De novo mammalian prion synthesis

Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases. They can be sporadic, genetic, or infectious disorders involving post-translational modifications of the cellular prion protein (PrP^C). Prions (PrP^Sc) are characterized by their infectious property and intrinsic ability to convert the physiological PrP^C into the pathological form, acting as a template. The “protein-only” hypothesis, postulated by Stanley B. Prusiner, implies the possibility to generate de novo prions in vivo and in vitro. Here we describe major milestones towards proving this hypothesis, taking into account physiological environment/s, biochemical properties and interactors of the PrP^C.Key words: prion protein (PrP), prions, amyloid, recombinant prion protein, transgenic mouse, protein misfolding cyclic amplification (PMCA), synthethic prionPrions are responsible for a heterogeneous group of fatal neurodegenerative diseases (1 They can be sporadic, genetic or infectious disorders involving post-translational modifications of the cellular prion protein (PrP^C).² Prions are characterized by their infectious properties and by their intrinsic ability to encipher distinct biochemical properties through their secondary, tertiary and quaternary protein structures. In particular, the transmission of the disease is due to the ability of a prion to convert the physiological PrP^C into the pathological form (PrP^Sc), acting as a template.³ The two isoforms of PrP appear to be different in terms of protein structures, as revealed by optical spectroscopy experiments such as Fourier-transform infrared and circular dichroism.⁴ PrP^C contains 40% α-helix and 3% β-sheet, while the pathological isoform, PrP^Sc, presents approximately 30% α-helix and 45% β-sheet.^4,5 PrP^Sc differs from PrP^C because of its altered physical-chemical properties such as insolubility in non-denaturing detergents and proteinases resistance.^2,6,7

Table 1

The prion diseases

Mutant PrPSc conformers induced by a synthetic peptide and several prion strains

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP^Sc(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4°C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP^Sc(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a β-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP^Sc(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP^Sc in the brains of Tg196 mice. Our results argue that MoPrP^Sc(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP^C(P101L) as well as in Tg196 mice expressing low levels of MoPrP^C(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a β-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.The discovery that brain fractions enriched for prion infectivity contain a protein (rPrP^Sc) that is resistant to limited proteolytic digestion advanced prion research (8, 37). N-terminal truncation of rPrP^Sc produced a protease-resistant fragment, denoted PrP 27-30, that is readily measured by Western blotting, enzyme-linked immunosorbent assay, or immunohistochemistry. The measurement of PrP^Sc was dramatically changed with the development of the conformation-dependent immunoassay (CDI), which permitted detection of full-length rPrP^Sc as well as previously unrecognized protease-sensitive forms of PrP^Sc (39).The CDI depends on using anti-PrP antibodies that react with an epitope exposed in native PrP^C but that do not bind to native PrP^Sc. Upon denaturation, the buried epitope in PrP^Sc becomes exposed and readily reacts with anti-PrP antibodies. Using the CDI, we discovered that most PrP^Sc is protease sensitive, which we designate sPrP^Sc. Whether sPrP^Sc is an intermediate in the formation of rPrP^Sc remains to be determined. In Syrian hamsters inoculated with eight different strains of prions, the ratio of rPrP^Sc to sPrP^Sc was different for each strain and the concentration of sPrP^Sc was proportional to the length of the incubation time (39).In earlier studies, transgenic (Tg) mice, denoted Tg2866, expressing high levels of PrP(P101L) were used to model Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L point mutation. In the brains of several lines of mice expressing high levels of PrP(P101L), no rPrP^Sc(P101L) was detectable (26, 27, 47). This was particularly perplexing since these Tg mice expressing high levels of PrP(P101L) developed all facets of prion-induced neurodegeneration, including multicentric PrP amyloid plaques. Moreover, brain extracts from ill Tg2866 mice transmitted disease to Tg196 mice expressing low levels of PrP(P101L) that infrequently developed spontaneous neurodegeneration (29).In humans with GSS, several different mutations of the PrP gene (PRNP) resulting in nonconservative amino acid substitutions have been identified (23). In these patients, the clinical presentation, disease course, and amounts of rPrP^Sc in the brain are variable. Brain extracts from humans who died of GSS were inoculated into apes and monkeys, but the transmission rates were not correlated with the levels of PrP^Sc in the inoculum (1, 2, 9, 32). In a limited study, GSS(P102L) was transmitted to Tg mice expressing a chimeric mouse-human (MHu2 M) PrP transgene carrying the P102L mutation but not to Tg mice expressing MHu2M PrP without the mutation (47). In another study, GSS(P102L) human prions were transmitted to Tg mice expressing MoPrP(P101L) in which the transgene was incorporated through gene replacement (31). The use of gene replacement permits all of the regulatory elements that control the wild-type (wt) MoPrP gene to modulate the expression of MoPrP(P101L). In these mice, the expression level of MoPrP(P101L) in brain is likely to be similar to that in Tg196 mice.When we synthesized a 55-mer MoPrP peptide composed of residues 89 to 143 containing the P101L mutation and folded it under conditions favoring a β-structure, it induced neurodegeneration in Tg196 mice (29). When the peptide was not folded into a β-structure, it did not produce disease in Tg196 mice. We report here that the peptide-initiated disease in Tg196 mice could be serially transmitted to other Tg196 mice using brain extracts from the peptide-inoculated Tg196 mice. Using procedures derived from the CDI, brain extracts from inoculated Tg196 mice were found to contain sPrP^Sc(P101L), from which a 22- to 24-kDa PrP fragment was generated by limited digestion with proteinase K (PK) at 4°C and selective precipitation with phosphotungstate (PTA) (25, 39). In the interest of clarity, we have designated digestion at 4°C as “cold PK” and simply refer to standard digestion at 37°C as “PK.” To aid in distinguishing rPrP^Sc(P101L) from sPrP^Sc(P101L), their properties based on the work reported here and in other previously published papers are listed in Table ​Table11 (39, 40).

TABLE 1.

Characteristics of PrP(P101L) isoforms

Comparative analysis of Japanese and foreign L-type BSE prions

L-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE. To characterize the Japanese L-type BSE prion, we conducted a comparative study of the Japanese and foreign L-type BSE isolates. The L-type BSE isolates of Japan, Germany, France and Canada were intracerebrally inoculated into bovinized prion protein-overexpressing transgenic mice (TgBoPrP). All the examined L-type BSE isolates were transmitted to TgBoPrP mice, and no clear differences were observed in their biological and biochemical properties. Here, we present evidence that the Japanese and Canadian L-type BSE prions are identical to those from the European cases.Key words: prion, atypical BSE, L-type BSEBovine spongiform encephalopathy (BSE) is one of the transmissible spongiform encephalopathies (TSEs), or prion diseases, in cattle. TSE is characterized by spongiform changes in the central nervous system (CNS) and the accumulation of an abnormal prion protein (PrP^Sc) in the CNS.¹ PrP^Sc has been regarded as the major component of TSE pathogens.²BSE was detected in the UK in 1986,³ and subsequently spread to the other European countries, Japan and North America.^4–6 BSE is thought to be caused by a single prion strain, based on the analyses of its biological and biochemical characteristics.⁷ From 2003, however, several atypical neuropathological and molecular phenotypes of BSE (atypical BSE) have been detected in Japan, several European countries and North America.^6,8–17 Currently, based on the molecular size of the proteinase-digested non-glycosylated form of PrP^Sc, atypical BSE is classified into two groups (L-type and H-type).¹⁴L-type BSE cases have been identified in the European countries, including Italy, France, Germany, Netherland, Poland and in Canada and Japan.^8–15 Two L-type BSE cases have been identified in Japan. One case was detected in a healthy 23-mo-old Holstein steer (BSE/JP8),⁸ and the other was detected in a 14-y-old black Japanese beef cattle (BSE/JP24).⁹ The latter case was successfully transmitted to bovinized transgenic mice and cattle, and the biological and biochemical properties differed from that of classical BSE (C-BSE).^18,19 However, it is unclear whether Japanese L-type BSE prion is identical to that of L-type BSE isolates from other countries. To characterize the Japanese L-type BSE isolate, we performed a comparative study of the Japanese and foreign L-type BSE isolates.A transmission study using experimental animals is a useful approach for prion characterization. Therefore, we performed a transmission study of the L-type BSE isolates in bovinized prion protein (PrP)-overexpressing transgenic mice (TgBoPrP).²⁰ Brain samples of L-type BSE-affected cattle from Japan (BSE/JP24),⁹ France,¹⁰ Germany¹¹ and Canada¹² were used in this study. The brain homogenates were intracerebrally inoculated into TgBoPrP using previously described methods in reference ¹⁸. All animal experiments were reviewed by the Committee of the Ethics on Animal Experiment of the National Institute of Animal Health.All the examined L-type BSE isolates were transmitted to TgBoPrP, and the affected mice developed progressive neurological diseases. Japanese L-type BSE isolate-affected TgBoPrP exhibited a unique clinical sign, the circling behavior. The same phenotype was observed when TgBoPrP were inoculated with German, French and Canadian L-type BSE isolates. On the other hand, in the first passage the incubation period for the Japanese L-type BSE isolate was significantly different from that of the other L-type BSE isolates (

