- Email: [email protected]
Molecular biology of prion propagation
338
Molecular biology of prion propagation Jonathan DF Wadsworth, and John Collinge* The
occurrence
of new
variant
Creutzfeldt-Jakob
the experimental confirmation that prion strain as BSE has dramatically
disease
it is caused highlighted
precise understanding of the molecular basis propagation. The molecular basis of prion-strain previously
a major
challenge
becoming
clearer.
The
central fold
to prion to one
propagation,
reproduced aggregates
in vitro, provides
propagation.
These
biology
of prion
from
and
by the same the need for a of prion diversity,
protein-only change
model,
is now
thought
a predominantly
comprising
p-structure,
to be
a-helical can
now
and the ability of P-PrP to form fibrillar a plausible molecular mechanism for and
other
propagation
becoming arguably neurodegenerative development
to the
conformational
predominantly
Graham S Jackson, Andrew F Hill
prion
in the fundamental
are leading
the best conditions
of rational
advances
be
to prion
diseases
understood of the and strategies for the
therapeutics
are becoming
clearer.
Addresses MRC Pnon Unit and Department School of Medicine at St. Mary’s, *e-mail: [email protected]
Current
Opinion
in Genetics
of Neurogenetq lmpenal London W2 1 PG, UK
& Development
1999,
College
9:338-345
http://biomednet.com/elecref/O959437X00900338 9 Elsevier
Science
Ltd ISSN
0959-437X
Abbreviations BSE bovine spongiform CJD Creutzfeldt-Jakob CNS
FDC FFI GPI GSS PRNP
Pfnp vCJD
encephalopathy disease central nervous system follicular dendritic cell fatal familial insomnia glycosylphosphatidylinositol Gerstmann-Strtiussler-Scheinker human prion protein gene mouse prion protein gene new variant CJD
disease
conceivably severe threat to public health in the IJK and other countries. ‘The central feature of prion diseases is the post-translational conversion of a normal host-encoded. glycosylphosphatidylinositol (WI)-anchored glycoprotein, the cellular prion protein (PrF:), to an abnormal isoform. designated PrF’sC. This transition appears to involve only conformational change rather than covalent modification and confers PrPsc with partial resistance to proteolytic degradation and detergent insolubility. Priori diseases are biologically unique in that the disease process can be triggered through inherited germline mutations in the human prion protein gene (PHNP), infection - by inoculation, or in some cases by dietary exposure - with tissue containing I’rPsC or by rat-e sporadic events that generate PrPsC. A wealth of experimental evidence indicates that an abnormal PrP isoform is the principal, if not the sole, component of the transmissible infectious agent, or priori. ‘I’hc proteinonly hypothesis, first proposed by Griffith in 1967 [ 11, in its existing form argues that prion propagation occurs through I’rPSc acting to replicate itself with high fidelicy by recruiting endogenous PrP(: [Zj. Although tremendous advances have been made in our understanding of prion diseases, many key issues remain rmaddrcssed. For example, we still know little about the norm4 cellular function of I’rP(:. the precise structure of the transmissible agent, or the mechanism of neurodegenerdtion. Resolving these issues will be crucial to the development of rational chemotherapeutic approaches for treating prion diseases. the urgency of which is emphasised by the occurrence of new variant (:relttzfeldt-Jakob disease (v(:JII) in the IJK and France which is thought to be caused by HSE exposure. Incubation periods of acquired prion diseases in humans can be extremely prolonged, and it remains to be seen if a substantial epidemic of v(:JII will occur.
The molecular Introduction The priori diseases or transmissible spongiform encephalopathies are fatal neurodegenerative disorders that include scrapie in sheep, bovine spongiform encephalopathy (,RSE) in cattle. and the human prion diseases: (:relitzt’eldt-Jakob disease ((:JD), Gcrstmann-StrzusslcrSchcinker disease (GSS) and kuru. Although rare in humans, the prion diseases have become an area of intense research interest. ‘[‘his is firstly because of their unique biolog). in that the transmissible agent. ;I priori appears co be devoid of nucleic acid and to consist of a post-translationally modified host protein. Secondly, because of the ability of these and related animal diseases to cross from one species to another, sometimes by dietary exposure, there has been widespread concern that the expos~rc to the epidemic of a novel bovine priori disease. bo\.ine spongiform enccphalopathy (HSR) posed a distinct and
basis of prion strain
diversity
A major problem for the ‘protein-only’ hypothesis of prion propagation has been how to explain the existence of multiple isolates, or strains, of prions. Such strains arc distinguished bv their biological properties: they produce distinct incubation periods and patterns of neuropathological targeting in inbred mouse lines. As they can be serially propagated in inbred mice with the same Prq gcnocype, they cannot be encoded by differences in Prl’ primar) structllrc. l;urthermorc, strains can bc re-isolated in mice after passage in intermediate species with different PrP primary structures [3]. IJnderstanding how a protein-only infectious agent could encode such phenotypic information has been of considerable biological interest. Support for the contention that strain specificity is encoded by PrP alone was provided by studying two distinct strains of transmissible mink encephalopathy prions which
Molecular
can be serially propagated in hamsters, designated hyper (HY) and drowsy (DY). These strains can be distinguished by differing physiochemical properties of the accumulated 1’9°C in the brains of affected hamsters [4]. Following limited proteolysis, strain-specific migration patterns of PrPSC on polyacrylamide gels can be seen which relate to different amino-terminal ends of HY and DY PrPsC following protease treatment, implying differing conformations of HY and DY PrPSL‘ [S]. Recently. several human PrI XGOtypes have been identified which are associated with different phenotypes of CJD [6,7]. ‘l’he different fragment sizes seen on Western blots following treatment with proteinase K suggests that there are several different human PrPSC conformations (Figure 1) but to fulfil the criteria of strains, these biochemical properties must be retained after transmission to experimental animals of both the same and different species. This has been demonstrated in studies with CJD isolates, with both PrPsC fragment sizes and the ratios of the three PrP glycoforms (diglycosylated, monoglycosylated and unglycosylated PrP) maintained on passage in transgenic mice expressing human PrP [7]. Furthermore, transmission of human prions and bovine prions to wild-type mice results in murine PrV with fragment sizes and glycoform ratios which correspond to the original inoculum 171. vCJD is associated with
I’rI’Sc
glycoform
ratios
which
are
distinct
from
biology
of prion
propagation
Wadsworth
et al.
339
Molecular strain typing of prion isolates can now be applied to molecular diagnosis of vCJD [7,10] and to produce a new classification of human prion diseases with implications for epidemiological studies investigating the aetiology of sporadic CJD. Such methods allow strain typing that can be performed in days rather than the l-2 years often required for classic biological strain typing. This technique may also be applicable to determining whether HSE has transmitted to other species [7] -and thereby pose a threat to human health-for instance, to sheep [ 11,121. A novel conformation-dependent immunoassay has been reported [13] and shown to differentiate several classic rodent-adapted scrapie strains in the hamster model. It has not yet been shown to differentiate strains of naturally occurring prion diseases where PrPSC levels vary enormously between cases and different brain regions. The ability of a single polypeptide chain to encode information specifying distinct disease phenotypes raises intriguing evolutionary questions. Do other proteins behave in this way? The novel pathogenic mechanisms involved in prion propagation may be of far wider signif’icance and be relevant to other neurological and non-neurological illnesses, indeed other prion-like mechanisms have now been described [ 141 and the field of yeast and fungal prions has emerged [ 15,161.
those
seen in classic (:JD. Similar ratios are seen in BSE and HSE when transmitted to several other species [7]. *I’hese data strongly support the ‘protein only’ hypothesis of infectivity and suggest that strain variation is encoded by a combination of PrP conformation and glycosylation. Furthermore, polymorphism in PrP sequence can influcncc the generation of particular PrPSc conformers ([7]; l-‘igure 1). ‘liansmission of PrV fragment sizes from two different subtypes of inherited prion disease to transgenic mice expressing a chimaeric human mouse PrP has also been reported [HI. As PrP glycosylation occurs before conversion to PrPsc, the different glycoform ratios may represent selection of particular T’rP(: glycoforms by PrPSc of different conformations. According to such a hypothesis. IV conformation would be the primary determinant of strain type with glycosylation being involved as a sccondary process but, as it is known that different cell types may glycosylate proteins differently. PrPsc glycosylation patterns may provide a substrate for the neuropathological targeting that distinguishes different prion strains [7]. Particular IWsc glycoforms may replicate most favourably in neuronal populations with a similar PrP glycoform exprcsscd on the cell surface. Such targeting could also explain the different incubation periods which also discriminate strains, targeting of more critical brain regions, or regions with higher levels of PrP expression, producing shorter incubation periods. ITurther supportive evidence for the involvement of PrP glycosylation in priori strain propagation has come from the study of transgenic mice expressing PrP with mutations interfering with N-‘-linked glycosylation [9].
Conversion
of PrPc to PrPsc
Solution structures for recombinant prion proteins derived from both mouse [17] and hamster [18] have now been determined by NMK spectroscopy and indicate that PrP(: is composed mainly of a-helical elements. PrPsc Figure
1
Human M,(K) 30-
PrPSC types
Type1
2
Type
Type 3
-0
22-D
I
Sporadic
MM
latrogenic
MM
CzxT-
MM,MV,VV MM
MV,VV MV,VV
Variant L-
Type 4
MM Current Ophon
in Genetics & Development
Schematic representation of proteinase K digestion products generated from distinct PrPsc conformers associated with human prion diseases. PrP polymorphism at residue 129 (either methionine [Ml or valine [VI) contributes to genetic susceptibility to both sporadic [21j and acquired 1651 forms of CJD and to the generation of particular PrPsc conformers. Box sizes represent the relative differences in intensities of the three PrP glycoforms (corresponding to aminoterminally truncated cleavage products generated from di-,mono-, or non-glycosylated PrPsc).
