Of mice and (mad) cows — transgenic mice help to understand prions

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P rions cause

the transmissible spongifonn encephalopathies (TSE)’ (reviewed in Refs 2-41, which are fatal diseases characterized by neuronal vacuolation and loss, reactive astrocytosis and, sometimes, extracellular amyloidic plaques. Human prion diseases include Creutzfeldt-Jakob disease (CJD), Gerstmann-SttiusslerScheinker syndrome (GSS), fatal familial insomnia (FFI), and a novel variant of C. (KIJD) now observed in the UK (Ref. 5). Scrapie of sheep, long the experimental prototype of these disorders, seems now to have spread to cattle in the form of bovine spongiform encephalopathy (BSEYj.There are no known naturally occurring TSEs in mice or hamsters. The normal prion protein PrPc is encoded by rhe host gene Ptv2-p in the mouse. PrPc is a small glycoprotein that is anchored to the surface of neurons and other cells through a glycophosphoinositol (GPI) moiety (Box 1, Fig. 1). Prions appear to propagate by refolding mature PrPc into an aberrant conformation called PrP”‘, which, in turn, is the only known component of prions and serves as a ‘template’ to convert more PrPC molecules (Fig. 2). Thus, the host gene P)n-p provides both the ‘sub?*Tte’ CPrPC)and the ‘template’ (PrP% for further prion ‘replication’. This account of prion propagation as a proteinaceous chaiti reaction summarizes the views of the now popular *protein only’ model of prions, which explains most observed phenomena without resorting to many nd hoc hypotheses (however, see Box 2). Indeed, transgenic experiments did much to establish the ‘protein only’ paradigm as a plausible theory, and we use this model here to evaluate various Box 1. The prionpfotelns

Prions that cause the transmissible spongifonn encephalopathies are thought to propagate by converting a normal protein of the host, PrPC, into an abnormal conformation called PrP*. The PrpCand PrP* isoforms (collectively known as PrP) are encoded by the host gene. which in mice is called Pm-pand in humans PfW? Pm-p comprises two or three exons. but the whole open reading frame is contained within the iasr exon. PrPc is a small (231 amino acids) glycophosphoinositol-anchored giycoprotein {Fig. 1). The transformation of Prfc into PrP% appears to be solely conformational and appears to invoive the change of a-helices into &sheet&. While the brains of animals with prion dii synthesize PrPc and PrPsc, only PrpC is found in healthy animals. The PrP isoforms differ strikingly in theti properties. in contrast to PrpC, native PrPsc possesses a protease-resistant core (called PrP27-301, ts insoluble in all known detergents and has the propensity to form amyloidic structures called priori rods. Because there are no isoform-specific PrP an&o&is, these disparate properties are used to differentiate between PrPc and PrP*. However, there are often experimental situations in which ir is diicult to decide whether a specific PrP molecule is in the PrpCor the PfPk state. The conversion PrpC-3 PrPsc has been extensively studied in cultured cells inFectedwith priors. These are cells that have been incubated with brain extracts from animals with priori d&eases and produce more prions as well as PrP*. Itzuitm mixing experiments have also mimicked the conversion of PrPc into a PrP species with many of the properties ofPrPsc (Ref. 481, although generation of priori infectivity has yet to be demo-ted in these experiments.

Ofmiceand(mad)cowstransgenic micehelpto understandpriors

Primts

pm-sent a most fawbaatbt.. biological cmndmltl

Tbesejmotelnaceott.spartkles seemto pro$agate tbmugb a cbah macthta ia tub&ba bostpmteiq FrF, is post-tra&ationaUy ndz@&d fofm neaupims. By this mecbantsm tbey ?e@kate wubowt &mhmmt of spc@c nucleic acids Due to their nttiqw nmdks operafflu,ph.9 cause disoders tbat can be infect&w, h&r&d and sporadic Transgeneti bas bem hnwluable in bdpingto uaderstnntl t&isursiqrreptir Here we &scr&e some of the most salient cotitributi 4 of transgedc n&e to tbrSpeti experiments. Other views of the TSE agent, which range from virinos’ to unconventional \ Auses~, have been less successfuul, to date, in accounting for the existing experimental data. ptls-p genes contribute to the species bapier Incubation time is usually vastly lengthened when prions are transmitted across animal species: this is the species harrier. With further passages within the new species, the incubation time then decreases and stabilizes around a new figure that is characteristic for that specific prion strain (Box 2) in that particular species. For instance. the species barrier between Syrian hamsters