Prion interference with multiple prion isolates

Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.Key words: prion diseases, prion interference, prion strainsPrion diseases are fatal neurodegenerative diseases that are caused by an abnormal isoform of the prion protein, PrP^Sc.¹ Prion strains are hypothesized to be encoded by strain-specific conformations of PrP^Sc resulting in strain-specific differences in clinical signs, incubation periods and neuropathology.^2–7 However, a universally agreed upon definition of prion strains does not exist. Interspecies transmission and adaptation of prions to a new host species leads to the emergence of a dominant prion strain, which can be due to selection of strains from a mixture present in the inoculum, or produced upon interspecies transmission.^8,9 Prion strains, when present in the same host, can interfere with each other.Prion interference was first described in mice where a long incubation period strain 22C extended the incubation period of a short incubation period strain 22A following intracerebral inoculation.¹⁰ Interference between other prion strains has been described in mice and hamsters using rodent-adapted strains of scrapie, TME, Creutzfeldt-Jacob disease and Gerstmannn-Sträussler-Scheinker syndrome following intracerebral, intraperitoneal, intravenous and sciatic nerve routes of inoculation.^10–15 We previously demonstrated the detection of PrP^Sc from the long incubation period DY TME agent correlated with its ability to extend the incubation period or completely block the superinfecting short incubation period HY TME agent from causing disease and results in a reduction of HY PrP^Sc levels following sciatic nerve inoculation.¹² However, it is not known if a single long incubation period agent (e.g., DY TME) can interfere with more than one short incubation period agent or if interference can occur by a natural route of infection.To examine the question if one long incubation period agent can extend the incubation period of additional short incubation period agents, hamsters were first inoculated in the sciatic nerve with the DY TME agent 120 days prior to superinfection with the short-incubation period agents HY TME, 263K scrapie and HaCWD.^16–18 The HY TME and 263K scrapie agents have been biologically cloned and have distinct PrP^Sc properties.^19,20 The HaCWD agent used in this study is seventh hamster passage that has not been biologically cloned and therefore will be referred to as a prion isolate. Sciatic nerve inoculations were performed as previously described.^11,12 Briefly, hamsters were inoculated with 103.0 i.c. LD₅₀ of the DY TME agent or equal volume (2 µl of a 1% w/v brain homogenate) of uninfected brain homogenate 120 days prior to superinfection of the same sciatic nerve with either 10^4.6 i.c. LD₅₀ of the HY TME agent, 10^5.2 i.c. LD₅₀ of the HaCWD agent or 10^4.6 i.c. LD₅₀/g 263K scrapie agent (Bartz J, unpublished data).^16,18,21 Animals were observed three times per week for the onset of clinical signs of HY TME, 263K and HaCWD based on the presence of ataxia and hyperexcitability, while the clinical diagnosis of DY TME was based on the appearance of progressive lethargy.^16–18 The incubation period was calculated as the number of days between the onset of clinical signs of the agent strain that caused disease and the inoculation of that strain. The Student''s t-test was used to compare incubation periods.¹² We found that sciatic nerve inoculation of both the HaCWD agent and 263K scrapie agent caused disease with a similar incubation period to animals infected with the HY TME agent (12 In hamsters inoculated with the DY TME agent 120 days prior to superinfection with the HaCWD or 263K agents, the animals developed clinical signs of DY TME with an incubation period that was not different from the DY TME agent control group (12 The PrP^Sc migration properties were consistent with the clinical diagnosis and all co-infected animals had PrP^Sc that migrated similar to PrP^Sc from the DY TME agent infected control animal (Fig. 1, lanes 1–10). This data indicates that the DY TME agent can interfere with more than one isolate and that interference in the CNS may be a more generalized phenomenon of prion strains.Open in a separate windowFigure 1The strain-specific properties of PrP^Sc correspond to the clinical diagnosis of disease. Western blot analysis of 250 µg brain equivalents of proteinase K digested brain homogenate from prion-infected hamsters following intracerebral (i.c.), sciatic nerve (i.sc.) or per os inoculation with either the HY TME (HY), DY TME (DY), 263K scrapie (263K), hamster-adapted CWD (CWD) agents or mock-infected (UN). The unglycoyslated PrP^Sc glycoform of HY TME, 263K scrapie and hamster-adapted CWD migrates at 21 kDa. The unglycosylated PrP^Sc glycoform of DY PrP^Sc migrates at 19 kDa. Migration of 19 and 21 kDa PrP^Sc are indicated by the arrows on the left of the figure. n.a., not applicable.

Table 1

Clinical signs and incubation periods of hamsters inoculated in the sciatic nerve with either the HY TME, HaCWD or 263K scrapie agents, or co-infected with the DY TME agent 120 days prior to superinfection of hamsters with the HY TME, HaCWD or 263K agents

Is,indeed, the prion protein a Harlequin servant of “many” masters?