340
Genetics
of disease
differs markedly from PrP(:. Biophysical measurements show differences in secondary structure content between the two &forms, both circular dichroism and Fourier transform infrared spectroscopic methods indicating PrPSchas a higher P-sheet content [ 191. In addition, PrPsC exists as very large insoluble aggregates which exhibit substantial protease resistance, whereas PrPc is a protease-sensitive, soluble monomer.
isoform of PrP, which requires additional, as yet unknown, co-factors for the acquisition of infectivity. Defining the precise molecular events that occur during the conversion of benign PrPC to the infectious isoform is of paramount importance as this process is a prime target for therapeutic intervention. Seeded conversion has been used successfully to identify compounds which inhibit the in Z&-O generation of PrPKES [26,27’] but it is unknown whether compounds identified in this way will inhibit the production of infectivity.
‘I’he underlying molecular events during infection which lead to the conversion of PrP(: to PrPsc remain ill-defined. The most coherent and general model proposed to date is that the protein, PrP, fluctuates between a dominant native state, PrP(:, and a series of minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure, PrW, composed of misfolded I’# monomers. once a stable ‘seed’ structure is formed, PrP can then be recruited, leading to an explosive, auto-catalytic formation of PrPSC.Molecular genetic studies support the hypothesis that PrP(; and PrPSCinteract directly and indicate that a high level of complimentarity is required with the conversion process being most efficient when both proteins have identical amino acid sequences [20,21].
Recent work has identified conditions in which the PrP polypeptide can be converted between alternative folded conformations representative of PrP(: and PrPSc [28”]. .4t neutral or basic pH, PrP adopts an a-helical fold representative of PrP(: and this conformation is ‘locked’ by the presence of the native disulphide bond (Figure 2). IJpon reduction of the disulphide bond, PrP(: was shown to rearrange to a predominantly P-sheet structure. l’his alternative conformation is only populated at acidic pH with the PrP(: conformation predominating at neutral pH. p-PrP was further shown to possess additional properties of PrPsc, that is partial proteinase K resistance, and a propensity to aggregate into fibrils. ‘This new work suggests that P-P@ may be an intermediate on the pathway to formation of PrPSC.The conditions required to form P-PrP may bc relevant to the conditions PrP would encounter within the cell during internalisation and recycling. The low pH and reducing environment of the endosomal pathway M;ould be a probable candidate for such a conversion reaction although this has not yet been demonstrated.
Direct. although preliminary. evidence for such a process has been provided by itz uitr.o mixing experiments [Z--24]. In such experiments, an excess of PrPsc can used as a seed to convert recombinant PrP(: to a protease-resistant form (PrPKI? but bioassays of such conversion products have not shown any detectable infectivity [ZS’]. Despite the obvious limitations of such experiments they may represent an initial step in the generation of the infectious
Figure
2
r Neutral
or basic
pH
Denaturant
PrPC
Acidic
Schematic representation of the folding pathway to P-PrP. PrPc is protected from complete denaturation by the constramts
pH
Partiallyunfolded
Oxldising
P-PrP
monomers
Reducing
Unfolded
I
L-.-.
Fibrils ~
.--~~-
.~~
-.--
-
------___
Current Opinm
I” Genehcs B
of
the disulphide bond. It retains its native ahelical conformation at neutral or basic pH even in the absence of the disulphide bond but when reduced and acidified, PrPc converts slowly to /3-PrP, which lacks a-helical structure. At physiological ionic strength, P-PrP readily aggregates into material that contains fibrils indistinguishable from those obtained from diseased tissue and which exhibit marked protease resistance [28”].
Molecular
The discovery of p-PrP, and the demonstration that PrP is capable of slow inter-conversion between a native cx and a non-native p conformation which can be locked by intermolecular association, provides a plausible mechanism of propagation of a rare conformational state. It is possible that the a-PrP to P-PrP conversion, caused by reduction and mild acidification, is relevant to the conditions that PrP’: would encounter within the cell, following its internalisation during recycling. Such a mechanism could underlie prion propagation and account for the transmitted, sporadic and inherited aetiologies of prion disease. Initiation of a pathogenic self-propagating conversion reaction, with accumulation of aggregated P-PrP, may be induced by exposure to a ‘seed’ of aggregated fi-PrP following prion inoculation, or as a rare stochastic conformational change, or as an inevitable consequence of expression of a pathogenic PrP(: mutant which is predisposed to form P-Prl? It remains to be demonstrated whether monomeric, oligomeric or fibrillar forms of P-PrP are infectious in experimental animals under appropriate conditions, or whether other cellular cofactors are also required.
Inherited
human
prion
diseases
Further pathogenic PRNP mutations continue to be identified and the currently recognised list is summarised in Figure 3. ‘I’hese inherited forms account for -15% of all human prion diseases [29]. Although traditionally classified into (CSS, C:JD or fatal familial insomnia (l:FI), the degree of phenotypic overlap observed between different mutations and even in family members with the same mutation indicates that future classification will probably be based upon mutation alone [30,31]. How pathogenic mutations in PRA’P cause priori disease has yet to be resolved. In most cases, the mutation is thought to lead to an increased tendency of PrP(: to form PrPSC, although recent studies [32’,33’] suggest that this may not be attributable solely to Figure
biology
of prion
Wadsworth et al.
341
decreased thermodynamic stability of mutated PrP(:. Additionally, evidence has emerged indicating that experimentally manipulated mutations of the prion gene can lead to spontaneous neurodegeneration without the formation of detectable protease-resistant PrP [34,35-l. These findings raise the question of whether all inherited forms of human prion disease invoke disease through the same mechanism and, in this regard, it is currently unknown whether all are transmissible by inoculation.
New variant
CJD and BSE
Acquired prion diseases in humans can result from exposure to human prions through medical and surgical procedures (iatrogenic CJD) or cannibalism (kuru [29]). Lluring 19951996, the appearance of young-onset cases of vCJD with atypical clinical presentation led to extreme concern that BSE, epidemic in cattle in the LJK, had been transmitted to humans. Subsequently, both molecular strain typing [7,10] and transmission studies in wild-type [l&36] and transgenic mice [lo] have established that vCJD and BSE are caused by the same prion strain. PrPSc associated with vCJD generates a unique pattern of proteolytic digestion products, that can be readily distinguished from PrPsC associated with sporadic or iatrogenic (:JD [7,10] (Figures 1 and 4). ‘li) date, 40 cases of v<:JD have been confirmed in the IJK and, at present, because of the long incubation periods associated with these diseases, no clear epidemiological prediction can be made regarding the potential number of cases. As the pathogenesis of vCJD leads to l’rPsC deposition in lymphoreticular tissues [37,38”] and also the appendix [39’], in distinction to classic forms of CJD. this may now allow the estimation of pre-clinical vCJD prevalence [38”] by screening archival lymphoreticular material and in prospective studies of surgical tissues. The PrPsc type seen in tonsil in v(:JD differs in the proportions of the PrP glycoforms from that seen in the brain of the same patients, suggesting the
3
Pathogenic
mutations
Y 145stop
I
P105L
Polymorphic
mutations variants
R208H ,
T183A
I
variants
-~__---~__~
Pathogenic polymorphic
propagation
and polymorphisms are shown below.
in the human
prion
-
protein.
Pathogenic
mutations
are shown
above
Current Opmton in Genetics & Development
the schematic
representing
PrP;
342
Genetics
of disease
superimposition of tissue-specific effects on PrP glycosylation (Figure
Peripheral
pathogenesis
and 4).
is associated with defective FDC maturation, these investigators went on to demonstrate that mice deficient in tumour necrosis factor 1, which lack functional FDCs, were susceptible following peripheral inoculation - arguing that FDCs were not required for neuroinvasion [43]. As B lymphocytes are circulating cells that are known, in common with most bone marrow derived cells, to express PrP(:, these data highlighted the need to consider the risks posed by blood transfusions from donors incubating prion disease.
strain-specific
of prion disease
Peripheral inoculation of experimental animals with prions is typically followed by a prolonged, clinically silent, phase prior to detectable neuroinvasion and the subsequent appearance of neurological deficits. During this pre-clinical period, prions can be isolated from lymphoreticular tissues where they may replicate to high titres. Elucidating the cell types in which prions replicate in the periphery and, crucially, how prions are transported to the central nervous system (CNS) is of considerable interest and represents a plausible target for therapeutic and prophylactic regimes 1401.
As both prion replication [44] and transport to the CNS 1451 is dependent on PrPC: expression, it was anticipated that PrP(z expression on B cells would be required for their role in prion pathogenesis. This may not be the case, however, as haematopoetic stem cells derived from PrI’ knockout mice are just as efficient in removing the block to neuroinvasion in B-cell deficient mice as comparable cells from wild-type mice [46*-l.
‘I’he lack of effect of whole-body ionising radiation on prion pathogenesis argues against significant involvement of proliferating cells in the lymphoreticular phase of prion propagation 1411. Follicular dendritic cells (FDCs), which do not proliferate. have been the favoured candidate for some time [42] but evidence for a key role for B cells in prion propagation was produced using a panel of immunodeficient mice [43]. Although all of these mutants could be infected by intracerebral exposure to prions, mice with defects in B-lymphocyte differentiation were extremely resistant to prion infection following intrdperitoneal inoculation with scrapir prions. .4s an absence of mature B cells Figure
Although it is possible that B cells could transport prions to the CIKS via a non-PrP-dependent process, or that they might secrete factors which bind to prions and enhance their neuropathogenicity [46”], an alternative possibility is that FDCs - or a subset of cells not bearing the FCD-Ml or MZ immunophenotype - are in fact the cell type that promotes prion neuroinvasion. It is possible that prion propagation to high levels in FDCs simply increases the probability of prion uptake by the autonomic nerve terminals that extensively innervate lymphoid organs. Neuroinvasion via peripheral nerves has long been considered likely [47]. Thus, B cells need not be involved directly in either prion propagation or neuroinvasion but rather are one of a number of necessary factors that promote FDC development. It must also be considered that more than one mechanism may be involved and that different prion strains may preferentially use these different routes, complicating any therapeutic strategies. Such studies aiming to dissect the cell type(s) involved in the peripheral pathogenesis of prion disease by various reconstitution of P~,pO/O mice, may also be complicated by the known ability of GPI-anchored proteins to transfer from cell to cell (‘GPI-painting’) [4X].