(SHa) and mice (MO) is large. When mice of the FVB smin are inoculated with SHa prions @ion strain Sc237>, they become sick only after at least 400 days. whereas when they are inoculated with homologous mouse prions @ion strain RMLI,they die within 130 days. In a groundbreaking experiment performed in Stanley Prusiner’s laboratov, Scott et 61.9demonstrated that the species barrier is strongly modulated by the differences between the Pm-p open reading frames of tile two animal species. These investigators constructed mice expressing vari-ous levels of hamster PrPc, inoculated them with brain homogenates from either hamsters or mice with scrapie, and made several interesting observations. (1) The incubation time of hamster prions in these mice was reduced significantly when compared with that in wild-type mice; i’le higher the expression level of hamster PrPc, the shorter the incubation time. (2) Following inoculation of hamster prions, the transgenie mice produced a PrPsCderived from the SHa transgene. (3) Upon further passages, these prions infected hamstersmore easily than mice and so they were, therefore, of the hamster variety. Expressing SHaPrPc in mice seemed, therefore, to abolish the species barrier between hamsters and mice, suggesting that Pm-p specifies the species barrier (but not exclusively; see ‘Protein x’, below). From the point of view of the ‘protein only’ model, this happens

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s-s 129 #etNal r, J I

because heterologous hamster priConservedregion PrP ons now have a PrPc *substrate’ of CHO CHO their own kind to ad on in the 23 231 transgenic mouse. PrP27-30 181 197 I Conversely, when mouse prions D178N E209K P102L Signal peptide were inoculated into SHd’m-p (cleavedat ER) transgenic mice, the brains of these 215 230 Proposed‘Protein X mice produced prions of the mouse bindlng site kind: slow to cause disease in a hamster, but quick in a mouse, and MH2M their PrPsC was also of the mouse 96 131 133 sequence. Thus, of the two PrPc species (SHa and MO) expressed in the transgenic host, the prion species used for inoculation seemed to MHu2M interact with and select the PrPc of 96 131 its own species. This suggested to Prusinert” that PrPc and PrPsC must come in direct contact within the 1.Map of mature priori protein t PrP;see Box 1). The wild-type human priori host cell, perhaps to form meta- FIGUUE stable PrPc-PrPsc heterodimers, be- protein (PrP) isofonns. namely PrPc and PrPs’. possess identicai covalent structures. Several post-trandational modifications of the protein are depicted: f1J two N-Jinked fore forming more PrP%. carbohydrates (CHO); (2) a disulfide bridge between into cysteines (5-S): and Finally, the incubation time for (3) a C-terminal glycophosphoinositol anchor (CPU. Three pathogenic mutations causing mouse prions increased in proporCreutzfeldt-Jakob disease (D178N and EZOOK) or Gerstmann-Stdussler-Scheinker tion to the number of the hamster syndrome (P102L). and the polymorphism Ml2gV are also shown. PrP27-30 is the transgenes in these transgenic mice, protease-resistant core of Prl’~. hlH,M and MHu2Mare hybrid hamster-mouse and suggesting that transgenic hamster human-mouse PrP. respectively. These chime& proteins were used in the construction of PrPc inhibited the replication of diverse trxqenic mice in Table 1. Ahhreviations: ER. endoplasmic reticuhtm; Hu. human; mouse prions. These results, there- MO, mouse: SHa. Syrian hamster. fore, revealed the existence of a (yet uncharacterized) rate-limiting step The brain of patients with genetic priori diseases for which hamster and mouse prions both compete in usually contain infectious prionst6. Can a mere mutheir attempt to replicate. To avoid interference of entation in PRNP really cause the production of de nova dogenous mouse prions with the tmnsgenic prion proinfectious prions in the host? This question received a tein of interest, mice transgenic for Pm-p are now often persuasive answer when Hsiao et al. produced mice constructed on a null endogenous Pm-p background. that expressed a mutant mouse PrP with the substitution ProlOHeu, which mimics the pathological muCbimeric mouse-hamster Pm-p bridges the species tation of GSS (Ref. 17). All these mice spontaneously barrier developed a priori disease at a mean age of 70 days If the barrier between successive species is specified old and developed a characteristic neuronal vacuoby differences in their respective &n-p, might it not be lation, whereas their non-transgenic littermates remained possihle to ‘bridge’ this obstacIe by devising a hybrid healthy. Even more interestingly, a prion-like disease Pn2-p gene containing parts of the two species? To could be transmitted hy inoculating Syrian hamsters address this question, Scott el a/It constructed mice expressing a hybrid hamster-mouse Pm-p called MH,M (but, surprisingly, not wild-type mice) with brai;l samples from these spontaneously sick transgenic mice, (Fig. 1) and inoculated them either with Syrian hamster which, therefore, contained infectious prion@ (see abo prions or with mouse prions. Indeed, in either case a hybrid MH,M-PrPs’ was formed, and the resulting attifiRef. 19)‘. These results can be accounted for very simply by cial prions were equally infectious in both hosts. This the ‘protein only’ model. It suffices to assume that the type of study paves the way to mapping the residues amino acid substitution destabilizes the spatial folding within the prion protein that are required for a producof PrPc, or otherwise renders this protein more prone to tive interaction between PrPc and PrP”‘. converting spontaneously into PrPsC. The new prions would subsequently commence an ‘autoinfection’ in the Mimicking genetic and sporadic priori disases mouse (Fig. 2). Harris et al. have, indeed, observed that Hsiao et al. were the first to demonstrate a genetic when PrP molecules with pathological mutations are linkage hehveen a mutation in the human PRh’P gene expressed in cultured Chinese hamster ovary cells, they (resulting in the substitution of ProlO to i.eu) and an inherited prion disease (GSS)t?. Other mutations in PRlVP have abnormal properties reminiscent of those of PrPQ (Ref. 201, possibly pointing to their fundamental were subsequently linked to familial CJD (such as the substitution Glu200Lys in Lybian Jews’% FFI and GSS, tendency to fold abnormalty. while a silent polymorphism at codon 129 was found to modulate the susceptibility of patients to CJD (Ref. 14) as well as to determine whether carriers of a pathogenic sub stitution at codon 178 will develop CJD or FFI (Ref. 15). TfG JULY1997 VOL. 13 No. 7

