Strategies for eliminating PrP as substrate for prion conversion and for enhancing PrP degradation

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Sabine Gilch, Max Nunziante, Alexa Ertmer, Hermann M. Schätzl

To cite this version:

Sabine Gilch, Max Nunziante, Alexa Ertmer, Hermann M. Schätzl. Strategies for eliminating PrP as

substrate for prion conversion and for enhancing PrP degradation. Veterinary Microbiology, Elsevier,

2007, 123 (4), pp.377. �10.1016/j.vetmic.2007.04.006�. �hal-00532240�

Title: Strategies for eliminating PrP

as substrate for prion conversion and for enhancing PrP

degradation

Authors: Sabine Gilch, Max Nunziante, Alexa Ertmer, Hermann M. Sch¨atzl

PII: S0378-1135(07)00174-5

DOI: doi:10.1016/j.vetmic.2007.04.006

Reference: VETMIC 3651

To appear in: VETMIC

Please cite this article as: Gilch S., Nunziante M., Ertmer A. and Sch¨atzl H.M., Strategies for eliminating PrP

as substrate for prion conversion and for enhancing PrP

degradation, Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2007.04.006 This is a PDF file of an unedited manuscript that has been accepted for publication.

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Accepted Manuscript

Strategies for eliminating PrP

as substrate for prion conversion

1

and for enhancing PrP

degradation

2 3

Sabine Gilch

, Max Nunziante

, Alexa Ertmer and Hermann M. Schätzl

4

5

Institute of Virology, Prion Research Group, Technical University of Munich, 6

Trogerstr. 30, 81765 Munich, Germany 7

8

#

contributed equally 9

10

#

Corresponding author at: Institute of Virology, Prion Research Group, Technical University 11

of Munich, Trogerstr. 30, 81765 Munich, Germany 12

Tel.: +49 89 4140 6820; fax: +49 89 4140 6823.

13

E-mail address: schaetzl@lrz.tum.de (Hermann M. Schätzl) 14

15

Keywords: Cholesterol; gleevec; prion; prion protein; therapy; vaccination 16

17

18

Accepted Manuscript

Abstract 18

Prion diseases are fatal neurodegenerative infectious disorders for which no 19

therapeutic or prophylactic regimens exist. Our work aims to eliminate PrP

as substrate for 20

the conversion into PrP

and to increase the cellular clearance capacity of PrP

. In order to 21

achieve the first objective, we used chemical compounds which interfere with the subcellular 22

trafficking of PrP

, e.g. by intracellular re-routing. Recently, we found that PrP

requires 23

cholesterol for cell surface localization. Treatment with mevinolin significantly reduced the 24

amount of cell-surface PrP

and led to its accumulation in the Golgi compartment. These data 25

show that cholesterol is essential for the cell surface localisation of PrP

, which is in turn 26

known to be necessary for the formation of PrP

. Another anti-prion strategy uses RNA and 27

peptide aptamers directed against PrP

. We have selected peptide aptamers using a 28

constrained peptide library presented on the active-site loop of the E. coli protein TrxA in a 29

Y2H screen. Several peptides reproducibly binding to PrP

in several assays were identified.

30

Preliminary data indicate that selected peptide aptamers are able to interfere with prion 31

propagation in prion-infected cells. To obtain additive effects we have tried to clarify cellular 32

mechanisms that enable cells to clear prion infectivity. This goal was achieved by selective 33

interference in intracellular signalling pathways which apparently also increase the cellular 34

autophagy machinery. Finally, we have tried to establish an active auto-vaccination approach 35

directed against PrP, which gave some positive preliminary results in the mouse system. This 36

might open the door to classical immunological interference techniques.