4
W Diglycosylated 80
I
0 Monoglycosylated I
I
Unglycosylated I
I
70 -
n 60 50 t
New clues to PrPc function
0 I
;-
I
I
I
I
I
1I
Current Opmon in Genetics & Development -I
Relative proportions of di-, mono- and unglycosylated PrP following partial digestion with proteinase K. Types l-3 are seen in classic forms of CJD (either sporadic or iatrogenic in origin), type 4 is seen in new variant CJD [71. A distinct pattern is seen in the tons11 of vCJD patients. designated type 4t [38”]. Error bars where not visible were smaller than the symbols used to designate the means.
I
Gaining direct insight into the normal cellular function of I’rP(: through the generation of PrP-deficient (~-‘MP null) mice has proved frustrating. Although Prn~p null mice are completely resistant to prion disease [44], they develop and behave essentially normally [49]. Reported changes in synaptic activity [SO-.531 and circadian rhythms [54] have not pinpointed a precise role for PrP(: although intriguingly similar neiirophysiological features (and disturbances of sleep rhythm) are seen in priori disease itself, arguing that prion ncurodegeneration may result. at least in part, from PrP loss of function [SO]. ‘I’hese studies are complicated by the possibility that adaptivc changes during neuronal development may effectively mask the normal cellular role of PrP(:. (:omplete knockout of PI-T/~
Molecular
in adult mice, by conditional gene expression methods, may be revealing with respect to the normal cellular function of PrP, but has not yet been reported. Studies with synthetic peptides corresponding to the amino-terminal octapeptide repeats of PrP have demonstrated that this region can bind Cu2+ [55-571 and this has recently been extended to show Cu?+ binding to the entire amino-terminal domain of recombinant human PrP [SS] or full-length recombinant hamster PrP [59]. Furthermore, it has been demonstrated that membrane-rich brain fractions from P~np null mice possess reduced levels of Cuz+ compared to wild-type mice [SN] and also exhibit reduced activity of the C&+-dependent enzyme superoxide dismutase I [60]. There is also evidence suggesting that increased extracellular concentrations of
unless
I’rl’
also
binds
(:uz+
at
regions
biology
of prion
propagation
Wadsworth
343
et al.
important. The precise cell types involved in peripheral prion replication and transport to the CNS remain unclear. An important role for follicular dendritic cells is likely and multiple mechanisms may be able to mediate neuroinvasion following peripheral prion inoculation.
References
and recommended
Papers of particular interest, have been highlighted as:
published
reading
within the annual
period
of review,
l of special interest ‘*of outstanding interest
1.
Griffith JS: Self replication 215:1043-1044.
2.
Prusmer scrapie.
3.
Bruce M, Chree A, McConnell I, Foster J, Pearson G, Fraser H: Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. P/%/OS Fans R Sot Land 1994, 343:405-411.
4.
Bessen RA, Marsh RF: Biochemical and physical prion protein from two strains of the transmissible encephalopathy agent. J Viral 1992, 66:2096-2101.
5.
Bessen RA, Marsh RF: Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Wrol 1994, 66:7859-7868.
6.
Parchi P, Castellani R, Capellari S, Ghetti B, Young K, Chen SG, Farlow M, Dickson DW, Sims AAF, Troianowski JQ ef al.: Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease. Ann Neural 1996, 39:767-778.
7.
Collinge J, Sidle KCL, Meads J, lronslde J, HIII AF: Molecular of prion strain variation and the aetiology of ‘new variant’ Nature 1996, 363:685-690.
8.
Telling GC, Parchi P, DeArmond SJ, Cortelli P, Montagna P, Gablzon R, Mastrianni J, Lugaresl E, Gambetti P, Prusiner SB: Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity. Science 1996, 27412079.2082.
and screpie.
SB: Novel proteinaceous Science 1982, 216:136-l
Nature
infectious 44.
1967, particles
cause
properties mink
of the
analysis CJD.
other
than the octapeptide to note that whereas
repeats. In this regard, it is of interest transgenic mice expressing PrP lack-
9.
DeArmond SJ, Sanchez H, Yehlely F, Qiu Y, Ninchak-Casey A, Daggett V, Camerino AP, Cayetano J, Rogers M, Groth D et a/.: Selective neuronal targeting in prion disease. Neuron 1997, 19:1337-l 348.
ing
develop
10
Hill AF, Desbruslais same prion strain 389:448-450.
11
HIII AF, Sidle KCL, Joiner S, Keyes P, Martin TC, Dawson M, Collinge J: Molecular screening of sheep for bovine spongiform encephalopathy. Neurosci Lett 1998, 255159-l 62.
12
Hope J, Wood Goidmann W, prion protein experimental 80:1-4.
13.
Safar J, Wille H, ltri V, Groth D, Serban H, Torchia M, Cohen FE, Prusiner SB: Eight prion strains have PrPsc molecules with different conformations. Nat Med 1998.4:1 157-l 165.
14
Milner J, Medcalf EA: Cotranslation of activated mutant p53 with wild type drives the wild type ~53 protein into the mutant conformation. Cell 1991, 65:765-774.
15
Wickner RB, Masison DC: Evidence for two and [PSI]. Curr Top Microbial lmmunol1996,
16
Wickner RB: A new prion controls fungal cell fusion incompatibility. Proc Nat/ Acad Sci USA 1997, 94:10012-l
17.
Riek R, Hornemann S, Wider G, Billeter M, Glockshuber Wuthrich K: NMR structure of the mouse prion protein (121-231). Nature 1996, 382:180-182.
residues
32-106
and
behave
normally,
mice
expressing PrP ivith deletion of residues 32-121 develop severe ataxia and neuronal cell death in the cerebellum, an effect that can be abolished by the presence of one copy of a wild-type
PrP
gene
[64”].
Conclusions ‘I’hc existence of multiple prion strain types can be accommodated within a protein-only hypothesis of prion propagation. Kapid molecular methods are now available to tyI)c prion strains and to investigate their origins. ‘This abilit>- of a single polypeptide chain to encode phenotypic information may have wider biological and pathobiological significance. The i/l vitrn interconversion of recombinantderived PrP between native and fibrilogenic conformations provides a plausible molecular mechanism for prion propagation. New variant CJD is caused by a prion strain indistinguishable from that causing BSE and has a pathogenesis distinct from that of classic CJD, having a prominent involvement of lymphoreticular tissues. It remains unclear if ;I substantial
cise function accumulating
epidemic
of
human
RSI<
will
occur.
of cellular I’rl’ remains obscure evidence suggests that copper binding
‘I’he
prc-
although
may be
18
M, Joiner S, Sidle KCL, Gowland I, Collinge causes vCJD and BSE. Nature 1997,
J: The
SCER, Birkett CR. Chona A. Bruce ME. Cairns D. Hunter N, Bostock CJ: &le&lar analysis of ovine identifies similarities between BSE and en isolate of natural scrapie, CH1641. J Gen Viral 1999,
prions in yeast: 207:147-l 60.
lUREI
0014. R. domain
PrP
James TL. Liu H, Ulyanov NB, Farr-Jones S, Zhang H, Donne DG, Kaneko K, Groth D, Mehlhorn I, Prusiner SB ef a/.: Solution structure of a 142-residue recombinant prion protein corresponding to the
344
Genetics
of disease
infectious fragment of the scrapie USA 1997, 94:i 0086-l 0091. 19.
isoform.
Proc
Nat/ Acad
Sci
Pan K-M, Baldwin MA, Nguyen J, Gasset M, Serban A, Groth D, Mehlhom I, Huang Z, Fletterick RJ, Cohen FE et al.: Conversion of ahelices into p-sheets features in the formation of the scrapie prion proteins. froc Nat/ Acad Sci USA 1993, 90:10962-l 0966.
20.
Prusiner SB, Scott M, Foster D, Pan KM, Groth D, Mirenda C, Torchia M, Yang SL, Serban D, Carlson GA et al.: Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 1990, 63:673-686.
21.
Palmer MS, Dryden AJ, Hughes JT, Collinge protein genotype predisposes to sporadic disease. Nature 1991, 352:340-342.
22.
Kocisko DA, Come JH, Priola SA, Chesebro B, Raymond GJ, Lansbury PT, Caughey B: Cell-free formation of protease-resistant prion protein. Nature 1994, 370:471-474.
23.
Bessen RA, Kocisko DA, Raymond GJ, Nandan S, Lansbury PT, Caughey B: Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature 1995, 375698-700.
24.
J: Homozygous Creutzfeldt-Jakob
prion
Kocisko DA, Priola SA, Raymond GJ, Chesebro B, Lansbury PT Jr, Caughey B: Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. Proc Nat/ Acad Sci USA 1995, 92:3923-3927.
25. .
Hill AF, Antoniou M, Collinge J: Protease-resistant prion protein produced in vitro lacks detectable infectivity. I Gen viral 1999, 80:1 l-14. A demonstration that the acquisition of protease resistance by PrPc in vitro is not sufficient for the propagation of prion infectivity, highlighting the need for caution in interpreting the biological significance of experimentally generated protease-resistant PrP. 26.
Chabry J, Caughey B, Chesebro B: Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides. J Biol Chem 1998, 273:13203-i 3207.
27. .
Caughey WS, Raymond LD, Horiuchi M, Caughey B: Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines. Proc NaN Acad Sci USA 1998, 95:i 2117-l 2122. This study identifies a new class of compounds that interferes with the generation of protease-resistant PrP formation in vitro and suggests their use as lead compounds for the generation of therapeutic agents for treating prion diseases. 28. l 0
Jackson GS, Hosszu LLP, Power A, Hill AF, Kenney J, Saibil H, Craven CJ, Waltho JP, Clarke AR, Collinge J: Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations. Science 1999, 283:1935-l 937. Conversion of PrPC to PrPsc results in an increase in the amount of P-sheet structure in the protein. This study identifies conditions in which recombinant human PrP can switch between native u-helical conformation and a compact, soluble monomeric form rich in P-sheet structure @-PrP). The generation of P-PrP in suitable cellular compartments and subsequent stabilisation by inter-molecular association provides a molecular mechanism for prion propagation. 29.
Collinge J: Human prion encephalopathy (BSE).
30.
