Ex vivo propagation of transmissible spongiform encephalopathies agents

Oral Pwsrr~ution:

S58

EX VIVO PROPAGATION OF TRANSMISSIBLE ENCEPHALOPATHIES AGENTS

SPONGIFORM

Ex vivo transmission of the agents responsible for the transmlsalble rpongiform encephalapathies (TSEa), or prion diseases, in cultured cells is a useful model to understand the mechanisms of infection and replication of the agents. We have recently developed new systems using the murine neuronal cell lines GTI-7 and N2a (Nishida et al.. (2000) J Virol 74:320-5). In the latter model, we were able to increase \enTitivity of the cells to the infectious agent by means of over-expressing cellular prlon protein, PrP’. Our results corroborate the hypothesis that the successful transmission of agents ex viva depends on both expression levels of host PrPC and the wquence of PrP”‘. Interestingly. these models are strains specific and could be used to elucidate the factors involved in specxs harrier and cell tropism. The sensitivity of these ay,tems was determined hy an endpoint dilution assay of a Chandler-scrapie affected mouse brain (IO ” LDSO/g). Successful transmission of the agent from brain to the culture? was determined by Western blotting ofprotease-resistant priori protein. PrP”. after several passage?. Both cell culture systems could detect infectivity as low a’i 200 lD50. wggestinp that the ex viva transmiwion system could be a valuable diagnostic tool for the detection of infectivity. Importantly, this method it does not rely on the use of laboratory animals and it is le\a time and money consuming than animal assays. We are currently working toward< establishing cell lines expressing high amounts of bovine or human PrP that can be expected to be sensitive to the agents of bovine \pongiform encephalopathy and variant Creutzfeld-Jakob disease.

A DECAMER DUPLICATION IN THE BRI GENE ORIGINATES A DE-NOVO AMYLOID PEPTIDE THAT CAUSES DEMENTIA IN A DANISH KINDRED.

Ftlmilial Danish dementia (FDD). previously known as heredopathia ophthalmo-otoencephalica is an auto
INVESTIGATION OF THE INVOLVEMENT PROTEIN IN OXIDATIVE STRESS

OF THE

PRION

It is bchevrd that the agent responbiblr for Priori diseabea or transmissible spongiform encephalophathies is a protem termed PIP”‘. PrF”’ 15 a conformational variant ot the normal host protein, PrPC. Although PrP“ function remains elusive, this protein ha\ been implicated recently in cellular defense against oxidative stress. Interestingly, PIP’ was shown to bind copper (Co) and it has been proposed that PrPC may play a role in the metabolism of this lon. This is important considering the potential role of Cu and oxidative stress in neurodegenerativr diseases. In addition, cells derived from PrP ablated mice have in some models a lowered resistance to oxidative stress, which IS believed to be linked to an altered Cu/Zn superoxide dismutase (SOD) activity. One explanation for this rewlt it that PrP expreswn modulatea SOD activity. Fmally, the activity of another important oxidatrve enzyme, nitric oxide synthase (NOS), was reported to be reduced in the brains of Infected animals. It teems now essential to evaluate in details the consequence of the convewon of PIP” into PrP” on the cellular response to oxidative sue\<. To inveatigatr thir. we decided to abe infected cell line\ recently generated in the laboratory (Nlshida N. et al., J Vlrol 2000 74:320-S). WC are currently testing the sewitivity of these cell\ to oxidative stress, a\ well a\. the impact of the infection of the activity of variou rnrymatic actiwties (like

Alzhrimer’s

Diseuse and Related Disorders

(Prims)

SOD, catalase, gluthatione peroxydase activities). Thir approach may provide useful data on the pathogenic mechanism of Priori diseases.

ELECTRON MICROSCOPY AND X-RAY DIFFRACTION STUDIES FURTHER CONFIRM THE EFFICACY OF PT1-O0703TM (CAT’S CLAW DERIVATIVE) AS A POTENT INHIBITOR OF ALZHEIMER’S BETA-AMYLOID PROTEIN FIBRILLOGENESIS Gem-do Ho.\lm

M Custillo. Co/l,

Boston.