37

38

39

Accepted Manuscript

1. Introduction 39

The elucidation of the cellular metabolism and the trafficking of the cellular prion 40

protein (PrP

) remains a fundamental target for developing therapeutic and prophylactic 41

strategies against prion diseases. Since the central nervous system suffers severe damage 42

during the course of TSEs even in the early clinical phase of the disease, treatments become 43

of value especially when prion infection arises from peripheral sites in the body. This is the 44

case when accumulation and replication of the infectious agent are also seen before 45

neuroinvasion, particularly in the lymphoreticular system. The recent studies assessing the 46

role of the immune system in prion diseases (Klein et al., 1997; Klein et al., 2001; Mabbott et 47

al., 2001) have led to promising vaccination strategies against prion infection (Gilch et al., 48

2003; Heppner et al., 2001; Peretz et al., 2001; Weissmann et al., 2001; White et al., 2003).

49

Other anti-prion approaches have targeted the cellular prion protein with the aim of stabilising 50

its conformation, thereby inhibiting initial conformational changes leading to conversion to its 51

infectious isoform (PrP

). Different strategies were designed to enhance PrP

degradation or 52

to prevent interaction between the cellular and the pathogenic molecules, one of the key 53

events in prion biogenesis. These substances should prolong the incubation time and 54

decelerate the pace of the disease. Among them, Congo red (Caughey and Race 1992;

55

Caughey and Raymond 1993), polyene antibiotics (Adjou et al., 1996; Adjou et al., 1999), 56

anthracycline derivatives (Tagliavini et al., 1997), sulphated polyanions (Caughey and 57

Raymond 1993; Shyng et al., 1995), porphyrins (Priola et al., 2000) and branched polyamines 58

(Supattapone et al., 2001) have been shown to prolong the survival time of scrapie-infected 59

animals, to transiently reduce brain infectivity, or to inhibit PrP

accumulation in cell culture 60

models. The effects of some of these agents result primarily from their ability to interfere with 61

the trafficking of PrP

(Shyng et al., 1995).

62

63

Accepted Manuscript

2. Targeting PrP

for inhibition of prion conversion: Suramin and its derivatives as anti- 64

prion compounds 65

In line with the above-mentioned studies, we worked with different classes of 66

compounds that were shown to inhibit PrP

accumulation in cell culture and in in vivo 67

models by targeting different moments in PrP

synthesis and metabolism. Among these 68

compounds, the naphthylurea polysulphonated aromatic drug suramin, a urea derivative, and a 69

number of related compounds were tested as a potential class of new anti-prion molecules, 70

and the molecular mechanisms underlying these effects were analysed (Gilch et al., 2001;

71

Gilch et al., 2004; Nunziante et al., 2005). Suramin was initially developed to treat 72

trypanosomiasis (Dressel et al., 1961) and has previously been tested in a hamster scrapie 73

model for anti-prion effects (Ladogana et al., 1992). The compound is known to down- 74

regulate surface proteins and to interfere with the oligomerisation state of certain proteins (for 75

review see Stein et al., 1995). We showed that suramin interferes with the folding of mature 76

complex-glycosylated PrP

in a post-ER/Golgi compartment leading to its aggregation. The 77

further trafficking of the misfolded, full-length PrP molecules to the outer leaflet of the 78

plasma membrane is therefore prevented and the compartments of PrP

conversion bypassed.

79

Hence, PrP aggregates are efficiently re-targeted to acidic compartments for intracellular 80

degradation. In contrast to PrP

in prion-infected cells, PrP aggregates formed in the presence 81

of suramin do not accumulate, are sensitive to proteolytic digestion, and are not infectious.

82

The change in the folding state of PrP

induced by suramin and the consequent aggregation 83

seems to activate a novel Golgi/TGN-based cellular quality control mechanism for the cellular 84

prion protein (Ellgaard et al., 1999). Proteins that are labelled as “misfolded” are ready for 85

lysosomal transport and are subsequently efficiently degraded. When tested in mice for its 86

prophylactic effect after intraperitoneal infection with scrapie prions, peripheral application of 87

suramin around the time of inoculation significantly delays the onset of prion disease.

