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On scrapie interference and artificial prions
Medical Hypotheses (2004) 63, 838–840
http://intl.elsevierhealth.com/journals/mehy
On scrapie interference and artificial prions Lucio Tremolizzoa,*, Virginia Rodriguez-Menendeza, Carlo Ferraresea,b a
Laboratory of Neurobiology, D.N.T.B., University of Milano-Bicocca, S.Gerardo Hospital, Villa Serena 4 p. Sud, via Donizetti 106, 20052 Monza (MI), Italy b Department of Neurology, S.Gerardo Hospital, University of Milano-Bicocca, 20052 Monza (MI), Italy Received 27 January 2004; accepted 15 February 2004
Summary The mechanisms responsible for neuronal death in transmissible spongiform encephalopathies (TSEs) are still not completely understood, and at least two major hypotheses have been formulated, based on the peculiar aspects of prion protein biology. In fact, the neuronal spreading of the prion conformational change may lead either to gain toxic properties, or to loose the normal function of this protein. In order to investigate the relative contribution of these two opposite mechanisms, two theoretical approaches may be proposed: RNA interference (RNAi) and artificial prion engineering. In fact, RNAi techniques offer now an extremely exciting new tool for investigating the effects of gene silencing both in prion, and other neurological disorders. On the other hand, the gain-of-toxic-function hypothesis might be definitely evaluated by creating an artificial prion choosing a protein target whose loss of function could be bypassed in the experimental set. In this paper the two aforementioned strategies are outlined, briefly discussing the consequent implications for TSE therapy. c 2004 Elsevier Ltd. All rights reserved.
On prion dualism The name prion derives from the acronym “proteinaceous infective particle”, proposed by Prusiner with the specific intention of stressing the revolutionary idea that the responsible agent of the transmissible spongiform encephalopathies (TSEs) was of proteinaceous nature, lacking of nucleic acids [1]. In spite of extensive characterization work done on this protein in the attempt to understand the pathological mechanisms of the spongiform neurodegeneration, the specific functions of the prions remain still, at least in part, matter of discussion [2]. It is conceivable that a more precise identification of the physiological role of the prion protein (or PrP) would reasonably *
Corresponding author. Tel./fax: +39-039-233-3586. E-mail address: [email protected] (L. Tremolizzo).
allow improvement in our knowledge of the pathophysiology of the TSEs, with possible implications in the therapeutic approach to these dreadful diseases. Probably the more fascinating aspect of the pathophysiology of prions is their mean of replication. Briefly, the original theory enunciated by Prusiner postulated that the prion pathological proteins are altered in the secondary structure in absence of mutations involving misreading of the primary backbone. Moreover, it is widely believed that even the alteration of the structure of few prions would lead to the following conformational change of the normal cellular PrPs, in a chain reaction culminating in the clinical presentation of the spongiform encephalopathy [3]. Two theories contend each other over the predominant pathogenic role: the hypothesis that the structurally mutated prion protein could gain a
0306-9877/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2004.02.054
On scrapie interference and artificial prions toxic function [4], and the hypothesis that the conformational change could produce a loss of the physiologic role of the cellular PrP [5,6]. Several really convincing experiments have been done, strongly supporting both hypotheses, but a definitive answer has not been formulated because of the intrinsic difficulties in obtaining a model by which studying separately the respective contribution of these two mechanisms. Here are hypothesized two theoretical approaches that would possibly help to achieve a separated evaluation of the role of these two mechanisms in the genesis of the TSEs.
