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Article Type: Short Communication
Protein misfolding cyclic amplification (PMCA) induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis
Zhen Yuan1, Lifeng Yang1, Baian Chen2, Ting Zhu1, Mohammad Farooque Hassan1, Xiaomin Yin1, Xiangmei Zhou1, Deming Zhao*1 1 State Key Laboratories for Agrobiotechnology, Key Lab of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. 2 Department of Laboratory Animal Science, School of Basic Medical Science, Capital Medical University, Beijing, 100069, China.
Zhen Yuan and Lifeng Yang contributed to this work equally.
Correspondence to: Deming Zhao, China Agricultural University, Haidian District, Beijing, China. E-mail: [email protected]. Tel: +86-01062732975
Running title: PMCA induces PrP monomers to form oligomers
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jnc.13098 This article is protected by copyright. All rights reserved.
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Keywords: Prion protein; oligomers; protein misfolding cyclic amplification; cytotoxic; neuronal apoptosis
Abbreviations used: PMCA, protein misfolding cyclic amplification; PrP, prion protein; PrPC, cellular form of the prion protein; TSEs, transmissible spongiform encephalopathies; HaPrP, hamster prion protein; HuPrP, human prion protein; RaPrP, rabbit prion protein; MuRaPrP, mutant rabbit prion protein.
Abstract: The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β-state oligomers. Herein, we demonstrate that β-state oligomeric PrP can be generated through protein misfolding cyclic amplification (PMCA) from recombinant full-length hamster, human, rabbit and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP-induced neurotoxicity. We have characterized PMCA-induced monomer-to-oligomer conversion of PrP from three species using western blotting, circular dichroism, size exclusion chromatography, and resistance to proteinase K digestion. We have further shown that all of the resulting β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrPC in the neurons, while the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3 in both wild-type and PrP-/- cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain. Introduction: According to the “protein-only” hypothesis, the infectious PrPSc agent consists solely of a misfolded form of the host-encoded cellular prion protein (PrPC) (Prusiner 1998, Prusiner This article is protected by copyright. All rights reserved.
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showed that PrP oligomers could be formed from recombinant PrP in the absence of PrPSc by PMCA, that these oligomers are cytotoxic even in the absence of additional endogenous PrP, and that they are cytotoxic across species lines. This may also advance our understanding of the pathogenesis of prion diseases and help to delve into the molecular determinants for prion-like molecules, allowing the development of novel drugs to treat these diseases.
Acknowledgements We thank Dr. Charles Weissmann at the Scripps Research Institute for supplying the PrP-/knockout mice. This study was supported by 948 projects (2013-S11; 2014-S9) to BC. The authors have no conflicts of interest to declare.
References Anderson, M., Bocharova, O. V., Makarava, N., Breydo, L., Salnikov, V. V. and Baskakov, I. V. (2006) Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy. Journal of molecular biology, 358, 580-596. Büeler, H., Aguzzi, A., Sailer, A., Greiner, R.-A., Autenried, P., Aguet, M. and Weissmann, C. (1993) Mice devoid of PrP are resistant to scrapie. Cell, 73, 1339-1347. Biasini, E., Turnbaugh, J. A., Unterberger, U. and Harris, D. A. (2012) Prion protein at the crossroads of physiology and disease. Trends in neurosciences, 35, 92-103. Bocharova, O. V., Breydo, L., Parfenov, A. S., Salnikov, V. V. and Baskakov, I. V. (2005) In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP Sc. Journal of molecular biology, 346, 645-659. Chianini, F., Fernández-Borges, N., Vidal, E. et al. (2012) Rabbits are not resistant to prion infection. Proceedings of the National Academy of Sciences, 109, 5080-5085. Corsaro, A., Thellung, S., Villa, V., Nizzari, M. and Florio, T. (2012) Role of Prion Protein Aggregation in Neurotoxicity. International journal of molecular sciences, 13, 8648-8669. Couzin, J. (2004) An end to the prion debate? Don't count on it. Science, 305, 589-589.
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Animal Information and Ethics Statement Wild-type C57BL/6J suckling mice with no genomic modifications were acquired from Charles River Laboratories (USA). PrP-/- knock-out mice on a C57BL/6J×129Sv genetic background were supplied from Dr. Charles Weissmann in the Scripps Research Institute. Wild-type and PrP-/- mice were verified by western blotting (Fig. S1). Wild-type mice and PrP-/- knock-out mice used in this study were postnatal suckling female mice less than 24 hours old. All animal experiments were conducted according to the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the Beijing Municipality Administration Office of Laboratory Animals (BAOLA).
Expression and Purification of Recombinant Prion Proteins Full-length hamster, human and rabbit recombinant PrP (23-230) were used in this study. Plasmids for expression of PrP were created from hamster, human or rabbit cDNA as described previously (Bocharova et al. 2005, Anderson et al. 2006, Wang et al. 2011b). The cDNAs were cloned into the Escherichia coli expression vector pPROEX-HTb (Invitrogen, USA). Purification of the recombinant PrP and cleavage of the 6-histidine tags were performed according to previously published procedures(Wang et al. 2011a). The mutant RaPrP (23–230) was constructed using site-directed mutagenesis PCR (Wen et al. 2010c, Hosszu et al. 2010). Purified PrP in PBS was kept at −80°C. The purity of recombinant protein was monitored by coomassie brilliant blue staining on SDS-PAGE gel. The concentration of purified PrP was calculated using OD280 reading.
In Vitro Generation of PrP Oligomers Preparation of PMCA Substrate Purified monomeric PrP was thawed and centrifuged at 100,000 X g for 1 h at 4°C. The supernatant was diluted with sterile deionized water to 0.50 mg/ml. In a 15 ml centrifuge This article is protected by copyright. All rights reserved.
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tube, 180 μl of diluted PrP was mixed with 500 μl of 5% Triton X-100, 900 μl of 10 × TN buffer (100mM Tris-HCl, pH 7.5, 1.5M NaCl) and 7420 μl of nuclease-free H2O. The mixture was mixed thoroughly and incubated at room temperature for 10 min. The prepared PMCA substrate was distributed into PCR tubes (90 μl per tube) and stored at −80°C until sonication. POPG was not essential in the generation of PrP oligomers (Fig.S1).
PMCA Instrument Setup A sonicator with a microplate horn (model S-4000, Misonix, Inc.) was fitted with a homemade PCR-tube rack and used for all PMCA reactions. Reactions were carried out in 200 μl PCR tubes; tubes were placed in the rack and partially submerged in sterile 37.0°C water (to 3 mm above the horn surface). The sonication cycle consisted of 30 s of sonication at an amplitude of 80, followed by a 29.5-min incubation at 37.0°C. The water was changed after every 24 cycles.