88

Suramin-induced intracellular re-routing can therefore be used for prophylactic approaches

89

Accepted Manuscript

against prion diseases in vivo. The high and persistent blood concentrations that can be 90

achieved by suramin application might result in a transient and therefore conditioned 91

knockout of peripheral surface PrP

, thereby eventually preventing the replication and/or the 92

transport of pathological prions to the central nervous system. Our data also demonstrate that 93

insolubility of PrP in non-ionic detergents, its relative PK resistance and the formation of 94

infectious PrP

are clearly distinct entities which can co-exist within a scrapie-infected cell 95

and suggest that cellular quality control mechanisms can play a protective role against the 96

formation of infectious prions. This might be of special interest for the pathogenesis of 97

sporadic prion diseases and might also be relevant in a more general scenario for other 98

cellular proteins that are hallmarks of the pathogenesis of a variety of neurodegenerative 99

disorders.

100

Our findings raise the possibility that suramin alone or in combination with other 101

drugs could be used for prophylactic purposes, especially in cases of unintentional or 102

accidental exposure to prions. Further analysis using PrP deletion mutants highlighted that the 103

intracellular re-routing phenotype, but not aggregation, is mediated by the N-terminal PrP (aa 104

23-90) and, more precisely, by the pre-octarepeat domain (aa 23-50) (Gilch et al., 2004).

105

Chimeric proteins containing the N-terminus of PrP

do not aggregate and are not re-targeted 106

upon treatment of cells with suramin, indicating that the N-terminus is only active in re- 107

routing when prion protein aggregation occurs. Interestingly, the re-targeting phenotype can 108

be re-established by inserting a region of similar primary structure contained in the PrP 109

paralogue prnd/doppel (aa 27-50) into N-terminally deleted PrP

. These data reveal an 110

important role for the conserved pre-octarepeat region of PrP, namely controlling the 111

intracellular trafficking of misfolded PrP.

112

With the aim of better elucidating action mechanisms and molecular requirements 113

underlying effective clearance of persistently prion infected cells by suramin, we undertook 114

the screening of a larger number of polyanionic compounds with aromatic molecular structure

115

Accepted Manuscript

closely related to suramin (Nunziante et al., 2005). These derivatives have been studied for 116

their anti-angiogenic activity (Stein 1993), and are known to interfere with signal transduction 117

pathways (Kassack et al., 2002; McCain et al., 2004). When applied on prion infected 118

neuronal cells, only compounds containing symmetric aromatic sulfonic acid substitutions 119

inhibited the de novo synthesis of PrP

and induced PrP molecules to form aggregates which 120

were rapidly degraded by the cells. These effects correlated with direct binding to PrP at the 121

cell surface (as seen in in vitro studies with recombinant PrP) and can be explained either by a 122

cross-linking mechanism, as previously described for PrP-antibodies (Enari et al., 2001; Gilch 123

et al., 2003; Peretz et al., 2001), or by a change in the folding of PrP

. Binding of suramin 124

derivatives to PrP molecules would thereby hinder the docking of PrP

or that of a cofactor 125

critical for the conversion of PrP

to PrP

, deterring the essential substrate for prion 126

propagation. Of note, plasma membrane localisation of PrP

seems to be mandatory for the 127

inhibitory activity created by such drugs. Our data reveal an anti-prion effect mechanism 128

different from those characterising other sulphated polyanions that depend on the presence of 129

the symmetric anionic structure of these molecules.

130

It will be necessary to test their prophylactic and therapeutic potential in in vivo 131

scenarios, especially taking into consideration the involvement of PrP

in signalling pathways 132

and the fact that cross-linking PrP

in vivo was found to trigger apoptosis in hippocampal and 133

cerebellar neurons (Mouillet-Richard et al., 2000; Solforosi et al., 2004). The pronounced 134

inhibitory effect of suramin derivatives on prion conversion seen in cell culture models and 135

the availability of an enormous number of related substances make these compounds 136

attractive candidates for further studies.

