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5.5.4. CD Spectroscopy
The pepttdes were suspended m 200 mM phosphate buffer, pH 5.0 and 7.0,
or in deionized water at a concentratton of 0.5, 1.5, and 3.0 mg/mL. Following
incubation at room temperature for 5 mm or 1 h, CD spectra were recorded in
quartz cells with an optical path of 0.01 or 0. I cm, using a Jasco (Toyko,
Japan) J-500 dtchrograph at a scan speed of 1 nm/min. Mean residue elliptt-
ctttes were calculated using the followmg equatton.
(6JM = A x 3300 x M/C x I, (1)
where A = observed dtchrotsm absorbance, 1= path length m cm, C = concen-
tration of peptide m g/L, and M = mean restdue weight. The percentages of the
Prion Protein Amyloids 279

secondary structure of the peptldes were calculated according to Yang et al
(31). To investigate the influence of a-helix stabilizing solvents on the second-
ary structure of the peptldes, l,l, I-trifluoroethanol was added up to a final
concentration of 50% to suspensions of peptides In phosphate buffer or delon-
lzed water To analyze the effects of SDS mlcelles on peptlde conformation,
CD spectra were collected from suspensions of peptides m 5% SDS m delon-
lzed water or in 5 mM phosphate buffer, pH 7.4
5.6. Biological Effects of PrP Pepticies In Vitro
5.6.1. Neuronal Cultures
Hlppocampal neuron cultures were prepared usmg the followmg protocol
I Remove brams from rat fetuses on embryonic d 17
2 Dissect out hippocampus and dissociate cells m serum-free medium contammg
0 1% trypsm (Dlfco, Surrey, UK) and 25 pg/mL deoxyrlbonuclease for 5 mm at
room temperature
3 Plate cells at 5 x lo5 cells/well m Primarla (Falcon, Becton Dlckmson, Plymouth,
UK) 15-mm wells, precoated with poly-D-lysine (50 pg/mL, Sigma)
4 Culture cells m basal medium Eagle (BME-Hank™s salt, Gibco-BRL, Galthersburg,
MD) supplemented with 10% fetal calf serum (FCS, Glbco) and 2 mMglutamme
Keep cultures at 37°C m a humidified 5% CO, atmosphere
5 After 5 d m vitro, halt nonneuronal cell dlvlslon by exposure to IO-˜M cytosme
arabinoslde to prevent overgrowth of gllal cells
Additional cultures were prepared in tissue culture chamber slides (Nunc,
Naperville, IL) to analyze culture composition immunocytochemlcally, using
antibodies that selectively label astrocytes, mlcrogllal cells, or neurons.
Cultures were exposed either acutely or chronically to PrP peptldes at a con-
centration of 20, 40, 60, and 80 @4. For short-term treatment, peptides were
applied once at the time of plating or after 9 d of culture, for long-term treat-
ment, the peptldes were added to the medium on d 0,2,4, 6, and 8 of culture.
Control cultures were exposed to scrambled peptides or to vehicle only
On d 10 cell viablllty was evaluated by light and electron microscopy or
assessed quantitatively by a colortmetrlc method as follows Neurons cultured
m microtiter dishes were stained with crystal violet (0.5% in water/methanol
4: 1). After washing, cells were dried, solubilized in sodium citrate/ethanol 1: 1,
and spectrophotometrlcally analyzed at 540 nm wrth an automated microplate
reader (Perkin-Elmer [Norwalk, CT] lambda reader). The absorbance of the
solution is proportional to the number of viable cells.
5.6.2 Glial Cell Cultures
Glial cell cultures were prepared using the followmg protocol.
1 Remove brains from neonatal rats and remove leptomemnges.
280 Taglia vrni et al