Western Blot Protein samples were loaded onto a 12% SDS-PAGE gel. After electrophoresis, the SDS-PAGE gel was soaked in transfer buffer for 10 min. Meanwhile, 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA) was soaked sequentially in pure methanol for 60 seconds, in deionized water for 2 min, and in transfer buffer for 10 min. Transfer was carried out with cooling for 4 h with a constant voltage of 80 volts. After transfer, the PVDF membrane was blocked with 5% nonfat milk (CWBIO) for 1 h at 37°C. The membrane was incubated at room temperature with primary antibody in TBST with shaking at 4℃ overnight, washed three times with TBST (10 min each), incubated with secondary antibody and washed 5 times with TBST (10 min each). The signal was detected by exposure to chemiluminescence (Versadoc; Bio-Rad, Hercules, CA, USA).
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Statistics All assays were performed on three separate occasions. Data were expressed as means ± SD of three experiments. All comparisons for parametric data were made using Student’s t-test or one-way ANOVA followed by post hoc Tukey’s test using the SPSS software (version 13.0: SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 software (La Jolla, CA, USA). P < 0.05 was considered statistically significant.
Results: PMCA induces recombinant PrPC to form β-sheet rich oligomers Western blots were initially used to determine whether the PMCA treatment generated PrP oligomers. For all forms of PrP, PMCA treatment produced an oligomer with an apparent molecular weight of twice that of the monomeric PrP, evident on western blots (Fig.1A). A similar change in molecular weight was observed in three replicate experiments for each PrP. To further verify that the PMCA treatment was responsible for the formation of the observed oligomers, we also ran several controls that were identical to the test samples in all ways except that they were not sonicated; oligomers were not formed in these samples (Fig.1C). In addition, we found that POPG is not essential to the formation of oligomers (Fig. S2). Interestingly, 144 cycles of PMCA were sufficient for HaPrP and HuPrP to achieve complete conversion to the oligomeric form (over 99% of the total protein), while 216 cycles were required for RaPrP (Fig.1B). We proposed that this difference in the rate of oligomer formation may be related to differences in structural stability of the monomers. The proteins differ at several key amino acids which are thought to contribute to their structural stability (Yuan et al. 2013a, Julien et al. 2011, Fernandez-Funez et al. 2011a, Wen et al. 2010b). The relative stability of the PrP monomers of these three species is: RaPrP exhibit stronger stability than HuPrP and HaPrP. Therefore we created a mutant rabbit PrP (MuRaPrP) model in which certain stability-inducing amino acids in the RaPrP were replaced with the amino acids found at the same position in the HaPrP. The MuRaPrP contained the following This article is protected by copyright. All rights reserved.
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substituions: Ser107 to Asn, Ser173 to Asn, Gln219 to Lys, and Ala224 to Tyr. When MuRaPrP was subjected to PMCA, MuRaPrP monomers were fully converted to oligomers after 168 cycles (Fig.1D).
Size-exclusion chromatography is a simple and direct assay to determine whether PrP exist as monomers or oligomers(Huang et al. 2010, Simoneau et al. 2007a). The PrP oligomers generated by PMCA were clearly distinguished from the monomers based on elution time; the elution time for monomers was about 20.6 min, and for oligomers, about 8.5 min (Fig.2A). Circular dichroism spectroscopy was used to further analyze the oligomers that were formed by PMCA. The results showed that the oligomers possess a β-sheet structure (Fig.2B) more extensive than that found in the corresponding monomers. Secondary structure content of these PMCA-induced oligomers was found to be about 29% β-sheet and 18% α-helix. In contrast, the four monomeric recombinant prion proteins contained nearly 41% α-helix and only 13% β-sheet.
Oligomeric PrP showed partial resistance to proteinase K digestion We subjected our PMCA-induced oligomeric PrP to PK digestion, in parallel with their monomeric forms. As shown in Figure 3, the β-oligomeric PrP oligomers each exhibited partial resistance to PK digestion compared to their monomeric counterparts. At PK concentrations of 0.1 and 0.25 μg/ml, both types of PrP showed resistance to PK digestion. However, at PK concentrations of 0.5 and 1 μg/ml, oligomeric PrP showed an elevated PK resistance, whereas monomeric PrP was completely digested. At the highest PK concentration of 10 μg/ml, both monomers and oligomers were digested completely (Fig.3). There was no clear shift in the molecular weight of the PK treated protein. This result was further confirmed by using PrP antibody AH6 (Fig.S3).
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β-PrP oligomers are cytotoxic to primary cortical neurons from wild-type and PrP-/- Mice We first analyzed the cytotoxic properties of the PrP oligomers in primary mouse cerebral cortex neurons isolated from wild-type animals. For each of the oligomers, exposure of primary neurons to a dose of 100 μg/ml PrP resulted in a loss of nearly 30% of the neurons compared to the control cells treated with monomers or with buffer alone. No obvious loss of cells occurred at either 25 or 50 μg/ml. Furthermore, we did not observe any obvious difference in the cytotoxicity of the four proteins. Therefore, the PMCA-generated PrP β-oligomers showed obvious cytotoxicity to primary cerebral cortex neurons (Fig.4A). In order to determine whether the cell death induced by the PrP oligomers was dependent on endogenous PrPC, cerebral cortex neurons from PrP-/- mice were treated as described for the wild-type cortex neurons, above. PrPC were not observed in PrP-/- samples as expected by western blot (data not shown). The PrP oligomers exhibited the same levels of toxicity to PrP-/- neurons as to the wild-type neurons, indicating that the cytotoxicity of PrP oligomers was not dependent on the expression of endogenous PrP in neurons (Fig.4B).
β-PrP oligomers induce cellular apoptosis by activating caspase-3 and increasing the Bcl-2/Bax ratio Previous studies demonstrated that PrPSc, PrP oligomers or synthetic peptides such as PrP106-126 could induce cellular apoptosis in mouse cerebral cortex neurons, as measured by levels of Bcl-2, Bax, and caspase-3 (Song et al. 2013, Kovacs & Budka 2008, Simoneau et al. 2007a, Pan et al. 2014). We treated primary cultured cortical neurons with PrP (100 μg/ml) or PMCA conversion buffer for 24h respectively. As shown in Figure 5A, cleaved caspase-3 was clearly detected in wild-type cells treated with each of the four PMCA-generated oligomers, whereas the controls (treated with substrate buffer or PrPC monomers) all showed inconspicuous levels of cleaved caspase-3. Western blot analysis showed significant increases in cleavage of caspase-3 compared with the control groups in PrP-/- neurons as well (Fig.5B).
