J Mol Neurosci DOI 10.1007/s12031-015-0546-1
The Unexposed Secrets of Prion Protein Oligomers Gailing Wang & Mingcheng Wang & Chuanfeng Li
Received: 15 January 2015 / Accepted: 4 March 2015 # Springer Science+Business Media New York 2015
Abstract According to the Bprotein-only^ hypothesis, the misfolding and conversion of host-derived cellular prion protein (PrPC) into pathogenically misfolded PrP are believed to be the key procedure in the pathogenesis of prion diseases. Intermediate, soluble oligomeric prion protein (PrP) aggregates were considered a critical process for prion diseases. Several independent studies on PrP oligomers gained insights into oligomers’ formation, biophysical and biochemical characteristics, structure conversion, and neurotoxicity. PrP oligomers are rich in β-sheet structure and slightly resistant to proteinase K digestion. PrP oligomers exhibited more neurotoxicity and induced neuronal apoptosis in vivo and/or in vitro. In this review, we summarized recent studies regarding PrP oligomers and the relationship between misfolded PrP aggregates and neuronal death in the course of prion diseases. Keywords Prion diseases . Prion protein . Oligomers . Neurotoxicity
Introduction Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are fatal neurodegenerative diseases of humans and animals, including Creutzfeldt–Jakob disease, Gerstmann–Sträussler–Scheinker syndrome, kuru and fatal familial insomnia in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk (Prusiner 1982; Prusiner 1998; Yuan et al. G. Wang (*) : M. Wang : C. Li Department of Bioengineering, Huanghuai University, 463000 Zhumadian, China e-mail: [email protected]
2013b). The agent that causes TSEs was termed by Stanley B. Prusiner as Bprion^ and is defined as Ba small proteinaceous infectious particle^ (Prusiner 1982). According to Bproteinonly^ hypothesis, the central event in prion pathogenesis is the conformational conversion of normal soluble cellular prion protein (PrPC) into misfolded insoluble and partially protease-resistant isoform (PrPSc) (Kim et al. 2010; Wang et al. 2010). However, to date, no investigation has yielded direct experimental proof for this stringent hypothesis. Indeed, neuronal death can occur without detectable PrPSc during prion infection, and there is no detectable PrPSc in some cases of prion-infected diseases (Gambetti et al. 1995; Lasmézas et al. 1997; Manson et al. 1999; Manuelidis et al. 1997). Until now, the exact pathological mechanism of the prion diseases is still unclear. In neurodegenerative diseases, such as Alzheimer’s, Huntington, Parkinson, and prion diseases, the deposition of misfolded aggregated proteins is believed to be responsible for the neurotoxicity that characterizes these diseases (Bucciantini et al. 2004; Laganowsky et al. 2012; Nicoll et al. 2013; Tomic et al. 2009; Walsh and Teplow 2012). The accumulations of abnormal PrP forms usually colocalize with prion neuropathology (Caughey and Lansbury 2003a). Prion protein has been deeply studied for the peculiar feature of its misfolded oligomers, inducing the conversion of the natively folded PrP into the pathological conformation. Subsequent researchers reported that monomeric PrPC could be transformed into various forms of oligomers that showed neurotoxicity in vitro or in vivo (Huang et al. 2010; Ladner-Keay et al. 2014; Malchiodi-Albedi et al. 2011; Simoneau et al. 2007a; Swietnicki et al. 2000). Intermediate, soluble oligomeric PrP aggregates were soon considered a critical process for prion diseases. Several independent studies on PrP oligomers gained insights into oligomers’ formation, biophysical and biochemical characteristics, structure conversion, and neurotoxicity
J Mol Neurosci
mechanism. In this review, we summarized studies on PrP oligomers and the relationship between misfolded PrP aggregates and neuronal death in the course of prion diseases.
