Impact of the Hydrophobic Core on PrP Function and Conversion
Dissertation
Zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) des Fachbereichs Biologie der Universität Konstanz
vorgelegt von Jens Lutz
Tag der mündlichen Prüfung: 11.07.2008 Referent: Prof. A. Bürkle
Referentin: Prof. C.A. Stürmer
Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/6124/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-61240
„Zwei Seelen wohnen, ach!
in meiner Brust“
Prion Protein; nach Johann Wolfgang von Goethe, Faust I, Vers 1112 f.
„Habe nun, ach! Pflanzenphysiologie, molekulare Toxikologie und Neurobiologie, und leider auch
Biophysik! Durchaus studiert, mit heißem Bemüh‘n.
Da steh ich nun, ich armer Tor! Und bin so klug als wie zuvor.“
Absolut anonymer Doktorand; frei nach Johann Wolfgang von Goethe, Faust I,
Vers 354 ff.
Danksagung
I
I Danksagung
„Freunde kommen und gehen – aber Feinde sammeln sich an“ Murphys Gesetz
Kein Gesetz ohne Ausnahme: Über die Jahre meines Studiums haben sich, dem Herrn sei Dank, eigentlich nur Freunde angehäuft; und ich kann mich nur bei einer „kleinen“ Auswahl all derer bedanken, die mich in diesen Jahren unterstützt haben oder die mit mir einfach nur
„eine gute Zeit“ verbracht haben:
Ich möchte mich zuerst bei meiner Familie, Klaus, Ilse und Ralf Lutz für ihre Unterstützung über die Jahre bedanken, allen voran bei meiner Mutter – nicht nur für das beste Essen der Welt…
Danke bei allen Freunden und Kommilitonen während des Studiums, v.a. bei Marco, Wolf und Sylvia, aber auch beim „harten Kern“ der Freunde in Freiburg; bei Alex, Andy, Frank, Stephan, Stephie, Tobi und v.a. bei Thorsten, der immer ohne zögern da ist, „wenn’s brennt“ – im Lexikon müsste unter „wahrer Freund“ ein Bild von ihm gezeigt werden! Ein hoch auf die Mörchen!!!
Danke allen Mitgliedern der „Fürsten-WG“; Carolina, Carsten, Dominik, Karin und Konrad für die durchaus – abwechslungsreiche – Zeit, meist jedoch geprägt von einer Atmosphäre, die sich wie das Leben in einer zweiten Familie angefühlt hat.
Vielen Dank bei dem kleinen aber feinen Häuflein an Menschen, die noch tapfer in Konstanz ausharren bzw. sich schon nach Ulm verkrümelt haben, und mit denen man so ziemlich jeden Sch… anstellen kann, die mir viel geholfen haben und denen ich das Leben auch nicht immer leicht gemacht habe! Danke v.a. an Nathalie und Jörg, unter anderem dafür, dass sie sich bereit- und freiwillig durch dieses Opus gekämpft haben und besonders an Anja. Danke Euch dreien nicht nur für unvergessliche Touren in höchsten Höhen, zwischen Lawinen, mitten unter wilden Raubtieren oder trotz leicht gehandicapter physischer Konstitution auf den größten Bergen Afrikas… Ich schmeiß‘ mich gerne auf jeden von Euch, falls mal wieder einer mit Karacho auf die Felsen zudonnert…
Danke auch an alle Mitglieder der AG Bürkle für die stets hilfsbereite, freundliche Atmosphäre; v.a. an meine Ex-„Mitbewohnerin“ Tine, mit der ich mich zusammen durch die faszinierende Welt der Prionenforschung gekämpft habe, an meine jetzt auch schon wieder Ex-„Mitbewohnerin“ Gosi – Dein nächster Job ist schon vermerkt!!! An Anna und Benni, die
II während ihres Vertiefungskurses bei mir auch zu Teilen dieser Arbeit beigetragen haben, und natürlich an den Engels-Baustein der AG, an Katharina – Aldddar, good job!!!
Besonderer Dank gilt auch Herrn Mende und den Ladies der TFA, v.a. Birgitt und Heidi, die mit mir und meinen Excel-Listen viel „Spaß“ hatten; danke für die stets freundliche und kompetente Art in „Mausdingen“.
Großer Dank gebührt natürlich Vishu, dass er mich ein halbes Jahr in seinem Labor hat werkeln lassen, mir immer hilfsbereit und kompetent zur Seite stand und somit einen entscheidenden Teil dieser Doktorarbeit erst ermöglicht hat; ich habe noch niemanden getroffen, der wissenschaftliche Brillanz und Menschlichkeit so vereint, wie er. Danke auch dem Team bei CPMC, allen voran Genia, Arie und Armin für die Unterstützung und v.a. dank an Jean. Vielen Dank auch besonders an Andrew, der mir spontan in Bangkok schon das Zimmer für SF zur Verfügung gestellt hat – wobei; er hat dank mir ja auch was nettes aus Deutschland bekommen - und an all die, mit denen ich eine tolle, prägende Zeit im Lande der unbegrenzten Möglichkeiten verbringen durfte, obwohl sie mir eines meiner bis dato Lieblingsfeindbilder kaputt gemacht haben – ja; es gibt tatsächlich auch nette, intelligente und nicht oberflächliche Amis… Danke an Aurore, Sasha, Eric und David.
Vielen Dank auch den Leuten des FLI in Tübingen für die erfolgreiche Kooperation, danke an Karina, Lothar und besonders Eli, für die stets freundliche Unterstützung; und auch an Petra und Frank von der Aguzzigruppe für die Hilfe bei neuen Methoden.
Nicht zuletzt möchte ich den Gutachtern und Prüfern meiner Doktorarbeit danken, Herrn Scheffner, Lothar und v.a. Frau Stürmer, die mir dank des Transregio-SFBs erst den Aufenthalt bei Vishu ermöglicht hat.
Zum Schluss bleibt mir noch demjenigen zu danken, der mich all die Jahre stets kompetent unterstützt hat, der trotz seines überübervollen Terminkalenders immer Zeit für meine Anliegen hatte, und dem ich schlussendlich diese Doktorarbeit verdanke:
Vielen Dank Alexander! Und weiterhin viel Spaß beim „Altern“…
Deutsche Zusammenfassung
III
II Deutsche Zusammenfassung
Das zelluläre Prionprotein (PrPC) ist ein GPI-verankertes Zelloberflächenprotein, welches an der Pathologie verschiedener neuordegenerativer Erkrankungen, wie z.B. Scrapie bei Schafen, bovine spongiforme Enzephalopathie (BSE) bei Rindern oder Creutzfeldt-Jakob-Krankheit beim Menschen beteiligt ist.