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Apoptosis is regulated by families of pro-apoptotic factors (e.g., Bax) and anti-apoptotic factors (e.g., Bcl-2) (Malla et al. 2010, Mao et al. 2014, Song et al. 2013). We confirmed the cytotoxicity mechanism of β-PrP oligomers by testing the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax by western blot in cells that were treated with oligomeric PrP. The results showed a clear down-regulation of Bcl-2 and up-regulation of Bax in the oligomer-challenged groups compared with the control group (Fig.5C). Similar expression changes were observed in PrP-/- neurons (Fig.5D). Collectively, the detection of cleaved caspase-3 and Bcl-2/Bax levels on western blots showed that treatment with exogenous β-PrP oligomers can induce cellular apoptosis regardless of the presence of endogenous PrPC in neurons.
Discussion As research into the mechanisms underlying the development of TSEs has increased, it has become clear that PrPSc is not the only pathogenic agent in these prion diseases (Telling et al. 1996, Manuelidis et al. 1997, Couzin 2004). Several additional forms of PrP-derived peptides have been shown to be able to induce prion diseases in animals (Corsaro et al. 2012). Researchers also found that oligomeric PrP are highly neurotoxic in vitro, where they have been shown to be toxic to cultured neurons (Huang et al. 2010), and in vivo, in mouse models (Simoneau et al. 2007b). Oligomers, aggregates and polymeric amyloids from recombinant full-length PrP monomers have been generated in vitro under conditions of low pH, high temperature, strong denaturants, shaking, sonication and PMCA (Nandi et al. 2006, Do et al. 2014, Ladner-Keay et al. 2014, Yang et al. 2013). In our 2013 paper (Yang et al. 2013), we found that HaPrP dimers cannot be produced after 96 PMCA cycles in the absence of POPG. However, in the current study, we found HaPrP dimers can be completely formed after 144 PMCA cycles without POPG. So we speculated that POPG may simply accelerate the transformation, and the addition of extra cycles could compensate for the lack of POPG. However, it is not clear whether the mechanism of oligomer formation is different under the two conditions. This may be a subject for future study. In addition, detailed studies about This article is protected by copyright. All rights reserved.
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Article Type: Short Communication
Protein misfolding cyclic amplification (PMCA) induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis
Zhen Yuan1, Lifeng Yang1, Baian Chen2, Ting Zhu1, Mohammad Farooque Hassan1, Xiaomin Yin1, Xiangmei Zhou1, Deming Zhao*1 1 State Key Laboratories for Agrobiotechnology, Key Lab of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. 2 Department of Laboratory Animal Science, School of Basic Medical Science, Capital Medical University, Beijing, 100069, China.
Zhen Yuan and Lifeng Yang contributed to this work equally.
Correspondence to: Deming Zhao, China Agricultural University, Haidian District, Beijing, China. E-mail: [email protected]. Tel: +86-01062732975
Running title: PMCA induces PrP monomers to form oligomers
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jnc.13098 This article is protected by copyright. All rights reserved.
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increases the percentage of β-sheet relative to α-helix, as shown by circular dichroism spectroscopy indicating the formation of PrPSc-like oligomers. These oligomers also showed slight PK resistance relative to monomeric PrP, although they were less PK resistant than PrPSc. Collectively, our PMCA-generated PrP oligomers fall between PrP monomers and PrPSc in state of aggregation, altered secondary structure, and PK resistance. We showed that β- PrP oligomers of three different species, but not PrP monomers, were cytotoxic to mouse cortical neurons in culture, in accordance with previous studies (Yuan et al. 2013a, Huang et al. 2010, Simoneau et al. 2007a, Novitskaya et al. 2006, Kazlauskaite et al. 2005). Although it has been widely accepted that the absence of endogenous PrPC renders host animals resistant to the toxic effects of PrPSc (Raeberi et al. 1996, Biasini et al. 2012, Büeler et al. 1993), the dependency of PrP-induced toxicity on the presence of PrPC has remained a matter of debate among some prion scientists (Novitskaya et al. 2006, Simoneau et al. 2007a). Our findings showed that PrP oligomers were highly cytotoxic (to similar degrees) to both wild-type and PrP-/- mouse cortex neurons, thus demonstrating that, at least in our system, endogenous PrPC is not required for toxicity.
Previous studies have shown that prions can induce apoptosis, the important cellular pathway leading to neurodegeneration in prion diseases (Song et al. 2013). Apoptosis leads to an increased ratio of Bcl-2 to Bax protein (Martin et al. 2001, Oltval et al. 1993), indicating that cell death is occurring by this pathway, rather than through necrotic death. Our data showing down-regulation of Bcl-2 and up-regulation of Bax in response to PrP oligomer treatment, as well as activation of caspase-3, support the hypothesis that apoptosis is the relevant cell death pathway induced by PMCA-generated β-PrP oligomers. We, along with others, have shown that synthetic and recombinant prion protein fragments and oligomers produced in vitro are suitable models to study the molecular and cellular mechanisms of PrPSc causing neurodegeneration. We believe that the identification of the mechanisms driving PrPC conversion into infectious and/or neurotoxic entities may be helpful for identifying new potential pharmacological approaches for TSEs. In our study, we This article is protected by copyright. All rights reserved.
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showed that PrP oligomers could be formed from recombinant PrP in the absence of PrPSc by PMCA, that these oligomers are cytotoxic even in the absence of additional endogenous PrP, and that they are cytotoxic across species lines. This may also advance our understanding of the pathogenesis of prion diseases and help to delve into the molecular determinants for prion-like molecules, allowing the development of novel drugs to treat these diseases.
Acknowledgements We thank Dr. Charles Weissmann at the Scripps Research Institute for supplying the PrP-/knockout mice. This study was supported by 948 projects (2013-S11; 2014-S9) to BC. The authors have no conflicts of interest to declare.