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SmPm-p

w-r

Transgenic mice sensitive to hamster prions

(Refs 9,10>

Hamsters sensitive to prions produced by transgenic mice inoculated with hamster prions Extended incubation time in mice overexpressing SHaPrPC when incubated with mouse prions

PtpC and PrPk interact directly; PrP* prefers cognate PrpC PrP in host and priorrs specilks species barrier SHaPrP and MoPrP compete for PrPk formation sites

Pm-p ProlOHelJ (Ref. 17)

wf

Mice develop spontaneous spongiform neurodegeneration Mice produce tnfecttous prioI@

ProlOlLeu mutant PrP induces spontaneous disease and production of infectious prions

WI+ Pm-p

wr

Neuromuscular dii in old mice (400 days) Mice produce low titer infectious prions

W PrpC can induce spontaneous production of priors when overexpressed

MHZM (Ref. 11)

wr

Mice sensitive to hamster and mouse prions Mice produce prions that infect hamster and mouse

Chimeric PrP ‘bridges’species barrier between hamster and mouse

PIWP

w-r

Mice not very sensitive to human priors

NuU

Incubation not shortened when incubated with BSE (Ref. 39) Mice susceprible to human prions

Endogenous mouse PrP interferes with formation of human PrPk (see ‘Protein X below) Not indicative regarding susceptibility of human PrP to BSEb Protein X bids HuPrP too, albeit at lower affinity as compared with mouse PrP Priori species barrier from cattle to human is very large

OVefe~resxd

(Ref. 21)

(Ref. 35)

Mice inoculated with BSE show no symptoms after more than 500 days37

wr

MHuaM (Ref. 351

Truncated

Pm-p

Null

Mice sensitive to human prions (in contrast to PRNF expression, above) When inoculated with different strains of familial CJD prions, they produce PrPk of a distinct molecular weight ‘signature“Q

A putative species-specific Protein X binds to MoPrP at residues 215-230 Resulo strongly suggest that priori strains are determined by protein conformation without an additional informational molecule