To interfere or not to? According to some authors the hypothesis of the loss of function is already invalidated by the evidence that the prion protein knock-out mice apparently reaches the adult age without important neurological problems [7], although some neurodegenerative phenomena were described in old PrP knock-out mice at the cerebellar level [8]. It is reasonable, in any case, if the role of this protein is really so important for the development and the homeostasis of the CNS, and considering that the null phenotype does not result in embryonic lethality, that we might have some form of compensation from other proteins with overlapping functions, possibly less effective during aging. In fact, one of the more documented among the proposed PrP functions, is to protect neurons from oxidative stress, since these proteins possess a superoxide dismutase (SOD) activity, as well as the capacity to regulate the heavy metal homeostasis (in particular chelating copper [9]). Both these roles could be hypothetically supplied by the upregulation of analogous proteins in PrP0=0 mice. Interestingly, a similar situation is reported for SOD-1 knock-out mice that reach adult age without overt motor deficits or histological signs of pathology [10], although exhibiting increased vulnerability to axonal injury and slow decline in the number of motor units with aging [11]. In light of this data, for a definitive rejection of the loss of function hypothesis, it would be important to investigate what happens in a more stable system, characterized by a low degree of homeostatic plasticity, as the adult CNS probably is. In this case, it is possible that the abrupt loss of function of the prion protein could not be compensated, triggering, or at least contributing, to neurodegeneration. An experiment that could answer to this question consists in using antisense technology, reversibly
839 blocking the mRNA for the prion protein. Unfortunately well known limiting factors of antisense technology are represented by its intrinsic toxicity, a possible bias in the interpretation of the results. A possible bypass is now offered by the recent advancements of a technique previously wellknown in the field of invertebrate development: RNA interference or RNAi [12]. This technique consists of post-transcriptional gene silencing by injection of specific oligoriboprobes, a mechanism not fully understood but probably linked to the defense against viral attacks. In comparison with the antisenses it would offer less toxicity and no dose-dependent effect, as it should be sufficient in minimal doses to get long lasting results even far from the inoculation point. Until now an insurmountable problem was the exclusive applicability of RNAi to invertebrates, but recently Elbashir et al. [13] reported a positive result in mammalian adult cells, opening new exciting perspectives for the future evaluation of the loss of function hypothesis in the TSEs and other neurodegenerative diseases. Currently, in vitro applications for this technique are increasingly becoming a reality in the laboratories of the entire world, and RNAi conditional knock-out mice are appearing on the scene [14]. Very interestingly, Hommel et al. [15], by using this strategy to knock down tyrosine hydroxylase expression in mouse midbrain, succeeded in recreating behavioral changes reminiscent of those observed in other Parkinson’s disease animal models. In light of this premise, it is not impossible to hypothesize the creation of a prion RNAi knock-out mouse by which obtaining precious data about the mechanisms responsible for neuronal death in TSEs [16,17].
Could prions be artificial? A much more complex problem consists in addressing the definitive evaluation of the hypothesis of the toxic gain of function by the prion protein. A possible consideration stems from the fact that, curiously, the term prion describes more an attribute that can be acquired by the protein (i.e., the infectivity following the structural change), than a physiological characteristic of the protein itself. Hence, it is not impossible to hypothesize that the prion attribute could be in particular circumstances bestowed onto other proteins, rendering possible thinking about the creation of an artificial prion. This hypothetical artificial prion should be generated selecting the na€ıve peptide from a pool of proteins whose loss of function could be
840 bypassed in experimental conditions. For example, we could consider a metabolic protein, in order to prevent the eventual loss of function with the supply of the downstream reaction product. Unfortunately, the former remains a mere speculation because the realization of such an experiment is extremely complex, implying, once defined a suitable target, to be able somehow to change its structure, for example by inserting point mutations (something similar has already been done, see Wang and Hecht [18]), thus modeling familial TSEs. Once generated such an artificial prion, a crucial experiment might consist in testing its capacity of replicating itself, i.e., its transmissibility, for example in laboratory animals. This data would be extremely valuable for discussing the importance of the toxic gain of function hypothesis in sporadic prion-linked neurodegenerative disorders. However, the recent report that a neuronal isoform of the aplysia CPEB, cytoplasmic polyadenylation element binding protein, has prion-like properties, possibly involved in long-term memory associated synaptic changes [19], allows to surmise that prion conformational switch may be a physiological mechanism and not unequivocally associated to neuronal degeneration.
Conclusions If it were possible to design an artificial prion endowed of the infectivity and neurotoxicity characteristic of the PrPsc , without any trace of loss-of-function effect, the therapeutic hopes could be addressed toward the gene silencing approach, hypothetically using the RNAi like a sort of prion mRNA “vaccine”. On the contrary, the demonstration that the loss of function could be the cause of the TSEs will point out the necessity of some sort of substitutive therapy, such as antioxidant medications or eventually gene therapy or epigenetic modulation, possibly with the induction of the expression of genes with overlapping functions. Obviously, the hypothesis that the coexistence of a loss and gain of function induces, in a synergic way, the neuronal loss, can not be rejected and should be carefully considered, justifying both kind of therapeutic approaches and future studies aimed in blending both aspects of prion protein pathophysiology in a single comprehensive hypothesis.
Tremolizzo et al.