Um die fundamentalen Mechanismen von Prionenerkrankungen besser zu verstehen und gleichzeitig die physiologische Funktion von PrPC zu untersuchen, wurden verschiedene Deletionsmutanten des Prionproteins in vitro und in vivo Experimenten unterzogen. Sämtliche Mutationen waren hierbei auf den am stärksten konservierten Teil des Prionproteins, dem sogenannten hydrophoben Kern, gerichtet.
Die Deletionsmutanten zeigten eine veränderte Membrantopologie in vitro, die mit dem hydrophoben Charakter des Kerns korrelierte: Die Reduzierung der Kernhydrophobie führte zu einer Verringerung transmembranärer PrPC-Topologieformen. Dieses Ergebnis unterstreicht die bedeutende Rolle des hydrophoben Kerns für die Integrierung von PrPC in die Membran.
Die Deletionen innerhalb des hydrophoben Kerns beeinflussten nicht nur die Membrantopologie des Prionproteins; sie übten darüber hinaus auch einen entscheidenden Einfluss auf die Generierung eines proteolytischen Proteinfragmentes aus, bezeichnet als C1.
Dies deutet auf die Beteiligung des hydrophoben Kerns an der sogenannten -Spaltung des Prionproteins hin; möglicherweise als Erkennungssequenz für die beteiligten Proteasen.
Neben der Erforschung der Funktion des hydrophoben Kerns in vitro, wurden transgene Mäuse auf pathologische Veränderungen untersucht. Diese Mäuse exprimierten eine Mutante des Prionproteins, welcher der hydrophobe Kern fehlte.
Junge und erwachsene Mäuse zeigten keine erkennbaren neurologischen Symptome, wobei alte Mäuse einen ataktischen Phänotyp entwickelten, begleitet von Lähmungen der Hinterläufe und starkem Kratzen. Neben diesen pathologischen Auswirkungen der Mutante im Alter zeigte die Deletion des hydrophoben Kerns jedoch auch einen positiven Einluss auf transgene Mäuse, indem sie diese teilweise vor den letalen Effekten einer Prioninokulation in dominant-negativer Weise schützte.
Die verschiedenen Effekte, die durch die Deletion des hydrophoben Kerns ausgelöst wurden, ermöglichten die Konzipierung eines allgemeinen Modells; sowohl für die physiologische, als
IV auch für die pathologische Funktion des Prionproteins, basierend auf verschiedenen Proteinkonformationen. Diese unterschiedlichen protektiven, toxischen oder infektiösen konformationellen Formen von PrP werden hierbei durch strukturelle Veränderungen einer flexiblen Region, welcher auch der hydrophobe Kern angehört, vermittelt.
Die Rolle des Kerns als ein entscheidender Teil dieses strukturbildenden Segmentes unterstreicht nicht nur seine herausragende Stellung für die Funktion des Prionproteins; sie hebt des Weiteren die Veränderung der Proteinstruktur als ein evolutionäres Konzept hervor, welche ein- und demselben Protein mehre, sogar gegensätzliche Funktionen ermöglicht.
Table of Content
V
III Table of Content
I DANKSAGUNG...I II DEUTSCHE ZUSAMMENFASSUNG...III III TABLE OF CONTENT...V
1 SUMMARY ... 1
2 INTRODUCTION ... 2
2.1 PRION DISEASES... 2
2.2 THE PRION - AN EXCEPTIONAL FORM OF INFECTIOUS AGE ... 4
2.3 INFECTIOUS, INHERITED AND SPONTANEOUS FORMS OF PRION DISEASES ... 4
2.4 PRION PROTEIN STRUCTURE ... 6
2.5 N-GLYCOSYLATION ... 8
2.6 PRPC DISTRIBUTION... 9
2.7 PRPC METABOLISM ... 9
2.8 PRPC -CLEAVAGE ... 10
2.9 TRANSMEMBRANE FORMS OF THE PRION PROTEIN ... 12
2.10 PRION PROTEIN CONVERSION ... 15
2.11 PRPSC PROPAGATION ... 19
2.12 PRPSCDISTRIBUTION ... 22
2.13 PRPSCMIGRATION ... 22
2.14 PRION STRAINS AND SPECIES BARRIER ... 23
2.15 PRION PROTEIN FAMILY ... 25
2.16 PRION PROTEIN IN OTHER SPECIES ... 27
2.17 PRION PROTEIN DEFICIENT MICE ... 27
2.18 FUNCTIONAL ASPECTS OF THE PRION PROTEIN ... 29
2.19 SIGNAL TRANSDUCTION ... 31
2.20 DOCTORAL THESIS - OUTLINE ... 34
3 MATERIALS AND METHODS ... 36
3.1 MATERIALS ... 37
3.1.1 Oligonucleotides... 37
3.1.2 Plasmids ... 37
3.1.3 Antibodies ... 38
VI
3.1.4 Bacteria ... 40
3.1.5 Mouse strains ... 40
3.1.6 DNA / Protein ladders ... 40
3.1.7 Enzymes... 41
3.1.8 Biochemical kits ... 41
3.1.9 Chemicals ... 42
3.1.10 Equipment ... 45
3.1.11 Software ... 47
3.1.12 Buffers and solutions ... 47
3.2 METHODS ... 51
3.2.1 Basic molecular biological methods ... 51
3.2.2 In vitro topology assay/ -cleavage ... 56
3.2.3 Mouse analysis ... 58
3.2.4 Statistical analyses ... 66
4 RESULTS ... 67
4.1 ASSESSMENT OF MUTANT PRPC TOPOLOGY ... 67
4.1.1 Mutants in the hydrophobic core region of PrPC ... 67
4.1.2 Topology assay ... 68
4.1.3 Characterization of PrPC mutant topology ... 69
4.1.4 Differences in the metabolism of PrPC fragments ... 71
4.1.5 Impact of the hydrophobic core on PrPC topology and -cleavage ... 72
4.1.6 Correlations of PrPC topology with HC sequence and hydrophobicity ... 74
4.1.7 Impact of PrPC topology and -cleavage on prion disease ... 76
4.2 CHARACTERIZATION OF TRANSGENIC MICE HARBORING PRNP 114-121 ... 77
4.2.1 Transgenic mice ... 77
4.2.2 Genotyping of transgenic mice ... 78
4.2.3 Assessment of Prnp 114-121 copy number ... 79
4.2.4 Spatial distribution of PrP 114-121 ... 80
4.2.5 Expression level of PrP 114-121 ... 82
4.2.6 Tissue and cell type specific expression of PrP 114-121 ... 83
4.2.7 PrP 114-121 lacks -cleavage in vivo ... 86
4.2.8 Alterations of apoptosis-related proteins in transgenic mice ... 88
4.2.9 Pathology of aged mice ... 89
4.3 PRION INOCULATION OF TRANSGENIC MICE ... 92
4.3.1 Inhibition of prion conversion by PrP 114-121 in vivo ... 92
4.3.2 PrP 114-121 is not convertible into a PK resistant form ... 94
Table of Content
VII
4.3.3 PrPres detection was not impaired by low PrP 114-121 expression ... 95
4.3.4 Histological assessment of PrPres in the mouse brain ... 96
4.4 DIFFERENCES IN THE HISTOPATHOLOGY OF INOCULATED AND PRION FREE MICE ... 98