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Kazlauskaite, J., Young, A., Gardner, C. E., Macpherson, J. V., Vénien-Bryan, C. and Pinheiro, T. J. (2005) An unusual soluble β-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells. Biochemical and biophysical research communications, 328, 292-305. Kovacs, G. G. and Budka, H. (2008) Prion diseases: from protein to cell pathology. The American journal of pathology, 172, 555-565. Ladner-Keay, C. L., Griffith, B. J. and Wishart, D. S. (2014) Shaking alone induces de novo conversion of recombinant prion proteins to beta-sheet rich oligomers and fibrils. PloS one, 9, e98753. Malla, R., Gopinath, S., Alapati, K., Gondi, C. S., Gujrati, M., Dinh, D. H., Mohanam, S. and Rao, J. S. (2010) Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas. PloS one, 5, e13731. Manuelidis, L., Fritch, W. and Xi, Y. G. (1997) Evolution of a strain of CJD that induces BSE-like plaques. Science, 277, 94-98. Mao, W., Yi, X., Qin, J., Tian, M. and Jin, G. (2014) CXCL12 inhibits cortical neuron apoptosis by increasing the ratio of Bcl-2/Bax after traumatic brain injury. International Journal of Neuroscience, 124, 281-290. Martin, S., Toquet, C., Oliver, L., Cartron, P.-F., Perrin, P., Meflah, K., Cuillère, P. and Vallette, F. M. (2001) Expression of bcl-2, bax and bcl-xl in human gliomas: a re-appraisal. Journal of neuro-oncology, 52, 129-139. Morales, R., Duran-Aniotz, C., Diaz-Espinoza, R., Camacho, M. V. and Soto, C. (2012) Protein misfolding cyclic amplification of infectious prions. Nature protocols, 7, 1397-1409. Nandi, P., Bera, A. and Sizaret, P.-Y. (2006) Osmolyte Trimethylamine< i> N-Oxide Converts Recombinant α-Helical Prion Protein to its Soluble β-Structured Form at High Temperature. Journal of molecular biology, 362, 810-820. Novitskaya, V., Bocharova, O. V., Bronstein, I. and Baskakov, I. V. (2006) Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons. Journal of Biological Chemistry, 281, 13828-13836. Oltval, Z. N., Milliman, C. L. and Korsmeyer, S. J. (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell, 74, 609-619. Pan, B., Yang, L., Wang, J., Wang, Y., Zhou, X., Yin, X., Zhang, Z. and Zhao, D. (2014) C-Abl tyrosine kinase mediates neurotoxic prion peptide-induced neuronal apoptosis via regulating mitochondrial homeostasis. Molecular neurobiology, 49, 1102-1116. Prusiner, S. B. (1998) Prions. Proceedings of the National Academy of Sciences, 95, 13363-13383.
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Prusiner, S. B. (2001) Neurodegenerative diseases and prions. New England Journal of Medicine, 344, 1516-1526. Qasim Khana, M., Sweetingb, B., Khipple Mulligana, V., Eli Arslanb, P. and Cashmanc, N. R. (2010) Prion disease susceptibility is affected by β-structure folding propensity and local side-chain interactions in PrP. PNAS, 107, 19808–19813. Raeberi, A., Fischert, M., Saileri, A., Kobayashit, Y. and Marino, S. (1996) Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature, 379, 25. Roucou, X. (2014) Regulation of PrP(C) signaling and processing by dimerization. Frontiers in cell and developmental biology, 2, 57. Simoneau, S., Rezaei, H., Salès, N. et al. (2007a) In vitro and in vivo neurotoxicity of prion protein oligomers. PLoS pathogens, 3, e125. Simoneau, S., Rezaei, H., Sales, N. et al. (2007b) In vitro and in vivo neurotoxicity of prion protein oligomers. PLoS pathogens, 3, e125. Song, Z., Zhao, D. and Yang, L. (2013) Molecular mechanisms of neurodegeneration mediated by dysfunctional subcellular organelles in transmissible spongiform encephalopathies. Acta biochimica et biophysica Sinica, gmt014. Telling, G. C., Haga, T., Torchia, M., Tremblay, P., DeArmond, S. J. and Prusiner, S. B. (1996) Interactions between wild-type and mutant prion proteins modulate neurodegeneration in transgenic mice. Genes & Development, 10, 1736-1750. Vorberg, I., Groschup, M. H., Pfaff, E. and Priola, S. A. (2003) Multiple Amino Acid Residues within the Rabbit Prion Protein Inhibit Formation of Its Abnormal Isoform. Journal of virology, 77, 2003-2009. Wang, F., Wang, X. and Ma, J. (2011a) Conversion of bacterially expressed recombinant prion protein. Methods, 53, 208-213. Wang, F., Wang, X. and Ma, J. (2011b) Conversion of bacterially expressed recombinant prion protein. Methods, 53, 208-213. Wang, F., Wang, X., Yuan, C.-G. and Ma, J. (2010) Generating a prion with bacterially expressed recombinant prion protein. Science, 327, 1132-1135. Wen, Y., Li, J., Xiong, M., Peng, Y., Yao, W., Hong, J. and Lin, D. (2010a) Solution structure and dynamics of the I214V mutant of the rabbit prion protein. PloS one, 5, e13273. Wen, Y., Li, J., Yao, W., Xiong, M., Hong, J., Peng, Y., Xiao, G. and Lin, D. (2010b) Unique structural characteristics of the rabbit prion protein. Journal of Biological Chemistry, 285, 31682-31693.
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Wen, Y., Li, J., Yao, W., Xiong, M., Hong, J., Peng, Y., Xiao, G. and Lin, D. (2010c) Unique structural characteristics of the rabbit prion protein. J Biol Chem, 285, 31682-31693. Yang, X., Yang, L., Zhou, X. et al. (2013) Using protein misfolding cyclic amplification generates a highly neurotoxic PrP dimer causing neurodegeneration. Journal of molecular neuroscience : MN, 51, 655-662. Yuan, Z., Zhao, D. and Yang, L. (2013a) Decipher the mechanisms of rabbit's low susceptibility to prion infection. Acta biochimica et biophysica Sinica, 45, 899-903. Yuan, Z., Zhao, D. and Yang, L. (2013b) Decipher the mechanisms of rabbit's low susceptibility to prion infection. Acta biochimica et biophysica Sinica, 45, 899-903. Zhang, Z., Zhang, Y., Wang, F., Wang, X., Xu, Y., Yang, H., Yu, G., Yuan, C. and Ma, J. (2013) De novo generation of infectious prions with bacterially expressed recombinant prion protein. The FASEB Journal, 27, 4768-4775. Zhu, T., Khan, S. H., Zhao, D. and Yang, L. (2014) Regulation of proteasomes in prion disease. Acta biochimica et biophysica Sinica, gmu031.