Null

Mice susceptible to, and produce, prions

N-terminal 90 residues dispensable For

Null

Upon intracerebral inoculation of prions, vacuolation and PrPsc develop in graft, but not in normal tissue Upon peripheral inoculation, graft untouched

Local PrPc expression necessary for development of cytopathology

PrPsc synthesis

(Ref. 33) 3iain @aft from WT mouse Pm-p

overexpressors31

PrPc expression needed to convey irktivity

From the periphery to the brain

%ow titer prions, perhaps of a different strain. bMore stringent conclusions about human susceptibility to

BSE prions can be drawn from transgenic mice expressing either the human PIP or the MJ+M chimera on a nuii background3’. Abbreviations: BSE, bovine spongiform encepha!op&y; CJD, Creutzfeldt-Jacob disease; MH2M, hybrid hamster-mouse prion gene; MHuzM, hybrid hu man-mouse prion gene tdefmed in Fig, 1); Pm-p, mouse prion gene; PRW. human priori gene; PrP, coU&ve term for prion isoforms (Box l), i.e. PrPc is the mature protein and PrPk is the aberrant form; SHa, Syrian hamster; MO, mouse; WT, wild type.

The unique modus operandi of prions, therefore, enables them to cause disorders that are either infectious or genetic. Another transgenic model appears to mimic the ‘sporadic’ aspect of prion diseases: transgenic mice overexpressing wild-type Pm-p developed, at advanced age, a degeneration of skeletal muscles, periphera! nerves and the central nervous systemal. Preliminary bioassay data indicate that infectious prions are also formed in these miceaa. These results argue that even wild-type PrpC can spontaneously convert into PrPk, albeit at a low rate. Alternatively, it has been suggested that large numbers of Pm-p transgenes can increase the

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chance of somatic pathological mutations, eventually mimicking the genetic diseases (Fig. 2). Transgenic mice, therefore, provide the first animal model for priori diseases of non-infectious etiology and might prove excellent systems for the development of therapeutic and prophylactic approaches.

Fvionsdo not propagate inm-p null mice If pnons are actually made principally or even solely of PrPsc, then they should be unable to replicate or cause disease in a mouse devoid of PrP. With this premise in mind, Charles Weissmann, Stanley Prusiner VOL. 13 No.

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and their collaborators created Pm-p knockout mice*3 and generated a crucial set of results. When these mice were inoculated with mouse prions, they, indeed, remained healthy, and prion titer failed to amplify in their brain+25. These results clearly fit the ‘protein only’ model.

(a) Infectious prions

Whatis thephykkgicd role of PrPc? Another motive for constructing the Pm-p knockout mice was to study the normal rote of PrpC, which had eluded other approaches. At fmt sight, the first Pm-p null mice showed no phenocypic abnormalities23. This type of negative result recurs in other systems where the inactivation of conserved and seemingly indispensable genes caused no phenotypic defect. Understanding how compensation for PrPc function is achieved in the null mice might be very informative about the role of this protein. Lately, minute effects were observed in the original Biieler line as well as other lines of Pml-pnull mice (reviewed in Ref. 26). The reported disorders include: Cl) impaired synaptic function in hippocampal slices27, although this finding has been debated%; (2) progressive ata.xia from 70 weeks of age with loss of cerebeilar Purkinje cells in another line of Pm-p nuI1 mice29; z.nd (3) altered circadian @hmj and .&ep pattern in two lines of null miceM. These abnormalities might all be related to compromised r-aminobutyric acid (GABAIdependent synapses, where PrPc could play a regulatory rolez6. Whether PrPc function is at all impaired during priori disease (e.g. as this protein is challenged by increasing amounts of abnormal PrP*) is unknown. Might such a putative functional loss be deteterious to brain cells and cause some of the pathology of priori diseases? Because of the probable compensation for loss of priori protein function in the Pm-p knockouts, these animals might not h a good system to answer this question. More realistic modeling of PrPc loss of function might be achieved in future transgenic mice where Pm-p expression can be regulated at will. A brain gift intoPmp null mice Besides the fundamental problems of what prions are and how they replicate, there are many other very important questions that need to be answered if one is to understand priori diseases, for example how do prions jump from cell to cell? How do prions kill cells? An ingenious system that addresses some of these questions was devised by Brandner et al.3’ who used brain grafts (implanted in the ventricIes) from transgenic priori protein overexpressors as (I) ‘Trojan horses’ to produce PrPsC in sitri within the brains of Pm-p-null mice, and as (2) sensors to detect the presence of infectious prions in their surroundings. When these mice were inoculated intracerebrally with mouse prions, the graft developed massive neuronal vacuolation and formed PrPk. However, no pathology was observed in the surrounding brain tissue (which does not express prion protein), although some PrPs’ did ‘spill over’ from the graft area. This suggested that endogenous PrPc expressed in brain cells is needed to mediate the pathogenic effects of PrP,k (Ref. 31). A possible reason for this is that exogenous PrPsc is handled differently by cells than endogenous PrPk. In scrapie-infected neurobiastoma cells, for instance,