4.4.1 Characterization of brain pathology... 98
4.4.2 Distinctive differences in the pathology of Prnp-wt and -ko mice ... 103
4.4.3 Characteristic astrocyte expression at the corpus callosum ... 105
4.4.4 Quantitative parameters for the evaluation of brain pathology ... 107
4.4.5 PrP 114-121 does not elicit PrPSc pathology upon prion inoculation ... 109
5 DISCUSSION ... 110
5.1 THE INFLUENCE OF THE HYDROPHOBIC CORE (HC) ON PRPC METABOLISM ... 110
5.1.1 HC influences PrPC topology by its hydrophobic character ... 110
5.1.2 HC supports the integration of PrP into the lipid bilayer ... 112
5.1.3 The deletion of HC leads to the loss of PrPC -cleavage ... 115
5.1.4 The role of HC in PrPC -cleavage ... 115
5.1.5 HC exerts two independent functions on PrPC metabolism ... 117
5.2 CHARACTERIZATION OF TRANSGENIC MICE EXPRESSING PRP 114-121 ... 118
5.2.1 PrP 114-121 expression level and distribution ... 118
5.2.2 Alterations in the expression of apoptosis-related proteins ... 118
5.2.3 Involvement of Bax and Bcl-2 in the pathology of aged mice ... 119
5.3 PRION INOCULATION OF TRANSGENIC MICE ... 121
5.3.1 Dominant negative inhibition of prion conversion by PrP 114-121 ... 121
5.3.2 PrP 114-121 is not convertible into PrPSc ... 122
5.3.3 PrP 114-121 contains no infectivity ... 122
5.3.4 A model for HC function in the prion conversion process ... 123
5.3.5 Different disease phenotypes in inoculated mice ... 127
5.4 PRP CONFORMATIONS - THE BASIC CONCEPT ... 127
5.4.1 Prion toxicity and infectivity reflect different PrP conformations ... 127
5.4.2 HC mediates the transmission of toxic and protective PrP signals ... 129
5.5 PRP CONFORMATIONS – THE DECISIVE FUNCTION OF HC ... 131
5.5.1 HC switches PrP from good to evil – and beyond ... 131
5.5.2 Complex interactions of different conformations lead to PrP disease ... 134
5.5.3 Distinct pathological phenotypes by different prion conformations ... 139
5.6 FINAL CONCLUSIONS ... 140
5.6.1 PrPC – Doctor Jekyll and Mister Hyde ... 140
5.6.2 One protein, multiple conformations, various functions – an evolutionary concept ... 141
VIII
6 LITERATURE ... 145
7 ABBREVIATIONS ... 167
8 APPENDIX ... 171
8.1 PUBLICATIONS ... 171
8.2 PRESENTATIONS AT CONFERENCES ... 171
8.3 INDEX OF FIGURES ... 172
8.4 INDEX OF TABLES ... 174
Summary
1
1 Summary
The cellular prion protein (PrPC) is a GPI-anchored cell surface protein, which is involved in the pathology of various neurodegenerative disorders, such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jakob disease (CJD) in man.
In order to better understand the underlying mechanisms of prion diseases, as well as to further elucidate the physiological function of PrPC, a set of micro-deletions within the most highly conserved regions of PrPC, termed the hydrophobic core (HC), was applied in different in vitro and in vivo experiments.
The deletion mutants exhibited altered membrane topology in an in vitro translation assay, correlating with the hydrophobic character of HC: The reduction of HC hydrophobicity resulted in a decrease of transmembrane PrPC forms, indicating that HC promotes PrPC membrane integration. Micro-deletions within HC did not only affect transmembrane topology, they also exerted a substantial influence on the generation of a proteolytic fragment, termed C1, which points to the involvement of HC in PrP cleavage, possibly functioning as the recognition site for proteases.
In addition to the assessment of HC function in vitro, transgenic mice, expressing a prion protein mutant, which lacks HC, were examined for pathological alterations.
Young and adult transgenic mice did not show overt neurological symptoms, whereas aged mice developed an ataxic phenotype, accompanied by hind-limb paresis and itching.
Furthermore, the deletion of HC protected from prion inoculation in a dominant negative manner, confirming previous cell culture results (Hölscher et al., 1998).
The various effects of HC deletion observed in this study led to a model for the physiological as well as the pathological function of the prion protein, based on different PrP conformations. These different protective, toxic or infectious conformational states of the protein are mediated by structural alterations of a flexible region, encompassing HC. The role of HC as a decisive part of this conformation determining segment not only underlines its pivotal impact on PrP function but also highlights the change in protein structure as an evolutionary concept, which enables one protein to exert multiple, even opposing functions.
2
2 Introduction
2.1 Prion diseases
Prion diseases, also referred to as transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders affecting numerous mammalian species including humans. In terms of pathogenesis, there are three different types of prion diseases: Besides infectious forms as for example scrapie in sheep, there are heritable forms of prion diseases (i.e., Gerstmann-Sträussler-Scheinker disease [GSS] in humans) and forms that occur sporadically without any infectious or familial background (i.e., sporadic Creutzfeldt-Jakob disease [sCJD] in humans).
Differences in the disease pattern of affected individuals, e.g., vacuolation of different brain areas or varying clinical symptoms, led to a classification system of prion disorders (Table 1).
In humans, prion diseases produce symptoms of dementia, loss of memory, motor dysfunctions such as cerebellar ataxia, or sleep disturbances. In sporadic and inherited forms, these symptoms occur mainly in late adult life and last from months (CJD, fatal familial insomnia [FFI], kuru) to years (GSS), leading invariably to death (Harris, 1999).
