliation: Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America

    X
  • Gabor G. Kovacs,

    Affiliation: Institute of Neurology, Medical University of Vienna, Vienna, Austria

    X
  • Regina Savtchenko,

    Affiliation: Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America

    X
  • Irina Alexeeva,

    Affiliation: Medical Research Service, Veterans Affairs Medical Center, University of Maryland, Baltimore, Maryland, United States of America

    X
  • Herbert Budka,

    Affiliation: Institute of Neurology, Medical University of Vienna, Vienna, Austria

    X
  • Robert G. Rohwer,

    Affiliation: Medical Research Service, Veterans Affairs Medical Center, University of Maryland, Baltimore, Maryland, United States of America

    X
  • Ilia V. Baskakov mail

    Baskakov@umaryland.edu

    Affiliations: Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America, Department of Anatomy and Neurobiology, University of Maryland, Baltimore, Maryland, United States of America

    X
  • Published: December 01, 2011
  • DOI: 10.1371/journal.ppat.1002419

Reader Comments (1)

Post a new comment on this article

PrP immunohistochemistry of 3rd passage is identical with 263K

Posted by yervand on 11 Dec 2011 at 01:43 GMT

Authors conclude : "...clinical and neuropathological features, both synthetic strains ...were remarkably different from all previously known rodent strains including those generated in sPMCA "

However, the comparison of PrP immunoreactivity graph (fig3B) for 3rd passage of LOTSS with that of 263K reported in their first paper (fig4b) shows that they are virtually identical.

Same is true for the description of some peculiar pathologic features:

A quote from the first paper: "Both strains displayed immunoreactivity in subependymal regions, whereas perivascular PrP deposits were found only in hamsters infected by the 263K strain."

Now a piece from the description of the 3rd passage of LOTSS in this paper: " In addition, at the terminal stage, perivascular miniplaques appeared in the cerebellum and brainstem (Figure 6 C, D and E)."

No competing interests declared.

RE: PrP immunohistochemistry of 3rd passage is identical with 263K

Baskakov replied to yervand on 12 Dec 2011 at 18:50 GMT

Dear Dr. Karapetyan,

We are grateful for your comments that allow us to reiterate the differences between properties of synthetic strains generated in our laboratory and those of previously known rodent strains including 263K. Below is a brief list of strain-specific features that led to the conclusions in our manuscript:

1) The PrP immunoreactivty graphs were not identical for LOTSS and 263K. Although PrP immunoreactivity appeared in the cerebellum and hippocampus in LOTSS 3d passage, however, it was less pronounced than in 263K.

2) Both SSLOW and LOTSS are characterized by striking accumulation of very large PrP plaques in subependymal regions of brain and spinal cord. Large subependymal plaques were absent in 263K. In contrast, 263K shows so called “miniplaques” in several anatomical regions including the cerebellum. These plaques were not observed in LOTSS and SSLOW, apart from some small perivascular plaques in the cerebellum and brainstem in LOTSS 3rd (still much fewer and not as widespread as in 263K).

3) The lesion profiles of SSLOW and LOTSS were different from that of 263K. In particular, in the following regions the spongiform changes were absent or much less pronounced in SSLOW and LOTSS relative to 263K: the hippocampus, the cerebellum, and the upper cortical layers.

4) LOTSS and SSLOW display different set of clinical signs. Severe obesity was one of the most prominent clinical signs in SSLOW and LOTSS-inoculated animals, which is not the case for 263K.

5) The incubation time to the first clinical signs exceeds 300 days for LOTSS and SSLOW, but is only ~80 days for 263K.

6) The progression of clinical disease after the first signs is very slow in SSLOW- or LOTSS-inoculated animals. It takes 4 to 6 months for the clinical disease to progress to the terminal stage for SSLOW or LOTSS, but only a few days for 263K-inoculated animals.

Sincerely,

Gabor Kovacs
Herbert Budka
Ilia Baskakov

No competing interests declared.

RE: RE: PrP immunohistochemistry of 3rd passage is identical with 263K

yervand replied to Baskakov on 14 Dec 2011 at 19:41 GMT


Dear colleagues,

Thank you for your response to my brief comment. The points that you outlined are taken into account in the following context:

It is well-known for TSEs that inoculation of animals with limiting or higher dilutions (low doses) of the agent can cause sub-clinical infection in few of the inoculated animals with minor lesions that are different from the pathological picture seen in the brains of animals inoculated with high doses.