References [1] Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science 1982;216:136–44. [2] Brown DR. Prion and prejudice: normal protein and the synapse. Trends Neurosci 2001;24:85–90. [3] Brandner S. CNS pathogenesis of prion diseases. Br Med Bull 2003;66:131–9. [4] Thompson AJ, Barrow CJ. Protein conformational misfolding and amyloid formation: characteristics of a new class of disorders that include Alzheimer’s and Prion diseases. Curr Med Chem 2002;9:1751–62. [5] Wong BS, Pan T, Liu T, et al. Prion disease: a loss of antioxidant function? Biochem Biophys Res Commun 2000;275:249–52. [6] Hetz C, Maundrell K, Soto C. Is loss of function of the prion protein the cause of prion disorders? Trends Mol Med 2003;9:237–43. [7] Bueler H, Fischer M, Lang Y, et al. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 1992;356:577–82. [8] Sakaguchi S, Katamine S, Nishida N, et al. Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature 1996;380:528–31. [9] Hijazi N, Shaked Y, Rosenmann H, Ben-Hur T, Gabizon R. Copper binding to PrP (C) may inhibit prion disease propagation. Brain Res 2003;993:192–200. [10] Reaume AG, Elliott JL, Hoffman EK, et al. Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet 1996;13:43–7. [11] Shefner JM, Reaume AG, Flood DG, et al. Mice lacking cytosolic copper/zinc superoxide dismutase display a distinctive motor axonopathy. Neurology 1999;53:1239– 46. [12] Kittler R, Buchholz F. RNA interference: gene silencing in the fast lane. Semin Cancer Biol 2003;13:259–65. [13] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–8. [14] Van den Haute C, Eggermont K, Nuttin B, Debyser Z, Baekelandt V. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain. Hum Gene Ther 2003;14:1799–807. [15] Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med 2003;9:1539–44. [16] Love R. First disease model created by RNA interference. Lancet Neurol 2004;3:7. [17] Tilly G, Chapuis J, Vilette D, Laude H, Vilotte JL. Efficient and specific down-regulation of prion protein expression by RNAi. Biochem Biophys Res Commun 2003;305:548–51. [18] Wang W, Hecht MH. Rationally designed mutations convert de novo amyloid-like fibrils into monomeric beta-sheet proteins. Proc Natl Acad Sci USA 2002;99:2760–5. [19] Si K, Lindquist S, Kandel ER. A neuronal isoform of the aplysia CPEB has prion-like properties. Cell 2003;115:879–91.
http://intl.elsevierhealth.com/journals/mehy
On scrapie interference and artificial prions Lucio Tremolizzoa,*, Virginia Rodriguez-Menendeza, Carlo Ferraresea,b a
Laboratory of Neurobiology, D.N.T.B., University of Milano-Bicocca, S.Gerardo Hospital, Villa Serena 4 p. Sud, via Donizetti 106, 20052 Monza (MI), Italy b Department of Neurology, S.Gerardo Hospital, University of Milano-Bicocca, 20052 Monza (MI), Italy Received 27 January 2004; accepted 15 February 2004
Summary The mechanisms responsible for neuronal death in transmissible spongiform encephalopathies (TSEs) are still not completely understood, and at least two major hypotheses have been formulated, based on the peculiar aspects of prion protein biology. In fact, the neuronal spreading of the prion conformational change may lead either to gain toxic properties, or to loose the normal function of this protein. In order to investigate the relative contribution of these two opposite mechanisms, two theoretical approaches may be proposed: RNA interference (RNAi) and artificial prion engineering. In fact, RNAi techniques offer now an extremely exciting new tool for investigating the effects of gene silencing both in prion, and other neurological disorders. On the other hand, the gain-of-toxic-function hypothesis might be definitely evaluated by creating an artificial prion choosing a protein target whose loss of function could be bypassed in the experimental set. In this paper the two aforementioned strategies are outlined, briefly discussing the consequent implications for TSE therapy. c 2004 Elsevier Ltd. All rights reserved.
On prion dualism The name prion derives from the acronym “proteinaceous infective particle”, proposed by Prusiner with the specific intention of stressing the revolutionary idea that the responsible agent of the transmissible spongiform encephalopathies (TSEs) was of proteinaceous nature, lacking of nucleic acids [1]. In spite of extensive characterization work done on this protein in the attempt to understand the pathological mechanisms of the spongiform neurodegeneration, the specific functions of the prions remain still, at least in part, matter of discussion [2]. It is conceivable that a more precise identification of the physiological role of the prion protein (or PrP) would reasonably *
Corresponding author. Tel./fax: +39-039-233-3586. E-mail address: [email protected] (L. Tremolizzo).