Tens of putative interacting partners of the cellular prion protein (PrP^C) have been identified, yet the physiologic role of PrP^C remains unclear. For the first time, however, a recent paper has demonstrated that the absence of PrP^C produces a lethal phenotype. Starting from this evidence, here we discuss the validity of past and more recent literature supporting that, as part of protein platforms at the cell surface, PrP^C may bridge extracellular matrix molecules and/or membrane proteins to intracellular signaling pathways.Key words: prion protein, PrP^C, extracellular matrix, cell adhesion molecules, neuritogenesis, p59fyn, Ca²⁺Initially, the discovery that the prion protein was the major, if not the unique, component of the prion agent causing transmissible spongiform encephalopathies (TSE)¹ has placed the protein in an extremely unfavorable light. Thereafter, however, a wealth of evidence has supported the notion that the protein positively influences several aspects of the cell physiology, and that its duality—in harboring both lethal and beneficial potentials—could be rationalized in terms of a structural switch. Indeed, the protein exists in at least two conformational states: the cellular, α helix-rich isoform, PrP^C, and the prion-associated β sheet-rich isoform, PrP^Sc.² If it is now unquestionable that the presence of PrP^C in the cell is mandatory for prion replication and neurotoxicity to occur,^3,4 nonetheless its physiologic function is still debatable, despite the long lasting effort, and the numerous, frequently genetically advanced, animal and cell model systems dedicated to the issue. From these studies the picture of an extremely versatile protein has emerged, whereby PrP^C acts in the cell defense against oxidative and apoptotic challenges, but also in cell adhesion, proliferation and differentiation, and in synaptic plasticity.^5,6 In an effort to converge these multiple propositions in an unifying functional model, different murine lines devoid of PrP^C have been studied. These animals, however, displayed no obvious phenotype,^7–9 suggesting that either PrP^C is dispensable during development and adult life or that compensative mechanisms mask the loss of PrP^C function in these paradigms. Thus, identifying the exact role of PrP^C in the cell would not only resolve an important biological question, but would also help elucidate the cellular steps of prion pathogenesis necessary for designing early diagnostic tools and therapeutic strategies for TSE.As is often the case, the employment of a model system unprecedented in prion research has recently disclosed a most interesting scenario with regards to PrP^C physiology, having unravelled, for the first time, a lethal phenotype linked to the absence of the protein.¹⁰ The paradigm is the zebrafish, which expresses two PrP^C isoforms (PrP1 and PrP2). Similarly to mammalian PrP^C, they are glycosylated and attached to the external side of the plasma membrane through a glycolipid anchor. PrP1 and PrP2 are, however, expressed in distinct time frames of the zebrafish embryogenesis. Accordingly, the knockdown of the PrP1, or PrP2, gene very early in embryogenesis impaired development at different stages, bypassing putative compensatory mechanisms. By focusing on PrP1, Malaga-Trillo et al. showed that the protein was essential for cell adhesion, and that this event occurred through PrP1 homophilic trans-interactions and signaling. This comprised activation of the Src-related tyrosine (Tyr) kinase p59fyn, and, possibly, Ca²⁺ metabolism, leading to the regulation of the trafficking of E-cadherin, a member of surface-expressed cell adhesion molecules (CAMs) responsible for cell growth and differentiation.¹¹ It was also reported that overlapping PrP1 functions were performed by PrP^Cs from other species, while the murine PrP^C was capable to replace PrP1 in rescuing, at least in part, the knockdown developmental phenotype. Apart from providing the long-sought proof for a vital role of PrP^C, the demonstration that a mammalian isoform corrected the lethal zebrafish phenotype strongly reinforces previous results—mainly obtained in a variety of mammalian primary neurons and cell lines—pointing to a functional interplay of PrP^C with CAMs, or extra cellular matrix (ECM) proteins, and cell signaling, to promote neuritogenesis and neuronal survival. A revisit of these data is the main topic of the present minireview.As mentioned, the capacity of PrP^C to act as a cell adhesion, or recognition, molecule, and to entertain interactions with proteins implicated in growth and survival, has already been reported for the mammalian PrP^C. A case in point is the interaction, both in cis- and trans-configurations, with the neuronal adhesion protein N-CAM12 that led to neurite outgrowth.¹³ Like cadherins, N-CAM belongs to the CAM superfamily. Following homo- or heterophylic interactions, it can not only mediate adhesion of cells, or link ECM proteins to the cytoskeleton, but also act as a receptor to transduce signals ultimately resulting in modulating neurite outgrowth, neuronal survival and synaptic plasticity.¹¹ Another example is the binding of PrPC to laminin, an ECM heterotrimeric glycoprotein, which induced neuritogenesis together with neurite adhesion and maintenance,^14,15 but also learning and memory consolidation.¹⁶ Further, it has been described that PrPC interacted with the mature 67 kDa-receptor (67LR) (and its 37 kDa-precursor) for laminin, and with glycosamminoglycans (GAGs), each of which is involved in neuronal differentiation and axon growth.^17–21 More recently, Hajj et al.²² have reported that the direct interaction of PrP^C with another ECM protein, vitronectin, could accomplish the same process, and that the absence of PrP^C could be functionally compensated by the overexpression of integrin, another laminin receptor.²³ Incidentally, the latter finding may provide a plausible explanation for the absence of clear phenotypes in mammalian PrP-null paradigms. By exposing primary cultured neurons to recombinant PrPs, others have shown that trans-interactions of PrP^C are equally important for neuronal outgrowth,^24,25 including the formation of synaptic contacts.²⁵ Finally, it has been demonstrated that the binding of PrP^C with the secreted co-chaperone stress-inducible protein 1 (STI1) stimulated neuritogenesis.²⁶ This same interaction had also a pro-survival effect, as did the interaction of PrP^C with its recombinant form.²⁴ Notably, the involvement of PrP^C in cell protection has been heightened by experiments with whole animals. By applying transient or permanent focal cerebral ischemia to the animals, it was found that their reduced brain damage correlated with spontaneous or adenoviral-mediated, upregulation of PrP^C,^27–29 (reviewed in ref. ³⁰), and that PrP^C deficiency aggravated their ischemic brain injury.^30,31 Thus, now that data are available from phylogenetically distant paradigms (zebrafish and mammalian model systems), it acquires more solid grounds the advocated engagement of PrP^C in homo/heterophilic cis/trans interactions to trigger signaling events aiming at neuronal—or, in more general terms, cell—survival and neuritogenesis. The latter notion is consistent with the delayed maturation of different types of PrP^C-less neurons, observed both in vitro and in vivo.^32,33If one assumes that the interaction of PrP^C with multiple partners (45 for PrP^C and PrP^Sc, as reviewed in Aguzzi et al.,⁵ or 46 considering the homophylic interaction) are all functionally significant, the most immediate interpretation of this “sticky” behavior entails that PrP^C acts as a scaffolding protein in different membrane protein complexes.^5,6 Each complex could then activate a specific signaling pathway depending on the type and maturation of cells, the expression and glycosylation of PrP^C, and availability of extra- and intra-cellular signaling partners. At large, all these signals have been shown to be advantageous to the cell. However, because in a cell only a subtle line divides the “good” from the “bad,” instances can be envisioned in which a pro-life signal turns into a pro-death signal. A typical example of this possibility is glutamate excitotoxicity resulting in dangerous, glutamate receptor-linked, Ca²⁺ overload. Likewise, an excessive or over-stimulated signal elicited by PrP^C, or by the putative complex housing the protein could become noxious to the cell. This possibility may explain why the massive expression of PrP^C caused degeneration of the nervous system,³⁴ and of skeletal muscles,^34,35 in transgenic animals. More intriguing is the finding that—in a mouse line expressing anchorless PrP^C—PrP^Sc was capable to replicate without threatening the integrity of neurons.³⁶ This may suggest that native membrane-bound PrP^C acts as, or takes part into, a “receptor for PrP^Sc”, and that lasting PrP^Sc-PrP^C interactions distort the otherwise beneficial signal of the protein/complex and cause neurodegeneration.³⁷ Consistent with this hypothesis is the finding that the in vivo antibody-mediated ligation of PrP^C provoked apoptosis of the antibody-injected brain area.³⁸ Speculatively, the action of N-terminally, or N-proximally truncated PrPs whose expression in PrP-less transgenic mice induced extensive neurodegeneration,^39–41 may be traced back to the same hyper-activation of PrP^C signaling. Possibly, this may hold true also for the synaptic impairment that, recorded only in PrP^C-expressing neurons, was attributed to the binding of amyloid beta (Aβ) peptide oligomers implicated in Alzheimer disease, to PrP^C.^42,43But which is (are) the cellular signaling pathway(s) conveyed by the engagement of PrP^C in different signaling complexes? In line with its multifaceted behavior, several intracellular effectors have been proposed, including p59fyn, mitogen-activated kinases (MAPK) Erk1/2, PI3K/Akt and cAMP-PKA. p59fyn is the most reported downstream effector, suggesting that, in accordance with its behavior, p59fyn could serve as the sorting point for multiple incoming and outgoing signals also in the case of PrP^C. The initial evidence of the PrP^C-p59fyn connection came from cells subjected to antibody-mediated cross-linking of PrP^C.⁴⁴ Later, it was shown that the PrP^C-p59fyn signal converged to Erk1/2 through a pathway dependent on (but also independent of) reactive oxygen species generated by NADPH oxidase.⁴⁵ A PrP^C-dependent activation of p59fyn^13,25 and Erk1/2 (but also of PI3K and cAMP-PKA)²⁴ was evident in other neuronal cell paradigms and consistent with the almost ubiquitous expression of PrP^C, in non-neuronal cells such as Jurkat and T cells.⁴⁶ Not to forget that in zebrafish embryonic cells activated p59fyn was found in the same focal adhesion sites harboring PrP1.¹⁰ Regarding the activation of the ERK1/2 pathway promoted by the PrP^C-STI1 complex, and leading to neuritogenesis, the role of p59fyn was not investigated.²⁶ The same holds true for the transduction of a neuroprotective signal by the PrP^C-STI1 complex involving the cAMP-PKA pathway.²⁶ Interestingly, this is not the only example reporting that engagement of PrP^C activates simultaneously two independent pathways. In fact, possibly after transactivating the receptor for the epidermal growth factor, the antibody-mediated clustering of PrP^C was shown to impinge on both the Erk1/2 pathway, and on a protein (stathmin) involved in controlling microtubule dynamics.⁴⁷Yet, if p59fyn is implicated in mammalian PrP^C-activated signaling cascade, a protein linking extracellular PrP^C to p59fyn is needed, given the attachment of the enzyme to the inner leaflet of the plasma membrane through palmitoylated/myristoylated anchors. In this, the PrP^C partner N-CAM (isoform 140) seems ideal to fulfill the task, given that p59fyn is part of N-CAM-mediated signaling. Indeed, after recruitment of N-CAM to lipid rafts—which may also depend on PrP^C,¹³—together with the receptor protein Tyr phosphatase α (RPTPα), the Tyr-phosphate removing activity of RPTPα allows the subsequent activation of p59fyn through an autophosphorylation step.⁴⁸ This event recruits and activates the focal adhesion kinase (FAK),¹¹ another non-receptor Tyr kinase. Finally, formation of the FAK-p59fyn complex triggers neuritogenesis through both Erk1/2 and PI3K/Akt pathways.^49,50 Parenthetically, the FAK-p59fyn and PI3K/Akt connection would be suitable to explain why aggravation of ischemic brain injury in PrP-deficient brains was linked to a depressed Akt activation.³¹ FAK-p59fyn complex, however, may be also involved in the signal triggered by the still mysterious PrP^C partner, 67LR. This protein was reported not only to act as a laminin receptor but also to facilitate the interaction of laminin with integrins,⁵¹ thereby possibly activating (through integrins) FAK-p59fyn-regulated pathways.⁴⁹ Conversely, other data have supported the candidature of caveolin-1 for coordinating the signal that from PrP^C reaches Erk1/2 through p59fyn.^44,45,52 Further scrutiny of this route has shown that it comprised players such as laminin and integrins (upstream), FAK-p59fyn, paxillin and the Src-homology-2 domain containing adaptor protein (downstream), and that caveolin-1, a substrate of the FAK-p59fyn complex, facilitated the interaction of these signaling partners by recruiting them in caveolae-like membrane domains.⁵³For the relevance they bear, we need to acknowledge recent propositions supporting the commitment of PrP^C with proteins whose function is unrelated from the above-mentioned cell adhesion or ECM molecules; namely, the β-site amyloid precursor protein (APP) cleaving enzime (BACE1) and the N-methyl-D-aspartate (NMDA)-receptor. BACE1 is a proteolytic enzyme involved in Aβ production. It has been shown that overexpressed PrP^C restricted, while depletion of PrP^C increased the access of BACE1 to APP, possibly because PrP^C interacts with BACE1 via GAGs.⁵⁴ Thus, native PrP^C reduces the production of Aβ peptides. A beneficial effect of PrP^C was also highlighted by Khosravani et al.⁵⁵ showing that, by physically associating with the subunit 2D of the NMDA-receptor, PrP^C attenuated neuronal Ca²⁺ entry and its possible excitotoxic effect. This clear example for the control of PrP^C on Ca²⁺ metabolism is particularly intriguing in light of previous reports linking Ca²⁺ homeostasis to PrP^C pathophysiology (reviewed in ref. ⁵⁶). Also, it is important to mention that a few partners of PrP^C or downstream effectors may initiate signals that increase intracellular Ca²⁺, and that, in turn, local Ca²⁺ fluctuations regulate some of the afore-mentioned pathways.^11,49,57,58In conclusion, although still somehow speculative, the implication of Ca²⁺ in PrP^C-dependent pathways raises the possibility that the different input signals originating from the interaction of PrP^C with diverse partners may all converge to the universal, highly versatile Ca²⁺ signaling. Were indeed this the case, then clearly the acting of PrP^C as Harlequin, the famous character of the 18^th century Venetian playwright Carlo Goldoni, who struggles to fill the orders of two masters, would be merely circumstantial.     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.     