Further passages of these brains with minor lesions are still going to give long incubation times if the animals are sacrificed before the terminal stage (and by terminal I mean the stage where the animal is so sick that is not able to feed and is going to die).
However, despite all these differences of lesion profile in the brain in initial stages, after few serial passages at the end it all comes back to the original picture (including shorter incubation time) that one gets with the injection of high dose in the first passage.

Therefore, in the above mentioned context, your results could be interpreted as a contamination of your injected materials with low doses of the 263K agent. This interpretation predicts that the incubation time, clinical and pathological picture will go back to that of the 263K in the further passages of your material.

Sicerely,

Yervand Karapetyan

No competing interests declared.

RE: RE: PrP immunohistochemistry of 3rd passage is identical with 263K

yervand replied to Baskakov on 15 Dec 2011 at 02:15 GMT

J Virol. 2002 Mar;76(5):2510-7.
Chronic subclinical prion disease induced by low-dose inoculum.
Thackray AM, Klein MA, Aguzzi A, Bujdoso R.
Source
Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, United Kingdom CB3 OES.
Abstract
We have compared the transmission characteristics of the two mouse-adapted scrapie isolates, ME7 and Rocky Mountain Laboratory (RML), in tga20 mice. These mice express elevated levels of PrP protein compared to wild-type mice and display a relatively short disease incubation period following intracerebral prion inoculation. Terminal prion disease in tga20 mice induced by ME7 or RML was characterized by a distinct pattern of clinical signs and different incubation times. High-dose RML inoculated intracerebrally into tga20 mice induced the most rapid onset of clinical signs, with mice succumbing to terminal disease after only 58 +/- 3 days. In contrast, high-dose ME7 gave a mean time to terminal disease of 74 +/- 0 days. Histological examination of brain sections from prion-inoculated tga20 mice at terminal disease showed that ME7 gave rise to a more general and extensive pattern of vacuolation than RML. Low-dose inoculum failed to induce terminal disease but did cause preclinical symptoms, including the appearance of reversible clinical signs. Some mice oscillated between showing no clinical signs and early clinical signs for many months but never progressed to terminal disease. Brain tissue from these mice with chronic subclinical prion disease, sacrificed at >200 days postinoculation, contained high levels of infectivity and showed the presence of PrP(Sc). Parallel analysis of brain tissue from mice with terminal disease showed similar levels of infectivity and detectable PrP(Sc). These results show that high levels of infectivity and the presence of the abnormal isomer of PrP can be detected in mice with subclinical disease following low-dose prion inoculation.

No competing interests declared.

RE: RE: RE: PrP immunohistochemistry of 3rd passage is identical with 263K

yervand replied to yervand on 15 Dec 2011 at 03:21 GMT

A quote from the first paper "One of the most remarkable features of the prion disease
caused by SSLOW strain is its long clinical duration after the
first clinical signs are observed. In fact, the actual duration to
a death endpoint is not known as the animals, while seriously
compromised, were still mobile, eating and drinking at the
time of euthanasia. It is conceivable that animals may have
endured longer than 120 days after first clinical signs, if they
had not been euthanized."

No competing interests declared.

RE: RE: RE: RE: PrP immunohistochemistry of 3rd passage is identical with 263K

Baskakov replied to yervand on 17 Dec 2011 at 13:21 GMT

Dear Dr. Karapetyan,

First of all, the fact that SSLOW-or LOTSS-inoculated animals show very long clinical duration of the clinical disease after the first signs argues that these strains are not 263K. In animals inoculated with 263K, the duration of clinical disease is very short regardless of the dose.

Let us summarize your comments. You believe that cross-contamination of inoculums with 263K occurred in a laboratory, building and by personnel that have never been exposed to any TSEs. You also believe that 263K selectively contaminated inoculums with rPrP fibrils, but not inoculums with alpha-rPrP or inoculums containing age-matched control brain homogenates. Because animals from three serial passages of age-matched brain homogenates were all negative (Table S1), you believe that 263K cross-contamination did not target any control inoculums but only inoculums with rPrP amyloid fibrils.

You stated that “ your results could be interpreted as a contamination of your injected materials with low doses of the 263K agent”. This interpretation would be reasonable to consider if one could provide an example, where a third serial passage of a low dose of 263K produces an incubation time to clinical disease above 300 days with a 100% attack rate. We are unaware of such results, but appreciate if you could provide us with one.