Figure legends Figure 1: PMCA induces PrP oligomer formation from four recombinant prion proteins. (A) A representative western blot, showing that HaPrP and HuPrP were completely converted to oligomers after 144 cycles, while 216 cycles were required for RaPrP. Conversion of MuRaPrP to oligomers was complete after 168 cycles. (B) Densitometry analysis by Image J software shows the percent of de novo oligomers after definite cycles (time course) of PMCA. (C) Western blot demonstrating that sonication is necessary for oligomer formation. These four PrP preparations remained in the monomeric state when treated as samples in (A) but in the absence of sonication. (D) The number of PMCA cycles required for complete dimerization is significantly different for these proteins. Such difference above was significantly similar in five repeating tests. Data are presented as mean± SD of quintuplicate experiments; ***P < 0.0001 versus HaPrP, HuPrP and MuRaPrP groups. Figure 2: PMCA induces the conversion of an α-helical monomeric protein to a ß-sheet rich oligomers. (A) Size-exclusion chromatography of PrP monomers and oligomeric PrP. The two forms of PrP can be clearly distinguished according to elution time of about 20.6 min for monomers and 8.5
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min for oligomers. (B) Circular dichroism spectrum analysis indicates that the oligomers adopt a ß-sheet-enriched conformation. Figure 3: PrP monomers and PrP oligomers showed different PK sensitivities when digested with 0, 0.1, 0.25, 0.5, 1 and 10 ug/ml of enzyme. The digestion products were analyzed by western blot with the monoclonal antibody 6D11. The monomeric PrPC was sensitive to PK digestion at lower concentrations while PrP oligomers exhibited partial protease resistance. There is no clear shift in the molecular weight of the PK treated protein. Figure 4: Cytotoxicity of Oligomeric PrP. (A) PrP oligomers were cytotoxic to primary cortical neurons. (B) The neurotoxicity of PrP oligomers is independent of endogenous PrP expression. Such difference above was significantly similar in three repeating tests. Data are presented as mean± SD of triplicate experiments; *P < 0.05 versus cells untreated group. Figure 5: Cleaved caspase-3, Bcl-2 and Bax expression in different groups. Primary cultured cortical neurons were treated with PrP (100 μg/ml) or PMCA conversion buffer for 24h respectively. Cleaved caspase-3 expression in wild-type (A) and PrP-/- (B) mouse cortical neurons was analyzed by western blot. Bcl-2 and Bax expression in wild-type (C) and PrP-/- (D) mouse cortical neurons was analyzed by western blot. All the blots were stripped and re-probed with β-actin antibody to verify equal loading. Similar results were observed in three replicate experiments. Bcl-2 antibody (sc-7382) and rabbit monoclonal Bax antibody (sc-493) were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Data are presented as mean of triplicate densitometric analysis by Image J software; **P < 0.01 versus cells untreated group.
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Article Type: Short Communication
Protein misfolding cyclic amplification (PMCA) induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis
Zhen Yuan1, Lifeng Yang1, Baian Chen2, Ting Zhu1, Mohammad Farooque Hassan1, Xiaomin Yin1, Xiangmei Zhou1, Deming Zhao*1 1 State Key Laboratories for Agrobiotechnology, Key Lab of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. 2 Department of Laboratory Animal Science, School of Basic Medical Science, Capital Medical University, Beijing, 100069, China.
Zhen Yuan and Lifeng Yang contributed to this work equally.
Correspondence to: Deming Zhao, China Agricultural University, Haidian District, Beijing, China. E-mail: [email protected]. Tel: +86-01062732975
Running title: PMCA induces PrP monomers to form oligomers
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jnc.13098 This article is protected by copyright. All rights reserved.
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Keywords: Prion protein; oligomers; protein misfolding cyclic amplification; cytotoxic; neuronal apoptosis
Abbreviations used: PMCA, protein misfolding cyclic amplification; PrP, prion protein; PrPC, cellular form of the prion protein; TSEs, transmissible spongiform encephalopathies; HaPrP, hamster prion protein; HuPrP, human prion protein; RaPrP, rabbit prion protein; MuRaPrP, mutant rabbit prion protein.
Abstract: The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β-state oligomers. Herein, we demonstrate that β-state oligomeric PrP can be generated through protein misfolding cyclic amplification (PMCA) from recombinant full-length hamster, human, rabbit and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP-induced neurotoxicity. We have characterized PMCA-induced monomer-to-oligomer conversion of PrP from three species using western blotting, circular dichroism, size exclusion chromatography, and resistance to proteinase K digestion. We have further shown that all of the resulting β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrPC in the neurons, while the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3 in both wild-type and PrP-/- cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain. Introduction: According to the “protein-only” hypothesis, the infectious PrPSc agent consists solely of a misfolded form of the host-encoded cellular prion protein (PrPC) (Prusiner 1998, Prusiner This article is protected by copyright. All rights reserved.
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showed that PrP oligomers could be formed from recombinant PrP in the absence of PrPSc by PMCA, that these oligomers are cytotoxic even in the absence of additional endogenous PrP, and that they are cytotoxic across species lines. This may also advance our understanding of the pathogenesis of prion diseases and help to delve into the molecular determinants for prion-like molecules, allowing the development of novel drugs to treat these diseases.
Acknowledgements We thank Dr. Charles Weissmann at the Scripps Research Institute for supplying the PrP-/knockout mice. This study was supported by 948 projects (2013-S11; 2014-S9) to BC. The authors have no conflicts of interest to declare.
References Anderson, M., Bocharova, O. V., Makarava, N., Breydo, L., Salnikov, V. V. and Baskakov, I. V. (2006) Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy. Journal of molecular biology, 358, 580-596. Büeler, H., Aguzzi, A., Sailer, A., Greiner, R.-A., Autenried, P., Aguet, M. and Weissmann, C. (1993) Mice devoid of PrP are resistant to scrapie. Cell, 73, 1339-1347. Biasini, E., Turnbaugh, J. A., Unterberger, U. and Harris, D. A. (2012) Prion protein at the crossroads of physiology and disease. Trends in neurosciences, 35, 92-103. Bocharova, O. V., Breydo, L., Parfenov, A. S., Salnikov, V. V. and Baskakov, I. V. (2005) In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP Sc. Journal of molecular biology, 346, 645-659. Chianini, F., Fernández-Borges, N., Vidal, E. et al. (2012) Rabbits are not resistant to prion infection. Proceedings of the National Academy of Sciences, 109, 5080-5085. Corsaro, A., Thellung, S., Villa, V., Nizzari, M. and Florio, T. (2012) Role of Prion Protein Aggregation in Neurotoxicity. International journal of molecular sciences, 13, 8648-8669. Couzin, J. (2004) An end to the prion debate? Don't count on it. Science, 305, 589-589.