Formation of PrP Oligomers PrPC dimers were detected in neuroblastoma N2a cells expressing Syrian hamster PrPC (Priola et al. 1995) and in a solution in a partially purified fraction from normal bovine thalamus (Meyer et al. 2000). Endogenous PrPC dimers were also detected in N2a cells by Rambold et al. Moreover, PrPC dimers were detected in crude membranes from human neuroblastoma SH-SY5Y cells and mouse brains (Rambold et al. 2008). These results convincingly demonstrated that PrPC has an intrinsic tendency to dimerize in native conditions and provided the in vivo evidence of PrP polymerization (Fig. 1). However, many recent studies were conducted to clarify the formation of PrP oligomers from recombinant PrP monomers in different conditions in vitro. These conditions include denaturants (guanidine hydrochloride or urea), high salts, and detergents (Baskakov et al. 2002; Morillas et al. 2001; Swietnicki et al. 2000). The methods including sizeexclusion chromatography (SEC), ultracentrifugation, dynamic light scattering (DLS), electron microscopy (EM), atomic force microscopy (AFM), immunochemical detection, or chemical cross-linking are required for accurate determination of oligomerization state (Bitan et al. 2005). In 2007, Steve Simoneau et al. (Simoneau et al. 2007a) generated dimeric ovine PrP oligomers by thermal refolding. For conversion, recombinant PrP (23–234) (pH 7.2 in 20 mM MOPS) was heated at 72 °C for 15 min and cooled at room temperature. SEC and Western blotting of the PrP preparations showed that they were exclusively in an oligomeric state. They also established that aging of PrP oligomers (extension of time to 1 month at 48 °C) makes oligomers polymerize into PrP fibrils. In 2010, Liqin Huang et al. (Huang et al. 2010) used 200 g/ L dextran 70 to mimic intracellular crowding condition to investigate its potential effects on recombinant human PrP. The oligomeric PrP sample was obtained by adding 0.4 mg/ mL PrP to crowding buffer. Light scattering, turbidity, and SEC confirmed the aggregation of the PrP samples.
In 2011, Zheng Zhou et al. (Zhou et al. 2011) reported that the crowding agents Ficoll 70 and dextran 70 have different effects on fibrillization of the recombinant PrPs from human, cow, and rabbit. These crowing agents dramatically promoted fibril formation of human and bovine PrP; they significantly inhibit fibrillization of the rabbit PrP. These results demonstrated that the sequence of PrP from different animals may affect both the kinetics and the inhibitory effect of crowding agents on rabbit PrP fibrillization. In 2013, Xiujin Yang et al. (Yuan et al. 2013a) generated dimeric hamster PrP oligomers by protein misfolding cyclic amplification (PMCA) consisting of recombinant hamster PrP (23–231). One cycle in PMCA was performed by sonication at amplitude of 80 for 30 s, followed by 29.5 min incubation at 37 °C. After 96 cycles, hamster PrP oligomers emerged. The assay for PMCA-induced oligomers was further supported by Carol L. Ladner-Keay et al. (Ladner-Keay et al. 2014). In 2014, Carol L. Ladner-Keay et al. (Ladner-Keay et al. 2014) found that de novo conversion of recombinant prion proteins to oligomers could be induced by shaking alone. The formation of oligomers occurred by shaking recombinant hamster PrP (90–232) and mouse PrP (90–231) at 37 °C and 350 rpm, in buffer at pH 5.5 for at least 1 day. The size of these PrP oligomers ranged from octamers to dodecamers as determined by RENAGE (Ladner and Wishart 2012) and EM. Essentially, the PrP fibrils determined by EM can be formed by shaking at 350 rpm at 37 °C for 5 days.
Structures of PrP Oligomers Prion diseases are caused by misfolding of α-helix-rich PrPC into the β-sheet-rich aggregated PrPSc. Early studies including low-resolution, crystallographic, and structural nuclear magnetic resonance spectroscopy indicated that PrPC had nearly 40 % α-helix and 3 % β-sheet (Hornemann et al. 1997; Knowles et al. 2007; Pan et al. 1993; Riek et al. 1996). In contrast to PrPC, the α-helix content of PrPSc comprises 30 % of the protein, whereas β-sheet comprises 40 % of the protein as measured by circular dichroism (CD) spectroscopy (Safar et al. 1993) and Fourier transform infrared spectroscopy (FTIR) (Pan et al. 1993). Studies found that oligomeric PrP derived from recombinant monomeric PrP has an increased
Fig. 1 Pathological processes of misfolded PrP aggregation. PrP fibril formation from native state is a polymerization process consisting of monomeric misfolding, protein oligomerization, and fibril elongation
J Mol Neurosci
percentage of β-sheet structure. Carol L. Ladner-Keay et al. found that CD spectroscopy analysis for recombinant PrPC gave 43 % α-helix and 10 % β-sheet and for PrP oligomers 16 % α-helix and 24 % β-sheet. The CD result was further confirmed by FTIR that shaking-induced oligomers adopted a β-sheet-enriched conformation. Essentially, oligomers can develop into fibrils under certain conditions in prion conversion (Caughey and Lansbury 2003b; Ladner-Keay et al. 2014; Simoneau et al. 2007a; Zhou et al. 2011). Zheng Zhou et al. used FTIR and CD to compare the secondary structures of rabbit/human/bovine PrP fibrils and found that rabbit PrP fibrils contained less β-sheet structure and more α-helix structure than human and bovine PrP fibrils.