allow improvement in our knowledge of the pathophysiology of the TSEs, with possible implications in the therapeutic approach to these dreadful diseases. Probably the more fascinating aspect of the pathophysiology of prions is their mean of replication. Briefly, the original theory enunciated by Prusiner postulated that the prion pathological proteins are altered in the secondary structure in absence of mutations involving misreading of the primary backbone. Moreover, it is widely believed that even the alteration of the structure of few prions would lead to the following conformational change of the normal cellular PrPs, in a chain reaction culminating in the clinical presentation of the spongiform encephalopathy [3]. Two theories contend each other over the predominant pathogenic role: the hypothesis that the structurally mutated prion protein could gain a
0306-9877/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2004.02.054
On scrapie interference and artificial prions toxic function [4], and the hypothesis that the conformational change could produce a loss of the physiologic role of the cellular PrP [5,6]. Several really convincing experiments have been done, strongly supporting both hypotheses, but a definitive answer has not been formulated because of the intrinsic difficulties in obtaining a model by which studying separately the respective contribution of these two mechanisms. Here are hypothesized two theoretical approaches that would possibly help to achieve a separated evaluation of the role of these two mechanisms in the genesis of the TSEs.
To interfere or not to? According to some authors the hypothesis of the loss of function is already invalidated by the evidence that the prion protein knock-out mice apparently reaches the adult age without important neurological problems [7], although some neurodegenerative phenomena were described in old PrP knock-out mice at the cerebellar level [8]. It is reasonable, in any case, if the role of this protein is really so important for the development and the homeostasis of the CNS, and considering that the null phenotype does not result in embryonic lethality, that we might have some form of compensation from other proteins with overlapping functions, possibly less effective during aging. In fact, one of the more documented among the proposed PrP functions, is to protect neurons from oxidative stress, since these proteins possess a superoxide dismutase (SOD) activity, as well as the capacity to regulate the heavy metal homeostasis (in particular chelating copper [9]). Both these roles could be hypothetically supplied by the upregulation of analogous proteins in PrP0=0 mice. Interestingly, a similar situation is reported for SOD-1 knock-out mice that reach adult age without overt motor deficits or histological signs of pathology [10], although exhibiting increased vulnerability to axonal injury and slow decline in the number of motor units with aging [11]. In light of this data, for a definitive rejection of the loss of function hypothesis, it would be important to investigate what happens in a more stable system, characterized by a low degree of homeostatic plasticity, as the adult CNS probably is. In this case, it is possible that the abrupt loss of function of the prion protein could not be compensated, triggering, or at least contributing, to neurodegeneration. An experiment that could answer to this question consists in using antisense technology, reversibly
839 blocking the mRNA for the prion protein. Unfortunately well known limiting factors of antisense technology are represented by its intrinsic toxicity, a possible bias in the interpretation of the results. A possible bypass is now offered by the recent advancements of a technique previously wellknown in the field of invertebrate development: RNA interference or RNAi [12]. This technique consists of post-transcriptional gene silencing by injection of specific oligoriboprobes, a mechanism not fully understood but probably linked to the defense against viral attacks. In comparison with the antisenses it would offer less toxicity and no dose-dependent effect, as it should be sufficient in minimal doses to get long lasting results even far from the inoculation point. Until now an insurmountable problem was the exclusive applicability of RNAi to invertebrates, but recently Elbashir et al. [13] reported a positive result in mammalian adult cells, opening new exciting perspectives for the future evaluation of the loss of function hypothesis in the TSEs and other neurodegenerative diseases. Currently, in vitro applications for this technique are increasingly becoming a reality in the laboratories of the entire world, and RNAi conditional knock-out mice are appearing on the scene [14]. Very interestingly, Hommel et al. [15], by using this strategy to knock down tyrosine hydroxylase expression in mouse midbrain, succeeded in recreating behavioral changes reminiscent of those observed in other Parkinson’s disease animal models. In light of this premise, it is not impossible to hypothesize the creation of a prion RNAi knock-out mouse by which obtaining precious data about the mechanisms responsible for neuronal death in TSEs [16,17].