-

+%-

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Spontaneous cvnversion

00

00

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‘Autoinfection

rare event)

1

Somatic mutation

0 0 Spontaneous conversion 0 0 (Rare- event) m00 OPfl

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‘Autoinfection

?? Mutant PrP%

FIGURE 2. Mechanismsproposed to explain infectious,genetic and sporadic propagation of prions in the ‘protein only’ model. The conversion of PrPCto PrPk is the major event in prion replication. (a) Infectious propagation of prions is achieved when PrPk ‘templates’ convert PrpC‘substrate’into more PrP*. Dinxt interaction between PrpCand PIP”, as well 35 mediation of other cellular facton. such as Protein X, are thought to feature in this conversion.(b, c) In the genetic and sporadic manifestations of prions. the initial conversion events are thought 10 Lx spontaneous, without intervening PrPk templates.Thisis then followed by ‘autoinfection’ in the patient’s brain. In genetic cases. mutations in the gene PEW might destabilize the conformation of PrPcand predispose it towards converting into PrP*. in sporadic cases. the initial formation of PrP from PrF molecules might either result from pathogenic somatic mutations in PRNP,or even be caux~I by a very rare spontaneous refolding of a few wild-type PrPc molecules into the pathological conformation. endogenous PrPsC is attached to cholesterol ‘rafts’within cellular membranes (probably via its GPI anchorY*, whereas exogenous PrPsc is endocytosed, but fails to attach to these rafts in large amounts (N. Naslavsky and A. Taraboulos, unpublished). It is also possible that endogenous PrPc is needed to activate the toxicity of PrPSC,perhaps by a receptor-ligand-like mechanism. In contrast, when the graft-bearing mice were inoculated peripherally, no PrPsc or pathology developed in the graft. This suggests that PrpC is also needed to convey prions through peripheral pathways to the cenual nenrous system, possibly by supporting local Prpsc’synthesis that can counter prion ‘signal attenuation’ on its way to the brain.

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REVIEWS a rate-limiting step for which mouse and human prions competed. In contrast to the result with the hamster transgene, however, this competition could not be overcome just by overexpressing PRNPin a wild-type mouse: it was also necessary to abolish the endogenous mouse Pm-p altogether. The reason for this difference became apparent when a mouse-human chime+ prion protein (Fig. 1) was expressed in Pm-p+/+ mice: in this case the transgenic mice were, indeed, susceptible to human prions. So, human and mouse-human hybrid priori proteins differ vastly in their ability to form PrP* and to support priori propagation in the presence of the mouse priori background. Observing that this contrast must stem from differences in the sequence of the transgenic products, Telling et g!.s hypothesized that another molecule of the host must interact with the priori protein in the course of priori replication. Dubbed Protein X, this molecule prefers sequences of the mouse-human hybrid protein to those found in the human prion protein, that is, it should interact with either of the mouse portions of the mouse- human protein sequence. Because the N-terminal portion of the priori protein is dispensable for prion replication, these authors assumed that Protein X interacts with the C-terminal part of the prion proteins. The species barrier might, therefore, not be determined exclusively by Pm-p, but is also modulated by additional host factors, such as Protein X. This further complicates the construction of reliable transgenic mice models for human prion diseases.