Collinge J, Owen F, Poulter M, Leach M, Crow TJ, Rossor MN, Hardy J, Mullan MJ, Janota I, Lantos PL: Prion dementia without characteristic pathology. Lancet 1990, 336:7-g.
31.
Collinge J, Brown J, Hardy J, Mullan M, Rossor MN, Baker H, Crow TJ, Lofthouse R, Poulter M, Ridley R et a/.: Inherited prion disease with 144 base pair gene insertion: II: clinical and pathological features. Brain 1992, 115:687-710.
diseases and bovine spongiform Hum MO/ Genetics 1997, 6:1699-l
705.
32. .
Riek R, Wider G, Billeter M, Hornemann S, Glockshuber R, Wuthrich K: Prion protein NMR structure and familial human spongiform encephalopathies. Proc Nat/ Acad Sci USA 1998, 95:i 1667-l 1672. How pathogenic mutations in the prion gene cause disease is unknown. One simple mechanism would be that the mutations lead to decreased thermodynamic stability of mutated PrPC and concomitant propensity to form PrPsc. This study questions the likelihood of such a universal mechanism and suggests that subtle structural differences in the mutant proteins may affect intermolecular signalling in a variety of ways. Swietnicki W, Petersen RB, Gambetti P, Surewicz WK: Familial mutations and the thermodynamic stability of the recombinant human prion protein. J Biol Chem 1998, 273:31048-31052. See annotation [32-l.
34.
Muramoto T, DeArmond SB. Heritable disorder mice expressing prion 1997, 3:750-755.
SJ, Scott M, Telling GC, Cohen FE, Prusiner resembling neuronal storage disease in protein with deletion of an &helix. Nat Med
35. .
Hegde RS, Mastrianni JA, Scott MR, DeFea KA, Tremblay P, Torchia M, DeArmond SJ, Prusiner SB, Lingappa VR: A transmembrane form of the prion protein in neurodegenerative disease. Science 1998, 279:827-834. Examlnatlon of different topological forms of PrP generated at the endoplasmic reticulum identifies a particular transmembrane form of PrP whose presence is associated with neurodegeneration in mice. The absence of detectable PrPsc indicates that abnormal PrP biogenesis and topology may result in neurodegeneration and suggests that this mechanism may underlie pathogenesis in certain genetically inherited forms of human prion disease. 36.
Bruce ME, Will RG, lronside JW, McConnell I, Drummond D, Suttie A, McCardle L, Chree A, Hope J, Birkett C et al.: Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 1997, 389:498-501.
37.
Hill AF, Zeidler M, lronside J, Collinge J: Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. Lancef 1997, 349:99-l
00.
38. ..
Hill AF, Butterworth RJ, Joiner S, Jackson GS, Rossor MN, Thomas DJ, Frosh A, Tolley N, Bell JE, Spencer M et a/.: Investigation of variant Creutzfeldt Jakob disease and other human prion diseases with tonsil biopsy samples. Lancet 1999,-353:183-l 89. This study reveals the deposition of PrPsC In lymphoreticular tissues of patients with new variant CJD but not in other forms of human prion disease. These findings demonstrate that new variant CJD has a different pathogenesis to classic CJD and that, in the appropriate clinical context, characteristic PrPsC positivity in tonsil biopsy is diagnostic of new variant CJD. Screening of surgical tonsil and other lymphoreticular tissues [39’] can now be used to estimate the prevalence of pre-clinical vCJD. 39. .
Hilton DA, Fathers immunoreactivity Creutzfeldt-Jakob See annotation [38*-l.
E, Edwards P, lronside JW, Zajicek J: Prion in appendix before clinical onset of variant disease. Lancef 1998, 352:703-704.
40.
Aguzzi A, Collinge prion inoculation.
J: Post-exposure prophylaxis after Lancef 1997, 350:1519-l 520.
41.
Fraser H, Farquhar scrapie incubation
42.
Clarke MC, Kimberlin RH: Pathogenesis of mouse scrapie: distribution of agent in the pulp and stroma of infected spleens. Vet Microobiol1984, 9:215-225.
43.
Klein MA, Frigg R, Flechsig E, Raeber AJ, Kalinke U, Bluethmann H, Bootz F, Suter M, Zinkernagel RM, Aguui A: A crucial role for B cells in neuroinvasive scrapie. Nature 1997, 390:687-690.
44.
Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet Weissmann C: Mice devoid of PrP are resistant to scrapie. 1993, 73:1339-l 347.
45.
Blattler T, Brandner S, Raeber AJ, Klein MA, Voigtlander T, Weissmann C, Aguzzi A: PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 1997, 389:69-73.
accidental
CF: lonising radiation has no influence on period in mice. Vet Microbial 1987, 13:21 l-223.
M, Cell
46. ..
Klein MA, Frigg R, Raeber AJ, Ftechsig E, Hegyi I, Zinkernagel RM, Weissmann C, Aguzzi A: PrP expression in B-lymphocytes is not required for prion neuroinvasion. Nat Med 1998,4:1429-l 433. This study demonstrates that the critical role of B cells in neuroinvasion by scrapie [431 does not require B-cell expression of PrPC. These data suggest that B cells may simply allow maturation of follicular dendritic cells. Other interpretations are discussed in [48]. 47.
Kimberlin RH, Walker CA: Pathogenesis evidence for neural spread of infection 1980, 51 :I 83-I 87.
of mouse scrapie: to the CNS. J Gen
l/ho/
48.
Collinge J, Hawke S: B lymphocytes in prion neuroinvasion: Central or peripheral players. Nat Med 1998,4:1369-l 370.
49.
Bueler H, Fischer M, Lang Y, Bluethmann H, Lipp H-P, DeArmond SJ, Prusiner SB, Aguet M, Weissmann C: Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 1992, 356:577-582.
50.
Collinge J, Whittington MA, Sidle KCL, Smith CJ, Palmer MS, Clarke AR, Jefferys JGR: Prion protein is necessary for normal synaptic function. Nature 1994, 370:295-297.
51.
Whittington MA, Sidle KCL, Gowland I, Meads J, Hill AF, Palmer MS, Jefferys JGR, Collinge J: Rescue of neurophysiological phenotype
33. .
Molecular
seen
in PrP null mica by transgene 1995,9:197-201.
encoding
human
prion
protein.
60.
Manson
JC, Hope
gene dosage 53
Coking
SB, Collinge
protein
null mice:
potentiation.
J, Jefferys
Disrupted
JGR: Hippocampal slices Ca*+-activated K+ currents.
terminal protein.
from prion Neurosci
MP, McDermott
tandem
repeat
Biochem
Biophys
in mice devoid JR, Candy
regions
of prion
JM: Copper
of mammalian
Res Commun
protein.
binding to the Nand avian prion
Miura T, Hori-i A, Takeuchi
58.
59.
Left 1996,
H: Metal-dependent
rich octapeptide
u-helix formation region of prion protein.
396:248-252.
Brown Fraser
DR, Gin K, Herms JW, Madlung A, Manson J, Strome R, PE, Kruck T, von Bohlen A, Schulz-Schaeffer W et a/.: The cellular prion protein binds copper in v&o. Nature 1997, 390:684-687. Stockel
selectively
WJ, Schmidt
B, Kretzschmar
cells show altered response SOD-l activity. Exp Neural
AC, Cohen FE, Prusrner SB: Prion protein copper(h) ions. Biochemistry 1998, 37:7185-7193.
J, Safar J, Wallace
binds
Pattison
IH, Jebbett
scrapie
and cuprizone
et a/.
345
HA: Prion
to oxldative
stress
1997,146:104-l
12.
of the prion
64. ..
JN: Histopathological
toxicity
in mice.
similarities between Nature 1971, 230:115-i
17.
Fischer M, Rulicke T, Raeber A, Sailer A, Moser M, Oesch 8, Brandner S, Aauzzi A, Weissmann C: Prion protein (PrP) with amino-proximal EMBO
restoring J 1996,
susceptibility
15:1255-l
of PrP knockout
mice to scrapie.
264.
Shmerling D, Hegyi I, Fischer M, Blamer T, Brandner S, Giitz J, Riilicke Flechsig E, Couio A, von Mering C et al.: Expression of amino-
terminally truncated specific cerebellar
1995,207:621-629.
57.
FEBS
Wadsworth
Pauly PC, Harris DA: Copper stimulates endocytosis J Biol Chem 1998, 273:33107-33110.
deleiions
Nature
Homshaw MP, McDermott JR, Candy JM, Lakey JH: Copper binding to the N-terminal tandem repeat region of mammalian and avian prion protein: structural studies using synthetic peptides. Biochem Biophys Res Commun 1995, 214:993-999.
by the glycine-
62.
P, Frscher M, Rulicke T, JC: Altered circadian
56.
promoted
61.
63.
I, Gaus SE, Deboer T, Achermann M, Oesch 8, McBride PA, Manson
Hornshaw
1995,
209:49-52.
activity rhythms and sleep 1996, 380:639-642. 55.
N: PrP
A, Black C, MacLeod Neurodegeneration
propagation
protein.
14.
Tobler Moser
AR, Johnston
and long term
4:113-l
Leff 1996, 54
J, Clarke
of prion
DR, Schulz-Schaeffer
protein-deficient due to decreased
Nat Genet 52
Brown
biology
PrP in the mouse leading lesions. Cell 1998,93:203-214.
to ataxia
T,
and
Transgenrc mice expressing amino-terminally truncatea rrr- racxmg resroues 32-l 06 develop and behave normally whereas those with longer deletions of 32-l 21 or 32-134 develop ataxia and specific neurodegeneration 2-3 months after birth. These data suooest that an essential PrP function may resrde within residues 106-l 21 a% that truncated mutant proteins lacking this region may be non-functional and may compete with another molecule possessing PrP-like function for a common ligand. 65.
Collinge
iatrcgenic
J, Palmer MS, Dryden
Creutzfeldt-Jakob
AJ: Genetic
disease.
predisposition to 1991,337:1441-l
Lancet
442.
Other recommended reading 66.
Moore RC, Hope J, McBride PA, McConnell, Selfridge J, Melton DW, Manson JC: Mica with gene targetted prion protein alterations show that Prnp, Sine and Prini are congruent Nat Genet 18:118-l 25.