2 Drssoctate nervous ttssue by trrturatron wtth a Pasteur prpet
3 Grow cells m poly+-lysme coated Prtmarta (Falcon) dashes contammg Dulbecco™s
modtfied mmrmal essenttal medmm (DMEM. Gtbco) supplemented with 10%
FCS and 2 mM glutamme, at 37°C m a humtdtfied 5% CO2 atmosphere
4. Change medmm every 3 d
Addmonal cultures were prepared m tissue culture chamber slides (Nunc) to
analyze culture composmon nnmunocytochemrcally, using antrbodres that
selectrvely label astrocytes, mtcroghal cells, or neurons.
The cultures were treated wrth the pepttdes (10, 25, and 50 pA4 concentra-
tion) for 1 or 14 d. For short-term treatment, the PrP peptides were deltvered 2
wk after plating, and the cultures were exammed 24 h later. For long-term
treatment, the PrP pepttdes were added to the medmm every 3 d for 2 wk start-
mg from d 0 or 7, and the cultures were analyzed on d 14 or 2 1, respecttvely
Control cultures were exposed to scrambled pepttdes or to vehicle only.
After treatments, cell vrabrlity and growth were estimated by light and elec-
tron microscopy, or assessedquantttattvely by a colorlmetrtc method as fol-
lows Cultures were supplied with 3-(4,5-drmethylthlazol-2yl)-2,5-dlphenyl
tetrazohum bromide (MTT; 1.5 mg/mL), which 1sknown to be converted mto an
insoluble blue formazan product by hvmg cells but not by dying cells or then
lytlc debris (32,33). The blue product was then solubthzed by adding 80 n-&I
HCl m rsopropanol (500 pL/well) and the color intensity was measured at
570 nm using an automated mtcroplate reader (Perkm-Elmer lambda reader).
The optical density of the solution in the microwell is proportronal to the num-
ber of viable cells. Cell culture growth was also quantitatively assessedby
spectrophotometrlc determination of DNA (34) or E3H]-thymidme uptake (35)

Acknowledgments
This work was supported by the Italian Mmtstry of Health, Department of
Social Services, and by the US Natronal Institutes of Health (Grant NS29822).