Characterization of PrP Oligomers Previous studies showed that thioflavin T (ThT), a common dye, can bind to amyloid aggregations and also be applied to determine the oligomeric PrP (Huang et al. 2010; Nandi et al. 2006; Simoneau et al. 2007a). Steve Simoneau et al. treated various kinds of PrP with ThT to compare their interaction with ThT. PrP oligomers showed slightly enhanced ThT binding. This finding was further confirmed by Liqin Huang et al. ANS, a polarity-sensitive fluorescent probe, was usually used to compare various hydrophobic surfaces of soluble monomers and oligomers. Liqin Huang et al. found that ANS fluorescence intensity increased by nearly 3.5-fold at 473 nm compared with PrPC, further indicating that oligomers have an increase in hydrophobic surfaces than monomers (Huang et al. 2010). This result is consistent with previous findings that PrP oligomers have a larger hydrophobic surface than monomeric PrP (Nandi et al. 2006; Rezaei et al. 2005). Naturally existing infectious prions, as well as many in vitro generated PrPSc-like forms or oligomers, are known to show resistance to proteinase K digestion (Bocharova et al. 2005; Weissmann 2004). PrPC is a proteinase K-sensitive monomer, whereas PrPSc is resistant to proteinase K digestion (Song et al. 2013a; Yao et al. 2013; Zhu et al. 2014). Proteinase K resistance, in fact, has been considered to be a hallmark for the presence of PrPSc (Ladner-Keay et al. 2014). Recently, many studies found that the formation of the soluble PrP oligomers was accompanied by a substantial increase in resistance to proteinase K digestion. For instance, Liqin Huang et al. subjected oligomeric PrPs to a range of proteinase K concentrations in parallel with their monomeric forms. The PrP monomer was sensitive to proteinase K digestion. In contrast, PrP oligomers exhibited partial protease resistance and displayed increased resistance to degradation by proteinase K. In addition, there were several bands approximately 12∼ 14 kDa corresponding to proteinase K (PK)-resistant fragments. These observations were consistent with other studies
that PrP oligomers are partially resistant to PK digestion (Kaimann et al. 2008; Ladner-Keay et al. 2014; Nandi et al. 2006; Simoneau et al. 2007a).
Neurotoxicity of PrP Oligomers In Vitro and In Vivo Recent studies suggested that the PrP oligomers are neurotoxic (Huang et al. 2010; Kazlauskaite et al. 2005; Ladner-Keay et al. 2014; Novitskaya et al. 2006; Simoneau et al. 2007a). Steve Simoneau et al. first analyzed the cytotoxic properties of the PrP oligomers in primary mouse cerebral cortex neurons isolated from wild-type animals. They exhibited a roughly approximately 30-fold higher toxic effect than PrP peptide 105–132. To investigate the toxicity of PrP oligomers in vivo, stereotaxic subcortical injections of oligomeric PrP were carried out in the right hemispheres of mice. Gallocyanine staining of brain sections was used to detect the toxicity. The result that the pyramidal layer of neurons underneath the injection site was completely destructed showed that PrP oligomers were highly toxic in wild-type and PrPC knockout mice. The neurotoxicity of PrP oligomers was further confirmed by subsequent studies. Liqin Huang et al. found that the soluble oligomers formed in macromolecular crowding were neurotoxic to the human neuroblastoma SK-N-SH and SH-SY5Y cell lines. Xiujin Yang et al. showed that PMCA-induced hamster PrP oligomers were neurotoxic to mouse neuroblastoma N2a cells. Although it has been widely accepted that the absence of endogenous PrPC renders host animal resistant to the toxic effects of PrPSc (Büeler et al. 1993; Biasini et al. 2012; Raeberi et al. 1996), the dependency of PrP-induced toxicity on the presence of PrPC has remained a matter of debate among some prion scientists (Song et al. 2013b). One study showed that the toxicity of PrP oligomers in vitro was dependent on the expression of PrPC (Novitskaya et al. 2006), and this study was supported by Kudo et al. that PrPC is essentially involved in the oligomeric amyloid-β-induced neuronal cell death. However, Steve Simoneau et al. proved that the toxicity of PrP oligomers was independent of endogenous PrPC expression in vivo and in vitro (Simoneau et al. 2007b). Recently, Steve Simoneau et al. found that the hydrophobic domain of PrP oligomers is essential for toxicity. They identified the domains of PrP oligomers by occluding different regions by incubating PrP oligomers with a series of domain-specific PrP antibodies. The neurotoxicity of murine or ovine PrP oligomers to neurons was fully prevented by the monoclonal antibody Pri303 directed against the domain 106– 126 of PrP. In contrast, incubation with the PrP monoclonal antibodies SAF84 (aa161–170), Pri917 (aa 217–221), or SAF32 (aa 59–92) had no protective effect. These results indicated that the exposure of hydrophobic domain of PrP at the surface of the PrP oligomers is required for the neurotoxic
J Mol Neurosci
effect. Especially, the effect of the various antibodies on neurons was similarly observed in PrP-expressing and nonexpressing cells. Kudos et al. (Kudo et al. 2012) found the antibody 6D11 binding to PrPC (93–109) could prevent neuronal death caused by the Aβ oligomers. On the contrary, antibody 6H4 binding to PrPC (144–152) failed to block the cytotoxicity. These data showed that the residues 93–109 might be responsible for binding the Aβ oligomers and causing the neurotoxicity. Lauren et al. (Laurén et al. 2009) certainly showed that antibody 6D11 could prevent the interaction of PrPC with the Aβ oligomers and stop the oligomerinduced synaptic dysfunction.