Photodegradation illuminates the role of polyanions in prion infectivity

Understanding the mechanism by which prion infectivity is encoded by the misfolded protein PrP^Sc remains a high priority within the prion field. Work from several groups has indicated cellular cofactors may be necessary to form infectious prions in vitro. The identity of endogenous prion conversion cofactors is currently unknown, but may include polyanions and/or lipid molecules. In a recent study, we manufactured infectious hamster prions containing purified PrP^Sc, co-purified lipid and a synthetic photocleavable polyanion. The polyanion was incorporated into infectious PrP^Sc complexes and then specifically degraded by exposure to ultraviolet light. Light-induced in situ degradation of the incorporated polyanion had no effect on the specific infectivity of the samples as determined by end-point dilution sPMCA and scrapie incubation time assays. Furthermore, prion strain properties were not changed by polyanion degradation, suggesting that intact polyanions are not required to maintain the infectious properties of hamster prions. Here, we review these results and discuss the potential roles cofactors might play in encoding prion infectivity and/or strain properties.Key words: prion, polyanion, photodegradation, incorporation, PrPThe prion diseases are infectious diseases that are believed to be caused by the conformational change of a host-encoded protein, PrP^C, to a pathogenic conformer PrP^Sc. The controversial “protein-only” hypothesis posits that the infectious agent is composed solely of the misfolded conformer PrP^Sc. There have been many attempts to create infectious prions from purified recombinant PrP protein. However, all of the samples generated in these experiments display relatively low levels of specific infectivity when inoculated intracerebrally into wild-type animals.^1–4 Several lines of evidence suggest that cellular cofactors, such as polyanionic molecules, facilitate the formation of the infectious conformation.^5–14The first in vitro PrP conversion assay used radiolabeled PrP^C substrate purified from mammalian cells mixed with a stoichiometric excess of unlabeled PrP^Sc. This cell free assay produced a protease-resistant, radioactive product termed PrP-res.¹⁵ These pioneering studies showed for the first time that PrP could be specifically transformed in vitro, but the yield using purified substrates was low. Using a modification of the cell free assay in which crude brain homogenate replaced purified PrP^C as the substrate, our laboratory was able to amplify PrP^Sc 6-fold over input prion seed, suggesting that non-PrP constituents of crude brain homogenate might be required for efficient PrP^Sc formation in vitro.¹⁶ Using this system, we discovered that nuclease treatment of hamster brain homogenates abolished PrP^Sc amplification in vitro, and that reconstituting the nuclease-treated reactions with purified mammalian RNA rescued the amplification process.⁵ PrP^Sc amplification could also be obtained by adding certain synthetic homopolymeric nucleic acids to immunopurified PrP^C.⁶ Taken together, these surprising results argue that non-proteinaceous, host-encoded cofactors such as RNA molecules might facilitate prion conversion through a structural (as opposed to encoding) mechanism.⁸ The high efficiency of the serial protein misfolding amplification (sPMCA) technique developed by Soto and colleagues has allowed researchers to amplify prion infectivity as well as PrP^Sc molecules.^17,18 Using sPMCA, we showed that infectious PrP^Sc molecules could be formed from immunopurified PrP^C, co-purified lipid and synthetic RNA molecules. Moreover, even unseeded reactions containing these defined components were capable of generating prions with high specific infectivity in a prion-free environment, showing for the first time that wild type infectious prions could be produced de novo.⁷Additional studies in this purified system showed that PrP^C molecules undergo a time-dependent conformational change upon interaction with RNA. When this change occurs, PrP^C adopts an intermediate conformation that mimics some of the characteristics of PrP^Sc, such as detergent insolubility and reactivity to PrP^Sc-specific antibodies, but remains sensitive to proteinase K digestion.⁸ When incubated with a heterogeneous size mixture of homopolymeric [³²P] poly(A) molecules during PMCA, hamster PrP^C molecules incorporated a specific size subset (1–2.5 kb) of the RNA molecules into nuclease-resistant complexes. The physical interaction between RNA and PrP^Sc was confirmed by fluorescence microscopy experiments showing that fluorescein-labeled RNA molecules became integrated into nuclease-resistant complexes with PrP^Sc molecules. Interestingly, neuropathologic analysis of scrapie-infected hamsters revealed that endogenous RNA molecules stained with acridine orange co-localized with large extracellular PrP aggregates.⁸ Taken together, these studies suggest that PrP interacts specifically with polyanionic molecules in vitro and in situ, and raised the possibility that polyanions might be a necessary component of infectious prions.Jeong et al. investigated whether endogenous RNA molecules might be required for prion infectivity by treating scrapie brain homogenates with LiAlH4 (lithium aluminum hydride), a strong reducing agent that can cleave the phosphodiester bond in RNA molecules.¹⁹ Interestingly, treatment of hamster scrapie brain homogenates with LiAlH₄ caused an ∼3-fold increase in scrapie incubation period measured by bioassay, suggesting that RNA may be an important component of infectious prions and therefore may play a role in stabilizing PrP^Sc structure. However, LiAlH₄ is not a specific reagent, and can damage a variety of other macromolecules, including proteins. Therefore, the decrease in infectivity measured in this study cannot be specifically ascribed to degradation of the polyanion.¹⁹We recently reinvestigated the potential role of polyanion in maintaining prion infectivity by using a more targeted approach.²⁰ Specifically, we utilized a synthetic oligonucleotide that could be selectively hydrolyzed by treatment with ultraviolet (UV) light. The photocleavable oligonucleotide was synthesized by inserting a photocleavable linker in between every fives bases of a poly(dT) 100-mer. Exposure to UV light quantitatively converted the oligonucleotide into five base fragments. During incubation with excess recombinant PrP, the photocleavable oligonucleotide became incorporated into a nuclease-resistant nucleoprotein complex, but remained sensitive to photocleavage. This novel system allowed us to study the role of a polyanion molecule incorporated into infectious prions in situ (Fig. 1).Open in a separate windowFigure 1Selective photodegradation of an incorporated polyanion in situ.We used PMCA to create PrP^Sc molecules that contained either the photocleavable oligonucleotide or a non-photocleavable control analog. After treatment with UV light, the infectivity of each sample was measured using a combination of end-point dilution sPMCA and animal bioassays. The end-point dilution PMCA assay showed a ∼1 log decrease in the seeding ability of PrP^Sc samples treated with UV light, but this effect was not specific since a similar decrease was measured in samples containing the control nucleic acid. In the bioassay, there was no change in the incubation periods of animals inoculated with PrP^Sc samples treated either in the presence or absence of UV light. Neuropathological analysis of inoculated animals also showed no differences in neurotropism between the two groups. Degradation of the nucleic acid had no effect on the molecular migration or structural stability of PrP^Sc samples as determined by SDS-PAGE and urea denaturation assays, respectively. There were also no differences in the molecular migration or glycosylation profile of the PrP^Sc molecules produced in the brains of animals inoculated with light- versus mock-treated inocula, and urea denaturation assays showed no differences in PrP^Sc stability. These results collectively demonstrate that the presence of intact polyanion molecules is not required to maintain the infectious, biochemical or strain properties prions generated in vitro.These results are consistent with the stringent “protein-only” hypothesis, but do not yet provide definitive proof. The purified PrP^C molecules used as substrate in these experiments contain a stoichiometric amount of co-purified lipid⁷ that may play a role in the generation of prion infectivity.⁹ Also, although the efficacy of photocleavage conditions was carefully confirmed in control reactions, it is possible that some intact oligonucleotide survived UV treatment at a level below detection. Alternatively, the remnant five base nucleic acid fragments may remain incorporated within the PrP^Sc molecule and play a role in maintaining the infectious conformation. Even in this scenario, our results would place a significant geometric constraint on the role of incorporated polyanion. While polyanions ≥40 bases facilitate the formation infectious prions in vitro,⁸ our results suggest that polyanions >5 bases are not necessary to maintain the infectious properties the prion. The exact role polyanions play in prion formation is still unclear, but it is tempting to speculate that they may serve as scaffolds that facilitate prion conversion by (a) bringing PrP^C and PrP^Sc seed together for templating to occur or (b) acting as a catalyst which is necessary to reduce the activation energy of refolding to the PrP^Sc form. Future studies will need to be performed to differentiate between these two hypotheses. It is also possible that polyanions are completely dispensable for maintaining PrP^Sc structure, and it is the co-purified lipid molecules that serve this role instead. Consistent with this possibility, we recently discovered that mouse PrP^Sc can be serially propagated in vitro in the absence of nucleic acids.²¹ Finally, it is possible that either polyanions or lipids can function equally well as stabilizers of the infectious PrP^Sc conformation. More work is required to distinguish between these possibilities.Generating high levels of specific infectivity solely using purified recombinant PrP remains the ultimate proof of the “protein-only” hypothesis. To date, evidence suggests that cellular cofactors are necessary to create infectious prions but may or may not be required to maintain infectivity once formed. Significantly, Wang et al. showed that bona fide prions could be formed from recombinant PrP, synthetic lipid and RNA molecules.⁹ Although no completely pure preparations of misfolded PrP possessing significant levels of specific infectivity have yet been produced, it should eventually be possible to produce such a preparation if the “protein-only” hypothesis is correct. On the other hand, a rigorous refutation of the hypothesis would require demonstrating that PrP^Sc and infectivity can be dissociated.     