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Animal Information and Ethics Statement Wild-type C57BL/6J suckling mice with no genomic modifications were acquired from Charles River Laboratories (USA). PrP-/- knock-out mice on a C57BL/6J×129Sv genetic background were supplied from Dr. Charles Weissmann in the Scripps Research Institute. Wild-type and PrP-/- mice were verified by western blotting (Fig. S1). Wild-type mice and PrP-/- knock-out mice used in this study were postnatal suckling female mice less than 24 hours old. All animal experiments were conducted according to the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the Beijing Municipality Administration Office of Laboratory Animals (BAOLA).
Expression and Purification of Recombinant Prion Proteins Full-length hamster, human and rabbit recombinant PrP (23-230) were used in this study. Plasmids for expression of PrP were created from hamster, human or rabbit cDNA as described previously (Bocharova et al. 2005, Anderson et al. 2006, Wang et al. 2011b). The cDNAs were cloned into the Escherichia coli expression vector pPROEX-HTb (Invitrogen, USA). Purification of the recombinant PrP and cleavage of the 6-histidine tags were performed according to previously published procedures(Wang et al. 2011a). The mutant RaPrP (23–230) was constructed using site-directed mutagenesis PCR (Wen et al. 2010c, Hosszu et al. 2010). Purified PrP in PBS was kept at −80°C. The purity of recombinant protein was monitored by coomassie brilliant blue staining on SDS-PAGE gel. The concentration of purified PrP was calculated using OD280 reading.
In Vitro Generation of PrP Oligomers Preparation of PMCA Substrate Purified monomeric PrP was thawed and centrifuged at 100,000 X g for 1 h at 4°C. The supernatant was diluted with sterile deionized water to 0.50 mg/ml. In a 15 ml centrifuge This article is protected by copyright. All rights reserved.
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tube, 180 μl of diluted PrP was mixed with 500 μl of 5% Triton X-100, 900 μl of 10 × TN buffer (100mM Tris-HCl, pH 7.5, 1.5M NaCl) and 7420 μl of nuclease-free H2O. The mixture was mixed thoroughly and incubated at room temperature for 10 min. The prepared PMCA substrate was distributed into PCR tubes (90 μl per tube) and stored at −80°C until sonication. POPG was not essential in the generation of PrP oligomers (Fig.S1).
PMCA Instrument Setup A sonicator with a microplate horn (model S-4000, Misonix, Inc.) was fitted with a homemade PCR-tube rack and used for all PMCA reactions. Reactions were carried out in 200 μl PCR tubes; tubes were placed in the rack and partially submerged in sterile 37.0°C water (to 3 mm above the horn surface). The sonication cycle consisted of 30 s of sonication at an amplitude of 80, followed by a 29.5-min incubation at 37.0°C. The water was changed after every 24 cycles.
Western Blot Protein samples were loaded onto a 12% SDS-PAGE gel. After electrophoresis, the SDS-PAGE gel was soaked in transfer buffer for 10 min. Meanwhile, 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA) was soaked sequentially in pure methanol for 60 seconds, in deionized water for 2 min, and in transfer buffer for 10 min. Transfer was carried out with cooling for 4 h with a constant voltage of 80 volts. After transfer, the PVDF membrane was blocked with 5% nonfat milk (CWBIO) for 1 h at 37°C. The membrane was incubated at room temperature with primary antibody in TBST with shaking at 4℃ overnight, washed three times with TBST (10 min each), incubated with secondary antibody and washed 5 times with TBST (10 min each). The signal was detected by exposure to chemiluminescence (Versadoc; Bio-Rad, Hercules, CA, USA).
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Statistics All assays were performed on three separate occasions. Data were expressed as means ± SD of three experiments. All comparisons for parametric data were made using Student’s t-test or one-way ANOVA followed by post hoc Tukey’s test using the SPSS software (version 13.0: SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 software (La Jolla, CA, USA). P < 0.05 was considered statistically significant.
Results: PMCA induces recombinant PrPC to form β-sheet rich oligomers Western blots were initially used to determine whether the PMCA treatment generated PrP oligomers. For all forms of PrP, PMCA treatment produced an oligomer with an apparent molecular weight of twice that of the monomeric PrP, evident on western blots (Fig.1A). A similar change in molecular weight was observed in three replicate experiments for each PrP. To further verify that the PMCA treatment was responsible for the formation of the observed oligomers, we also ran several controls that were identical to the test samples in all ways except that they were not sonicated; oligomers were not formed in these samples (Fig.1C). In addition, we found that POPG is not essential to the formation of oligomers (Fig. S2). Interestingly, 144 cycles of PMCA were sufficient for HaPrP and HuPrP to achieve complete conversion to the oligomeric form (over 99% of the total protein), while 216 cycles were required for RaPrP (Fig.1B). We proposed that this difference in the rate of oligomer formation may be related to differences in structural stability of the monomers. The proteins differ at several key amino acids which are thought to contribute to their structural stability (Yuan et al. 2013a, Julien et al. 2011, Fernandez-Funez et al. 2011a, Wen et al. 2010b). The relative stability of the PrP monomers of these three species is: RaPrP exhibit stronger stability than HuPrP and HaPrP. Therefore we created a mutant rabbit PrP (MuRaPrP) model in which certain stability-inducing amino acids in the RaPrP were replaced with the amino acids found at the same position in the HaPrP. The MuRaPrP contained the following This article is protected by copyright. All rights reserved.
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substituions: Ser107 to Asn, Ser173 to Asn, Gln219 to Lys, and Ala224 to Tyr. When MuRaPrP was subjected to PMCA, MuRaPrP monomers were fully converted to oligomers after 168 cycles (Fig.1D).
Size-exclusion chromatography is a simple and direct assay to determine whether PrP exist as monomers or oligomers(Huang et al. 2010, Simoneau et al. 2007a). The PrP oligomers generated by PMCA were clearly distinguished from the monomers based on elution time; the elution time for monomers was about 20.6 min, and for oligomers, about 8.5 min (Fig.2A). Circular dichroism spectroscopy was used to further analyze the oligomers that were formed by PMCA. The results showed that the oligomers possess a β-sheet structure (Fig.2B) more extensive than that found in the corresponding monomers. Secondary structure content of these PMCA-induced oligomers was found to be about 29% β-sheet and 18% α-helix. In contrast, the four monomeric recombinant prion proteins contained nearly 41% α-helix and only 13% β-sheet.