PrP Oligomers Can Induce Cellular Apoptosis Previous studies demonstrated that prion or synthetic peptides such as PrP106-126 could induce cellular apoptosis in primary cultures of hippocampal (Forloni et al. 1993), cortical (Pan et al. 2014; Song et al. 2013a; Song et al. 2014b; Thellung et al. 2000b), and cerebellar neurons (Gellman and Gibson 1996; Jobling et al. 1999; Thellung et al. 2000a). Steve Simoneau et al. used nuclear staining and ApopTag BrdU kit to understand the type of neuronal death induced by murine or ovine PrP oligomers. Neuronal cultures treated with PrP oligomers revealed a number of cells exhibiting condensed and fragmented chromatin, a hallmark of apoptosis. Neurons exposed to the toxic PrP oligomers exhibited intense BrdU labeling, which indicates that the neurons underwent apoptosis. Xiujin Yang et al. used terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay and mitochondrial membrane potential assay (JC-1) to determine the type of neuronal death induced by dimeric PrP oligomers. The results of TUNEL and JC-1 showed that PrP oligomers can cause neuronal apoptosis in vivo and in vitro. The regulatory pathway to apoptosis induced by PrPSc has been well studied. The cardinal apoptotic-regulatory pathways include caspases (Degterev et al. 2003) and ER stress (Nakagawa et al. 2000). Caspase-dependent apoptosis can be initiated by the activation of death receptors or by mitochondrial stress. ER stress-induced apoptosis can be triggered by the activation of the ER-resident caspase-12 owing to alteration of calcium homeostasis or the accumulation of misfolded proteins (Soto and Satani 2011). Recent reports found that apoptosis induced by aggregated PrP peptides or mutant PrP variants proceeds via the mitochondrial pathway (Hachiya et al. 2005; Nicolas et al. 2007; O’Donovan et al. 2001; Pan et al. 2014). More recently, studies found that activation of Wnt signaling can rescue neuronal dysfunction in neurodegenerative diseases (Jeong et al. 2014; Song et al. 2014a). However, so far, few researches were performed to study the specific pathway to PrP oligomer-induced apoptosis. Therefore, a detailed understanding of the exact regulatory
pathway to apoptosis induced by oligomeric PrP awaits further investigation.
Conclusion Even though it is clear that prion pathology is generally related to PrP aggregation and abnormal PrP deposits, there are multiple disease-related PrP forms, including protease-sensitive species, which might account for the majority of infectivity in some isolates (Cronier et al. 2008; Hosszu et al. 2005; Safar et al. 1998). In particular, an increasing body of evidence demonstrated that non-fibrillar oligomers are implicated as neurotoxic species in prion diseases. Consequently, the focus of research on pathologic mechanisms underlying amyloidosis has shifted to oligomers. As was shown in this review, great advance in oligomer research has been achieved. However, it is still not clear about oligomers’ transmissibility. Although a number of high-β-sheet PrPSc-like oligomers have been formed in different conditions, none has proven to produce serially transmissible prion disease when inoculated into experimental animals. In addition, attainment and biophysical characterization of the in vivo oligomers are rather difficult due to their metastable nature. It seems certain that exploring PrP conformation and/or aggregation state is of much neuropathologic significance whether or not aggregated PrP is the major neurotoxic factor in prion diseases. Therapeutic strategies may then reasonably be committed to reducing or eliminating those PrP isoforms. Therefore, further studies about the pathologic mechanisms of the various types of TSE diseases will be needed.