Could prions be artificial? A much more complex problem consists in addressing the definitive evaluation of the hypothesis of the toxic gain of function by the prion protein. A possible consideration stems from the fact that, curiously, the term prion describes more an attribute that can be acquired by the protein (i.e., the infectivity following the structural change), than a physiological characteristic of the protein itself. Hence, it is not impossible to hypothesize that the prion attribute could be in particular circumstances bestowed onto other proteins, rendering possible thinking about the creation of an artificial prion. This hypothetical artificial prion should be generated selecting the na€ıve peptide from a pool of proteins whose loss of function could be
840 bypassed in experimental conditions. For example, we could consider a metabolic protein, in order to prevent the eventual loss of function with the supply of the downstream reaction product. Unfortunately, the former remains a mere speculation because the realization of such an experiment is extremely complex, implying, once defined a suitable target, to be able somehow to change its structure, for example by inserting point mutations (something similar has already been done, see Wang and Hecht [18]), thus modeling familial TSEs. Once generated such an artificial prion, a crucial experiment might consist in testing its capacity of replicating itself, i.e., its transmissibility, for example in laboratory animals. This data would be extremely valuable for discussing the importance of the toxic gain of function hypothesis in sporadic prion-linked neurodegenerative disorders. However, the recent report that a neuronal isoform of the aplysia CPEB, cytoplasmic polyadenylation element binding protein, has prion-like properties, possibly involved in long-term memory associated synaptic changes [19], allows to surmise that prion conformational switch may be a physiological mechanism and not unequivocally associated to neuronal degeneration.
Conclusions If it were possible to design an artificial prion endowed of the infectivity and neurotoxicity characteristic of the PrPsc , without any trace of loss-of-function effect, the therapeutic hopes could be addressed toward the gene silencing approach, hypothetically using the RNAi like a sort of prion mRNA “vaccine”. On the contrary, the demonstration that the loss of function could be the cause of the TSEs will point out the necessity of some sort of substitutive therapy, such as antioxidant medications or eventually gene therapy or epigenetic modulation, possibly with the induction of the expression of genes with overlapping functions. Obviously, the hypothesis that the coexistence of a loss and gain of function induces, in a synergic way, the neuronal loss, can not be rejected and should be carefully considered, justifying both kind of therapeutic approaches and future studies aimed in blending both aspects of prion protein pathophysiology in a single comprehensive hypothesis.
Tremolizzo et al.
References [1] Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science 1982;216:136–44. [2] Brown DR. Prion and prejudice: normal protein and the synapse. Trends Neurosci 2001;24:85–90. [3] Brandner S. CNS pathogenesis of prion diseases. Br Med Bull 2003;66:131–9. [4] Thompson AJ, Barrow CJ. Protein conformational misfolding and amyloid formation: characteristics of a new class of disorders that include Alzheimer’s and Prion diseases. Curr Med Chem 2002;9:1751–62. [5] Wong BS, Pan T, Liu T, et al. Prion disease: a loss of antioxidant function? Biochem Biophys Res Commun 2000;275:249–52. [6] Hetz C, Maundrell K, Soto C. Is loss of function of the prion protein the cause of prion disorders? Trends Mol Med 2003;9:237–43. [7] Bueler H, Fischer M, Lang Y, et al. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 1992;356:577–82. [8] Sakaguchi S, Katamine S, Nishida N, et al. Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature 1996;380:528–31. [9] Hijazi N, Shaked Y, Rosenmann H, Ben-Hur T, Gabizon R. Copper binding to PrP (C) may inhibit prion disease propagation. Brain Res 2003;993:192–200. [10] Reaume AG, Elliott JL, Hoffman EK, et al. Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet 1996;13:43–7. [11] Shefner JM, Reaume AG, Flood DG, et al. Mice lacking cytosolic copper/zinc superoxide dismutase display a distinctive motor axonopathy. Neurology 1999;53:1239– 46. [12] Kittler R, Buchholz F. RNA interference: gene silencing in the fast lane. Semin Cancer Biol 2003;13:259–65. [13] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–8. [14] Van den Haute C, Eggermont K, Nuttin B, Debyser Z, Baekelandt V. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain. Hum Gene Ther 2003;14:1799–807. [15] Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med 2003;9:1539–44. [16] Love R. First disease model created by RNA interference. Lancet Neurol 2004;3:7. [17] Tilly G, Chapuis J, Vilette D, Laude H, Vilotte JL. Efficient and specific down-regulation of prion protein expression by RNAi. Biochem Biophys Res Commun 2003;305:548–51. [18] Wang W, Hecht MH. Rationally designed mutations convert de novo amyloid-like fibrils into monomeric beta-sheet proteins. Proc Natl Acad Sci USA 2002;99:2760–5. [19] Si K, Lindquist S, Kandel ER. A neuronal isoform of the aplysia CPEB has prion-like properties. Cell 2003;115:879–91.