Molecular biology of prion propagation Jonathan DF Wadsworth, and John Collinge* The
occurrence
of new
variant
Creutzfeldt-Jakob
the experimental confirmation that prion strain as BSE has dramatically
disease
it is caused highlighted
precise understanding of the molecular basis propagation. The molecular basis of prion-strain previously
a major
challenge
becoming
clearer.
The
central fold
to prion to one
propagation,
reproduced aggregates
in vitro, provides
propagation.
These
biology
of prion
from
and
by the same the need for a of prion diversity,
protein-only change
model,
is now
thought
a predominantly
comprising
p-structure,
to be
a-helical can
now
and the ability of P-PrP to form fibrillar a plausible molecular mechanism for and
other
propagation
becoming arguably neurodegenerative development
to the
conformational
predominantly
Graham S Jackson, Andrew F Hill
prion
in the fundamental
are leading
the best conditions
of rational
advances
be
to prion
diseases
understood of the and strategies for the
therapeutics
are becoming
clearer.
Addresses MRC Pnon Unit and Department School of Medicine at St. Mary’s, *e-mail: [email protected]
Current
Opinion
in Genetics
of Neurogenetq lmpenal London W2 1 PG, UK
& Development
1999,
College
9:338-345
http://biomednet.com/elecref/O959437X00900338 9 Elsevier
Science
Ltd ISSN
0959-437X
Abbreviations BSE bovine spongiform CJD Creutzfeldt-Jakob CNS
FDC FFI GPI GSS PRNP
Pfnp vCJD
encephalopathy disease central nervous system follicular dendritic cell fatal familial insomnia glycosylphosphatidylinositol Gerstmann-Strtiussler-Scheinker human prion protein gene mouse prion protein gene new variant CJD
disease
conceivably severe threat to public health in the IJK and other countries. ‘The central feature of prion diseases is the post-translational conversion of a normal host-encoded. glycosylphosphatidylinositol (WI)-anchored glycoprotein, the cellular prion protein (PrF:), to an abnormal isoform. designated PrF’sC. This transition appears to involve only conformational change rather than covalent modification and confers PrPsc with partial resistance to proteolytic degradation and detergent insolubility. Priori diseases are biologically unique in that the disease process can be triggered through inherited germline mutations in the human prion protein gene (PHNP), infection - by inoculation, or in some cases by dietary exposure - with tissue containing I’rPsC or by rat-e sporadic events that generate PrPsC. A wealth of experimental evidence indicates that an abnormal PrP isoform is the principal, if not the sole, component of the transmissible infectious agent, or priori. ‘I’hc proteinonly hypothesis, first proposed by Griffith in 1967 [ 11, in its existing form argues that prion propagation occurs through I’rPSc acting to replicate itself with high fidelicy by recruiting endogenous PrP(: [Zj. Although tremendous advances have been made in our understanding of prion diseases, many key issues remain rmaddrcssed. For example, we still know little about the norm4 cellular function of I’rP(:. the precise structure of the transmissible agent, or the mechanism of neurodegenerdtion. Resolving these issues will be crucial to the development of rational chemotherapeutic approaches for treating prion diseases. the urgency of which is emphasised by the occurrence of new variant (:relttzfeldt-Jakob disease (v(:JII) in the IJK and France which is thought to be caused by HSE exposure. Incubation periods of acquired prion diseases in humans can be extremely prolonged, and it remains to be seen if a substantial epidemic of v(:JII will occur.
The molecular Introduction The priori diseases or transmissible spongiform encephalopathies are fatal neurodegenerative disorders that include scrapie in sheep, bovine spongiform encephalopathy (,RSE) in cattle. and the human prion diseases: (:relitzt’eldt-Jakob disease ((:JD), Gcrstmann-StrzusslcrSchcinker disease (GSS) and kuru. Although rare in humans, the prion diseases have become an area of intense research interest. ‘[‘his is firstly because of their unique biolog). in that the transmissible agent. ;I priori appears co be devoid of nucleic acid and to consist of a post-translationally modified host protein. Secondly, because of the ability of these and related animal diseases to cross from one species to another, sometimes by dietary exposure, there has been widespread concern that the expos~rc to the epidemic of a novel bovine priori disease. bo\.ine spongiform enccphalopathy (HSR) posed a distinct and
basis of prion strain
diversity
A major problem for the ‘protein-only’ hypothesis of prion propagation has been how to explain the existence of multiple isolates, or strains, of prions. Such strains arc distinguished bv their biological properties: they produce distinct incubation periods and patterns of neuropathological targeting in inbred mouse lines. As they can be serially propagated in inbred mice with the same Prq gcnocype, they cannot be encoded by differences in Prl’ primar) structllrc. l;urthermorc, strains can bc re-isolated in mice after passage in intermediate species with different PrP primary structures [3]. IJnderstanding how a protein-only infectious agent could encode such phenotypic information has been of considerable biological interest. Support for the contention that strain specificity is encoded by PrP alone was provided by studying two distinct strains of transmissible mink encephalopathy prions which
Molecular
can be serially propagated in hamsters, designated hyper (HY) and drowsy (DY). These strains can be distinguished by differing physiochemical properties of the accumulated 1’9°C in the brains of affected hamsters [4]. Following limited proteolysis, strain-specific migration patterns of PrPSC on polyacrylamide gels can be seen which relate to different amino-terminal ends of HY and DY PrPsC following protease treatment, implying differing conformations of HY and DY PrPSL‘ [S]. Recently. several human PrI XGOtypes have been identified which are associated with different phenotypes of CJD [6,7]. ‘l’he different fragment sizes seen on Western blots following treatment with proteinase K suggests that there are several different human PrPSC conformations (Figure 1) but to fulfil the criteria of strains, these biochemical properties must be retained after transmission to experimental animals of both the same and different species. This has been demonstrated in studies with CJD isolates, with both PrPsC fragment sizes and the ratios of the three PrP glycoforms (diglycosylated, monoglycosylated and unglycosylated PrP) maintained on passage in transgenic mice expressing human PrP [7]. Furthermore, transmission of human prions and bovine prions to wild-type mice results in murine PrV with fragment sizes and glycoform ratios which correspond to the original inoculum 171. vCJD is associated with
I’rI’Sc
glycoform
ratios
which
are
distinct
from
biology
of prion
propagation
Wadsworth
et al.
339
Molecular strain typing of prion isolates can now be applied to molecular diagnosis of vCJD [7,10] and to produce a new classification of human prion diseases with implications for epidemiological studies investigating the aetiology of sporadic CJD. Such methods allow strain typing that can be performed in days rather than the l-2 years often required for classic biological strain typing. This technique may also be applicable to determining whether HSE has transmitted to other species [7] -and thereby pose a threat to human health-for instance, to sheep [ 11,121. A novel conformation-dependent immunoassay has been reported [13] and shown to differentiate several classic rodent-adapted scrapie strains in the hamster model. It has not yet been shown to differentiate strains of naturally occurring prion diseases where PrPSC levels vary enormously between cases and different brain regions. The ability of a single polypeptide chain to encode information specifying distinct disease phenotypes raises intriguing evolutionary questions. Do other proteins behave in this way? The novel pathogenic mechanisms involved in prion propagation may be of far wider signif’icance and be relevant to other neurological and non-neurological illnesses, indeed other prion-like mechanisms have now been described [ 141 and the field of yeast and fungal prions has emerged [ 15,161.
those
seen in classic (:JD. Similar ratios are seen in BSE and HSE when transmitted to several other species [7]. *I’hese data strongly support the ‘protein only’ hypothesis of infectivity and suggest that strain variation is encoded by a combination of PrP conformation and glycosylation. Furthermore, polymorphism in PrP sequence can influcncc the generation of particular PrPSc conformers ([7]; l-‘igure 1). ‘liansmission of PrV fragment sizes from two different subtypes of inherited prion disease to transgenic mice expressing a chimaeric human mouse PrP has also been reported [HI. As PrP glycosylation occurs before conversion to PrPsc, the different glycoform ratios may represent selection of particular T’rP(: glycoforms by PrPSc of different conformations. According to such a hypothesis. IV conformation would be the primary determinant of strain type with glycosylation being involved as a sccondary process but, as it is known that different cell types may glycosylate proteins differently. PrPsc glycosylation patterns may provide a substrate for the neuropathological targeting that distinguishes different prion strains [7]. Particular IWsc glycoforms may replicate most favourably in neuronal populations with a similar PrP glycoform exprcsscd on the cell surface. Such targeting could also explain the different incubation periods which also discriminate strains, targeting of more critical brain regions, or regions with higher levels of PrP expression, producing shorter incubation periods. ITurther supportive evidence for the involvement of PrP glycosylation in priori strain propagation has come from the study of transgenic mice expressing PrP with mutations interfering with N-‘-linked glycosylation [9].
Conversion
of PrPc to PrPsc
Solution structures for recombinant prion proteins derived from both mouse [17] and hamster [18] have now been determined by NMK spectroscopy and indicate that PrP(: is composed mainly of a-helical elements. PrPsc Figure
1
Human M,(K) 30-
PrPSC types
Type1
2
Type
Type 3
-0
22-D
I
Sporadic
MM
latrogenic
MM
CzxT-
MM,MV,VV MM
MV,VV MV,VV
Variant L-
Type 4
MM Current Ophon
in Genetics & Development
Schematic representation of proteinase K digestion products generated from distinct PrPsc conformers associated with human prion diseases. PrP polymorphism at residue 129 (either methionine [Ml or valine [VI) contributes to genetic susceptibility to both sporadic [21j and acquired 1651 forms of CJD and to the generation of particular PrPsc conformers. Box sizes represent the relative differences in intensities of the three PrP glycoforms (corresponding to aminoterminally truncated cleavage products generated from di-,mono-, or non-glycosylated PrPsc).