You argument about incubation times at low doses of 263K is incorrect for the following reasons. For the 263K, the clinical onset extends from 60 days to 145 days post inoculation in a predictable dose response. The endpoint or “limiting” dilutions of 263K produce incomplete transmissions that are scattered randomly at ever diminishing frequency from 100 days PI to end of life. In the fraction of animals that do develop clinical disease al limiting dilutions, only very small percentage develops the clinical onsets after 300 days post inoculation. Moreover, these animals produce a clinical course and disease phenotype typical for the 263K strain. In contrast, SSLOW- or LOTSS- inoculated animals showed incubation time >300 days with 100% attack rate and disease phenotype remarkably distinct from that of 263K (video clips in Makarava et al 2010 Acta Neiropath).

As you stated, your “interpretation predicts that the incubation time, clinical and pathological picture will go back to that of the 263K in the further passages of your material”. They do not. The third and fourth passages of SSLOW have been completed. In both passages, 100 % attack rate, >300 days incubation time to disease, an additional 4 to 6 months duration of clinical disease to terminal stage, SSLOW-specific clinical symptoms and SSLOW-specific PrPSc biochemical properties were observed. 4th passage of LOTSS is on its way. No clinical signs have been detected so far by 200 days pi. If this material would represents 263K, all animals should have been terminally sick by 80 days pi. These results put your arguments to rest.

No competing interests declared.

RE: RE: RE: RE: RE: PrP immunohistochemistry of 3rd passage is identical with 263K

yervand replied to Baskakov on 18 Dec 2011 at 03:09 GMT


Dear Dr. Baskakov,

First of all, in your animals injected with rPrP fibrils there is no any clinical disease in the first passage. This means if there is any infection going on it is subclinical regardless of whether it is caused by contamination or anything else. As you correctly note, only a fraction of the animals inoculated with "limiting" dilutions of 263K develops characteristic clinical disease. The rest of the animals inoculated with "limiting" dilutions of the 263K agent never produce any symptoms and clinical disease. Therefore there is a possibility of subclinical infection in these animals at low levels. These animals are the "equivalents" of your first passage - no disease with low levels of infectivity - subclinical infection at low rate. And these group of animals are the ones that should be compared with regard to the second and third passages.

Only in the second passage of selected animals you describe a set of signs which you call clinical disease. As you note ( Makarava et al 2010 Acta Neiropath) : "In fact, the actual duration to
a death endpoint is not known as the animals, while seriously
compromised, were still mobile, eating and drinking at the
time of euthanasia. It is conceivable that animals may have
endured longer than 120 days after first clinical signs, if they
had not been euthanized."
In fact, it is also not known if this set of signs would have led to lethality at all.
Therefore it is not clear what you mean by "terminal stage" when you describe the results of the 3rd and 4th passages of "SSLOW". These additional, new results only can be interpreted after the full picture is presented, including the neuropathology.

You stated in the supplement of Makarava et al 2010 Acta Neiropath, that: "However, the transmissions were conducted in an animal BSL3 laboratory that has been dedicated for use with TSEs. ... The vast majority of the work in this facility has been conducted using the 263K strain of hamster scrapie or the 301v strain of mouse-adapted BSE. However, TSE strains from mice, rats, cervids, sheep, cattle, monkeys and humans including field isolates are also present and have occasionally been used. "

I believe that cross-contamination of your inoculums occurred in the facility where the transmissions were conducted.

The possibility for molecular components of the infectious agent selectively incorporating only into inoculums with preheated rPrP but not the others is in agreement with a report where only preheated rPrP (and not the other forms) complexed with RNA from infected brain transmitted scrapie disease into hamsters.

Simoneau, Steve, Ruchoux, Marie-Madeleine, Vignier, Nicolas, Lebon, Pierre, Freire, Sophie, Comoy, Emmanuel, Deslys, Jean-Philippe, and Fournier, Jean-Guy. Small critical RNAs in the scrapie agent. Available from Nature Precedings <http://hdl.handle.net/101...> (2009)





No competing interests declared.

RE: RE: RE: RE: RE: PrP immunohistochemistry of 3rd passage is identical with 263K

yervand replied to Baskakov on 18 Dec 2011 at 04:04 GMT

Clarification: "When induced by rPrP fibrils, which are known to be structurally different from PrPSc [14]–[17], the clinical disease developed only at the third serial passage."

No competing interests declared.