137 138

3. Studying cellular cholesterol metabolism to inhibit prion formation 139

Numerous studies have shown that both isoforms of the prion protein are associated 140

with lipid rafts, membrane microdomains (DRMs) enriched in cholesterol and sphingolipids

141

Accepted Manuscript

(Gorodinsky and Harris 1995; Taraboulos et al., 1995; Vey et al., 1996; Sarnataro et al., 142

2002). Although the exact cellular compartment of prion synthesis remains to be established, 143

localization of PrP at the plasma membrane seems to be mandatory for this process.

144

Numerous studies hint at lipid rafts as possible sites of prion conversion or interaction 145

between PrP

and PrP

(Borchelt et al., 1990; Caughey et al., 1989; Caughey and Raymond 146

1991) and (Prusiner et al., 1998). Generally, GPI-anchored proteins localise within these 147

microdomains. Association with raft is enabled by the interaction of the GPI-anchor with 148

membrane lipids or by the binding of a non-raft protein to a raft-resident protein (Conese et 149

al., 1995). Of note for PrP

, it has also been put forward that the N-terminal domain may 150

contain an exoplasmic raft localization signal (Walmsley et al., 2003). Based on the 151

importance of elucidating factors that influence the trafficking and the subcellular localisation 152

of PrP

for understanding prion conversion (Caughey et al., 1989; Caughey and Raymond 153

1991) and (Borchelt et al., 1990) and for discovering novel therapeutic strategies (Cashman 154

and Caughey 2004), we investigated the effect of metabolic inhibition of cholesterol synthesis 155

by the HMG-CoA-reductase inhibitor mevinolin on PrP trafficking in neuronal cells able to 156

propagate prions (Solassol et al., 2003).

157

In previous studies, interference with raft formation/association of PrP either 158

pharmacologically, by perturbation of cholesterol synthesis (Taraboulos et al., 1995; Mange et 159

al., 2000; Bate et al., 2004), or by expression of transmembrane PrPs (Kaneko et al., 1997a;

160

Taraboulos et al., 1995), counteracted PrP

formation. It was assumed that upon dissociation 161

from rafts, PrP localises in non-raft membrane compartments not eligible for prion 162

conversion. In contrast, reduced sphingolipid content supports prion conversion (Naslavsky et 163

al., 1999). In addition, manipulation of cells with the cholesterol-binding agent amphotericin 164

B indicates the importance of cholesterol for the prion conversion process (Mange et al., 165

2000). The fundamental role played by cholesterol and rafts in PrP

/PrP

metabolism and 166

trafficking seems to be specific to neuronal cells (Paquet et al., 2004; Sarnataro et al., 2002;

167

Accepted Manuscript

Uelhoff et al., 2005). Taken together, these results underline the importance of cholesterol, 168

which participates in the maintenance of lipid rafts in prion conversion.

169

In our study, cholesterol depletion by mevinolin treatment hampered the secretory 170

transport of PrP

to the cell surface (Gilch et al., 2006). This leads to PrP

accumulation in the 171

Golgi compartment. These results provide a new explanation for inhibition of prion 172

conversion in cholesterol-depleted neuronal cells, namely, the lack of surface-located PrP

173

(Gilch et al., 2006). Analysis of mutant PrPs highlights the importance of the GPI-anchor for 174

raft localization and provides information about domains implicated in lipid raft association of 175

PrP in the secretory pathway. Our data show that cholesterol is essential for the cell surface 176

localization of PrP

, known to be necessary for prion conversion. Further analysis of PrP 177

trafficking employing different PrP-mutants revealed that the GPI-anchor acts as a dominant 178

signal for recruiting PrP into lipid rafts along the secretory pathway. All these investigations 179

provide us with evidence that cholesterol is absolutely essential for the transport of PrP

to the 180

cell surface in neuronal cells. In terms of the anti-prion effect, this finding is extremely 181

important, since it elucidates the underlying mechanism and sheds new light on the role of 182

cholesterol for PrP trafficking.