The pathophysiological changes that lead to the characteristic neurological symptoms are mainly manifested by vacuolation of the neuropil (i.e., the axon-synapse-dendrite complex between nerve cell bodies) in the grey matter of the brain (DeArmond et al., 1987) accompanied by apoptotic cell death of neurons. Vacuolation and neuronal loss finally lead to a spongy appearance of the affected brain area, associated with activation and proliferation of glial cells, mainly astrocytes (astrocytic gliosis) (Prusiner, 2001).
With increasing numbers of neurons undergoing cell death, the function of the affected brain areas gets more and more impaired, leading to the typical neurological symptoms such as dementia and ataxia. When the disease process finally strikes vital regions, the individual dies.
In most cases, these neuropathological changes are accompanied by the accumulation of intra- and extracellular deposits of an abnormal isoform of the normal cellular prion protein (PrPC),forming aggregates in the central nervous system (CNS) comparable to the amyloid plaques in Alzheimer’s disease (DeArmond et al., 1985; Meyer et al., 1986; Price et al., 1993).
Introduction
3 This abnormally folded isoform of PrPC, termed PrPSc, is postulated to be the infectious agent in TSEs (Prusiner, 1982).
Disease Host Mechanism of pathogenesis
Kuru Fore people, Papua New
Guinea
Infection through ritualistic cannibalism
Iatrogenic Creutzfeldt-Jakob-disease (iCJD) Humans Infection via prion-contaminated growth
hormone preparations, dura mater grafts, etc.
Variant CJD (vCJD) Humans Infection with bovine prions
Familial CJD (fCJD) Humans Germ line mutations in PRNP
Gerstmann-Sträussler-Scheinker-Syndrome (GSS)
Humans Germ line mutations in PRNP
Fatal familial insomnia Humans Germ line mutations in PRNP
Sporadic CJD (sCJD) Humans Somatic mutation or spontaneous
conversion of PrPC into PrPSc?
Scrapie Sheep Infection of genetically susceptible sheep
Bovine spongiform encephalopathy (BSE) Cattle Infection with prion-contaminated meat
and bone meal (MBM)
Transmissible mink encephalopathy (TME) Mink Infection with prions from sheep or cattle
Chronic wasting disease (CWD) Mule deer, elk Unknown
Feline spongiform encephalopathy (FSE) Cats Infection with prion-contaminated beef
Exotic ungulate encephalopathy Greater kudu, Nyla, Oryx Infection with prion-contaminated MBM Table 1: Different types of transmissible spongiform encephalopathies
Modified from: (Prusiner, 1997)
4
2.2 The prion - an exceptional form of infectious age
The nature of the prion particle and its resultant diseases is remarkable in many aspects. After a long and controversial debate, the hypothesis of Stanley B. Prusiner is today the most widely accepted explanation for the pathogenesis of transmissible spongiform encephalopathies, regarding the so-called prion as the cause of these kinds of disorders:
The prion is characterized as “a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids” (Prusiner, 1982). A prion is thereby defined as one unit of infectious agent that causes a prion disease. Given that a 10-9 dilution of brain homogenate from a scrapie sick animal is sufficient to cause disease upon inoculation in a healthy animal, the prion titer of this brain is 109.
This prion unit is considered to be a misfolded form (PrP scrapie or PrPSc) of the cellular prion protein (PrPC), replicating itself by converting the normal prion protein (PrPC) into PrPSc. PrPSc might not only be the infectious agent but could also possess some toxic properties at the same time. Its ability to transmit an infectious disease as a misfolded protein represents a novel type of infectious agent that differs from the conventional nucleic acid-containing infectious agents like bacteria, viruses, viroids or plasmids (Harris, 1999; Prusiner et al., 1998).
2.3 Infectious, inherited and spontaneous forms of prion diseases
In the infectious forms of prion diseases, the misfolded prion particle (PrPSc) is transmitted from one individual to another, e.g., by food intake, whereupon it converts the normal cellular isoform of the prion protein (PrPC) into PrPSc (Huang et al., 1996a; Prusiner, 1982). The question arises, however, how this conversion process is initiated in inherited and sporadic types of TSEs where no infectious particle has been introduced into the organism.
The inheritable forms of prion diseases have their origin in a variety of mutations of the prion protein gene (PRNP). Over 50 point mutations in PRNP are known to be casually involved in dominantly inherited forms of prion diseases like familial CJD (fCJD), GSS or FFI (Wadsworth et al., 2003).
Four mutations have been genetically linked to GSS: One of these missense mutations replaces proline with leucine at codon 102 (P102L) (Hsiao et al., 1989). The three others take place at codon 117, where alanine is replaced with valine (A117V); at codon 198, where
Introduction
5 phenylalanine is substituted with serine (F198S); and at codon 217, where glutamine is replaced with arginine (Q217R) (Dlouhy et al., 1992; Hsiao et al., 1989; Hsiao et al., 1992;
Tranchant et al., 1992).
In fCJD, mutations occur at codon 200, where glutamate is changed to lysine (E200K), and at codon 178, where aspartic acid is substituted with asparagine (D178N) (Gabizon et al., 1993);
(Bosque et al., 1992). Deletions or insertions in the octapeptide repeat region of PrP are also linked to fCJD and to FFI. One example is a 144 bp insert at codon 53, resulting in six octapeptide repeats in addition to the normal five (Baker et al., 1991; Collinge et al., 1992;
Hsiao et al., 1989).
In some subtypes of fCJD and FFI, a polymorphism decides which kind of prion disease will develop: Methionine at codon 129 determines FFI whereas the amino acid valine results in fCJD, while both diseases have a mutation at amino acid 178 (D178N) in common (Goldfarb et al., 1992).
In genetic and sporadic disease, the major issue is how the first PrPSc-like moleculecan form, which would then be able to replicate by converting PrPC into PrPSc.
Several lines of evidence suggest that PrPSc is thermodynamically more stable than its physiological form but a high kinetic barrier prevents the spontaneous conversion from PrPC to PrPSc. Mutations that lower this barrier might lead to a spontaneous change into a more stable PrPSc-like conformation. Once this form has been created, the self-replication of PrPSc can proceed as in the infectious diseases.
Interestingly, patients suffering from fCJD who are heterozygous for the point mutation E200K in PRNP, display a wt-form of PrP that is detergent soluble but protease resistant, resembling an intermediary conformational form of prion protein, termed PrP* (Gabizon et al., 1996).
The sporadic forms of prion diseases might be the result of an extremely rare spontaneous PrPC => PrPSc conversion or of somatic mutations that have the same effect as the inherited types by lowering the kinetic barrier and therefore enabling a spontaneous conversion of PrPC into a pathologic conformation (Telling et al., 1995; Telling et al., 1996).