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The Unexposed Secrets of Prion Protein Oligomers Gailing Wang & Mingcheng Wang & Chuanfeng Li
Received: 15 January 2015 / Accepted: 4 March 2015 # Springer Science+Business Media New York 2015
Abstract According to the Bprotein-only^ hypothesis, the misfolding and conversion of host-derived cellular prion protein (PrPC) into pathogenically misfolded PrP are believed to be the key procedure in the pathogenesis of prion diseases. Intermediate, soluble oligomeric prion protein (PrP) aggregates were considered a critical process for prion diseases. Several independent studies on PrP oligomers gained insights into oligomers’ formation, biophysical and biochemical characteristics, structure conversion, and neurotoxicity. PrP oligomers are rich in β-sheet structure and slightly resistant to proteinase K digestion. PrP oligomers exhibited more neurotoxicity and induced neuronal apoptosis in vivo and/or in vitro. In this review, we summarized recent studies regarding PrP oligomers and the relationship between misfolded PrP aggregates and neuronal death in the course of prion diseases. Keywords Prion diseases . Prion protein . Oligomers . Neurotoxicity
Introduction Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are fatal neurodegenerative diseases of humans and animals, including Creutzfeldt–Jakob disease, Gerstmann–Sträussler–Scheinker syndrome, kuru and fatal familial insomnia in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk (Prusiner 1982; Prusiner 1998; Yuan et al. G. Wang (*) : M. Wang : C. Li Department of Bioengineering, Huanghuai University, 463000 Zhumadian, China e-mail: [email protected]
2013b). The agent that causes TSEs was termed by Stanley B. Prusiner as Bprion^ and is defined as Ba small proteinaceous infectious particle^ (Prusiner 1982). According to Bproteinonly^ hypothesis, the central event in prion pathogenesis is the conformational conversion of normal soluble cellular prion protein (PrPC) into misfolded insoluble and partially protease-resistant isoform (PrPSc) (Kim et al. 2010; Wang et al. 2010). However, to date, no investigation has yielded direct experimental proof for this stringent hypothesis. Indeed, neuronal death can occur without detectable PrPSc during prion infection, and there is no detectable PrPSc in some cases of prion-infected diseases (Gambetti et al. 1995; Lasmézas et al. 1997; Manson et al. 1999; Manuelidis et al. 1997). Until now, the exact pathological mechanism of the prion diseases is still unclear. In neurodegenerative diseases, such as Alzheimer’s, Huntington, Parkinson, and prion diseases, the deposition of misfolded aggregated proteins is believed to be responsible for the neurotoxicity that characterizes these diseases (Bucciantini et al. 2004; Laganowsky et al. 2012; Nicoll et al. 2013; Tomic et al. 2009; Walsh and Teplow 2012). The accumulations of abnormal PrP forms usually colocalize with prion neuropathology (Caughey and Lansbury 2003a). Prion protein has been deeply studied for the peculiar feature of its misfolded oligomers, inducing the conversion of the natively folded PrP into the pathological conformation. Subsequent researchers reported that monomeric PrPC could be transformed into various forms of oligomers that showed neurotoxicity in vitro or in vivo (Huang et al. 2010; Ladner-Keay et al. 2014; Malchiodi-Albedi et al. 2011; Simoneau et al. 2007a; Swietnicki et al. 2000). Intermediate, soluble oligomeric PrP aggregates were soon considered a critical process for prion diseases. Several independent studies on PrP oligomers gained insights into oligomers’ formation, biophysical and biochemical characteristics, structure conversion, and neurotoxicity
J Mol Neurosci
mechanism. In this review, we summarized studies on PrP oligomers and the relationship between misfolded PrP aggregates and neuronal death in the course of prion diseases.