PrP assemblies: Spotting the responsible regions in prion propagation

The “protein only” hypothesis states that the key phenomenon in prion pathogenesis is the conversion of the host protein (PrP^C) into a β-sheet enriched polymeric and pathogenic conformer (PrP^Sc). However the region of PrP bearing the information for structural transfer is still controversial. In a recent report, we highlighted the role of the C terminal part i.e., the helixes H2 and H3, using mutation approaches on recombinant PrP. The H2H3 was shown to be the minimal region necessary to reproduce the oligomerization pattern of the full-length protein. The oligomers produced from isolated H2H3 domain presented the same structural characteristics as the oligomers formed from the full-length PrP. Combining other groups'' results, this paper further discusses the relative, direct or indirect role of different PrP regions in assembly. The H2H3 region represents the core of PrP oligomers and fibrils, whereas the N terminus could explain divergences among different aggregates. Finally this review evocates the possibility to separate the domain involved in prion information transference (i.e., prion replication) from the domain bearing the cytotoxicity properties.Key words: prion, H2H3, amyloid, domain of replication, unfolding, strain, polymer, fibersTransmissible spongiform encephalopathies (TSE), fatal neurodegenerative diseases affecting humans and other mammalians, induce in most cases loss of motor control and dementia. PrP is a protein physiologically present in parts of the animal kingdom (in mammals, birds, reptiles and fishes). According to the “protein-only” hypothesis,^1,2 the key phenomenon in the pathogenesis is the conversion of the α-helix rich host-encoded PrP form (PrP^C) into a pathogenic conformer (PrP^Sc) characterized by a higher content in β-sheet and a polymeric state. The conversion to an enriched β-sheet structure is supposed to be due to the modification—induced only by a PrP^Sc-like state acting as a template- of PrP^C into the PrP^Sc conformer. This hypothesis was first proposed by Griffith in 1967³ and revisited by Lansbury et al. in 1993.⁴ The prion hypothesis has now found increasing support from experimental evidence based on the synthetic production of β-sheeted recombinant PrP which shows pathogenic properties in a wide variety of physico-chemical conditions.^5–7 However, the molecular basis of prion conversion remains unclear, especially the various structural landscape of the PrP^Sc, which is the basis of the strain phenomenon.⁸To understand the mechanisms of transfer of the structural information, two mains issues have to be addressed: (1) we need to understand which region(s) of the protein act as template for conversion and (2) what is the “pathogenic” state of this domain. In this review, we shall assume that the region bearing the infectious information for replication and the region responsible for polymerization are identical. However, the link between the propensity of a domain to form aggregates and the ability to contain the necessary information for prion replication is far from being trivial. Generally the formation of amyloid assemblies results from the aggregation of disordered peptides or in some cases from disordered regions of a folded protein.⁸ If we consider that prion replication is only supported by the globular part of PrP⁹ the currently available model involves the folded domain. Since all structural transitions need at least a partial unfolding and refolding process, pre-required structural events should be considered prior to the conversion process.     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

Antagonistic roles of the N-terminal domain of prion protein to doppel

Prion protein (PrP)-like molecule, doppel (Dpl), is neurotoxic in mice, causing Purkinje cell degeneration. In contrast, PrP antagonizes Dpl in trans, rescuing mice from Purkinje cell death. We have previously shown that PrP with deletion of the N-terminal residues 23–88 failed to neutralize Dpl in mice, indicating that the N-terminal region, particularly that including residues 23–88, may have trans-protective activity against Dpl. Interestingly, PrP with deletion elongated to residues 121 or 134 in the N-terminal region was shown to be similarly neurotoxic to Dpl, indicating that the PrP C-terminal region may have toxicity which is normally prevented by the N-terminal domain in cis. We recently investigated further roles for the N-terminal region of PrP in antagonistic interactions with Dpl by producing three different types of transgenic mice. These mice expressed PrP with deletion of residues 25–50 or 51–90, or a fusion protein of the N-terminal region of PrP with Dpl. Here, we discuss a possible model for the antagonistic interaction between PrP and Dpl.Key words: prion protein, doppel, neurotoxic signal, neurodegeneration, neuroprotection, prion diseaseThe normal prion protein, termed PrP^C, is a membrane glycoprotein tethered to the outer cell surface via a glycosylphosphatidylinositol (GPI) anchor moiety.^1,2 It is ubiquitously expressed in neuronal and non-neuronal tissues, with highest expression in the central nervous system, particularly in neurons.³ The physiological function of PrP^C remains elusive. We and others have shown that PrP^C functionally antagonizes doppel (Dpl), a PrP-like GPI-anchored protein with ∼23% identity in amino acid composition to PrP, protecting Dpl-induced neurotoxicity in mice.^4–7 Dpl is encoded on Prnd located downstream of the PrP gene (Prnp) and expressed in the testis, heart, kidney and spleen of wild-type mice but not in the brain where PrP^C is actively expressed.^4,5,8 However, when ectopically expressed in brains, particularly in cerebellar Purkinje cells, Dpl exerts a neurotoxic activity, causing ataxia and Purkinje cell degeneration in Ngsk, Rcm0 and Zrch II lines of mice devoid of PrP^C (Prnp^0/0).^4,9,10 In these mice, Dpl was abnormally controlled by the upstream Prnp promoter.^4,5 This is due to targeted deletion of part of Prnp including a splicing acceptor of exon 3.¹¹ Pre-mRNA starting from the residual exon1/2 of Prnp was abnormally elongated until the end of Prnd and then intergenically spliced between the residual Prnp exons 1/2 and the Prnd coding exons.^4,5 As a result, Dpl was ectopically expressed under the control of the Prnp promoter in the brain, particularly in neurons including Purkinje cells.^4,5 In contrast, in other Prnp^0/0 lines, such as Zrch I and Npu, the splicing acceptor was intact, resulting in normal Purkinje cells without ectopic expression of Dpl in the brain.⁴The molecular mechanism of the antagonistic interaction between PrP^C and Dpl remains unknown. We recently showed that the N-terminal half of PrP^C includes elements that might mediate cis or trans protection against Dpl in mice, ameliorating Purkinje cell degeneration.¹² We also showed that the octapeptide repeat (OR) region in the N-terminal domain is dispensable for PrP^C to neutralize Dpl neurotoxicity in mice.¹² Here, possible molecular mechanisms for the antagonism between PrP^C and Dpl will be discussed.     

Tunnelling nanotubes: A highway for prion spreading?