340
Genetics
of disease
differs markedly from PrP(:. Biophysical measurements show differences in secondary structure content between the two &forms, both circular dichroism and Fourier transform infrared spectroscopic methods indicating PrPSchas a higher P-sheet content [ 191. In addition, PrPsC exists as very large insoluble aggregates which exhibit substantial protease resistance, whereas PrPc is a protease-sensitive, soluble monomer.
isoform of PrP, which requires additional, as yet unknown, co-factors for the acquisition of infectivity. Defining the precise molecular events that occur during the conversion of benign PrPC to the infectious isoform is of paramount importance as this process is a prime target for therapeutic intervention. Seeded conversion has been used successfully to identify compounds which inhibit the in Z&-O generation of PrPKES [26,27’] but it is unknown whether compounds identified in this way will inhibit the production of infectivity.
‘I’he underlying molecular events during infection which lead to the conversion of PrP(: to PrPsc remain ill-defined. The most coherent and general model proposed to date is that the protein, PrP, fluctuates between a dominant native state, PrP(:, and a series of minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure, PrW, composed of misfolded I’# monomers. once a stable ‘seed’ structure is formed, PrP can then be recruited, leading to an explosive, auto-catalytic formation of PrPSC.Molecular genetic studies support the hypothesis that PrP(; and PrPSCinteract directly and indicate that a high level of complimentarity is required with the conversion process being most efficient when both proteins have identical amino acid sequences [20,21].
Recent work has identified conditions in which the PrP polypeptide can be converted between alternative folded conformations representative of PrP(: and PrPSc [28”]. .4t neutral or basic pH, PrP adopts an a-helical fold representative of PrP(: and this conformation is ‘locked’ by the presence of the native disulphide bond (Figure 2). IJpon reduction of the disulphide bond, PrP(: was shown to rearrange to a predominantly P-sheet structure. l’his alternative conformation is only populated at acidic pH with the PrP(: conformation predominating at neutral pH. p-PrP was further shown to possess additional properties of PrPsc, that is partial proteinase K resistance, and a propensity to aggregate into fibrils. ‘This new work suggests that P-P@ may be an intermediate on the pathway to formation of PrPSC.The conditions required to form P-PrP may bc relevant to the conditions PrP would encounter within the cell during internalisation and recycling. The low pH and reducing environment of the endosomal pathway M;ould be a probable candidate for such a conversion reaction although this has not yet been demonstrated.
Direct. although preliminary. evidence for such a process has been provided by itz uitr.o mixing experiments [Z--24]. In such experiments, an excess of PrPsc can used as a seed to convert recombinant PrP(: to a protease-resistant form (PrPKI? but bioassays of such conversion products have not shown any detectable infectivity [ZS’]. Despite the obvious limitations of such experiments they may represent an initial step in the generation of the infectious
Figure
2
r Neutral
or basic
pH
Denaturant
PrPC
Acidic
Schematic representation of the folding pathway to P-PrP. PrPc is protected from complete denaturation by the constramts
pH
Partiallyunfolded
Oxldising
P-PrP
monomers
Reducing
Unfolded
I
L-.-.
Fibrils ~
.--~~-
.~~
-.--
-
------___
Current Opinm
I” Genehcs B
of
the disulphide bond. It retains its native ahelical conformation at neutral or basic pH even in the absence of the disulphide bond but when reduced and acidified, PrPc converts slowly to /3-PrP, which lacks a-helical structure. At physiological ionic strength, P-PrP readily aggregates into material that contains fibrils indistinguishable from those obtained from diseased tissue and which exhibit marked protease resistance [28”].
Molecular
The discovery of p-PrP, and the demonstration that PrP is capable of slow inter-conversion between a native cx and a non-native p conformation which can be locked by intermolecular association, provides a plausible mechanism of propagation of a rare conformational state. It is possible that the a-PrP to P-PrP conversion, caused by reduction and mild acidification, is relevant to the conditions that PrP’: would encounter within the cell, following its internalisation during recycling. Such a mechanism could underlie prion propagation and account for the transmitted, sporadic and inherited aetiologies of prion disease. Initiation of a pathogenic self-propagating conversion reaction, with accumulation of aggregated P-PrP, may be induced by exposure to a ‘seed’ of aggregated fi-PrP following prion inoculation, or as a rare stochastic conformational change, or as an inevitable consequence of expression of a pathogenic PrP(: mutant which is predisposed to form P-Prl? It remains to be demonstrated whether monomeric, oligomeric or fibrillar forms of P-PrP are infectious in experimental animals under appropriate conditions, or whether other cellular cofactors are also required.
Inherited
human
prion
diseases
Further pathogenic PRNP mutations continue to be identified and the currently recognised list is summarised in Figure 3. ‘I’hese inherited forms account for -15% of all human prion diseases [29]. Although traditionally classified into (CSS, C:JD or fatal familial insomnia (l:FI), the degree of phenotypic overlap observed between different mutations and even in family members with the same mutation indicates that future classification will probably be based upon mutation alone [30,31]. How pathogenic mutations in PRA’P cause priori disease has yet to be resolved. In most cases, the mutation is thought to lead to an increased tendency of PrP(: to form PrPSC, although recent studies [32’,33’] suggest that this may not be attributable solely to Figure
biology
of prion
Wadsworth et al.
341
decreased thermodynamic stability of mutated PrP(:. Additionally, evidence has emerged indicating that experimentally manipulated mutations of the prion gene can lead to spontaneous neurodegeneration without the formation of detectable protease-resistant PrP [34,35-l. These findings raise the question of whether all inherited forms of human prion disease invoke disease through the same mechanism and, in this regard, it is currently unknown whether all are transmissible by inoculation.
New variant
CJD and BSE
Acquired prion diseases in humans can result from exposure to human prions through medical and surgical procedures (iatrogenic CJD) or cannibalism (kuru [29]). Lluring 19951996, the appearance of young-onset cases of vCJD with atypical clinical presentation led to extreme concern that BSE, epidemic in cattle in the LJK, had been transmitted to humans. Subsequently, both molecular strain typing [7,10] and transmission studies in wild-type [l&36] and transgenic mice [lo] have established that vCJD and BSE are caused by the same prion strain. PrPSc associated with vCJD generates a unique pattern of proteolytic digestion products, that can be readily distinguished from PrPsC associated with sporadic or iatrogenic (:JD [7,10] (Figures 1 and 4). ‘li) date, 40 cases of v<:JD have been confirmed in the IJK and, at present, because of the long incubation periods associated with these diseases, no clear epidemiological prediction can be made regarding the potential number of cases. As the pathogenesis of vCJD leads to l’rPsC deposition in lymphoreticular tissues [37,38”] and also the appendix [39’], in distinction to classic forms of CJD. this may now allow the estimation of pre-clinical vCJD prevalence [38”] by screening archival lymphoreticular material and in prospective studies of surgical tissues. The PrPsc type seen in tonsil in v(:JD differs in the proportions of the PrP glycoforms from that seen in the brain of the same patients, suggesting the
3
Pathogenic
mutations
Y 145stop
I
P105L
Polymorphic
mutations variants
R208H ,
T183A
I
variants
-~__---~__~
Pathogenic polymorphic
propagation
and polymorphisms are shown below.
in the human
prion
-
protein.
Pathogenic
mutations
are shown
above
Current Opmton in Genetics & Development
the schematic
representing
PrP;
342
Genetics
of disease
superimposition of tissue-specific effects on PrP glycosylation (Figure
Peripheral
pathogenesis
and 4).
is associated with defective FDC maturation, these investigators went on to demonstrate that mice deficient in tumour necrosis factor 1, which lack functional FDCs, were susceptible following peripheral inoculation - arguing that FDCs were not required for neuroinvasion [43]. As B lymphocytes are circulating cells that are known, in common with most bone marrow derived cells, to express PrP(:, these data highlighted the need to consider the risks posed by blood transfusions from donors incubating prion disease.
strain-specific
of prion disease
Peripheral inoculation of experimental animals with prions is typically followed by a prolonged, clinically silent, phase prior to detectable neuroinvasion and the subsequent appearance of neurological deficits. During this pre-clinical period, prions can be isolated from lymphoreticular tissues where they may replicate to high titres. Elucidating the cell types in which prions replicate in the periphery and, crucially, how prions are transported to the central nervous system (CNS) is of considerable interest and represents a plausible target for therapeutic and prophylactic regimes 1401.
As both prion replication [44] and transport to the CNS 1451 is dependent on PrPC: expression, it was anticipated that PrP(z expression on B cells would be required for their role in prion pathogenesis. This may not be the case, however, as haematopoetic stem cells derived from PrI’ knockout mice are just as efficient in removing the block to neuroinvasion in B-cell deficient mice as comparable cells from wild-type mice [46*-l.
‘I’he lack of effect of whole-body ionising radiation on prion pathogenesis argues against significant involvement of proliferating cells in the lymphoreticular phase of prion propagation 1411. Follicular dendritic cells (FDCs), which do not proliferate. have been the favoured candidate for some time [42] but evidence for a key role for B cells in prion propagation was produced using a panel of immunodeficient mice [43]. Although all of these mutants could be infected by intracerebral exposure to prions, mice with defects in B-lymphocyte differentiation were extremely resistant to prion infection following intrdperitoneal inoculation with scrapir prions. .4s an absence of mature B cells Figure
Although it is possible that B cells could transport prions to the CIKS via a non-PrP-dependent process, or that they might secrete factors which bind to prions and enhance their neuropathogenicity [46”], an alternative possibility is that FDCs - or a subset of cells not bearing the FCD-Ml or MZ immunophenotype - are in fact the cell type that promotes prion neuroinvasion. It is possible that prion propagation to high levels in FDCs simply increases the probability of prion uptake by the autonomic nerve terminals that extensively innervate lymphoid organs. Neuroinvasion via peripheral nerves has long been considered likely [47]. Thus, B cells need not be involved directly in either prion propagation or neuroinvasion but rather are one of a number of necessary factors that promote FDC development. It must also be considered that more than one mechanism may be involved and that different prion strains may preferentially use these different routes, complicating any therapeutic strategies. Such studies aiming to dissect the cell type(s) involved in the peripheral pathogenesis of prion disease by various reconstitution of P~,pO/O mice, may also be complicated by the known ability of GPI-anchored proteins to transfer from cell to cell (‘GPI-painting’) [4X].