RE: RE: RE: RE: RE: PrP immunohistochemistry of 3rd passage is identical with 263K

yervand replied to Baskakov on 18 Dec 2011 at 04:08 GMT

Is RNA critical for the production of TSE infection?
Jean-Guy Fournier*
CEA/DSV/IMETI/SEPIA, Fontenay aux Roses, France
A commentary on

Recombinant prion protein induces a new transmissible prion disease in wild-type animals.
by Makarava, N., Kovacs, G. G., Bocharova, O., Savtchenko, R., Alexeeva, I., Budka, H., Rohwer, R. G., and Baskakov, I. V. (2010). Acta Neuropathol. 119, 177–187.

Generating a prion with bacterially expressed recombinant prion protein.
by Wang, F., Wang, X., Yuan, C. G., and Ma, J. (2010). Science 327, 1132–1135.

Mammalian prions generated from bacterially expressed prion protein in the absence of any mammalian cofactors.
by Kim, J., Kim, J. I., Cali, I., Surewicz, K., Kong, Q., Raymond, G. J., Atarashi, R., Race, B., Qing, L., Gambetti, P., Caughey, B., and Surewicz, W. K. (2010). J. Biol. Chem. 285, 14083–14087.

The prion hypothesis proposed by Prusiner (1998) stipulates that the causal agent of transmissible spongiform encephalopathies (TSEs) is composed exclusively of an infectious protein (PrPsc) produced after structural conversion of a cellular protein (PrPc). Recently, three studies attempting to test predictions arising from this hypothesis have been published by Makarava et al. (2010),Wang et al. (2010), and Kim et al. (2010).

Paying particular attention to the molecular composition of inocula inducing TSE disease reported in these investigations, I plan to discuss here, how these novel findings complement our previous results (Simoneau et al., 2009). In a pioneer work, we substantiated the idea that the requirement of a cofactor may be indispensable for the acquisition of converted recombinant prion protein (recPrP) infectivity. Our results indicate that this crucial cofactor extracted from 263 K PrPsc is composed of two populations of small RNA having an average length of about 27 and 55 nucleotides. The association of these RNAs with recPrP reconstituted an infectious TSE agent showing in hamster brain strain characteristics slightly different from 263 K infectious agent (Simoneau et al., 2009).

As currently recognized, the demonstration of the prion hypothesis should be performed in wild-type (wt) animals. However I suggest that comprehensive proof of the causal agent of TSEs is composed exclusively of an infectious PrPsc protein can only be obtained when experimentation meets four requirements. This tetrad of rules is equivalent to Koch’s postulates for microbial agents, namely that:

1. In vitro conversion of pure (e.g., recombinant) PrPc to PrPsc is required.

2. Experiments should be performed using wt animals.

3. Infected animals should develop fatal TSEs.

4. Transmissibility of the disease at the second passage should be demonstrated.

In the first study (Makarava et al., 2010) the authors inoculated different preparations of recPrP in hamsters. Though they use wt animals in accord with the second requirement, they fail to meet the first criterion. Indeed the key inoculum produced from a pure solution of recPrP fibrils remains innocuous, i.e., fails to cause disease. This recPrP is effective only if it is “activated” by an “annealing” process involving normal hamster brain homogenate (NHB). Other components may well have been incorporated from NHB. Moreover, the preparation induces a TSE-like disease only on second passage: the original inoculum did not have the ability to cause the TSE disease characterized at second pass; therefore the third condition is not met. Though a new scrapie agent has been created, the experimental context does not allow to know for sure that it is derived from converted recPrP alone. Indeed, the inocula have had to undergo a long maturation step during the first passage (661 days). One could presume that converted recPrP, associated in a first step with endogenous RNA (present in NHB), is capable of inducing in the first brain the progressive synthesis of PrPsc and small RNA. The association (PrPsc–small RNA) we have reported (Simoneau et al., 2009) would form the infectious agent which inoculated in the second brain would induce overt TSE.

In a similar way to Makarava’s work, the approach by Wang et al. (2010) of using wt animals to test the prion hypothesis is compromised by the incorporation of RNA into the inoculum. Infection is only achieved by adding some critical components to the recPrP solution, such as RNA (extracted from mouse liver) and synthetic phospholipid molecules, followed by a very extensive protein misfolding cyclic amplification (PMCA). The putative importance of the host RNA in this sophisticated preparation has not been explored or even debated. We must ask whether it is catalytic or not, imposing control on whether recPrP is associated or not with RNA. We can also ask what its molecular characteristics are: single or double stranded, short or long lengths and, eventually, what sequence? The role of RNA could be evaluated simply using inoculum containing recPrP without RNA.