The discovery of tunnelling nanotubes (TNTs) and their proposed role in long intercellular transport of organelles, bacteria and viruses have led us to examine their potential role during prion spreading. We have recently shown that these membrane bridges can form between neuronal cells, as well as between dendritic cells and primary neurons and that both endogenous and exogenous PrP^Sc appear to traffic through these structures between infected and non-infected cells. Furthermore, prion infection can be efficiently transmitted from infected dendritic cells to primary neurons only in co-culture conditions permissive for TNT formation. Therefore, we propose a role for TNTs during prion spreading from the periphery to the central nervous system (CNS). Here, we discuss some of the key steps where TNTs might play a role during prion neuroinvasion.Key words: tunnelling nanotubes, TNTs, prion, PrP^Sc, prion spreading, dendritic cells, neuroinvasionPrion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that have been found in a number of species, including scrapie in sheep, bovine spongiform encephalopathy in cattle (BSE), Chronic wasting disease in deer and Creutzfeldt-Jacob, the Gerstmann-Straüssler-Scheinker syndrome, fatal familial insomnia and kuru in humans (reviewed in ref. ¹). Human TSEs can be sporadic, genetic or acquired by infection. A new variant of Creutzfeldt-Jakob disease (termed vCJD) was reported from the UK in 1996.² The majority of vCJD cases diagnosed to date resulted from a peripheral exposure via the consumption of BSE-contaminated food. Pathological features of TSE diseases can include gliosis, neuronal cell loss and spongiform changes, but the common feature of all members of this group of diseases is the build-up of an aberrant form of the host cellular protein PrP^C, named PrP^Sc (from scrapie). The normal cellular isoform, PrP^C, is an endogenous glycosylphosphatidyl inositol (GPI)-anchored protein present in numerous tissues in mammals, including neurons and lymphoid cells. While the exact function of PrP^C remains unclear, evidence suggest putative roles in neuroprotection, cell adhesion and signal transduction (reviewed in refs. ³ and ⁴). According to the ‘protein-only hypothesis,’ the causative agents of prion diseases are proteinaceous infectious particles (‘prions’), which are composed essentially of misfolded PrP^C, or PrP^Sc.^5,6 Prions replicate through a molecular mechanism in which abnormally folded PrP^Sc acts as a catalyst and serves as a template to convert normal PrP^C molecules into PrP^Sc.^5,6 PrP^Sc differs from PrP^C in the conformation of its polypeptide chain, which is enriched in β-sheets and is protease resistant. Although the conversion process is believed to have a predominant role in the pathogenesis of prion diseases, the cellular and molecular basis for the pathogenic conversion of PrP are still unknown.Another important question is how PrP^Sc spreads to and within the brain. After oral exposure, PrP^Sc accumulates into lymphoid tissues, such as the spleen, lymph nodes or Peyer''s patches, prior to neuroinvasion.^7–9 The exact mechanisms and specific cells involved in the spreading from the gastrointestinal track to the lymphoid system and to the peripheral nervous system (PNS), leading to neuroinvasion of the CNS remain to be elucidated. However, a range of evidence suggests that the accumulation of PrP^Sc within lymphoid tissues is necessary for efficient neuroinvasion.^9–11 In particular it has been shown that PrP^Sc accumulates first within follicular dendritic cells (FDCs)¹² and macrophages.¹³ FDCs are stromal-differentiated cells in the germinal centres of activated lymphoid follicles. A number of studies have demonstrated that FDCs play a critical role during spreading of infection since their absence greatly impaires the neuroinvasion process.^8,11,14,15 However, because FDCs are immobile cells, it is not clear how they acquire PrP^Sc and how it spreads from the FDCs to the PNS. FDCs and nerve synapses occupy different anatomical sites^16,17 and therefore the lack of physical contact between the gut and FDCs and between FDCs and the nerve periphery imply the presence of intermediate mechanisms for the transport of PrP^Sc. Dendritic cells (DCs) have been proposed to play a critical role in the transport of PrP^Sc from the gut to FDCs.¹⁸ DCs function as sentinels for incoming pathogens. Bone-marrow dendritic cells (BMDCs) are migratory cells that are able to transport proteins within Peyer''s patches and into mesenteric lymph nodes.¹⁹ Interestingly, mucosal dendritic cells which play a role in the transport of intestinal antigen for presentation to Peyer''s patches and to mesenteric lymph nodes, can also extend trans-epithelial dendrites to directly sample bacteria in the gut.^20,21 However, the transport of PrP^Sc from FDCs to the PNS remains controversial and evidence for a direct role of DCs during this process has been debated.^22,23 Several mechanisms have been proposed for the intercellular transfer of PrP^Sc, including cell-cell contact, transfer via exosomes or by GPI-painting.^24–26 For example, similar to other types of pathogens such as HIV-1, which was proposed to follow the “exosomal” pathway to be released from the cells,²⁷ it has been shown that the supernatant of prion infected cells contain large amount of PrP^Sc in membranous vesicles known as exosomes.^25,28 Thus, it was suggested that exosomes might be a way to spread prion infection in vivo.^25,28 Recently, a different type of vesicles known as plasma membrane-derived microvesicles, were also described as a potential spreading mechanism during neuroinvasion.²⁹In 2004, Rustom and colleagues discovered a new mechanism of long distance intercellular communication in mammalian cells, called tunnelling nanotubes (TNTs).³⁰ TNTs are transient, long, actin-rich projections that allow for long-distance intercellular communication (reviewed in refs. ^31–33). TNT-like structures have been described to form in vitro between numerous cell types, including neuronal and immune cells.^30,34,35 These studies demonstrated that TNT-like structures formed bridges or channels between distant cells that can be used to transfer material between cells, including Lysotracker positive or endosomal vesicles, calcium fluxes, bacteria or viruses through their cytoplasms or along the surface of the nanotubes.^31–33 Interestingly, a model GPI-anchored protein, GFP-GPI, was found to move at the surface of these tubes³⁴ and while studying the neuritic transport of prions in neuronal cells, Magalhães and colleagues noticed a strong correlation between internalized PrP-res and Lysotracker positive vesicles in neurites,³⁶ suggesting that PrP-res might also be able to transfer through TNTs during prion cell-cell spreading.The results from the studies mentioned above and random observations of TNT-like structures in neuronal model cell cultures first led us to study whether these structures could in fact provide an efficient mechanism for prion cell-cell spreading.³⁷ We initially characterized TNT-like structures in the mouse catecholaminergic neuronal cell line, Cath.a-Differentiated cells (CAD cells) a well-recognized neuronal cell model for prion infection.³⁸ Under our culturing conditions, over 40% of the CAD cells could efficiently form actin-rich TNT-like structures between differentially labelled cell populations. In CAD cells, these nanotubes were very heterogeneous, both in length and in diameters. Indeed, TNT-like structures had lengths ranging from 10 to 80 µm and while over 70% of the nanotubes had diameters smaller than 200 nm, the remaining TNT-like structures had larger diameters (200 to 800 nm). We demonstrated that vesicles of lysosomal origins, a fluorescent form of PrP (GFP-PrP), infectious Alexa-PrP^Sc, as well as both endogenous and exogenous PrP^Sc could traffic within TNTs between neuronal cells (Fig. 1). The lysosomal and GFP-PrP vesicles observed to move through TNTs had a directed movement with a speed in the range of actin-mediated motors,³⁷ consistent with previous studies suggesting the involvement of an actomyosin-dependent transport.³⁹ Interestingly, active transfer of endogenous PrP^Sc, lysosomal or GFP-PrP vesicles occurred through TNTs with larger diameters, suggesting distinct roles for the different TNT-like structures observed.³⁷ These results do not seem to be specific to CAD cells since the transfer of GFP-PrP throught TNTs was observed in different types of transfected cells, including HEK293 cells (unpublished data). Furthermore, these results were in agreement with previous observations by Onfelt and colleagues showing the presence of a fluorescent GPI model protein (GFP-GPI) in TNTs formed between EBV-transformed human B cells³⁴ suggesting that different GPI-anchored proteins can be transferred along the surface and inside vesicles within TNTs. In order to determine the relevance of this type of intercellular communication in the case of prion diseases, it was necessary to evaluate the trafficking of the pathological form of PrP (PrP^Sc) within TNTs, by analyzing the transfer of endogenous PrP^Sc between chronically infected ScCAD cells and non-infected CAD cells. By immunofluorescence after guanidium treatment, endogenous PrP^Sc was found inside TNTs and in the cytoplasm of recipient non-infected CAD cells. Similar to exogenous PrP^Sc, endogenous PrP^Sc particles were not present in non-infected CAD cells not in contact with ScCAD cells after overnight co-cultures, thus excluding exosomal transfer or protein shedding.³⁷ Similarly, no transfer was observed between cells in direct contact with one another or upon treatment with latrunculin, which inhibits TNT formation. Strikingly, the transfer of endogenous PrP^Sc was visible only when TNTs were present, demonstrating that in vitro, PrP^Sc can efficiently exploit TNTs to spread between cells of neuronal origin. These data suggested that TNTs could be a mechanism for prion spreading within the cells of the CNS.Open in a separate windowFigure 1Endogenous PrP^Sc transfer from ScCAD cells to CAD cells via TNTs. Endogenous PrP^Sc is found in punctate structures inside TNTs and in the cytoplasms of recipient cells. CAD cells were transfected with Cherry-PLAP (red) and co-cultured with ScCAD for 24 h. Cells were fixed, treated with Gnd and immunostained for PrP using SAF32 Ab (green). (A) Merge projection of Z-stacks obtained with a confocal Andor spinning-disk microscope. (B) Three-dimensional reconstruction of (A) using OsiriX software. (C) Zoom in on TNT-like structures. PrP^Sc is found in vesicular structures inside TNTs and in the cytoplasm of the recipient non-infected CAD cells (see blue arrow heads). Scale bar represents 10 µm.Interestingly, DCs were shown to form networks of TNTs both in vitro⁴⁰ and in vivo.⁴¹ In an elegant study, Watkins and Salter demonstrated that DCs could propagate calcium flux upon cell stimulation to other cells hundreds of microns away through TNTs, both between DCs and between DCs and THP-1 monocytes.⁴⁰ These data suggested the possibility that DCs could form tubular connections with neuronal cells in order to transport PrP^Sc to the PNS via TNTs. Using BMDCs in co-cultures with both cerebellar granular neurons (CGNs) and primary hippocampal neurons, we showed that BMDCs could form networks of TNTs with both types of neurons. Furthermore, these TNTs appeared to be functional, allowing for the transport of Lysotracker positive vesicles and infectious Alexa-PrP^Sc between loaded BMDCs and primary neurons, suggesting that DCs could transfer the infectious prion agent to primary neuronal cultures through TNTs. By using filters and conditions unfavorable for other mechanisms of transport, we found that moRK13 cells,²⁸ as well as CGNs (unpublished data), could be infected by co-cultures with BMDCs loaded with infectious brain homogenate.³⁷ Overall, these data indicate that TNTs could be an efficient mechanism of prion transmission between immune cells and neuronal cells, as well as between neuronal cultures. Since DCs can interact with peripheral neurons,⁴² we propose that TNTs could be involved in the process of neuroinvasion at multiple stages, from the peripheral site of entry to the PNS by neuroimmune interactions with DCs, allowing neurons to retrogradely transport prions to the CNS, and within the CNS (Fig. 2).Open in a separate windowFigure 2Transport of PrP^Sc via TNTs, an alternative spreading mechanism during neuroinvasion. Studies in our laboratory suggest that TNTs allow for the intracellular transport of PrP^Sc between dendritic cells and neurons and between neurons (see inset). The exact mechanism of transport remains to be determined. For instance, it is still not clear, whether PrP^Sc is strictly transported within endocytic vesicles, or whether it can slide along the surface or be transported as aggregosomes within the tubes. Similarly, the types of motors used, as well as the possible gated mechanisms to enter the recipient cells are not known. Because of the high propensity of DCs to form TNTs with different cell types, we propose that TNTs could play important roles in delivering PrP^Sc to the proper cell types along the neuroinvasion route. For instance, DCs could deliver PrP^Sc from the peripheral entry sites to FDCs in the secondary lymphoid tissues (2) or in a less efficient manner, they might occasionally directly transport PrP^Sc to the PNS (1). They could also bridge the immobile FDC networks and the PNS (3), since we have shown that DCs can form TNTs with nerve cells. Finally, once PrP^Sc has reached its final destination within the CNS, TNTs might play a final role in the spreading of PrP^Sc within the brain between neurons and possibly between neuronal cells and astrocytes (4).Recently, it was demonstrated that the distance between FDCs and the neighbouring PNS was critical for prion neuroinvasion.⁴³ Indeed, in the spleen of CD19^−/− mice, FDC networks were found to be 50% closer to the nerve fibers compared to wild-type mice.⁴³ The authors suggested that the increase in prion spreading efficiency in these mice was directly dependent on the reduction in the distance between the FDC networks and the PNS in these mice. These results would be consistent with a mechanism of transfer such as exosomes release. However, shortening the distance between FDCs and the PNS would also reduce the route of transport that mobile cells would have to travel and increase the chances for transfer of prions to the PNS, resulting in an increase in prion spreading efficiency. While the importance of FDCs in prion replication during the spreading to the CNS seems to be clear,^11,14,15 their specific role in the transfer of prions and their possible interactions with other mobile cells are much more debated.^22,23 In order to bridge the gap between FDCs and the PNS, a role for DCs as possible carriers of PrP^Sc has been postulated. Aucouturier and colleagues have previously shown, using RAG-1^−/− mice, which are deficient in FDCs, that CD11c⁺ DCs infected with 139A were able to carry prion infection to the CNS, without accumulation and replication in lymphoid organs,²² thus suggesting that DCs are able to transport prions from the periphery to nerve cells. Recently, however, another study using TNFR1^−/− mice, deficient for FDCs, suggested that DCs were unlikely candidates in the transport of prion to the PNS.²³ In this study, the authors showed that in TNFR1^−/− mice, ME7 or 139A infected DCs were inefficient in transferring infection to the PNS. The authors suggested that the differences between the results obtained with RAG-1^−/− mice and TNFR1^−/− mice could be due to the differences in the levels of innervation of the spleen in RAG-1^−/−mice compared to TNFR1^−/− mice. They suggested that in RAG-1^−/− mice, DCs could spread prion infection because their spleens are highly innervated, compared to TNFR1^−/− or wild-type mice, therefore increasing the propensity of DCs to encounter nerve cells and tranfer the prion agents. Because of the reduction in the levels of innervation in the spleens of wild-type mice, the authors concluded that DCs are unlikely candidate for the transport of prions directly to the PNS [see (1) in Fig. 2]. However, since these studies are using mice deficient for FDCs, it remains unclear what type of interactions might occur between FDCs and DCs, and how DCs might be able to transport prions from FDCs to the PNS in wild-type mice [see (2) in Fig. 2]. Indeed, both studies show that under the right circumstances, DCs can interact with nerve cells, similar to what was recently shown in infected mice⁴² and in agreement with our findings that DCs can form TNTs with neurons.³⁷ Within this scenario, it is clear that to determine the specific role of DCs during the spreading of prions from the gut to the PNS, the transfer mechanisms between DCs and other cell types, especially FDCs and peripheral neurons, need to be better characterized.Overall, these in vitro data strongly point toward TNTs as one possible mechanism of prion spreading. The next step will be to identify these structures in vivo and to determine whether prion spreading in vivo is the result of passive mechanisms, such as exosome release, active intercellular transport along and within TNTs or whether prions will use any means available to reach their targets. Recently, TNT-like structures were imaged in a mouse cornea,⁴¹ suggesting that while challenging, the visualization of in vivo trafficking of prions in lymphoid tissues such as in lymph nodes or in the spleen as well as in brain organotypic cultures might be possible and could be used to reveal the presence of TNTs.The discovery of the existence of nanotubular membrane bridges in vitro has opened-up a new field of research. Channels, called plasmodesmata,⁴⁴ connecting plant cells have long been known to play crucial roles in the transport of nutrients, molecules and signals during development and some of their functions were recently compared with some of the recently proposed functions of TNTs.⁴⁵ Furthermore, in vivo long, actin-rich filopodia like structures were found to be crucial during development.^46–49 For example, these structures exist in developing sea urchin embryos and were proposed to play a role in signalling and patterning during gastrulation.⁴⁷ Similar roles were proposed for thin filopodia-like structures observed during dorsal closure in drosophila.⁴⁹ In addition, TNT-like structures were observed in the mouse cornea between DCs and were shown to increase under inflammatory conditions.⁴¹ The authors postulated that these TNT-like structures could play a role in Ag-specific signalling, especially as a response to eye inflammation. Therefore, the possibility that TNTs might play numerous roles during cell development, in the immune system and as conduits for the spreading of pathogens could lead to major changes in the way we view animal cell interactions. Specifically, understanding how pathogens usurp these cellular connections to spread could allow for the screening and the identification of new therapeutic inhibitors. To this aim, characterizing the basic mechanism of TNT formation within cell model systems will be necessary to improve the knowledge of TNTs in general, to analyze the transfer of pathogens more specifically, and to identify key molecules during this process. In the case of prions, whether they hijack nanotubes to spread between cells or whether prions increase the formation of filopodia and TNT-like structures similar to some viruses^33,50 and/or the efficiency of transfer remain to be determined. Overall, in this specific field, the constant improvement of cell imaging techniques and the emergence of imaging tools to study prion spreading^{36,37,51–53} could lead to exciting new insights both in the physiology of these intercellular connections and in the pathology of these devastating diseases.     