Formation of PrP Oligomers PrPC dimers were detected in neuroblastoma N2a cells expressing Syrian hamster PrPC (Priola et al. 1995) and in a solution in a partially purified fraction from normal bovine thalamus (Meyer et al. 2000). Endogenous PrPC dimers were also detected in N2a cells by Rambold et al. Moreover, PrPC dimers were detected in crude membranes from human neuroblastoma SH-SY5Y cells and mouse brains (Rambold et al. 2008). These results convincingly demonstrated that PrPC has an intrinsic tendency to dimerize in native conditions and provided the in vivo evidence of PrP polymerization (Fig. 1). However, many recent studies were conducted to clarify the formation of PrP oligomers from recombinant PrP monomers in different conditions in vitro. These conditions include denaturants (guanidine hydrochloride or urea), high salts, and detergents (Baskakov et al. 2002; Morillas et al. 2001; Swietnicki et al. 2000). The methods including sizeexclusion chromatography (SEC), ultracentrifugation, dynamic light scattering (DLS), electron microscopy (EM), atomic force microscopy (AFM), immunochemical detection, or chemical cross-linking are required for accurate determination of oligomerization state (Bitan et al. 2005). In 2007, Steve Simoneau et al. (Simoneau et al. 2007a) generated dimeric ovine PrP oligomers by thermal refolding. For conversion, recombinant PrP (23–234) (pH 7.2 in 20 mM MOPS) was heated at 72 °C for 15 min and cooled at room temperature. SEC and Western blotting of the PrP preparations showed that they were exclusively in an oligomeric state. They also established that aging of PrP oligomers (extension of time to 1 month at 48 °C) makes oligomers polymerize into PrP fibrils. In 2010, Liqin Huang et al. (Huang et al. 2010) used 200 g/ L dextran 70 to mimic intracellular crowding condition to investigate its potential effects on recombinant human PrP. The oligomeric PrP sample was obtained by adding 0.4 mg/ mL PrP to crowding buffer. Light scattering, turbidity, and SEC confirmed the aggregation of the PrP samples.
In 2011, Zheng Zhou et al. (Zhou et al. 2011) reported that the crowding agents Ficoll 70 and dextran 70 have different effects on fibrillization of the recombinant PrPs from human, cow, and rabbit. These crowing agents dramatically promoted fibril formation of human and bovine PrP; they significantly inhibit fibrillization of the rabbit PrP. These results demonstrated that the sequence of PrP from different animals may affect both the kinetics and the inhibitory effect of crowding agents on rabbit PrP fibrillization. In 2013, Xiujin Yang et al. (Yuan et al. 2013a) generated dimeric hamster PrP oligomers by protein misfolding cyclic amplification (PMCA) consisting of recombinant hamster PrP (23–231). One cycle in PMCA was performed by sonication at amplitude of 80 for 30 s, followed by 29.5 min incubation at 37 °C. After 96 cycles, hamster PrP oligomers emerged. The assay for PMCA-induced oligomers was further supported by Carol L. Ladner-Keay et al. (Ladner-Keay et al. 2014). In 2014, Carol L. Ladner-Keay et al. (Ladner-Keay et al. 2014) found that de novo conversion of recombinant prion proteins to oligomers could be induced by shaking alone. The formation of oligomers occurred by shaking recombinant hamster PrP (90–232) and mouse PrP (90–231) at 37 °C and 350 rpm, in buffer at pH 5.5 for at least 1 day. The size of these PrP oligomers ranged from octamers to dodecamers as determined by RENAGE (Ladner and Wishart 2012) and EM. Essentially, the PrP fibrils determined by EM can be formed by shaking at 350 rpm at 37 °C for 5 days.
Structures of PrP Oligomers Prion diseases are caused by misfolding of α-helix-rich PrPC into the β-sheet-rich aggregated PrPSc. Early studies including low-resolution, crystallographic, and structural nuclear magnetic resonance spectroscopy indicated that PrPC had nearly 40 % α-helix and 3 % β-sheet (Hornemann et al. 1997; Knowles et al. 2007; Pan et al. 1993; Riek et al. 1996). In contrast to PrPC, the α-helix content of PrPSc comprises 30 % of the protein, whereas β-sheet comprises 40 % of the protein as measured by circular dichroism (CD) spectroscopy (Safar et al. 1993) and Fourier transform infrared spectroscopy (FTIR) (Pan et al. 1993). Studies found that oligomeric PrP derived from recombinant monomeric PrP has an increased
Fig. 1 Pathological processes of misfolded PrP aggregation. PrP fibril formation from native state is a polymerization process consisting of monomeric misfolding, protein oligomerization, and fibril elongation
J Mol Neurosci
percentage of β-sheet structure. Carol L. Ladner-Keay et al. found that CD spectroscopy analysis for recombinant PrPC gave 43 % α-helix and 10 % β-sheet and for PrP oligomers 16 % α-helix and 24 % β-sheet. The CD result was further confirmed by FTIR that shaking-induced oligomers adopted a β-sheet-enriched conformation. Essentially, oligomers can develop into fibrils under certain conditions in prion conversion (Caughey and Lansbury 2003b; Ladner-Keay et al. 2014; Simoneau et al. 2007a; Zhou et al. 2011). Zheng Zhou et al. used FTIR and CD to compare the secondary structures of rabbit/human/bovine PrP fibrils and found that rabbit PrP fibrils contained less β-sheet structure and more α-helix structure than human and bovine PrP fibrils.