183 184

4. Signal transduction pathways as potential anti-prion targets 185

We have screened almost 50 prototype substances known to inhibit various signal 186

transduction pathways in prion-infected cells for a potential effect on the PrP

content 187

(Ertmer et al., 2004). Only one substance appeared to interfere with PrP

propagation without 188

exhibiting any effect on the amount, the trafficking or the subcellular localization of PrP

. The 189

inhibitor we identified was the tyrosine kinase inhibitor STI571, also known as Gleevec or 190

imatinib mesylate. In prion-infected cells, STI571 cured the cells in a dose- and time- 191

dependent manner with an IC

below 1 mM. When we analysed the amount and subcellular 192

distribution of PrP

, we found no difference between mock-treated cells and cells treated with

193

Accepted Manuscript

STI571. PrP

was still localized at the cell surface and, in contrast to many other anti-prion 194

compounds, did not form insoluble aggregates. Surprisingly, STI571 treatment did not inhibit 195

the PrP

de novo generation, as revealed by metabolic labelling and immunoprecipitation 196

experiments. In contrast, STI571 treatment accelerated the cellular degradation of PrP

and 197

decreased its half-life, from the usual span of more than 24 hours, to about 9 hours. By 198

inhibiting lysosomal degradation using ammonium chloride we were able to prevent the 199

intracellular PrP

digestion induced by STI571, indicating that this compound triggers the 200

lysosomal degradation of PrP

. The known targets of STI571 are the tyrosine kinases c-Abl, 201

the PDGF receptor and c-Kit (Buchdunger et al., 1996; Heinrich et al., 2000). In order to 202

identify the target kinase involved in the induction of PrP

degradation, we employed an 203

inhibitor of PDGF receptor and c-Kit autophosphorylation (CT52923) (Yu et al., 2001). When 204

ScN2a cells were incubated with this compound, no effect on the amount of PrP

could be 205

observed. Moreover, when ScN2a cells were treated with STI571 and in parallel were 206

transiently transfected with a trans-dominant negative mutant of c-Abl, the degradation of 207

PrP

induced by STI571 could be partially prevented. Similar results were obtained when 208

using c-Abl targeting siRNA. Taken altogether, these findings indicate that the target involved 209

in the increased lysosomal degradation of PrP

was the tyrosine kinase c-Abl. With STI571 210

we describe an example for a new class of compounds with a significant effect on PrP

in 211

prion-infected cells. The drug has been developed and is successfully used for the treatment 212

of chronic myeloid leukaemia (CML). It has also been prescribed against gastrointestinal 213

stromal tumours caused by deregulation of c-Kit activity (Sawyers et al., 1994). In addition, it 214

has been shown that STI571 inhibits β-amyloid production, suggesting that STI571 might also 215

be a useful tool for the development of novel therapies against Alzheimer's disease (Netzer et 216

al., 2003).

217

A huge variety of compounds with anti-prion activity have already been identified.

218

Unfortunately, only a few drugs are effective against prion disease in mice. One prerequisite

219

Accepted Manuscript

for a substance with therapeutic effects is that it has to reach the CNS and therefore must 220

cross the blood-brain barrier (Gilch and Schätzl 2003). In mice, about 2.8 % of orally 221

administered STI571 is found in the cerebrospinal fluid (Petzer et al., 2002) and additionally, 222

the biological half-life of STI571 on oral application in mice is less than 4 hours (le Coutre et 223

al., 1999). Since these amounts might not be sufficient to elicit a therapeutic anti-prion effect, 224

the screening for more applicable STI571 derivatives which might overcome these problems, 225

is desirable.

226

Most of the described anti-prion compounds interfere with the de novo synthesis of 227

prions. For branched polyamines, however, it has been reported that they affect pre-existing 228

PrP

and that they act in lysosomes. These studies also raise the possibility that lysosomal 229

proteases normally degrade PrP

in cultured cells. The authors claimed that branched 230

polyamines could render PrP

molecules protease-sensitive by dissociating PrP

aggregates 231

or that they could facilitate the transport of PrP

from the membrane into secondary 232

lysosomes (Supattapone et al., 1999; Supattapone et al., 2001; Winklhofer and Tatzelt, 2000).