References
1 Prusmer, S B (199 1) Molecular biology of prion dtseases Sczence 252, 15 15-l 522
2 Prusmer, S. B (1993) Genetrc and mfectrous prlon drseases Arch Neurol 50,
1129-l 153
3 Caughey,B W., Dong, A, Bhat,K S.,Ernst,D , Hayes,S F., andCaughey,W S
(1991) Secondary structure analysts of the scrapte-assoctated protein PrP 27-30
in water by infrared spectroscopy Blochemlstry 30,7672-7680
4 Pan, K -M., Baldwm, M , Nguyen, J , Gasset, M., Serban, A , Groth, D., Mehlhorn,
I , Huang, Z , Fletterrck, R. J., Cohen, F. E , and Prusmer, S. B. (1993) Conver-
sion of a-hehces mto P-sheets features in the formation of the scrapie prton pro-
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5 Safar, J , Roller, P P , GaJdusek, D C , and Gtbbs, C J. (1993) Conformational
transtttons, dissoctatton, and unfolding of scrapte amyloid (prton) protem J Bzol
Chem 268,20,276-20.2&I
6 Ghetto, B , Dlouhy, S R , Gtaccone, G , Bugtam, O., Frangtone, B , Farlow, M
R , and Tagliavmt, F. (1995) Gerstmann-Straussler-Schemker disease and the
Indiana kmdred Bram Pathol 5,61-75
7 Ghetto, B., Ptccardo, P , Spillantmt, M. G., Ichtmlya, Y , Porro, M , Perim, F.,
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F , Goedert, M., Dlouhy, S R , and Tagltavmt, F. ( 1996) Vascular variant of prton
protem cerebral amylotdosis with r-postttve neurofibrtllary tangles the phenotype of
the stop codon 145 mutation m PRNP Proc Nat1 Acad Scl USA, 93,744-748
8 Ghetto, B , Tagliavmt, F , Giaccone, G , Bugtam, 0 , Frangtone, B , Farlow, M
R., and Dlouhy, S. R (1994) Familial Gerstmann-Strdussler-Schemker disease
with neurofibrtllary tangles A401 Neuroblol 8,41-48
9. Gtaccone, G., Taghavmt, F , Verga, L., Frangtone, B , Farlow, M R , Bugtam,
O., and Ghetto, B (1990) Neurofibrillary tangles of the Indiana kmdred of
Gerstmann-Straussler-Schemker disease share anttgemc determinants with those
of Alzhetmer dtsease Brazn Res 530, 325-329
10 Tagliavmr, F , Gtaccone, G., Prelh, F., Verga, L , Porro, M , TroJanowskt, J ,
Farlow, M R , Franglone, B , Ghetti, B , and Bugtam, 0. (1993) A68 is a compo-
nent of patred helical filaments of Gerstmann-Strdussler-Scheinker disease, Indt-
ana kindred Bram Res 616, 325-328
11 Taghavini, F , Prelh, F , Ghtso, J , Bugtani, 0, Serban, D , Prusmer, S B , Farlow,
M R , Ghetto, B , and Frangtone, B (1991) Amylotd protem of Gerstmann-
Straussler-Schemker disease (Indiana kindred) 1san 11 kd fragment of prton pro-
tem with an N-termmal glycme at codon 58 EMBO J 10, 5 13-5 19
12 Tagliavmt, F , Prelh, F , Porro, M., Rossi, G , Gtaccone, G , Farlow, M R ,
Dlouhy, S. R., Ghettt, B., Bugtam, O., and Frangione, B. (1991) Amyloid fibrtls
m Gerstmann-Straussler-Schemker disease (Indtana and Swedish kmdreds)
express only PrP pepttdes encoded by the mutant allele. Cell 79,695-703.
13 Dlouhy, S. R., Hstao, K , Farlow, M R., Foroud, T , Conneally, P M , Johnson,
P , Prusmer, S. B., Hodes, M E , and Ghetto, B (1992) Lmkage of the Indiana
kindred of Gerstmann-Straussler-Schemker disease to the prton protein gene.
Nature Genet 1,64--67.
14 Hstao, K , Dlouhy, S R , Farlow, M R , Cass, C , DaCosta, M., Conneally, P. M ,
Hodes, M. E , Ghetto, B., and Prusmer, S B. (1992) Mutant prton proteins m
Gerstmann-Straussler-Schemker disease with neuroftbrtllary tangles Nature
Genet 1,68-71
15 Gtaccone, G , Verga, L , Bugtani, O., Frangtone, B., Serban, D., Prusiner, S B.,
Farlow, M R , Ghetto, B , and Tagltavmr, F (1992) Prlon protem preamylotd and
amylotd deposits m Gerstmann-Straussler-Schemker dtsease, Indiana kindred
Proc Nat1 Acad Scl USA 89,9349-9353.
16. Taghavmt, F., Prelh, F., Porro, M., ROSSI, G , Gtaccone, G., Bud, T D., Dlouhy,
S R , Young, K., Ptccardo, P., Ghetto, B , Bugtani, 0 , and Frangtone, B (199 1)
282 Tagliavini et al