6
2.4 Prion protein structure
Human PrP, consisting of 253 amino acids (aa), is the product of a single gene (PRNP) comprising two exons of which only the second harbors the coding region. It is located on chromosome 20 (Liao et al., 1986). Mouse PrP consists of 254 amino acids; its gene (Prnp) is located on chromosome 2 and consists of 3 exons, of which exon 3 contains the coding region (Basler et al., 1986; Oesch et al., 1985; Sparkes et al., 1986).
The prion protein is composed of an N-terminal signal peptide (aa 1-22), directing it into the endoplasmic reticulum (ER) (Basler et al., 1986; Turk et al., 1988), followed by five glycine- and proline-rich octapeptide repeats [P(Q/H)GG(T/G/S)WGQ] which are able to bind metal ions, especially copper; a stop transfer effector domain (STE) preceding a highly conserved transmembrane domain (TM); a disulfide bridge between cysteines 179 and 214 and two sites of glycosylation, with N-linked oligosaccharide chains at asparagine residues 180 and 196 (Caughey et al., 1989; Rivera-Milla et al., 2006; Schatzl et al., 1995; Wopfner et al., 1999) (Figure 1a).
During protein processing on its way through the ER and the Golgi, the signal peptide is cleaved off from the N-terminus, the protein gets glycosylated, the disulfide bridge forms, and a glycosyl-phosphatidylinositol-(GPI) anchor is attached to the C-terminus after cleavage of the hydrophobic carboxy-terminal segment. Upon fusion of trans-Golgi network vesicles with the plasma membrane, PrP is found linked to the outer leaflet of the plasma membrane by its GPI-anchor (Basler et al., 1986; Harris, 1999; Oesch et al., 1985; Stahl et al., 1987) (Figure 1b).
Introduction
7
Figure 1: Prion protein structure/ processing (a) Structural features of PrPC. Modified from: A. Bürkle
(b) During its way to the plasma membrane, nascent prion protein gets modified in the ER and Golgi and is finally attached to the outer leaflet of the cell membrane by a GPI-anchor.
From: (Harris, 1999)
Depending on the species, the status of protein processing and the mode of glycosylation, the size of the prion protein ranges between 25 kDa (N-terminus cleaved, unglycosylated), 28 kDa (full-length, unglycosylated) up to 41 kDa (di-glycosylated with long carbohydrate chains).
Extraction of the endogenous murine protein normally yields a size of 33-35 kDa (mono- and di-glycosylated forms of PrP) (Basler et al., 1986; Caughey et al., 1989).
Analysis of the PrPC structure by nuclear magnetic resonance (NMR) revealed a protein conformation consisting of three -helices and an anti-parallel β-sheet whereby the N- terminal end (aa 23 to 121) did not feature any specific structural motives (Donne et al., 1997;
James et al., 1997; Zahn et al., 2000) (Figure 2).
One of the most highly conserved parts of PrP is the region of the hydrophobic transmembrane domain (aa 111-134), encompassing the palindromic amino acid sequence AGAAAAGA, also termed the hydrophobic core (HC), which is implicated in possible functional properties of PrPC (see Chapters 1.16, 1.20). A point mutation within this region at codon 117 (A117V) causes GSS in humans (Hegde et al., 1998a).
8
Figure 2: Structure of PrPC
Three-dimensional illustration of amino acids 23-230 of the human prion protein (without N-terminal signal peptide). The three -helices are designated as α1 (residues 144-154), α2 (173-194) and α3 (200-228). The anti- parallel β-strands are displayed as blue arrows consisting of amino acid residues 128-131 and 161-164. Amino acids 23-121 show a flexible, disordered structure.
From: (Zahn et al., 2000)
2.5 N-Glycosylation
Both PrPC and PrPSc carry polysaccharide chains that are attached to specific glycosylation sites. The two-step glycosylation process begins with an N-linked core glycosylation at the two consensus sequences at asparagines 180 and 196 in the ER. On their way to the cell surface, the core glycans are further processed into complex-type glycans in the ER and Golgi. This process is in part controlled by the first -helix and the C-terminal GPI-signal (Winklhofer et al., 2003).
Loss of glycosylation either by mutations or by incubating cells with tunicamycin revealed PrPSc -like features of PrPC and a higher susceptibility for conversion into PrPSc, suggesting that during maturation, PrPC has a tendency to acquire PrPSc -like features and that glycosylation has a protective effect against this tendency, preventing PrPC from getting misfolded (Lehmann & Harris, 1997). Further evidence for protection by the polysaccharide chains comes from an inherited form of spongiform encephalopathy where a threonine to alanine substitution at codon 183 (T183A) results in the loss of one glycosylation site (Nitrini et al.,
Introduction
9 1997). Mutants that lack glycosylation exhibit enhanced apoptosis in cell culture by altering the expression of apoptosis related proteins (Chen et al., 2007). In addition, diverse types of prion strains (see Chapter 1.12) differ in their oligosaccharide chains, thus glycosylation can serve as a molecular marker, for example to classify different variants of sCJD (Parchi et al., 1996).
2.6 PrP
Cdistribution
PrPC is primarily found in the central nervous system (CNS) and to a smaller extend in heart, lung, spleen, kidney, adrenal, ovary, testis, skeletal muscle, intestinal tract, lymphoid organs and in the blood but not in the liver. Within the brain, PrPC occurs mainly in the hippocampus but also in other brain areas such as cortex, the caudate nucleus, thalamus, brain stem, the septal nuclei or in the Purkinje cells of the cerebellum and, less prominent, in motor neurons of the spinal cord (Bendheim et al., 1992; DeArmond et al., 1987; Paltrinieri et al., 2004;
Rocchi et al., 2007).
PrPC is expressed to the highest extent in neurons of the CNS where it is widely distributed on the neuronal plasma membrane (Hetz et al., 2003a; Kretzschmar et al., 1986; Mironov et al., 2003). It can be axonally transported to nerve terminals (Borchelt et al., 1994) where it is mainly localized in cholesterol-rich cavaeolae-like microdomains of the cell membrane (Gorodinsky & Harris, 1995; Harris, 1999; Taraboulos et al., 1995).
2.7 PrP
Cmetabolism
As mentioned above, PrPC travels via the ER and the Golgi, where it is post-translationally processed by the addition of a GPI-anchor and the glycan chains, to so-called “lipid rafts” on the cell membrane (Figure 3). After 3-6 h, it is taken up into endosomal compartments and rapidly degraded in lysosomes whereas PrPSc accumulates in the cell (Borchelt et al., 1990;
Caughey et al., 1989; Peters et al., 2003; Prado et al., 2004). During its normal metabolism, PrPC can be cleaved at two different sites, one being located within the GPI-anchor and the other within the highly conserved hydrophobic domain. Cell culture experiments suggest that PrPC constitutively travels between the cell membrane and endosomal compartments. During
10 this cycle, approximately 5% of PrPC molecules undergo C- or N-terminal cleavage. Both full- length PrPC and the N-terminally truncated form are recycled to the plasma membrane.
