A transgenic model of a familial prion disease

Symposium: Prinn Disease.5

(9471 MOLECULAR

S209

STUDIES OF PRION PROPAGATION

John Collinge, Imperial College School of Medicine, London, W2 En&md, John CoIlin&, Graham S Jwkson’, Lmzlo L P Hosszu’~ ‘. Aisling Power’, Andrew F Hill’, John Kenney’. Helen Saibrl’, C. Jeremy Craven2, Jonathan P Waltho and Anthony R Clarke’ ’ MRC Pi-ion Unit, Department qf Neurogeneticv, Imperial College School @Medicine at St. Mary s, London, W2 INY, UK ’ Krebs Institute for Moleculur Biology and Biotechnology, Univecsity ofSh&eld, Shefield SIO 2TN, UK ’Department of C~stalloRraphy. Birkbeck Collqe, London, WCIE 7HX. UK Priori diseases are associated with the accumulation in affected braina of a conformational isomer (PrPs’) of host-derived pi-ion protein (PrPc). According to the protein-only hypothesis, F’rP”‘ iy the principal or sole component of transmissible prions. The existence of multiple priori strains has been difficult to explain in terms of a protein-only infectious agent but recent studies of human priori diseases suggest that strain specific phenotypes can be encoded by different PrP conformations and glycorylation patterns. PrP” fomation ir associated with an increase in Its P-sheet secondary structure content and PrP SC is recognised biochemically by Its acquisition of pamal resistance to digestion with proteinaae K. Whilst the Structure of PrPc has been determined. the structure of PrPs’, essential to an understanding of priori propagation, is unknown. The insolubility of PrPs’, which is often present in brain as amyloid fib& and which is isolated from tissue in a highly aggregated state, has to date precluded high-resolution structural analyaia.We have recently establiched conditions in which recombmant human PrP could switch between the native u-conformation, characteristic of PrPC, and a compact, highly soluble, monomeric form nch in p-structure (@PrP) which is stable in aqueous solution9. Such an interconversion of a protein chain between two, discrete backbone topologies, while remaming m the monomeric state in the absence of denaturants, appears unprecedented. Soluble P-PrP possesses partial protease resistance and is a direct precursor of fibrillar structures closely similar to those isolated from diseared brains. The conversion of PrPC to p-PrP, and its subsequent stabilisation by intermolecular association, prowdea a molecular mechanism for priori propagation.

pii3J

PRION PROTEIN INTERCONVERSIONS

Byron Cmghey.

Rmky Mounmin Luborutorirs,

AND TSE DISEASES

NIAID. NIH, Harwilton. MT

Change\ in the conformation and aggregation atate of priori protein (PrP) are fundnmenial to the transmisible spongiforrn encephalopathies (TSE) or priori diseases. In ml’ectious TSE diseases, at least, the conversion of the normal. protease-sensitwe PrP (PrP-sen) to the disease-awxiated partially protease-resistant form (PrP-re\) I\ driven by preexisting PrP-re\ via direct interactions. We have drwloped cell-free models of this conversion reaction. These models reflect many important biological parameters of TSE diseases and have provided insight into its ~nolecular mechanism. In cell lywte\ and cell-culture cupematants. highly selective hmdmg between PrP-wn and PrP-rc\ wab observed. The binding oi PrP-sen and its conversion to the proteaae-reslstant state can be separated kinetically. We have begun mapping the sites of interaction between PrP-sen and PrP-res using site-specific antibodies and found evidence that contact ia initiated VM sites that are close in space to C-termmal residues 218.232. In combinations of PrP-sen and PrP-res from different species, binding can occur without conversion and, at the same time. interfere with conversion reactions between PrP-iaoforms of the same species. This interference phenomenon may help to explain the barriers that are often observed in mterspecies transmiaslons of TSE agents. Finally, we have used in vitro model\ to identify inhibitors of PrP conversion which serve as lead compounds in the development of anti-TSE drugs.