233

Similarly, the two anti-prion compounds quinacrine and chloroquine were reported to act in 234

lysosomes, eventually by raising the lysosomal pH (Doh-Ura et al., 2000). STI571 also 235

induces the lysosomal degradation of PrP

, probably by inhibiting the tyrosine kinase c-Abl, 236

notably without influencing quantity, distribution and biochemical features of PrP

. Although 237

the exact signalling pathways involved in this process need to be clarified, our results 238

confirmed that PrP

can be degraded by lysosomal proteases.

239 240

5. Vaccination approach against prion disease 241

One of the most powerful regimens against infectious diseases is active immunization 242

employing for example recombinantly expressed proteins or attenuated viruses as antigens. In 243

prion diseases, this approach is hampered by the pronounced auto-tolerance against the self- 244

protein PrP. Therefore, the design of improved immunogens is crucial for the development of

245

Accepted Manuscript

an effective active immunization strategy. In our experimental vaccination studies (Gilch et 246

al., 2003), a novel antigen consisting of two covalently linked murine PrP molecules not 247

containing N- and C-terminal signal peptides was introduced. This protein was recombinantly 248

expressed in E. coli and purified by metal affinity chromatography. Upon immunization of 249

PrP-expressing wild-type mice, high antibody titres could be achieved. The best results were 250

obtained when CpG-rich oligonucleotides (CpG) were used as adjuvant. In order to prove the 251

efficacy of the obtained antisera against PrP

, we used them for the treatment of persistently 252

prion-infected cultured cells. The rate of PrP

de novo synthesis was analysed as a read-out.

253

Under our experimental conditions, more than 40 % of all tested antisera raised against 254

dimeric PrP inhibited PrP

generation. Interestingly, the effects exerted on PrP

biogenesis 255

did not directly correlate with the anti-prion titres of the sera. In contrast, only ~5 % of 256

antisera obtained by immunization with monomeric PrP interfered with PrP

synthesis. In 257

addition, when cells were treated with polyclonal mouse serum for a period of 7 days, PrP

258

was completely abolished and did not reappear after further cultivation for 7 days without 259

antiserum. These results indicated that it is possible to overcome auto-tolerance against PrP in 260

wild-type mice by using dimeric PrP as immunogen. Although antibody titres were also 261

observed when monomeric PrP was used for immunization, these sera exhibited almost no 262

effect on the de novo generation of PrP

. 263

In further experiments, we compared the efficacy of full IgG molecules and Fab 264

fragments thereof, raised against either dimeric or monomeric PrP, in respect of their anti- 265

prion activity. Due to the high amount of sera necessary for the preparation of Fab fragments, 266

rabbit sera were used. Long-term treatment revealed that only treatment with IgG molecules 267

raised against dimeric PrP led to cross-linking and reduction of PrP

and cleared ScN2a cells 268

from PrP

. Neither IgG obtained upon immunization with monomeric PrP nor any Fab 269

fragments exhibited any effect on PrP

. In summary, we could confirm that antibodies raised 270

against dimeric PrP have a superior anti-prion effect compared to antibodies raised against

271

Accepted Manuscript

monomeric PrP. Furthermore, only full IgG exerted a significant effect on PrP

levels due to 272

cross-linking of PrP

. When the epitopes recognized by dimer-induced mouse and rabbit sera 273

were mapped, we found that residues 159-178 were the main targets, comprising a PrP region 274

involved in PrP-PrP interactions or the binding of a cellular protein that supports prion 275

conversion (Kaneko et al., 1997b; Horiuchi et al., 2001).