These observations hint to an involvement of the prion protein in uptake mechanisms or adhesion processes of the cell. The cleaved 10-kDa N-terminal fragment, resulting from PrP cleavage, is proposed to have signaling function as a released ligand (Checler & Vincent, 2002; Chen et al., 1995; Harris et al., 1993; Shyng et al., 1993).
Figure 3: Prion protein trafficking
Nascent prion protein gets processed on its way to the plasma membrane in ER and Golgi. After several hours on the cell surface, PrPc is endocytosed and may then be recycled to the cell surface.
During PrPC trafficking, the protein can be truncated by -cleavage, resulting in an N-terminal soluble fragment (N1; dark box) and a C-terminal, GPI-anchored segment (white box; C1).
From: (Harris, 1999)
2.8 PrP
C-cleavage
During its normal metabolism, mature PrPC can be cleaved at two sites, i.e., -cleavage at amino acids 89/90 or -cleavage at position 110/111 or 111/112, resulting in small N-terminal fragments (N1 or N2, respectively) and larger C-terminal fragments (C1 or C2, respectively) (Chen et al., 1995). These cleavages are reminiscent of cleavage events in the Alzheimer precursor protein (APP), and it has thus been hypothesized that similar mechanisms may be triggered by this cleavage in Alzheimer and prion disease (Figure 4). In Alzheimer’s disease, - cleavage, together with subsequent cleavage by -secretase, leads to the formation of
Introduction
11 neurotoxic A -peptide. The -cleavage of APP however leads to the destruction of A - peptide and to the release of the N-terminal APP-fragment acting as neuroprotective and trophic factor (Checler & Vincent, 2002). The site for PrPC -cleavage resides in the region crucial for PrPC => PrPSc conversion (Kaimann et al., 2008; Peretz et al., 1997). Furthermore, the toxic PrP fragment 105-125 also comprises this site. Thus, -cleavage of PrPC might protect from the generation of pathologic PrPSc and/ or destroy the toxic properties comprised in the hydrophobic core region of PrP, which is substantiated by the observation that PrPSc molecules of sCJD and GSS patients still comprise an intact C1-cleavage site (Jimenez-Huete et al., 1998). Interestingly, the same proteases of the ADAM (a disintegrin and metalloproteinase) family of zinc metalloproteases, notably ADAM10 and TACE (ADAM17) are responsible for both, cleavage of APP as well as of PrP (Vincent et al., 2001), and PrPC, localized in lipid rafts, enhances -cleavage of APP, thus preventing the formation of toxic A (Parkin et al., 2007), whereas ectopic expression of C1 in cell lines can lead to a higher susceptibility to staurosporine induced apoptosis, mediated by the p53/ caspase-3 pathway (Sunyach et al., 2007).
Figure 4: Protein -cleavage in PrP and APP
Cleavage of both PrPC and APP is performed by the same proteases, suggesting a common mechanism by preventing the formation of toxic fragments, such as A or the conversion into PrPSc.
Modified from: (Checler & Vincent, 2002)
12
2.9 Transmembrane forms of the prion protein
The vast majority of prion protein molecules are translocated in the ER lumen and then attached to the outer leaflet of the plasma membrane by the GPI anchor. This topology is designated secretory (“sec”) topology (secPrP). In vitro-translation assays revealed however, that PrPC can in addition adopt a transmembrane topology, where the protein is incorporated in the ER-membrane via its hydrophobic domain comprising amino acid residues 111-134 (TM).
In its transmembrane form, PrPC can be inserted in the ER membrane with either its N- terminus (= type I transmembrane protein; NtmPrP) or its C-terminus (= type II; CtmPrP) being translocated in the ER-lumen (Ntm/ Ctm topology) (Figure 5).
In cells expressing wild-type PrPC (PrP-wt), over 90% of the cellular prion protein is secPrP.
Figure 5: Membrane topologies of the prion protein
PrPC is present in the cell in different topological forms. Besides its exclusively GPI-anchored form, which is secreted in the ER-lumen and afterwards attached to the outer leaflet of the plasma membrane (sec), the prion protein can also be generated with the TM-1 domain (grey box) being anchored in the membrane, with either its N-terminus (Ntm) or its C-Terminus (Ctm) directed into the ER-lumen.
From: (Stewart & Harris, 2001)
Besides TM, a 24 amino acid hydrophilic domain (STE= stop transfer effector) preceding the transmembrane domain (Yost et al., 1990) and the N-terminal signal sequence play a crucial role in determining the different topologies of the prion protein. Mutations in these domains significantly alter the ratio of the different PrPC topologies.
Introduction
13 A mutation within the signal peptide for example, replacing leucine with arginine at codon 9 (L9R), combined with 3 substitutions of alanine into valine in the TM domain (A112V, A114V, A117V), resulted in 90% CtmPrP. By contrast, other mutations in the signal peptide resulted in no transmembrane forms at all (Hegde et al., 1998a; Stewart & Harris, 2003).
A combination and competition of the signal peptide, STE and TM on the translocon channel of the ER seems to direct PrPC in one of the three different topological forms (Figure 6):
The N-terminal signal has two functions. Firstly, it is responsible for the targeting of the nascent protein to the ER-translocon channel by binding to the signal recognition particle (SRP). Secondly, it directs the N-terminus in the ER-lumen, thus favoring either NtmPrP or
SecPrP topology. The positive STE on the other hand competes with the signal peptide for the C-terminus to be translocated to ER-lumen (“positive inside rule”), therefore favoring either the Ctm topology or cytosolic PrPC, whereas intrinsic properties of TM stabilize Ntm or Sec topology. Furthermore, it is responsible for the incorporation of PrPC into the cell membrane (Kim et al., 2001; Kim & Hegde, 2002; Ott et al., 2007).
Whereas the N-terminus and STE/TM mainly determine the different topology forms, C- terminal signals contribute to the initiation of the translocation into the ER-lumen. Prion proteins lacking the N-terminus plus STE/TM or just the N-terminal signal peptide are still processed post-translationally in the ER-lumen, mediated by the GPI-signal peptide, thereby acquiring Ctm-topology (Gu et al., 2008; Hölscher et al., 2001), and mutations, linked with human inherited prion diseases that lead to C-terminally truncated PrP (W145Stop;
Q160Stop), show a reduced translocation of PrP in the ER-lumen. This non-translocated PrP, residing in the cytosol, might exhibit toxic effects, ultimately leading to cell death (Heske et al., 2003; Winklhofer et al., 2003).