(949) A TRANSGENIC

MODEL OF A FAMILIAL

PRION DISEASE

David A. Hurrir, Roberto Chirsa, Bettinu Drisaldi, Elena Quuglio, Washington Universrty School of Medicine. St. Louis, MO: Antonio Migheli, University of Turin, Turin Italy; Pedro Piccardo, Benmrdino Ghrtti, Indiana University School of Medicine, Indinnupoli.~, IN A nine-octapeptide insertional mutation in the priori protein (PrP) gene is associated with an inherited form of Creutrfeldt-Jakob disease in humans. Transgenic mice that express the mouse PrP homologue of this mutation under control of a PrP promoter display a progressive neurological disorder characterized by ataxia, cerebellar atrophy, non-amyloid deposition of PrP, astrogliosis. and the presence by Western blotting of a detergent-insoluble and weakly protease-resistant form of PrP that bears some similarities to the pathogenic isoform, PrPs‘(Chiesa et al. Neuron 21: 1339.1351 11998)). We have now further characterized the clinical, neuropathological, and biochemical characteristics of these mice. Clinical symptoms begin at 65 days of age

when the tramgene array is homozygous and at 240 days of age when the transgene array ia hemizygous, suggesting that expression level influences the time coune of the illness. The PrPs’-like form of mutant PrP is detectable at birth, and accumulates nearly lO@fold as the animals age. The total amount of mutant PrP also increases dramatically with age, although the amount of the corresponding mRNA remains constant. lmmunohistochemicai staining of brain sections after guanidine thiocyanate den&ration and hydrolytic autoclaving reveals synaptic-like deposits of PrP that are most prominent in the molecular layer of the cerebellum and in the hippocampus. Histoblotting of brain sections reveals a widespread distribution of protease-resistant PrP. There is a progressive degeneration of cerebellar granule cells that correlates with the development of clinical symptoms, and that displays the characteristics of apoptosis, including nuclear fragmentation and pyknosis, DNA fragmentation revealed by in situ end-labeling and Southern blotting, and positive staining for activated caspase 3. Our results indicate that a threshold level of mutant PrP is necessary to produce disease, and that the mutant protein accumulates in the brain because it is degraded or cleared inefficiently. In addition, the PrP%ike form of mutant PrP is likely to be neurotoxic, triggering apoptotic degeneration of some neuronal populations.

1950( THERAPEUTIC

APPROACHES

TO PRION DISEASES

Fubrizicj Tagliavmi, Otituto Nazionnle Neurologico “Carlo Bestrr “, Miluno Ita1.v; Mario Suhnona, Istituto di Ricerche Farmrrcologiche “Mano Negri”, Miluno Italy; Orso Bu,qioni, lstituto Nazionalr Neurolo~ico “Carlo Bestu”, Milano Italy; GiunluiRi Forloni, I,stituro di Ricerchr Funnucologichr “Murio Negri”, Miluno It& The priori diseases are transmissible neurodegenerative disorder5 marked by the conversion of the cellular priori protein (PrPC) into disease-specific species (PIP”‘). Unlikr PrPr, PrP” isoforma have high content of p-sheet secondary structure, are protease-reristant, and form insoluble aggregates and amyloid fibrils. Evidence Indicates that they are responsible for neuropathnlogical changes and disease tranamissiblity. thw representing a primary target for therapeutxs. Although a variety of compounds has been tested on cellular and animal models of priori disease, only 8 few molecule\ including sulphated polyanions, amphotericin B. Congo red. lododoxorubicm. tetrapyrroles and modified PrP peptides effectively reduce the accumulation of PrP” in acrapie-infected neuroblastoma cells and/or prolong wrvival of rodents experimentally infected with scrapie. This group of compounds is structurally heterogeneous and the mechanism of action of individual molecules in experimental priori discase ib poorly undentood. However, Condo red, iododoxoruhlcin and tetrapyrrole\ share common chemicophysul propelties in that they contain an extended hydrophobic core formed by aromatic moieties with a large number of bydrophihc wbstituents conferring an amphiphilic character. Since PrP’PrPs’ conversion results in increased hydrophobicity due to solvent exposure of hpophilic residues and in the ability to form insoluble aggregates and amyloid fibrils, it I\ conceivable that the antI-priori activity of the compounds i? related to their hydrophobic character allowing for a strong interaction with PrP”‘. Thi\ view i\ supported by the recent observation that tetracycline which shares structural 15able to (I) bind and disrupt analogies with Congo red and iododoxorubicin fibrilluy assemblies of PrP peptldes, (ir) revert the protease resistance of P@ extracted from brain tissue of patients with Creutzfeldt-Jakob disease, (iii) prevent neuronal death and aatmcyte proliferation induced by PrP peptldes in vitro. and (iv) delay the onset of symptom\ and prolong survival of experimental hamster xnlpie. Comparative analysis of these cla\ses of compounds will help to understand the tnolecular requrements for antl-priori activity of drug\.