The third study (Kim et al., 2010) used the classical PMCA reaction combined with PrPsc to convert recombinant PrP. Again the solution destined to be injected is not as the authors believe, of absolute recPrP purity. They used PrPsc partially purified from 263 K-infected hamster brain. In these conditions, native PrPsc is known to be highly contamined by nucleic acids particularly RNA a key factor for the acquisition of PMCA product infectivity (Geoghegan et al., 2007). On this basis, the crucial control would have been to assess the response of RNAse treatment on the preparative solutions.

So the fundamental question as suggested by Supattapone (2010), is what phenomenon metamorphoses an innocuous recPrP preparation into an infectious one? It seems that a conversion step alone is not sufficient to obtain this objective. Rather, co-factors, RNA in some cases, appear to be necessary.

In conclusion, if one considers that the tetrad of rules that should be respected for a definitive demonstration of the prion hypothesis, ambiguities remain that should be clarified, with respect to the involvement of RNA.
Acknowledgments
The author would like to thank Laura Manuelidis for her kind encouragements and express his sincerest gratitude to Robert Somerville for discussions and his help in writing of the paper.
References
Geoghegan, J. C., Valdes, P. A., Orem, N. R., Deleault, N. R., Williamson, R. A., Harris, B. T., and Supattapone S. (2007). Selective incorporation of polyanionic molecules into hamster prions. J. Biol. Chem. 282, 36341–36353.
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Kim, J., Kim, J. I., Cali, I., Surewicz, K., Kong, Q., Raymond, G. J., Atarashi, R., Race, B., Qing, L., Gambetti, P., Caughey, B., and Surewicz, W. K. (2010). Mammalian prions generated from bacterially expressed prion protein in the absence of any mammalian cofactors. J. Biol. Chem. 285, 14083–14087.
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Makarava N., Kovacs G. G., Bocharova O., Savtchenko R., Alexeeva I., Budka H., Rohwer R. G., and Baskakov I. V. (2010). Recombinant prion protein induces a new transmissible prion disease in wild-type animals. Acta Neuropathol. 119, 177–187.
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Prusiner S. B. (1998). Prions. Proc. Natl. Acad. Sci. U.S.A. 95, 13363–13383.
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Simoneau, S., Ruchoux, M. M., Vignier, N., Lebon, P., Freire, S., Comoy, E., Deslys, J. P., and Fournier, J. G. (2009). Small critical RNAs in the scrapie agent. Nature Precedings. Available at http://hdl.handle.net/101....
Supattapone, S. (2010). What makes prion infectious? Science 327, 1091–1092.
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Wang F., Wang X., Yuan C. G., and Ma, J. (2010). Generating a prion with bacterially expressed recombinant prion protein. Science 327, 1132–1135.
Pubmed Abstract | Pubmed Full Text | CrossRef Full Text
Citation: Fournier J-G (2010) Is RNA critical for the production of TSE infection? Front. Psychiatry 1:24. doi: 10.3389/fpsyt.2010.00024
Received: 06 May 2010; Accepted: 06 July 2010;
Published online: 01 September 2010
Copyright: © 2010 Fournier. This is an open-access article subject to an exclusive license agreement between the authors and the Frontiers Research Foundation, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited.
*Correspondence: jean-guy.fournier@cea.fr

No competing interests declared.

RE: RE: RE: RE: RE: PrP immunohistochemistry of 3rd passage is identical with 263K

yervand replied to Baskakov on 22 Dec 2011 at 21:29 GMT

"Severe obesity was one of the most prominent clinical signs in SSLOW and LOTSS-inoculated animals, which is not the case for 263K."

Prominent pancreatic endocrinopathy and altered control of food intake disrupt energy homeostasis in prion diseases.
Bailey JD, Berardinelli JG, Rocke TE, Bessen RA.
J Endocrinol. 2008 May;197(2):251-63.

"The 139H scrapie agent causes preclinical obesity in hamsters (Carp et al. 1990, Hecker et al. 1992) and has an incubation period (i.e., the time to clinical disease) that ranges from 20 to 30 weeks (Kimberlin et al. 1989, Hecker et al. 1992, Prusiner 1998)."

No competing interests declared.