Only mutant PrP parttclpates m amylotd formatron m Gerstmann-Straussler-
Schemker dtsease with Ala + Val substmmon at codon 117. J Neuropathol Exp
New-01 54,4 16
17 Taghavmi, F , Prelh, F , Verga, L , Giaccone, G , Sarma, R , Gorevtc, P , Ghetto, B ,
Passermt, F , Ghtbaudt, E , Forlorn G , Salmona, M , Buglam, 0 , and Frangione,
B (1993) Synthetic peptldes homologous to prton protein residues 106-147 form
amylotd-hke fibrlls zn vztro Proc Nat1 Acad SCI USA 90, 9678-9682
18 Selvaggmt, C , De Gtota, L , Cant& L , Ghtbaudr, E , Dtomede, L , Passerim, F ,
Forlorn, G , Bugtam, 0 , Tagllavmt, F , and Salmona, M (1993) Molecular char-
acteristics of a protease-resistant, amylotdogemc and neurotoxlc peptide homolo-
gous to residues 106126 of the prton protem Bzochem Bzophys Res Commun
194, 138&1386
19 De Gtoia, L , Selvaggmt, C , Ghlbaudi, E , Dtomede, L , Bugtam, 0 , Forlorn, G ,
Tagliavmi, F , and Salmona, M (1994) Conformational polymorphism of the
amyloidogemc and neurotoxic peptide homologous to residues 106126 of the
prton protein J Bzol Chem 269,7859-7862
20 Forlorn G , Angerettt, N , Chtesa, R , Monzam, E , Salmona, M , Bugtam, 0 , and
Taghavml, F (1993) Neurotoxicity of a priori protein fragment Nature 362,543-545
21 Forlorn, G., Del Bo, R , Angerettt, N , Chtesa, R , Smtroldo, S , Dom, R ,
Ghtbaudi, E , Salmona, M , Porro, M , Verga, L , Giaccone, G , Buglam, 0 , and
Taghavmr, F (1994) A neurotoxtc priori protein fragment induces rat astroglral
proliferation and hypertrophy Eur J Neuroscz 6, 14 15-1422
22 Florto, T , Grtmaldt, M , Scorztello, A , Salmona, M , Buglam, 0 , Taghavmr, F ,
Forlorn, G , and Schettmi, G (1995) The priori protein fragment 106126 m-
creases intracellular calcium levels through a dthydropyrtdme-sensltlve mecha-
nism and induces corttcal type I astrocyte prohferatton zn vztro Sot Neuroscz
Abstr 21(l), 494
23 Posnett, D N , McGrath, H , and Tam, J P. (1988) A novel method for producing
anti-pepttde antibodies J Bzol Chem 256,495-497
24 Barry, R A , Vincent, M T , Kent, S B , Hood, L. E , and Prusmer, S B. (1988)
Charactertzatton of priori proteins with monospecific antisera to synthetic pep-
tides J Immunol 140, 1188-l 193
25 Kascsak, R J , Rubenstem, R , Merz, P A , Tonna-DeMast, M , Fersko, R , Carp,
R I , Wtsmewskt, H M , and Dumger, H (1987) Mouse polyclonal and mono-
clonal antrbody to scrapte-associated fibril protems J Ylro/ 61, 3688-3693
26 Rogers, M., Serban, D., Gyurts, T., Scott, M., Torchta, T , and Prusmer, S B
(1991) Epitope mapping of the Syrian hamster priori protein utihzmg chtmeric and
mutant genes m a vaccmia vu-us expression system. J Immunol 147, 356&3574
27 Serban, D , Taraboulos, A , DeArmond, S J , and Prusmer, S B (1990) Rapid
detection of Creutzfeldt-Jakob disease and scrapie priori proteins Neurology 40,
110-117
28 Schagger, H. and von Jagow, G (1987) Trtcme-sodium dodecyl sulfate-polyacry-
lamtde gel electrophoresls for the separation of proteins m the range from 1 to 100
kDa Anal. Bzochem 166,368-379
Pnon Protein Amyloids 283
29 Houghten, R A. and LI, C H (1983) Reductton of sulfoxtdes m pepttdes and
protems, m Methods znEnzymology, Vol. 91 Enzyme Structure Part I (Hers, C. H
W. and Ttmasheff, S N , eds ), Academic, New York, pp. 549-559
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31 Yang, J T , Wu, C -S C , and Martmez, H. M. (1986) Calculation of protem
conformation from circular dichroism, m Methods zn Enzymology, vol 130, Aca-
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35 Florto, T , Pan, M G., Newman, B., Hershberger, R. E., Ctvelh, O., and Stork, P
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17
Methods for Studying Prion Protein (PrP) Metabolism
and the Formation of Protease-Resistant PrP
in Cell Culture and Cell-Free Systems
Byron Caughey, David A. Kocisko, Suzette A. Priola,
Gregory J. Raymond, Richard E. Race, Richard A. Bessen,
Peter T. Lansbury, Jr., and Bruce Chesebro