4
W Diglycosylated 80
I
0 Monoglycosylated I
I
Unglycosylated I
I
70 -
n 60 50 t
New clues to PrPc function
0 I
;-
I
I
I
I
I
1I
Current Opmon in Genetics & Development -I
Relative proportions of di-, mono- and unglycosylated PrP following partial digestion with proteinase K. Types l-3 are seen in classic forms of CJD (either sporadic or iatrogenic in origin), type 4 is seen in new variant CJD [71. A distinct pattern is seen in the tons11 of vCJD patients. designated type 4t [38”]. Error bars where not visible were smaller than the symbols used to designate the means.
I
Gaining direct insight into the normal cellular function of I’rP(: through the generation of PrP-deficient (~-‘MP null) mice has proved frustrating. Although Prn~p null mice are completely resistant to prion disease [44], they develop and behave essentially normally [49]. Reported changes in synaptic activity [SO-.531 and circadian rhythms [54] have not pinpointed a precise role for PrP(: although intriguingly similar neiirophysiological features (and disturbances of sleep rhythm) are seen in priori disease itself, arguing that prion ncurodegeneration may result. at least in part, from PrP loss of function [SO]. ‘I’hese studies are complicated by the possibility that adaptivc changes during neuronal development may effectively mask the normal cellular role of PrP(:. (:omplete knockout of PI-T/~
Molecular
in adult mice, by conditional gene expression methods, may be revealing with respect to the normal cellular function of PrP, but has not yet been reported. Studies with synthetic peptides corresponding to the amino-terminal octapeptide repeats of PrP have demonstrated that this region can bind Cu2+ [55-571 and this has recently been extended to show Cu?+ binding to the entire amino-terminal domain of recombinant human PrP [SS] or full-length recombinant hamster PrP [59]. Furthermore, it has been demonstrated that membrane-rich brain fractions from P~np null mice possess reduced levels of Cuz+ compared to wild-type mice [SN] and also exhibit reduced activity of the C&+-dependent enzyme superoxide dismutase I [60]. There is also evidence suggesting that increased extracellular concentrations of
unless
I’rl’
also
binds
(:uz+
at
regions
biology
of prion
propagation
Wadsworth
343
et al.
important. The precise cell types involved in peripheral prion replication and transport to the CNS remain unclear. An important role for follicular dendritic cells is likely and multiple mechanisms may be able to mediate neuroinvasion following peripheral prion inoculation.
References
and recommended
Papers of particular interest, have been highlighted as:
published
reading
within the annual
period
of review,
l of special interest ‘*of outstanding interest
1.
Griffith JS: Self replication 215:1043-1044.
2.
Prusmer scrapie.
3.
Bruce M, Chree A, McConnell I, Foster J, Pearson G, Fraser H: Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. P/%/OS Fans R Sot Land 1994, 343:405-411.
4.
Bessen RA, Marsh RF: Biochemical and physical prion protein from two strains of the transmissible encephalopathy agent. J Viral 1992, 66:2096-2101.
5.
Bessen RA, Marsh RF: Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Wrol 1994, 66:7859-7868.
6.
Parchi P, Castellani R, Capellari S, Ghetti B, Young K, Chen SG, Farlow M, Dickson DW, Sims AAF, Troianowski JQ ef al.: Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease. Ann Neural 1996, 39:767-778.
7.
Collinge J, Sidle KCL, Meads J, lronslde J, HIII AF: Molecular of prion strain variation and the aetiology of ‘new variant’ Nature 1996, 363:685-690.
8.
Telling GC, Parchi P, DeArmond SJ, Cortelli P, Montagna P, Gablzon R, Mastrianni J, Lugaresl E, Gambetti P, Prusiner SB: Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity. Science 1996, 27412079.2082.
and screpie.
SB: Novel proteinaceous Science 1982, 216:136-l
Nature
infectious 44.
1967, particles
cause
properties mink
of the
analysis CJD.
other
than the octapeptide to note that whereas
repeats. In this regard, it is of interest transgenic mice expressing PrP lack-
9.
DeArmond SJ, Sanchez H, Yehlely F, Qiu Y, Ninchak-Casey A, Daggett V, Camerino AP, Cayetano J, Rogers M, Groth D et a/.: Selective neuronal targeting in prion disease. Neuron 1997, 19:1337-l 348.
ing
develop
10
Hill AF, Desbruslais same prion strain 389:448-450.
11
HIII AF, Sidle KCL, Joiner S, Keyes P, Martin TC, Dawson M, Collinge J: Molecular screening of sheep for bovine spongiform encephalopathy. Neurosci Lett 1998, 255159-l 62.
12
Hope J, Wood Goidmann W, prion protein experimental 80:1-4.
13.
Safar J, Wille H, ltri V, Groth D, Serban H, Torchia M, Cohen FE, Prusiner SB: Eight prion strains have PrPsc molecules with different conformations. Nat Med 1998.4:1 157-l 165.
14
Milner J, Medcalf EA: Cotranslation of activated mutant p53 with wild type drives the wild type ~53 protein into the mutant conformation. Cell 1991, 65:765-774.
15
Wickner RB, Masison DC: Evidence for two and [PSI]. Curr Top Microbial lmmunol1996,
16
Wickner RB: A new prion controls fungal cell fusion incompatibility. Proc Nat/ Acad Sci USA 1997, 94:10012-l
17.
Riek R, Hornemann S, Wider G, Billeter M, Glockshuber Wuthrich K: NMR structure of the mouse prion protein (121-231). Nature 1996, 382:180-182.
residues
32-106
and
behave
normally,
mice
expressing PrP ivith deletion of residues 32-121 develop severe ataxia and neuronal cell death in the cerebellum, an effect that can be abolished by the presence of one copy of a wild-type
PrP
gene
[64”].
Conclusions ‘I’hc existence of multiple prion strain types can be accommodated within a protein-only hypothesis of prion propagation. Kapid molecular methods are now available to tyI)c prion strains and to investigate their origins. ‘This abilit>- of a single polypeptide chain to encode phenotypic information may have wider biological and pathobiological significance. The i/l vitrn interconversion of recombinantderived PrP between native and fibrilogenic conformations provides a plausible molecular mechanism for prion propagation. New variant CJD is caused by a prion strain indistinguishable from that causing BSE and has a pathogenesis distinct from that of classic CJD, having a prominent involvement of lymphoreticular tissues. It remains unclear if ;I substantial
cise function accumulating
epidemic
of
human
RSI<
will
occur.
of cellular I’rl’ remains obscure evidence suggests that copper binding
‘I’he
prc-
although
may be
18
M, Joiner S, Sidle KCL, Gowland I, Collinge causes vCJD and BSE. Nature 1997,
J: The
SCER, Birkett CR. Chona A. Bruce ME. Cairns D. Hunter N, Bostock CJ: &le&lar analysis of ovine identifies similarities between BSE and en isolate of natural scrapie, CH1641. J Gen Viral 1999,
prions in yeast: 207:147-l 60.
lUREI
0014. R. domain
PrP
James TL. Liu H, Ulyanov NB, Farr-Jones S, Zhang H, Donne DG, Kaneko K, Groth D, Mehlhorn I, Prusiner SB ef a/.: Solution structure of a 142-residue recombinant prion protein corresponding to the
344
Genetics
of disease
infectious fragment of the scrapie USA 1997, 94:i 0086-l 0091. 19.
isoform.
Proc
Nat/ Acad
Sci
Pan K-M, Baldwin MA, Nguyen J, Gasset M, Serban A, Groth D, Mehlhom I, Huang Z, Fletterick RJ, Cohen FE et al.: Conversion of ahelices into p-sheets features in the formation of the scrapie prion proteins. froc Nat/ Acad Sci USA 1993, 90:10962-l 0966.
20.
Prusiner SB, Scott M, Foster D, Pan KM, Groth D, Mirenda C, Torchia M, Yang SL, Serban D, Carlson GA et al.: Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 1990, 63:673-686.
21.
Palmer MS, Dryden AJ, Hughes JT, Collinge protein genotype predisposes to sporadic disease. Nature 1991, 352:340-342.
22.
Kocisko DA, Come JH, Priola SA, Chesebro B, Raymond GJ, Lansbury PT, Caughey B: Cell-free formation of protease-resistant prion protein. Nature 1994, 370:471-474.
23.
Bessen RA, Kocisko DA, Raymond GJ, Nandan S, Lansbury PT, Caughey B: Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature 1995, 375698-700.
24.
J: Homozygous Creutzfeldt-Jakob
prion
Kocisko DA, Priola SA, Raymond GJ, Chesebro B, Lansbury PT Jr, Caughey B: Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. Proc Nat/ Acad Sci USA 1995, 92:3923-3927.
25. .
Hill AF, Antoniou M, Collinge J: Protease-resistant prion protein produced in vitro lacks detectable infectivity. I Gen viral 1999, 80:1 l-14. A demonstration that the acquisition of protease resistance by PrPc in vitro is not sufficient for the propagation of prion infectivity, highlighting the need for caution in interpreting the biological significance of experimentally generated protease-resistant PrP. 26.
Chabry J, Caughey B, Chesebro B: Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides. J Biol Chem 1998, 273:13203-i 3207.
27. .
Caughey WS, Raymond LD, Horiuchi M, Caughey B: Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines. Proc NaN Acad Sci USA 1998, 95:i 2117-l 2122. This study identifies a new class of compounds that interferes with the generation of protease-resistant PrP formation in vitro and suggests their use as lead compounds for the generation of therapeutic agents for treating prion diseases. 28. l 0
Jackson GS, Hosszu LLP, Power A, Hill AF, Kenney J, Saibil H, Craven CJ, Waltho JP, Clarke AR, Collinge J: Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations. Science 1999, 283:1935-l 937. Conversion of PrPC to PrPsc results in an increase in the amount of P-sheet structure in the protein. This study identifies conditions in which recombinant human PrP can switch between native u-helical conformation and a compact, soluble monomeric form rich in P-sheet structure @-PrP). The generation of P-PrP in suitable cellular compartments and subsequent stabilisation by inter-molecular association provides a molecular mechanism for prion propagation. 29.
Collinge J: Human prion encephalopathy (BSE).
30.