Oligomeric PrP showed partial resistance to proteinase K digestion We subjected our PMCA-induced oligomeric PrP to PK digestion, in parallel with their monomeric forms. As shown in Figure 3, the β-oligomeric PrP oligomers each exhibited partial resistance to PK digestion compared to their monomeric counterparts. At PK concentrations of 0.1 and 0.25 μg/ml, both types of PrP showed resistance to PK digestion. However, at PK concentrations of 0.5 and 1 μg/ml, oligomeric PrP showed an elevated PK resistance, whereas monomeric PrP was completely digested. At the highest PK concentration of 10 μg/ml, both monomers and oligomers were digested completely (Fig.3). There was no clear shift in the molecular weight of the PK treated protein. This result was further confirmed by using PrP antibody AH6 (Fig.S3).
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β-PrP oligomers are cytotoxic to primary cortical neurons from wild-type and PrP-/- Mice We first analyzed the cytotoxic properties of the PrP oligomers in primary mouse cerebral cortex neurons isolated from wild-type animals. For each of the oligomers, exposure of primary neurons to a dose of 100 μg/ml PrP resulted in a loss of nearly 30% of the neurons compared to the control cells treated with monomers or with buffer alone. No obvious loss of cells occurred at either 25 or 50 μg/ml. Furthermore, we did not observe any obvious difference in the cytotoxicity of the four proteins. Therefore, the PMCA-generated PrP β-oligomers showed obvious cytotoxicity to primary cerebral cortex neurons (Fig.4A). In order to determine whether the cell death induced by the PrP oligomers was dependent on endogenous PrPC, cerebral cortex neurons from PrP-/- mice were treated as described for the wild-type cortex neurons, above. PrPC were not observed in PrP-/- samples as expected by western blot (data not shown). The PrP oligomers exhibited the same levels of toxicity to PrP-/- neurons as to the wild-type neurons, indicating that the cytotoxicity of PrP oligomers was not dependent on the expression of endogenous PrP in neurons (Fig.4B).
β-PrP oligomers induce cellular apoptosis by activating caspase-3 and increasing the Bcl-2/Bax ratio Previous studies demonstrated that PrPSc, PrP oligomers or synthetic peptides such as PrP106-126 could induce cellular apoptosis in mouse cerebral cortex neurons, as measured by levels of Bcl-2, Bax, and caspase-3 (Song et al. 2013, Kovacs & Budka 2008, Simoneau et al. 2007a, Pan et al. 2014). We treated primary cultured cortical neurons with PrP (100 μg/ml) or PMCA conversion buffer for 24h respectively. As shown in Figure 5A, cleaved caspase-3 was clearly detected in wild-type cells treated with each of the four PMCA-generated oligomers, whereas the controls (treated with substrate buffer or PrPC monomers) all showed inconspicuous levels of cleaved caspase-3. Western blot analysis showed significant increases in cleavage of caspase-3 compared with the control groups in PrP-/- neurons as well (Fig.5B).
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Apoptosis is regulated by families of pro-apoptotic factors (e.g., Bax) and anti-apoptotic factors (e.g., Bcl-2) (Malla et al. 2010, Mao et al. 2014, Song et al. 2013). We confirmed the cytotoxicity mechanism of β-PrP oligomers by testing the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax by western blot in cells that were treated with oligomeric PrP. The results showed a clear down-regulation of Bcl-2 and up-regulation of Bax in the oligomer-challenged groups compared with the control group (Fig.5C). Similar expression changes were observed in PrP-/- neurons (Fig.5D). Collectively, the detection of cleaved caspase-3 and Bcl-2/Bax levels on western blots showed that treatment with exogenous β-PrP oligomers can induce cellular apoptosis regardless of the presence of endogenous PrPC in neurons.
Discussion As research into the mechanisms underlying the development of TSEs has increased, it has become clear that PrPSc is not the only pathogenic agent in these prion diseases (Telling et al. 1996, Manuelidis et al. 1997, Couzin 2004). Several additional forms of PrP-derived peptides have been shown to be able to induce prion diseases in animals (Corsaro et al. 2012). Researchers also found that oligomeric PrP are highly neurotoxic in vitro, where they have been shown to be toxic to cultured neurons (Huang et al. 2010), and in vivo, in mouse models (Simoneau et al. 2007b). Oligomers, aggregates and polymeric amyloids from recombinant full-length PrP monomers have been generated in vitro under conditions of low pH, high temperature, strong denaturants, shaking, sonication and PMCA (Nandi et al. 2006, Do et al. 2014, Ladner-Keay et al. 2014, Yang et al. 2013). In our 2013 paper (Yang et al. 2013), we found that HaPrP dimers cannot be produced after 96 PMCA cycles in the absence of POPG. However, in the current study, we found HaPrP dimers can be completely formed after 144 PMCA cycles without POPG. So we speculated that POPG may simply accelerate the transformation, and the addition of extra cycles could compensate for the lack of POPG. However, it is not clear whether the mechanism of oligomer formation is different under the two conditions. This may be a subject for future study. In addition, detailed studies about This article is protected by copyright. All rights reserved.
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Article Type: Short Communication
Protein misfolding cyclic amplification (PMCA) induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis
Zhen Yuan1, Lifeng Yang1, Baian Chen2, Ting Zhu1, Mohammad Farooque Hassan1, Xiaomin Yin1, Xiangmei Zhou1, Deming Zhao*1 1 State Key Laboratories for Agrobiotechnology, Key Lab of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. 2 Department of Laboratory Animal Science, School of Basic Medical Science, Capital Medical University, Beijing, 100069, China.
Zhen Yuan and Lifeng Yang contributed to this work equally.
Correspondence to: Deming Zhao, China Agricultural University, Haidian District, Beijing, China. E-mail: [email protected]. Tel: +86-01062732975
Running title: PMCA induces PrP monomers to form oligomers
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jnc.13098 This article is protected by copyright. All rights reserved.