Experimental Verification of a Traceback Phenomenon in Prion Infection

The clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD) correlate with the allelotypes (M or V) of the polymorphic codon 129 of the human prion protein (PrP) gene and the electrophoretic mobility patterns of abnormal prion protein (PrP^Sc). Transmission of sCJD prions to mice expressing human PrP with a heterologous genotype (referred to as cross-sequence transmission) results in prolonged incubation periods. We previously reported that cross-sequence transmission can generate a new prion strain with unique transmissibility, designated a traceback phenomenon. To verify experimentally the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into mice expressing human PrP with the 129M/M genotype. These 129M/M mice showed altered neuropathology and a novel PrP^Sc type after a long incubation period. We then passaged the brain homogenate from the 129M/M mouse inoculated with sCJD-VV2 prions into other 129M/M or 129V/V mice. Despite cross-sequence transmission, 129V/V mice were highly susceptible to these prions compared to the 129M/M mice. The neuropathology and PrP^Sc type of the 129V/V mice inoculated with the 129M/M mouse-passaged sCJD-VV2 prions were identical to those of the 129V/V mice inoculated with sCJD-VV2 prions. Moreover, we generated for the first time a type 2 PrP^Sc-specific antibody in addition to type 1 PrP^Sc-specific antibody and discovered that drastic changes in the PrP^Sc subpopulation underlie the traceback phenomenon. Here, we report the first direct evidence of the traceback in prion infection.Creutzfeldt-Jakob disease (CJD) is a lethal transmissible neurodegenerative disease caused by an abnormal isoform of prion protein (PrP^Sc), which is converted from the normal cellular isoform (PrP^C) (1, 23). The genotype (M/M, M/V, or V/V, where M and V are allelotypes) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of PrP^Sc in the brain are major determinants of the clinicopathological phenotypes of sporadic CJD (sCJD) (15-18). Type 1 and type 2 PrP^Sc are distinguishable according to the size of the proteinase K-resistant core of PrP^Sc (PrP^res) (21 and 19 kDa, respectively), reflecting differences in the proteinase K cleavage site (at residues 82 and 97, respectively) (15, 18). According to this molecular typing system, sCJD can be classified into six subgroups (MM1, MM2, MV1, MV2, VV1, or VV2).The homology of the PrP genes between inoculated animals and the inoculum determines the susceptibility to prion infection. Transmission of sCJD prions to mice expressing human PrP with a nonhomologous genotype (referred to as cross-sequence transmission) results in a relatively long incubation period (10, 12). Meanwhile, the cross-sequence transmission can generate a new prion strain. Transmission of sCJD-VV2 prions to mice expressing human PrP with the 129M/M genotype generates unusual PrP^res intermediate in size between type 1 and type 2 (10). We have designated this unusual PrP^res with an upward size shift (Sh+) from the inoculated type 2 template MM[VV2]2^Sh+ PrP^res, where the notation is of the following form: host genotype [type of inoculated prion] type of generated PrP^res.Similar to the MM[VV2]2^Sh+ PrP^res, the intermediate-sized PrP^res has been observed in the plaque-type of dura mater graft-associated CJD (p-dCJD) (10, 13). Furthermore, a transmission study using p-dCJD prions revealed that PrP-humanized mice with the 129V/V genotype were highly susceptible to p-dCJD prions despite cross-sequence transmission (10). In addition, these 129V/V mice inoculated with p-dCJD prions produced type 2 PrP^res (10). These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions to individuals with the 129M/M genotype. We have designated this phenomenon “traceback.” The traceback phenomenon was discovered for the first time by a transmission study using variant CJD (vCJD) prions (2). Mice expressing bovine PrP were highly susceptible to vCJD prions because vCJD was caused by cross-sequence transmission of bovine spongiform encephalopathy prions to human. These findings suggest that a traceback study can be a powerful tool to identify the origin of prions (2, 10, 11). However, the traceback phenomenon has not been verified experimentally despite the abundant circumstantial evidence described above.To verify the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into PrP-humanized mice with the 129M/M genotype as an experimental model of p-dCJD. Thereafter, we inoculated these MM[VV2]2^Sh+ prions into PrP-humanized mice with the 129M/M or 129V/V genotype and compared the incubation period, neuropathology, and the type of PrP^res in the brain. Here, we report the first direct evidence of the traceback in prion infection.     