1. Introduction
The pathogenesis of scrapte and other transmissible spongiform encephalop-
athtes(TSEs) appears to be based on the posttranslational conversion of the host™s
protease-sensitive prion protein (PrP-sen or PrPC)to abnormal protease-resis-
tant forms (PrP-res or PrPSC). vitro studies using both scrapte-infected tissue
In
culture cells and cell-free reactions have been used effectively to help define
the cellular and molecular details of this process and how it might be inhibited.
Here we discuss experimental approaches that we have used in these studtes
1.7. PrP-Sen and PrP-Res Metabolism
Striking differences in the cellular metabolism of PrP-sen and PrP-res have
been observed that may account for the pathogenic accumulation of PrP-res in
scrapie-infected animals (reviewed in ref. I). The abnormal accumulation of
PrP-res m humans with familial TSEs may also be potentiated by abnormal
metabolism of mutant PrP-sen molecules. Thus, comparative analyses of the
metabolism of PrP forms remain important in understanding the ettology and
pathogenesis of TSEs. PrP metabolism has been studied m Cl27 cells (2),
murine neuroblastoma (MNB) cells (reviewed in ref. 3), hamster HaB cells
(reviewed in ref. 4), fibroblasts (5), PC12 cells (R. Rubenstem and B. Caughey,
unpublished data), T-cells (6), and mixed leukocytes (5). The best character-
ized experimental models of PrP metabolism are the MNB cells and their

From Methods m Molecular Medune Prlon D&gases
Edlted by H Baker and R M Rldley Humana Press Inc , Totowa, NJ

285
286 Caughey et al

scrapie-Infected counterparts (Sc+MNB) (7-9). In this sectton we will focus on
methodologies used m metabolic studies with these cells.
1 I I Generatlon and Maintenance of Sc+MNB Cells
The Neuro 2A or Cl300 clones of MNB cells can be infected using bram
homogenates from Chandler scrapie-infected mice as an moculum (7-9) Typi-
cally, a 10% w/v suspension of scrapie-infected brain m physiologtcal buffer is
prepared by Dounce homogenization. The suspension 1s cleared of large debris
by low speed centrifugatton, diluted 1:4 in tissue culture medium (MEM with
10% fetal bovine serum [FBS]) and incubated over a 50% confluent cell mono-
layer with rocking for 4 h. The medtum/inoculum 1s diluted further to 2 mL
wtth medium and the cells are incubated for an additional 4 h. Six milliliters of
additional fresh medium is added and the cells are grown another l-2 d to con-
fluence. To obtain more homogeneously infected cultures, single-cell clones are
derived by limmng dilution and analyzed for scrapie mfection using PrP-res as
a marker. Often only l-2% of clones derived from the nnttal bulk culture rep-
licate scrapie mfectivity and produce PrP-res (8) The stabihty of the scrapie
infection varies m cultures dertved from individual clones. Some have pro-
duced PrP-res for over 100 serial passages, whereas others have unpredictably
lost scrapie infectivity (or PrP-res production) after only a few passages. Two
factors that can be Important m mamtammg the infection are the culture medium
and FBS We have identrfied mdividual lots of each that reproducibly cure the
Sc+MNB cultures of the scrapie mfection within one to two passages. Thus, mdi-
vidual lots of these components should be screened for use with Sc+MNB. In this
respect, Gibco (Gaithersburg, MD) OptrMEM (supplemented with 10% FBS)
has been the least problematic medium that we have used. Cell culture crises,
such as overgrowth and loss of CO2 in the mcubator, also can be detrimental to
the scrapie infection without permanently affecting the growth of the cells. Once
infected clones are identtfied, ahquots can be frozen m 10% dimethylsulfoxide,

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