Eox2.Prionstrahs Since the pioneering studies ofAllan Dickinsot@, the agents of the transmissible spongiform encephalopathies have been known to come in various ‘strati. When agents of different strains are inoculated into identical hosts, disparate spongiform encephalopathies develop, which diier in one or more of the following: (1) duration and presentation of the disease; (2) neuropathology; (3) distribution of PrPg in the brait@; (4) sensitivity of PrPk to proteolysis, and (5) size of the PrP protease-resistant core and its pattern of glycosyt ation (see Box 1). This last property was fti described in the two stmins oftransmissiile mink encephalopathy (dmqand &LX&~. and is now reported in additional prion diseases. For instance. brain samples from familial Creutzfeldt-Jakob dii patients have proteinase-K-resistant PrP bands characteristic to each mutation, and the banding ‘signature’ could be perpetuated when the brain samples were inoculated into chime& human-mouse mice*?. Strains usually ‘breed true’ when propagated (even when transmitted to different animal species). although they can ‘mutate’ now and then. More than five strains have been demonstrated _ii mice52 (the number of distinct strains is disputed). How the strain identity is encrypted within prior& and how it is transmitted with such a remarkable fidelity to the next ‘generation’, are still open questions. Tbe existence of prion .%Gns has been seen as proof that pfions

must can-ynucleic acid genomes, which can mediate inheritance and mutation@. As no such nucleic acid has been discovered, other hypotheses have been put forwarddl*Q.Priori strains have now emerged as the next major conundrum in priori research.

Mad cows

whataretheprm-psequencesnecessary for priori t+.iCdOlh? What is it that endows the prion protein with the competence to form infectious prions? Broadly speaking, one can discern two distinct functional components for infection: (1) the prion protein has adequate struc!~~rz!properties (e.g. it can fold into at least two altemaand can form amyloid fibrils); and tive conformations

(2) the host cell handles it appropriately (e.g. it provides a compartment for nascent PrPc to meet PrP?. Ultimately, these features must be determined by the prion protein sequence, which can be studied in transgenie mice. Trdnsgenic experiments have now shown that a truncated priori protein lacking all 90 N-terminal residues can restore the susceptibility of Pnz-p-null mice to prions33. These results extend previous observations obtained in cultured cell&. Other important results are awaited in this line of investigation. Human priori tmnsgene.s and Protein X In light of the previous successes with hamster priori protein, it was surmised that overexpressing human priori gene, PRh!P, in transgenic mice would decrease the incubation time of human prions in the mouse bioassay. The first set of PRNP transgenic mice was, therefore, puzzling: even considerable overexpression of PRNP did not decrease the incubation time of human prions in the transgenic mice when compared with their nontransgenie littermates. When the same PRNP was expressed in Pm-p-null mice, however, the incubation time for human prions was shortened very significantiy3j. As in the case of the SHaPm-p transgenic mice, this suggested

%!Veld findings suggest that the novel variant CJD, vCJD, in the UK (Ref. 51, originates from BSE, probably through the consumption of contaminated beef products: (1)vCJC has abnormal characteristics unseen before the BSE epidemic, such as an abnormal age distribution, and distinct clinical presentation and pathological findings; and (2) vCJD cases have a variant and characteristic PrPbc ‘banding signature’ (in SDS-PAGE) that resemble those of l3SE transmitted to mice, cats and macaques, rind might denote the involvement of a novel strain of prions (Box _‘)s-. How can transgenic mice help elucidate this issue? Wild-type mice are highly susceptible to inoculation with BSE prions*. Therefore, the finding that BSE incubation times are not shortened upon expression of human PRNP trdnsgene in Pm-p +I+mice39 does not necessarily indicate that the species barrier from cattle to human is large. From what was learned in the human-mouse tmnsgenic system, one can assume that the susceptibility of human prion protein to BSE can be best studied in mice expressing human priori protein in a null Pm-p background. Such mice have already been inoculated wirh BSE in several laboratories. In one laboratory, these animals remained free of clinical signs 500 days after inoculation, suggesting that BSE does not transmit easily, if at all, to these transgenic mice37. It will be interesting to compare the futute incidence of vCJD with the predictions of these transgenic experiments.