Role of prions in neuroprotection and neurodegeneration: A mechanism involving glutamate receptors?

There is increasing evidence that cellular prion protein plays important roles in neurodegeneration and neuroprotection. One of the possible mechanism by which this may occur is a functional inhibition of ionotropic glutamate receptors, including N-Methyl-D-Aspartate (NMDA) receptors. Here we review recent evidence implicating a possible interplay between NMDA receptors and prions in the context of neurodegenerative disorders. Such is a functional link between NMDA receptors and normal prion protein, and therefore possibly between these receptors and pathological prion isoforms, raises interesting therapeutic possibilities for prion diseases.Key words: NMDA, NR2D, glutamate, neuroprotection, calciumPrions are most often discussed in the context of transmissible spongiform encephalopathies (TSEs) which encompass a range of neurological disorders that include human Creutzfeldt-Jakob disease (among others), sheep scrapie and bovine spongiform encephalopathy.^1,2 It is well established that these disorders arise from a progressive conversion of the normal, mainly helical form of cellular prion protein (PrP^C) into a different PrP^Sc protein conformation with a high beta sheet content.³ In their PrP^Sc form, prions act as templates that catalyze misfolding of PrP^C to produce increasing levels of PrP^Sc, which likely represents several or even many different conformational states of the same source protein, resulting in diverse clinical phenotypes. This in turn leads to accumulation of PrP^Sc deposits in the brain that can appear as aggregates and amyloid-like plaques⁴ and which disrupt normal neurophysiology.⁵ While the neuropathology of TSE''s has been explored in great detail dating back to the 1920s,⁶ less effort has perhaps been expended on understanding the cellular and physiological function of PrP^C which is ubiquitously expressed, and found even in simple organisms.^5,7,8 A number of mouse lines either lacking PrP^C or overexpressing PrP^C have been created, including the widely used Zurich I PrP^C knockout strain.^9,10 Despite the wide distribution of PrP^C in the mammalian CNS, it perhaps surprisingly has only a relatively mild behavioral phenotype that appears to include some deficits in spatial learning at the behavioral level^11,12 as well as alterations in long term potentiation at the cellular level.^13–17 In addition, it has been shown that these mice show an increased excitability of hippocampal neurons.^13,18–20 In contrast, deletion of certain parts of the PrP^C protein in vivo can have serious physiological consequences. For example, deletion of a stretch of amino acids between just upstream of the octarepeat copper binding motifs produces a lethal phenotype, that can be rescued by overexpression of increasing levels of normal PrP^C.^21,22 Of particular note, these deletion mutants show degeneration of axons and myelin, both in the CNS and in peripheral nerves; indeed some mutants show a predilection for axomyelinic degeneration with little neuronal pathology,²¹ suggesting that certain mutated forms of PrP have a direct toxic effect on oligodendrocytes and/or myelin.²³ Moreover, activation of the Dpl1 gene in mice lacking PrP^C leads to an ataxic phenotype, that is not observed in the presence of PrP^C.²⁴ Collectively, this indicates that PrP^C may act in a protective capacity and in contrast, certain abnormal forms of PrP are “toxic”, promoting much more injury to various elements of the CNS and PNS than outright absence of wild-type PrP^C.This notion is further corroborated by a number of studies in PrP^C knockout mice, both in vivo and in cell culture models. Cultured hippocampal neurons from PrP^C null mice display greater apoptosis during oxidative stress.²⁵ Moreover, overexpression of PrP^C in rats protects them from neuronal damage during ischemic stroke, whereas PrP^C null mice show greater damage.^27–29 When PrP^C null mice are subjected to different types of seizure paradigms, they showed increased mortality and increased numbers of seizures.³⁰ This increased neuronal damage can be diminished by the NMDA receptor blocker MK-801,³¹ potentially implicating glutamate receptors in this process. Finally, it was recently shown that the absence of PrP^C protein protects neurons from the deleterious effects of beta amyloid, a protein involved in Alzheimer disease.³² It is important to note that NMDA receptors have been implicated in seizure disorders and in cell death during ischemic stroke.^33–35 Indeed, our own work has shown that NMDA receptors expressed endogenously in myelin contribute to myelin damage and may be one of the first steps leading to demyelination.³⁶ Furthermore, the NMDA receptor blocker memantine is used to treat Alzheimer disease, implicating NMDA receptors. The observations above suggest that there may be an interplay between NMDA receptor activity and the physiological function of PrP^C. In support of this hypothesis, our recent work has directly identified a common functional and molecular link between NMDA receptors and PrP^C.³⁷ Brain slices obtained from Zurich I PrP^C null mice showed an increased excitability of hippocampal slices, which could be ablated by blocking NMDA receptor activity with amino-5-phosphonovaleric acid. Removal of extracellular magnesium ions to enhance NMDA receptor activity resulted in stronger pro-excitatory effects in slices and cultured neurons from PrP^C null mice compared with those from normal animals. Synaptic recordings indicate that the amplitude and duration of NMDA mediated miniature synaptic currents is increased in PrP^C null mouse neurons, and evoked NMDA receptor currents show a dramatic slowing of deactivation kinetics in PrP^C null mouse neurons. The NMDA current kinetics observed in these neurons were qualitatively consistent with NMDA receptors containing the NR2D subunit.³⁸ Consistent with a possible involvement of NR2D containing receptors, siRNA knockdown of NR2D normalized current kinetics in PrP-null mouse neurons. Furthermore, a selective co-immunoprecipitation between PrP^C and the NR2D, but not NR2B subunits, was observed. This then may suggest the possibility that under normal circumstances, PrP^C serves to suppress NR2D function, but when PrP^C is absent, NR2D containing receptors become active, and because of their slow kinetics, may contribute to calcium overload under circumstances where excessive (or even normal) levels of glutamate are present. This would include conditions such as epileptic seizures, ischemia and Alzheimer disease, thus providing a possible molecular explanation for the link between PrP^C and neuroprotection under pathophysiological conditions. Indeed, NMDA promoted greater toxicity in PrP^C null mouse neurons, and upon injection into brains of PrP^C null mice. It is interesting to note that one of the major NMDA receptor subtypes expressed in myelin is NR2D, thus bridging the observations of Micu et al.³⁶ of NMDA receptor mediated cell death in ischemic white matter, and those of Baumann and colleagues²¹ showing that PrP^C deletion mutants can cause damage to myelin.How might PrP^C deletion mutants affect neuronal survival? One possibility may be that these deletion mutants compete with normal PrP^C for NMDA receptors, but are unable to functionally inhibit them. Alternatively, it is possible that the PrP^C deletion mutants, by virtue of binding to the receptors, may in fact increase receptor activity, thus causing increased cell death. In both cases, increasing the expression of normal PrP^C would be expected to outcompete the deletion variants, thus reestablishing the protective function. A similar mechanism could perhaps apply to TSEs. It is possible that the PrP^Sc form, perhaps in a manner reminiscent of the PrP^C deletion mutants, may be unable to inhibit NMDAR function, or perhaps would even enhance it. Any excess glutamate that may be released as a result of cell damage due to PrP^Sc aggregates, or even normally released amounts glutamate during the course of physiological neuronal signaling, could be sufficient to cause NMDAR mediated cell death and neuronal degeneration. In this context, it is interesting to note that chronic administration of the weakly NR2D selective inhibitor memantine delays death as a consequence of scrapie infection in mice.³⁹ In the context of Alzheimer disease, binding of PrP^C to beta amyloid may prevent the inhibitory action of PrP^C on NMDA receptor function, thus increasing NMDA receptor activity and promoting cell death. This then may perhaps explain the beneficial effects of memantine in the treatment of Alzheimer disease.In summary, despite the fact that PrP^C is one of the most abundantly expressed proteins in the mammalian CNS, its physiological role is uncertain. Recent observations from our labs have established an unequivocal functional link between normal prion protein and the ubiquitous excitatory NMDA receptor. Thus, one of the key physiological roles of PrP^C may be regulation of NMDA receptor activity. The presence of abnormal species of prion protein, whether acquired via “infection”, spontaneous conformational conversion or genetically inherited, may in turn alter normal function and regulation of NMDA receptors, leading to chronic “cytodegeneration” of elements in both gray and white matter regions of the CNS. This key functional link between PrP and glutamate receptors may provide our first opportunity for rational therapeutic design against the devastating spongiform encephalopathies and potentially other neurodegenerative disorders not traditionally considered as TSE''s.