Furthermore, the determination of prion protein topology is not only dependent on different signal domains, but requires also additional factors of the translocation machinery. Ablation of some of these translocation accessory factors (TrAFs) leads, for example, to an exclusive generation of CtmPrP (Hegde et al., 1998b).
14
Figure 6: Determination of PrPC topology at the ER-translocon channel
The nascent prion protein is directed by its N-terminal signal sequence (white box) to the translocon channel of the ER. The competition between the N-terminal signal peptide (SP), STE and TM (grey box) on the channel decides whether the N-terminus is directed into the ER-lumen or whether it stays outside in the cytosol. The positively charged STE favors the reorientation of the downstream TM and the translocation of TM with its C- terminus into the lumen of the ER, leading to CtmPrP or cytosolic PrP, whereas intrinsic features of TM and SP plus accessory factors of the translocon machinery counteract TM reorientation, thus driving PrP into Sec or Ntm conformation.
From: (Ott et al., 2007)
Interestingly, imbalances in the ratio of the different topological PrP-forms have been implicated in different inherited prion diseases. The A117V mutation in GSS for example, seems to be associated with the generation of PrPC in the Ctm topology, as brains from GSS patients show increased amounts of CtmPrP. Intriguingly, no PK-resistant prion protein has been detected in these brains, pointing to an additional, non-transmissible way of prion pathophysiology without PrPSc involvement (Hegde et al., 1998a; Hegde et al., 1999).
Therefore, PrPSc might be the infectious but not the toxic agent, eliciting either a toxic signal cascade or leading to the increase of another toxic molecule.
CtmPrP has been proposed to be a possible toxic intermediate (Hegde et al., 1999). The transit of CtmPrP to the cell surface is delayed compared to PrP-wt. It accumulates in the ER where it is recognized by the ER-quality control as an incorrect polypeptide, leading to its degradation by the cytosolic proteasome (ER-associated degradation; ERAD) and possibly to the induction of cellular stress pathways that may drive the cell into apoptosis (Drisaldi et al., 2003; Stewart et al., 2001). However, the idea of CtmPrP or cytosolic PrP being the actual toxic agent has been challenged by findings of Harris and colleagues, arguing for other as yet unidentified molecules besides PrPSc to play a crucial role in prion pathogenesis (Stewart & Harris, 2003).
Introduction
15 Transgenic mice, expressing a mutant that leads to enhanced CtmPrP levels exhibit ataxia and hind-limb paresis together with the pronounced loss of granular cells in the cerebellum and the hippocampus, whereas CtmPrP does not reach the plasma membrane but rather accumulates in the Golgi. Interestingly, CtmPrP needs the co-expression of PrP-wt in order to exhibit its toxic effect (Stewart et al., 2005). In addition, the CtmPrP-favoring mutation AV3 leads, like Dpl, to apical instead of normally basolateral localization of PrP in Madin-Darby canine kidney (MDCK) cells, implying that CtmPrP-mediated toxicity might result from PrP missorting (Uelhoff et al., 2005).
2.10 Prion protein conversion
A hallmark of most TSEs is the conversion of the physiological PrPC into its pathophysiological form, PrPSc. This process either occurs on the outer leaflet of the plasma membrane (Paquet et al., 2007) or intracellularly, after endocytosis of PrPSc (Peters et al., 2003). In cell culture experiments, the interaction between the cellular and the scrapie form of PrP is thereby dependent on accessory factors such as the laminin receptor and heparan sulfates (HS) (Figure 7). These factors directly bind to PrP and mediate the conversion as well as possibly the internalization of PrPSc into the cell (Gauczynski et al., 2001b; Horonchik et al., 2005;
Hundt et al., 2001; Paquet et al., 2007).
Figure 7: Prion conversion, assisted by co-factors
The conversion of PrP is mediated by accessory factors, such as heparan sulfates (grey), expressed by proteoglycans (green). These glycosaminoglycans bind both PrP conformations, thereby supporting PrPC and PrPSc interaction during the conversion process and also prion uptake into the cell.
From: (Horonchik et al., 2005)
16 During the conversion of PrPC into PrPSc, a portion of the -helices and the coil structure is refolded into β-sheets (Figure 8). Whereas PrPC consists of 42% α-helices and only 3% β- sheets, PrPSc is composed of 30% α-helical and 43% β-sheet structures (Pan et al., 1993).
Figure 8: Structural changes during the prion conversion process
Upon prion conversion, PrPC (a), mainly consisting of helical content, adopts a predominantly helical structure, becoming PrPSc (b). It should be noted that the structure shown in panel b is hypothetical.
From: http://www.sciscape.org/articles/madcow/
These structural changes render the protein partially resistant to proteinase K (PK) digestion, leading to an indigestible fragment of 27-30 kDa (= PrP 27-30). By contras the first 67 amino acids of the N-terminus remain digestible by proteinase K (Basler et al., 1986; Bolton et al., 1984; Oesch et al., 1985). The change in the tertiary structure of PrP is also responsible for other PrPSc-specific properties like detergent insolubility or loss of accessibility of the GPI- anchor to phosphatidylinositol-specific phospholipase C (PI-PLC) (Castle et al., 1987; Harris, 1999).
Of particular significance seems to be the folding of an N-terminal unstructured region between amino acids 90-120, comprising STE and HC, into β-sheet structure (Huang et al., 1996b; Peretz et al., 1997). Deletions in this region abrogate the convertibility of PrPC, and when overexpressed in cell culture, deletion mutants of the hydrophobic core exhibit trans-
Introduction
17 dominant inhibition of PrPSc accumulation in vitro (Hölscher et al., 1998; Norstrom &
Mastrianni, 2005). The importance of this region for PrP conversion is also supported by a truncated protein version, consisting of aa 105-125, which features PrPSc like properties (Singh et al., 2002).
This structural flexible hydrophobic core region may adopt short-lived intermediate conformations, which need further environmental influences in order to be stabilized (Liu et al., 1999). One possible intermediate conformation is the formation of -helical content between amino acids 90-124, including the hydrophobic core, which is maintained by the dimerization of two PrPC molecules (Kaimann et al., 2008). Due to its structural flexibility however, this region does not remain in one conformation, it rather swaps from a structured, -helical state into a variable, partially denatured state back and forth. The partial denaturing of the N-terminus turns PrPC into a preamyloid state, consequently lowering its activation barrier, which facilitates the conversion into a more stable -sheet structure (Stohr et al., 2008). This structural conversion of a predominantly -helical PrPC into a -sheet rich PrPSc needs two major steps: First, PrPC has to bind to a PrPSc molecule. This interaction leads in a second step to a structural change of PrPC resulting in the formation of a new PrPSc molecule.