ProteoTech MA;

A/m

inc, Kirkland, D Snow,

WA; Daniel

ProtroTech

A Kinrhner,

lnc, Kirkland.

Am

G Ye,

WA

PTI-00703’“(“703”)

is a derivative from the Amazon rain foreat woody vine, previously shown to inhibit the formation and growth of Alzheimer’s beta-amyloid protein CAP) deposits. In the present study, negative stain electron microscopy (EM) and x-ray diffraction further confirmed the efficacy of 703 as an inhibitor of AP fibril formation, compared to other herbals believed effective as cognitive enhancers. 50 PM AP l-40 was incubated at 37°C for 7 day\ m the prexnce or absence of 703, Ginkgo biloba, Gotu kola and Korean ginseng at an AP:compound wright ratio of 1: I with aliquots taken for EM analyria at 0. I, 3 and 7 dayb. Withm 3 days, AP alone formed an extensive network of amyloid fibrils. which was still observed in the presence of Ginkgo biloba, Gotu kola and Korean fm”eng extracts. However, AP in the presence of 703 primarily formed non-fibrillar amorphous material at all time points indicating that 703 ia a potent inhibitor of Aa fibril formatlon. In another study, x-ray diffraction patterns were collected from lyophilized AP l-40, or AP dissolved at I m&/ml, either in distilled water or in 703 (at an AD:703 weight ratio of 3: I or6: I ). The non-hydrated A@ peptide gave an x-ray pattern typical of lyophilired, synthelic amyloid peptides, with major reflections at -4.7A and - IOA, arlaing from the array of hydrogen (H)-bonded polypeptide chains and the packing of P-pleated sheets. When the lyophilized peptide was solubilized in wilter and dried, the H bonding reflection became darker and aharper, indicating a more extensive array of H-bonded chains, while the intersheet spacing became weaker and broader. Whereas 703 at an A@703 ratio of 6: I had little effect on AP fibril formation as demonstrated by x-ray diffraction, 703 at an A@703 ratio of 3:l markedly inhibited AP fibril formation as evidenced by a weakened and broadened H-bonding reflection. These studier confirm that PTI-00703 is a potent inhibitor of AP flbril formation, and suggests that one mechanism of AP fibril inhibition by 703 involves inhibition of H bonding important for growth and extension of amyloid libul\. Uncarin

tomentoso

(Cat’sclaw)

THE ARCTIC APP MUTATION (E693G) CAUSES ALZHEIMER’S DISEASE THROUGH A NOVEL MECHANISM: INCREASED AMYLOID OPROTFIBRIL FORMATION AND DECREASED AMYLOID PLEVELS IN PLASMA AND CONDITIONED MEDIA

Several pathogenic Al/heuner’\ disease (AD) mumtions have previously been identified, all leading to an increase in amyloid @peptIde (AP) levels. We have identified a novel pathogenic Pamyloid precursor protein (APP) mutation located within the APsequence at codon 693 (E693G). causing AD in a family from northern Sweden, the “Arctic” mutation. Clinical symptom\ of the patienl\ are typical of AD with slow maidious progression. Plasma levels of both AP40 and A642 were rignificantly lower m carrier< of the Arctic mutation compared to controls. This reduction was obwrved 20-30 years before the expected onwt of the disease in the youngest cases investigated and might thug follow these mdividuala throughout life. This finding was corroborated m vitro, where Ab42 %xretlon was reduced from cells trnnsfected with APPE693G. In addition, fibrilliration studie\ showed that the Arctic APpeptide formed larger quantities of protofibrils and at a much higher rate. To further investigate if the reduced APlevels were due to altered processing or mtracellular aggregation of A@, the APpeptide was charactcrired by mass spectroscopy and sequencing. Intracellular accumulation of peptldea harboring the Arctic mutation was also invwigated by immunopwcipitation. Taken together. our results indicate a new pathogenic mechanism for AD in the Arctic family where increawd ABprotofibril formation is a primary event in the pathogen&a. This highlights the need for therapeutic agent\ aimed at interfering with early event, in ABpolymerlr;ltion.