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increases the percentage of β-sheet relative to α-helix, as shown by circular dichroism spectroscopy indicating the formation of PrPSc-like oligomers. These oligomers also showed slight PK resistance relative to monomeric PrP, although they were less PK resistant than PrPSc. Collectively, our PMCA-generated PrP oligomers fall between PrP monomers and PrPSc in state of aggregation, altered secondary structure, and PK resistance. We showed that β- PrP oligomers of three different species, but not PrP monomers, were cytotoxic to mouse cortical neurons in culture, in accordance with previous studies (Yuan et al. 2013a, Huang et al. 2010, Simoneau et al. 2007a, Novitskaya et al. 2006, Kazlauskaite et al. 2005). Although it has been widely accepted that the absence of endogenous PrPC renders host animals resistant to the toxic effects of PrPSc (Raeberi et al. 1996, Biasini et al. 2012, Büeler et al. 1993), the dependency of PrP-induced toxicity on the presence of PrPC has remained a matter of debate among some prion scientists (Novitskaya et al. 2006, Simoneau et al. 2007a). Our findings showed that PrP oligomers were highly cytotoxic (to similar degrees) to both wild-type and PrP-/- mouse cortex neurons, thus demonstrating that, at least in our system, endogenous PrPC is not required for toxicity.
Previous studies have shown that prions can induce apoptosis, the important cellular pathway leading to neurodegeneration in prion diseases (Song et al. 2013). Apoptosis leads to an increased ratio of Bcl-2 to Bax protein (Martin et al. 2001, Oltval et al. 1993), indicating that cell death is occurring by this pathway, rather than through necrotic death. Our data showing down-regulation of Bcl-2 and up-regulation of Bax in response to PrP oligomer treatment, as well as activation of caspase-3, support the hypothesis that apoptosis is the relevant cell death pathway induced by PMCA-generated β-PrP oligomers. We, along with others, have shown that synthetic and recombinant prion protein fragments and oligomers produced in vitro are suitable models to study the molecular and cellular mechanisms of PrPSc causing neurodegeneration. We believe that the identification of the mechanisms driving PrPC conversion into infectious and/or neurotoxic entities may be helpful for identifying new potential pharmacological approaches for TSEs. In our study, we This article is protected by copyright. All rights reserved.
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showed that PrP oligomers could be formed from recombinant PrP in the absence of PrPSc by PMCA, that these oligomers are cytotoxic even in the absence of additional endogenous PrP, and that they are cytotoxic across species lines. This may also advance our understanding of the pathogenesis of prion diseases and help to delve into the molecular determinants for prion-like molecules, allowing the development of novel drugs to treat these diseases.
Acknowledgements We thank Dr. Charles Weissmann at the Scripps Research Institute for supplying the PrP-/knockout mice. This study was supported by 948 projects (2013-S11; 2014-S9) to BC. The authors have no conflicts of interest to declare.
References Anderson, M., Bocharova, O. V., Makarava, N., Breydo, L., Salnikov, V. V. and Baskakov, I. V. (2006) Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy. Journal of molecular biology, 358, 580-596. Büeler, H., Aguzzi, A., Sailer, A., Greiner, R.-A., Autenried, P., Aguet, M. and Weissmann, C. (1993) Mice devoid of PrP are resistant to scrapie. Cell, 73, 1339-1347. Biasini, E., Turnbaugh, J. A., Unterberger, U. and Harris, D. A. (2012) Prion protein at the crossroads of physiology and disease. Trends in neurosciences, 35, 92-103. Bocharova, O. V., Breydo, L., Parfenov, A. S., Salnikov, V. V. and Baskakov, I. V. (2005) In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP Sc. Journal of molecular biology, 346, 645-659. Chianini, F., Fernández-Borges, N., Vidal, E. et al. (2012) Rabbits are not resistant to prion infection. Proceedings of the National Academy of Sciences, 109, 5080-5085. Corsaro, A., Thellung, S., Villa, V., Nizzari, M. and Florio, T. (2012) Role of Prion Protein Aggregation in Neurotoxicity. International journal of molecular sciences, 13, 8648-8669. Couzin, J. (2004) An end to the prion debate? Don't count on it. Science, 305, 589-589.
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Figure legends Figure 1: PMCA induces PrP oligomer formation from four recombinant prion proteins. (A) A representative western blot, showing that HaPrP and HuPrP were completely converted to oligomers after 144 cycles, while 216 cycles were required for RaPrP. Conversion of MuRaPrP to oligomers was complete after 168 cycles. (B) Densitometry analysis by Image J software shows the percent of de novo oligomers after definite cycles (time course) of PMCA. (C) Western blot demonstrating that sonication is necessary for oligomer formation. These four PrP preparations remained in the monomeric state when treated as samples in (A) but in the absence of sonication. (D) The number of PMCA cycles required for complete dimerization is significantly different for these proteins. Such difference above was significantly similar in five repeating tests. Data are presented as mean± SD of quintuplicate experiments; ***P < 0.0001 versus HaPrP, HuPrP and MuRaPrP groups. Figure 2: PMCA induces the conversion of an α-helical monomeric protein to a ß-sheet rich oligomers. (A) Size-exclusion chromatography of PrP monomers and oligomeric PrP. The two forms of PrP can be clearly distinguished according to elution time of about 20.6 min for monomers and 8.5
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min for oligomers. (B) Circular dichroism spectrum analysis indicates that the oligomers adopt a ß-sheet-enriched conformation. Figure 3: PrP monomers and PrP oligomers showed different PK sensitivities when digested with 0, 0.1, 0.25, 0.5, 1 and 10 ug/ml of enzyme. The digestion products were analyzed by western blot with the monoclonal antibody 6D11. The monomeric PrPC was sensitive to PK digestion at lower concentrations while PrP oligomers exhibited partial protease resistance. There is no clear shift in the molecular weight of the PK treated protein. Figure 4: Cytotoxicity of Oligomeric PrP. (A) PrP oligomers were cytotoxic to primary cortical neurons. (B) The neurotoxicity of PrP oligomers is independent of endogenous PrP expression. Such difference above was significantly similar in three repeating tests. Data are presented as mean± SD of triplicate experiments; *P < 0.05 versus cells untreated group. Figure 5: Cleaved caspase-3, Bcl-2 and Bax expression in different groups. Primary cultured cortical neurons were treated with PrP (100 μg/ml) or PMCA conversion buffer for 24h respectively. Cleaved caspase-3 expression in wild-type (A) and PrP-/- (B) mouse cortical neurons was analyzed by western blot. Bcl-2 and Bax expression in wild-type (C) and PrP-/- (D) mouse cortical neurons was analyzed by western blot. All the blots were stripped and re-probed with β-actin antibody to verify equal loading. Similar results were observed in three replicate experiments. Bcl-2 antibody (sc-7382) and rabbit monoclonal Bax antibody (sc-493) were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Data are presented as mean of triplicate densitometric analysis by Image J software; **P < 0.01 versus cells untreated group.
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