These two steps could either occur at the same site, or PrPSc binding and the actual conversion might take place at two distinct regions of PrPC (Horiuchi et al., 2000). Fragments of PrP, inhibiting PrPSc binding downstream of HC, in combination with antibody binding and PrP-deletion mutant experiments, suggest that the first step, PrPSc binding, occurs not directly to but in the vicinity of the hydrophobic core, whereas this region is responsible for a major structural change upon prion conversion (Hölscher et al., 1998; Horiuchi & Caughey, 1999;
Horiuchi et al., 2001; Kaneko et al., 1997; Thaa et al., 2007). Concerning the kinetics of the conversion process, the rate-limiting step is not the binding of PrPC and PrPSc but the subsequent structural conversion (Horiuchi et al., 2000; Lee et al., 2007; Rigter & Bossers, 2005).
PrP mutants are not converted into PrPSc if parts of the hydrophobic core are missing, highlighting the HC as a focal point of prion conversion. Furthermore, these deletion mutants exhibit a dominant negative inhibition of the conversion process in persistently infected cell cultures (Holscher et al., 1998; Norstrom & Mastrianni, 2005). Next to this N-terminal region, the C-terminal globular domain also contributes to the conversion process. Different inherited
18 point mutations are located within or near the α-helices, thus leading to a destabilization of these motifs (Huang et al., 1994; Riek et al., 1996), and deletions in either of the three α- helical PrP elements lead to a resistance to PrPSc infection, indicating that PrPSc formation is dependent on structural changes within these regions (Muramoto et al., 1996). In addition, a single point mutation, Q218K, is sufficient to abrogate PrPSc formation in cell culture and in vivo (Kaneko et al., 1997; Perrier et al., 2002).
The abrogation or at least the slowdown of the conversion process by dominant negative mutants of the prion protein has made these constructs a possible therapeutic agent for prion diseases. The mutant Q218K has been used in lentiviral gene transfer (Crozet et al., 2004) and for the intracerebroventricular administration, which significantly prolonged the survival of infected mice (Furuya et al., 2006).
As shown for mutant prion proteins causing heritable CJD, PrPSc does not acquire its specific properties in a single step - it is rather a multilevel process whereby PrPSc is converted in three steps acquiring one special feature after another, thus passing through different intermediate states until “maturation” to PrPSc is complete.
At first, PrPC becomes PI-PLC resistant. Secondly, the prion protein gets detergent insoluble (ca. 1h after synthesis), which presumably correlates with PrP aggregation, and lastly, 1-6 hours after synthesis, the protein acquires PK-resistance, thus becoming a “mature” PrPSc. Whereas the first step might already occur within the cell, possibly during protein traveling through ER and Golgi, the last two steps happen either in lipid rafts on the cell membrane, where PrPC and PrPSc can both be co- localized, or in endosomal compartments after endocytosis.
The nature of this multilevel process suggests that besides PrPSc, additional proteins are involved in the conversion that direct the conversion process towards the “maturation” of the scrapie protein (Caughey & Raymond, 1991; Daude et al., 1997; Naslavsky et al., 1997).
Introduction
19
2.11 PrP
Scpropagation
Many lines of evidence support the theory of the prion particle being the actual cause of TSEs and rule out other proposals like viruses. Nevertheless, the properties of PrPSc and the mechanism of the conversion process are still poorly understood.
The model of ‘template assistance’ proposes PrPSc to be both, catalyst and template for its own replication. This model postulates the conversion from PrPC to PrPSc to take place in three steps: In the first, reversible step, PrPC binds to an as yet unknown adaptor protein termed protein X, changing its conformation into an insoluble intermediate PrP*, which is prone to undergo conversion into PrPSc. PrPSc then binds to this susceptible intermediate PrP* and promotes its irreversible conversion into PrPSc. After conversion, the complex, consisting of PrPSc and protein X, dissolves (Figure 9a).
The currently most accepted ‘nucleated polymerization theory’, on the other hand (Figure 9b), is based on in the polymerization of PrPSc as the crucial mechanism of prion disease: First, a nucleus of PrPSc oligomers has to be established. This would then recruit unstable PrPC monomers (= PrPU), transforming them into stable PrPSc upon accumulation to the oligomer.
In heritable forms of prion diseases, the nucleus would be established by a high level of mutated and thus unstable PrPU that could aggregate and thereby form a nucleus for PrPSc amyloid fibril formation. In infectious modes of prion disease, the already existing aggregation nucleus itself would be transmitted, leading to the accumulation and conversion of PrPU into PrPSc (Come et al., 1993; Masel et al., 1999; Rubenstein et al., 2007).
20
(a)
(b)
Figure 9: Schematic diagram of prion protein conversion (a) Template assisted theory; from: (Prusiner et al., 1998) (b) Nucleated polymerization theory; from: (Come et al., 1993)
The smallest infectious prion particle seems thereby not to be a single PrPSc molecule; it is rather a PrPSc oligomer, most probably a trimer. Recent studies suggest a trimeric PrPSc disc as the basic unit of PrP amyloid fibrils (Figure 10). This unit is formed by the conversion of structural flexible PrPC molecules into stable PrPSc trimers. After its initial formation, the PrPSc trimer recruits further PrPC molecules, which are converted into the more stable PrPSc form.
This gain in free energy by the stacking of further PrPC molecules leads to the establishment of a stable nucleus, which further drives fibril formation (DeMarco & Daggett, 2004; Govaerts et al., 2004; Stohr et al., 2008).
Introduction
21 The shortest incubation time for infection-induced prion disease is recorded when the amino acid sequences of PrPC and PrPSc are identical (Prusiner et al., 1990; Scott et al., 1989).
Transgenic mice expressing a chimeric human and mouse prion protein, differing from human PrPC in only 9 amino acids instead of 28, show a high susceptibility to human prions derived from FFI or CJD patients (Telling et al., 1995). Interestingly, infections with purified prions from FFI patients led to a phenotype specific for FFI, whereas the inoculation with CJD brain extract resulted in a typical CJD phenotype (Telling et al., 1996).
Figure 10: Prion fibril structure
(a) Side view of a prion fibril, consisting of five trimeric discs
(b) Top view of a single prion unit, consisting of three PrPSc molecules For details see Text. From: (Govaerts et al., 2004)