Collinge J, Owen F, Poulter M, Leach M, Crow TJ, Rossor MN, Hardy J, Mullan MJ, Janota I, Lantos PL: Prion dementia without characteristic pathology. Lancet 1990, 336:7-g.
31.
Collinge J, Brown J, Hardy J, Mullan M, Rossor MN, Baker H, Crow TJ, Lofthouse R, Poulter M, Ridley R et a/.: Inherited prion disease with 144 base pair gene insertion: II: clinical and pathological features. Brain 1992, 115:687-710.
diseases and bovine spongiform Hum MO/ Genetics 1997, 6:1699-l
705.
32. .
Riek R, Wider G, Billeter M, Hornemann S, Glockshuber R, Wuthrich K: Prion protein NMR structure and familial human spongiform encephalopathies. Proc Nat/ Acad Sci USA 1998, 95:i 1667-l 1672. How pathogenic mutations in the prion gene cause disease is unknown. One simple mechanism would be that the mutations lead to decreased thermodynamic stability of mutated PrPC and concomitant propensity to form PrPsc. This study questions the likelihood of such a universal mechanism and suggests that subtle structural differences in the mutant proteins may affect intermolecular signalling in a variety of ways. Swietnicki W, Petersen RB, Gambetti P, Surewicz WK: Familial mutations and the thermodynamic stability of the recombinant human prion protein. J Biol Chem 1998, 273:31048-31052. See annotation [32-l.
34.
Muramoto T, DeArmond SB. Heritable disorder mice expressing prion 1997, 3:750-755.
SJ, Scott M, Telling GC, Cohen FE, Prusiner resembling neuronal storage disease in protein with deletion of an &helix. Nat Med
35. .
Hegde RS, Mastrianni JA, Scott MR, DeFea KA, Tremblay P, Torchia M, DeArmond SJ, Prusiner SB, Lingappa VR: A transmembrane form of the prion protein in neurodegenerative disease. Science 1998, 279:827-834. Examlnatlon of different topological forms of PrP generated at the endoplasmic reticulum identifies a particular transmembrane form of PrP whose presence is associated with neurodegeneration in mice. The absence of detectable PrPsc indicates that abnormal PrP biogenesis and topology may result in neurodegeneration and suggests that this mechanism may underlie pathogenesis in certain genetically inherited forms of human prion disease. 36.
Bruce ME, Will RG, lronside JW, McConnell I, Drummond D, Suttie A, McCardle L, Chree A, Hope J, Birkett C et al.: Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 1997, 389:498-501.
37.
Hill AF, Zeidler M, lronside J, Collinge J: Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. Lancef 1997, 349:99-l
00.
38. ..
Hill AF, Butterworth RJ, Joiner S, Jackson GS, Rossor MN, Thomas DJ, Frosh A, Tolley N, Bell JE, Spencer M et a/.: Investigation of variant Creutzfeldt Jakob disease and other human prion diseases with tonsil biopsy samples. Lancet 1999,-353:183-l 89. This study reveals the deposition of PrPsC In lymphoreticular tissues of patients with new variant CJD but not in other forms of human prion disease. These findings demonstrate that new variant CJD has a different pathogenesis to classic CJD and that, in the appropriate clinical context, characteristic PrPsC positivity in tonsil biopsy is diagnostic of new variant CJD. Screening of surgical tonsil and other lymphoreticular tissues [39’] can now be used to estimate the prevalence of pre-clinical vCJD. 39. .
Hilton DA, Fathers immunoreactivity Creutzfeldt-Jakob See annotation [38*-l.
E, Edwards P, lronside JW, Zajicek J: Prion in appendix before clinical onset of variant disease. Lancef 1998, 352:703-704.
40.
Aguzzi A, Collinge prion inoculation.
J: Post-exposure prophylaxis after Lancef 1997, 350:1519-l 520.
41.
Fraser H, Farquhar scrapie incubation
42.
Clarke MC, Kimberlin RH: Pathogenesis of mouse scrapie: distribution of agent in the pulp and stroma of infected spleens. Vet Microobiol1984, 9:215-225.
43.
Klein MA, Frigg R, Flechsig E, Raeber AJ, Kalinke U, Bluethmann H, Bootz F, Suter M, Zinkernagel RM, Aguui A: A crucial role for B cells in neuroinvasive scrapie. Nature 1997, 390:687-690.
44.
Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet Weissmann C: Mice devoid of PrP are resistant to scrapie. 1993, 73:1339-l 347.
45.
Blattler T, Brandner S, Raeber AJ, Klein MA, Voigtlander T, Weissmann C, Aguzzi A: PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 1997, 389:69-73.
accidental
CF: lonising radiation has no influence on period in mice. Vet Microbial 1987, 13:21 l-223.
M, Cell
46. ..
Klein MA, Frigg R, Raeber AJ, Ftechsig E, Hegyi I, Zinkernagel RM, Weissmann C, Aguzzi A: PrP expression in B-lymphocytes is not required for prion neuroinvasion. Nat Med 1998,4:1429-l 433. This study demonstrates that the critical role of B cells in neuroinvasion by scrapie [431 does not require B-cell expression of PrPC. These data suggest that B cells may simply allow maturation of follicular dendritic cells. Other interpretations are discussed in [48]. 47.
Kimberlin RH, Walker CA: Pathogenesis evidence for neural spread of infection 1980, 51 :I 83-I 87.
of mouse scrapie: to the CNS. J Gen
l/ho/
48.
Collinge J, Hawke S: B lymphocytes in prion neuroinvasion: Central or peripheral players. Nat Med 1998,4:1369-l 370.
49.
Bueler H, Fischer M, Lang Y, Bluethmann H, Lipp H-P, DeArmond SJ, Prusiner SB, Aguet M, Weissmann C: Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 1992, 356:577-582.
50.
Collinge J, Whittington MA, Sidle KCL, Smith CJ, Palmer MS, Clarke AR, Jefferys JGR: Prion protein is necessary for normal synaptic function. Nature 1994, 370:295-297.
51.
Whittington MA, Sidle KCL, Gowland I, Meads J, Hill AF, Palmer MS, Jefferys JGR, Collinge J: Rescue of neurophysiological phenotype
33. .
Molecular
seen
in PrP null mica by transgene 1995,9:197-201.
encoding
human
prion
protein.
60.
Manson
JC, Hope
gene dosage 53
Coking
SB, Collinge
protein
null mice:
potentiation.
J, Jefferys
Disrupted
JGR: Hippocampal slices Ca*+-activated K+ currents.
terminal protein.
from prion Neurosci
MP, McDermott
tandem
repeat
Biochem
Biophys
in mice devoid JR, Candy
regions
of prion
JM: Copper
of mammalian
Res Commun
protein.
binding to the Nand avian prion
Miura T, Hori-i A, Takeuchi
58.
59.
Left 1996,
H: Metal-dependent
rich octapeptide
u-helix formation region of prion protein.
396:248-252.
Brown Fraser
DR, Gin K, Herms JW, Madlung A, Manson J, Strome R, PE, Kruck T, von Bohlen A, Schulz-Schaeffer W et a/.: The cellular prion protein binds copper in v&o. Nature 1997, 390:684-687. Stockel
selectively
WJ, Schmidt
B, Kretzschmar
cells show altered response SOD-l activity. Exp Neural
AC, Cohen FE, Prusrner SB: Prion protein copper(h) ions. Biochemistry 1998, 37:7185-7193.
J, Safar J, Wallace
binds
Pattison
IH, Jebbett
scrapie
and cuprizone
et a/.
345
HA: Prion
to oxldative
stress
1997,146:104-l
12.
of the prion
64. ..
JN: Histopathological
toxicity
in mice.
similarities between Nature 1971, 230:115-i
17.
Fischer M, Rulicke T, Raeber A, Sailer A, Moser M, Oesch 8, Brandner S, Aauzzi A, Weissmann C: Prion protein (PrP) with amino-proximal EMBO
restoring J 1996,
susceptibility
15:1255-l
of PrP knockout
mice to scrapie.
264.
Shmerling D, Hegyi I, Fischer M, Blamer T, Brandner S, Giitz J, Riilicke Flechsig E, Couio A, von Mering C et al.: Expression of amino-
terminally truncated specific cerebellar
1995,207:621-629.
57.
FEBS
Wadsworth
Pauly PC, Harris DA: Copper stimulates endocytosis J Biol Chem 1998, 273:33107-33110.
deleiions
Nature
Homshaw MP, McDermott JR, Candy JM, Lakey JH: Copper binding to the N-terminal tandem repeat region of mammalian and avian prion protein: structural studies using synthetic peptides. Biochem Biophys Res Commun 1995, 214:993-999.
by the glycine-
62.
P, Frscher M, Rulicke T, JC: Altered circadian
56.
promoted
61.
63.
I, Gaus SE, Deboer T, Achermann M, Oesch 8, McBride PA, Manson
Hornshaw
1995,
209:49-52.
activity rhythms and sleep 1996, 380:639-642. 55.
N: PrP
A, Black C, MacLeod Neurodegeneration
propagation
protein.
14.
Tobler Moser
AR, Johnston
and long term
4:113-l
Leff 1996, 54
J, Clarke
of prion
DR, Schulz-Schaeffer
protein-deficient due to decreased
Nat Genet 52
Brown
biology
PrP in the mouse leading lesions. Cell 1998,93:203-214.
to ataxia
T,
and
Transgenrc mice expressing amino-terminally truncatea rrr- racxmg resroues 32-l 06 develop and behave normally whereas those with longer deletions of 32-l 21 or 32-134 develop ataxia and specific neurodegeneration 2-3 months after birth. These data suooest that an essential PrP function may resrde within residues 106-l 21 a% that truncated mutant proteins lacking this region may be non-functional and may compete with another molecule possessing PrP-like function for a common ligand. 65.
Collinge
iatrcgenic
J, Palmer MS, Dryden
Creutzfeldt-Jakob
AJ: Genetic
disease.
predisposition to 1991,337:1441-l
Lancet
442.
Other recommended reading 66.
Moore RC, Hope J, McBride PA, McConnell, Selfridge J, Melton DW, Manson JC: Mica with gene targetted prion protein alterations show that Prnp, Sine and Prini are congruent Nat Genet 18:118-l 25.