FWqMtives The transgenetic technology has already yielded very important results in the TSE fie!d and its potential is certainly not exhausted. Results from mice constructed

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by novel techniques, such as inducible, ectopic, or cellrestricted expression of Pm-p (Ref. 401, are much awaited. While Pm-p has been the sole player in the field to date, it is to be expected that other genes will be added to the relevant pool in the future. For instance, $on-protein-binding proteins, such as Protein X, or other molecules that have been reported4t, are excellent candidates for studies in transgenic mice. These mice should help us to understand the mechanisms behind priori replication and should be instrumental in answering other important questions, such as how do prions gain access to the brain? How do they replicate in the host cell and subsequently infect neighboring cells? How do they cause neurodegeneration? Transgenic mice with chimetic genes will provide new models for studying priori strain&. Transgenic mice can be used in conjunction with molecular modeling to study the structural elements in the prion protein that are involved in: (1) its misfolding; (2) the interaction between PrPc and PrPk; and (3) in the species barrier. It seems that the time is ripe to use transgenic mice, which mimic the genetic and the sporadic forms of prion diseases, as a tool for prophylactic and therapeutic investigations. Several classes of anriprion compounds have already been described43~4* and could be assayed in these mice. The TSE prions are much more than a novel biological paradigm. They are emerging as a model for other diseases or biological processes that are transmitted and maintained epigenetically through protein conformational switches. For instance, several inherited metabolic disorders in yeast, long known for being nonmendelian,

are now thought to propagate through pathological protein refolding in a priori-- likefashiondj.+6.

2 Piusiner, S.B. (1991) Scimce

116. 13&l& 252. 1515-1522

3 Scotr, M.R.,Telling, G.C. and Prusiner, S.R.(1996) Crrw. Top. Microbial.Inlnlnrrol.207, 95-I 23 4 Prusiner, S.B. (1996) Czmrr.Top. .2licmbioi. 207, 5

Itnrturnol.

l-17

Will. R.G. d al. (1%) La~cel3+7,921-925 6 Wilesmith. J.W.. Ryan, J.B.M., Hueston, W.D. and Hoinville, L.J. (1992) vet. kkc. 130, 90-94 7 Kimberlin, R.H. (19%) Nutrrre297, 107-108 8 Diringer. H. (1991, Eur. J. Epidemiol.7, 562-566 9 Scott, M. etal.(1989) Cefi 59.847-857 IO Prusiner, S.B. da!. (1990) Celf63.673-686 II Scott, M. ef al. (1993) CeIi73.979-988 12 Hsiao, K. e1 nl. (1989) Nafrrve338, 342-345 13 Cabizon. R. d al. (1993) Am J. Him. Gene3.33. 828-835 14 Palmer, M.S., Dryden, A.J., Hughes, J.T. and Collinge, J. (1991) Natt~.e352,340-342 15 Medori. R. et al. (1992) Neul Engi. J. Med. 326. 444-449 16 Goldfarb, L.G., Brown, P. and Gajdusek. D.C. (1992) in Priorz D&euses of Hzrmarzs nnd Animal (Prusiner. SE., Collinge, J., Powell, J. and Anderton. B., e&I, pp. 139-153, Ellis Horwood 17 Hsiao, K.K. et al. (1990) Scie?zce250, 1587-1590

91269130 19 Telling, G.C. d ~1. (1996) Gerzes LIev. 10.1736-1750 20 Lehmann, S. and Han-is,D.A. Cl996,J. Biof. Chem. 271,

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Acknowledgements We thank the members of our laboratories for many helpful discussions. We apologize to authors whose work was not cited because of space limitations. References I Prusiner. S.B. ( 1982) Scimce

18 Hsiao, K.K. et ul. (1994) l%c. Nad Acad. Sri. V. S. A. 91,

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Nulltrc 370,471-474 49 Dickinson, A.G. and Meikle. V.M.H. (1971) +fol. Gezz. tietzer. 112,73-79 50 DeAnnond, SJ. er ai. (1993) Pm. Nar!.Acad. Sci. U. S. A. 90,6449-0453 51 Besxn. R.A. and Marsh. R.F. (1992) J. Viro[.66, 2096-2101 52 C&on, G.A. (1996) CUV.Top. Micmhiof. Imnunoi. 207, 35-47 53 Bruce. M.E.and Dickinson. A.G. t 19f$7)J Ge!z. Viml.68. 79-89 54 Hecker. R. ef rzl. (1992) GezzesDL’C.6. 1213-122s

R Gabtzom is in the Deparfment of Neum~ogy,Hahssah Uhmsiry Hospital,Jcrusaiem. Israel.

A. Tarabontos is in rhe Dq?arment of Mokcubir Biology, Hc&vr_w VnimWyHakmb MedicalSchool,Jenrsabem,&ael.

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