Characterization of PrP Oligomers Previous studies showed that thioflavin T (ThT), a common dye, can bind to amyloid aggregations and also be applied to determine the oligomeric PrP (Huang et al. 2010; Nandi et al. 2006; Simoneau et al. 2007a). Steve Simoneau et al. treated various kinds of PrP with ThT to compare their interaction with ThT. PrP oligomers showed slightly enhanced ThT binding. This finding was further confirmed by Liqin Huang et al. ANS, a polarity-sensitive fluorescent probe, was usually used to compare various hydrophobic surfaces of soluble monomers and oligomers. Liqin Huang et al. found that ANS fluorescence intensity increased by nearly 3.5-fold at 473 nm compared with PrPC, further indicating that oligomers have an increase in hydrophobic surfaces than monomers (Huang et al. 2010). This result is consistent with previous findings that PrP oligomers have a larger hydrophobic surface than monomeric PrP (Nandi et al. 2006; Rezaei et al. 2005). Naturally existing infectious prions, as well as many in vitro generated PrPSc-like forms or oligomers, are known to show resistance to proteinase K digestion (Bocharova et al. 2005; Weissmann 2004). PrPC is a proteinase K-sensitive monomer, whereas PrPSc is resistant to proteinase K digestion (Song et al. 2013a; Yao et al. 2013; Zhu et al. 2014). Proteinase K resistance, in fact, has been considered to be a hallmark for the presence of PrPSc (Ladner-Keay et al. 2014). Recently, many studies found that the formation of the soluble PrP oligomers was accompanied by a substantial increase in resistance to proteinase K digestion. For instance, Liqin Huang et al. subjected oligomeric PrPs to a range of proteinase K concentrations in parallel with their monomeric forms. The PrP monomer was sensitive to proteinase K digestion. In contrast, PrP oligomers exhibited partial protease resistance and displayed increased resistance to degradation by proteinase K. In addition, there were several bands approximately 12∼ 14 kDa corresponding to proteinase K (PK)-resistant fragments. These observations were consistent with other studies
that PrP oligomers are partially resistant to PK digestion (Kaimann et al. 2008; Ladner-Keay et al. 2014; Nandi et al. 2006; Simoneau et al. 2007a).
Neurotoxicity of PrP Oligomers In Vitro and In Vivo Recent studies suggested that the PrP oligomers are neurotoxic (Huang et al. 2010; Kazlauskaite et al. 2005; Ladner-Keay et al. 2014; Novitskaya et al. 2006; Simoneau et al. 2007a). Steve Simoneau et al. first analyzed the cytotoxic properties of the PrP oligomers in primary mouse cerebral cortex neurons isolated from wild-type animals. They exhibited a roughly approximately 30-fold higher toxic effect than PrP peptide 105–132. To investigate the toxicity of PrP oligomers in vivo, stereotaxic subcortical injections of oligomeric PrP were carried out in the right hemispheres of mice. Gallocyanine staining of brain sections was used to detect the toxicity. The result that the pyramidal layer of neurons underneath the injection site was completely destructed showed that PrP oligomers were highly toxic in wild-type and PrPC knockout mice. The neurotoxicity of PrP oligomers was further confirmed by subsequent studies. Liqin Huang et al. found that the soluble oligomers formed in macromolecular crowding were neurotoxic to the human neuroblastoma SK-N-SH and SH-SY5Y cell lines. Xiujin Yang et al. showed that PMCA-induced hamster PrP oligomers were neurotoxic to mouse neuroblastoma N2a cells. Although it has been widely accepted that the absence of endogenous PrPC renders host animal resistant to the toxic effects of PrPSc (Büeler et al. 1993; Biasini et al. 2012; Raeberi et al. 1996), the dependency of PrP-induced toxicity on the presence of PrPC has remained a matter of debate among some prion scientists (Song et al. 2013b). One study showed that the toxicity of PrP oligomers in vitro was dependent on the expression of PrPC (Novitskaya et al. 2006), and this study was supported by Kudo et al. that PrPC is essentially involved in the oligomeric amyloid-β-induced neuronal cell death. However, Steve Simoneau et al. proved that the toxicity of PrP oligomers was independent of endogenous PrPC expression in vivo and in vitro (Simoneau et al. 2007b). Recently, Steve Simoneau et al. found that the hydrophobic domain of PrP oligomers is essential for toxicity. They identified the domains of PrP oligomers by occluding different regions by incubating PrP oligomers with a series of domain-specific PrP antibodies. The neurotoxicity of murine or ovine PrP oligomers to neurons was fully prevented by the monoclonal antibody Pri303 directed against the domain 106– 126 of PrP. In contrast, incubation with the PrP monoclonal antibodies SAF84 (aa161–170), Pri917 (aa 217–221), or SAF32 (aa 59–92) had no protective effect. These results indicated that the exposure of hydrophobic domain of PrP at the surface of the PrP oligomers is required for the neurotoxic
J Mol Neurosci
effect. Especially, the effect of the various antibodies on neurons was similarly observed in PrP-expressing and nonexpressing cells. Kudos et al. (Kudo et al. 2012) found the antibody 6D11 binding to PrPC (93–109) could prevent neuronal death caused by the Aβ oligomers. On the contrary, antibody 6H4 binding to PrPC (144–152) failed to block the cytotoxicity. These data showed that the residues 93–109 might be responsible for binding the Aβ oligomers and causing the neurotoxicity. Lauren et al. (Laurén et al. 2009) certainly showed that antibody 6D11 could prevent the interaction of PrPC with the Aβ oligomers and stop the oligomerinduced synaptic dysfunction.