276

The aim of our work was to induce auto-antibodies targeting the cellular PrP, thereby 277

increasing the possibility of developing an active immunization strategy against prion 278

disorders. In mouse models for Alzheimer's disease (AD), a similar strategy, namely inducing 279

auto-antibodies directed against βA4 peptide, has been successfully tested (Ingram 2001;

280

Janus et al., 2000; Morgan et al., 2000). However, clinical trials in AD patients initially had to 281

be stopped because of side effects. Of note, in our study employing PrP-expressing wild-type 282

mice we did not observe any obvious side effects. Evidence that immunization in prion 283

diseases might be successful has already been supplied by passive immunization and in 284

transgenic mice expressing an IgM version of the anti-prion antibody 6H4 (Gilch and Schätzl 285

2003; Heppner et al., 2001; White et al., 2003). The latter was also used in cell culture 286

studies, and, in common with other antibodies or Fab fragments, treatment could cure prion- 287

infected cells of PrP

(Enari et al., 2001; Peretz et al., 2001; Perrier et al., 2004; Pankiewicz 288

et al., 2006). In contrast, we immunized wild-type mice and found auto-antibodies induced by 289

dimeric PrP to inhibit PrP

replication. In many immunization approaches using, for 290

example, recombinant PrP, PrP peptides or PrP displaying viral particles, antibody titres 291

against PrP could be achieved (Arbel et al., 2003; Nikles et al., 2004; Rosset et al., 2004;

292

Schwarz et al., 2003; Sigurdsson et al., 2002; Goni et al., 2005), but only in a limited number 293

of studies could a protective effect be produced upon intraperitoneal or oral prion infection 294

(Schwarz et al., 2003; Sigurdsson et al., 2002; Goni et al., 2005). When dimeric PrP was used 295

for immunization in combination with an anti-OX40 antibody as auto-tolerance breaker, in 296

most mice only a slight prolongation of incubation time was found, whereas two out of eight

297

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mice were completely protected (Polymenidou et al., 2004). Although these results have to be 298

confirmed, there is hope that with an improved antigen, immunisation against prion diseases 299

will finally be effective.

300 301

6. Outlook 302

In recent years there has been a tremendous effort in research oriented towards therapy 303

and/or prophylaxis against prion diseases. A huge number of compounds were tested in in 304

vitro assays, cell culture-based systems, and in animal studies, with some drugs also being 305

tested in humans. Problems encountered were not really unexpected: for example, non- 306

tolerable side effects and the often total lack of ability to cross the blood-brain barrier. There 307

was a great deal of excitement some years ago when experimental approaches for active and 308

passive immunisation were reported, although there is still no practical application. Very 309

probably, a sophisticated combination of compounds and approaches will be necessary to 310

solve the problem.

311 312

Acknowledgements 313

We thank all the members of the prion research group. Studies were performed partly 314

in collaboration with the groups of Prof. E. Wolf, Munich; Prof. M. Groschup, Riems; Prof.

315

D. Riesner, Düsseldorf; Prof. J. Tatzelt, Munich; Prof. I. Lasmezas, Paris; Dr. E. Kremmer, 316

Munich; Prof. M. Famulok, Bonn; Prof. A. Aguzzi, Zürich; Dr. Eckhard Flechsig, Würzburg 317

and Prof. M. Klein, Würzburg, Germany. Financial support was given by DFG (SCHA 594/3- 318

4), BMBF (01KO0108 and 01KO0202), the State of Bavaria (ForPrion; LMU5*), EU 319

(QLRT-2000-01924 and NoE Neuroprion), and SFBs 596 (Project A8) and 576 (Project B12).

320 321

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Figure legends 564

Figure 1. Targets for interference with prion infection.

565

According to the nucleation-dependent polymerisation model for prion synthesis (Brown et 566

al., 1990; Caughey et al., 1995), the host cellular PrP (PrP

) deposits on a polymerisation seed 567

consisting of an infectious PrP

oligomer. Upon binding, PrP

adopts the specific 568

conformation of the molecules forming the seed, leading to high molecular PrP

aggregates.

569

These can then fall apart and form new seeds.

570

571