PrP Oligomers Can Induce Cellular Apoptosis Previous studies demonstrated that prion or synthetic peptides such as PrP106-126 could induce cellular apoptosis in primary cultures of hippocampal (Forloni et al. 1993), cortical (Pan et al. 2014; Song et al. 2013a; Song et al. 2014b; Thellung et al. 2000b), and cerebellar neurons (Gellman and Gibson 1996; Jobling et al. 1999; Thellung et al. 2000a). Steve Simoneau et al. used nuclear staining and ApopTag BrdU kit to understand the type of neuronal death induced by murine or ovine PrP oligomers. Neuronal cultures treated with PrP oligomers revealed a number of cells exhibiting condensed and fragmented chromatin, a hallmark of apoptosis. Neurons exposed to the toxic PrP oligomers exhibited intense BrdU labeling, which indicates that the neurons underwent apoptosis. Xiujin Yang et al. used terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay and mitochondrial membrane potential assay (JC-1) to determine the type of neuronal death induced by dimeric PrP oligomers. The results of TUNEL and JC-1 showed that PrP oligomers can cause neuronal apoptosis in vivo and in vitro. The regulatory pathway to apoptosis induced by PrPSc has been well studied. The cardinal apoptotic-regulatory pathways include caspases (Degterev et al. 2003) and ER stress (Nakagawa et al. 2000). Caspase-dependent apoptosis can be initiated by the activation of death receptors or by mitochondrial stress. ER stress-induced apoptosis can be triggered by the activation of the ER-resident caspase-12 owing to alteration of calcium homeostasis or the accumulation of misfolded proteins (Soto and Satani 2011). Recent reports found that apoptosis induced by aggregated PrP peptides or mutant PrP variants proceeds via the mitochondrial pathway (Hachiya et al. 2005; Nicolas et al. 2007; O’Donovan et al. 2001; Pan et al. 2014). More recently, studies found that activation of Wnt signaling can rescue neuronal dysfunction in neurodegenerative diseases (Jeong et al. 2014; Song et al. 2014a). However, so far, few researches were performed to study the specific pathway to PrP oligomer-induced apoptosis. Therefore, a detailed understanding of the exact regulatory
pathway to apoptosis induced by oligomeric PrP awaits further investigation.
Conclusion Even though it is clear that prion pathology is generally related to PrP aggregation and abnormal PrP deposits, there are multiple disease-related PrP forms, including protease-sensitive species, which might account for the majority of infectivity in some isolates (Cronier et al. 2008; Hosszu et al. 2005; Safar et al. 1998). In particular, an increasing body of evidence demonstrated that non-fibrillar oligomers are implicated as neurotoxic species in prion diseases. Consequently, the focus of research on pathologic mechanisms underlying amyloidosis has shifted to oligomers. As was shown in this review, great advance in oligomer research has been achieved. However, it is still not clear about oligomers’ transmissibility. Although a number of high-β-sheet PrPSc-like oligomers have been formed in different conditions, none has proven to produce serially transmissible prion disease when inoculated into experimental animals. In addition, attainment and biophysical characterization of the in vivo oligomers are rather difficult due to their metastable nature. It seems certain that exploring PrP conformation and/or aggregation state is of much neuropathologic significance whether or not aggregated PrP is the major neurotoxic factor in prion diseases. Therapeutic strategies may then reasonably be committed to reducing or eliminating those PrP isoforms. Therefore, further studies about the pathologic mechanisms of the various types of TSE diseases will be needed.
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