<<

. 9
( 45 .)



>>

Jakob disease m a recipient of pituitary-derived human growth hormone. N Engl
J Med 313,734-738
42. Billette de Villemeur, T., Gelottt, A., Deslys, J. P., Dormont, D., Duyckaerts, C.,
Jardm, L., et al. (1994) Iatrogenic Creutzfeldt-Jakob disease m three growth hor-
mone recipients: a neuropathological study. Neuropathol Appl Neurobtol 20,
111-117
43. Goodbrand, I A., Ironside, J. W., Nicolson, D , and Bell, J. E. (1995) Priori pro-
tein accumulation in the spinal cords of patients with sporadic and growth hor-
mone associated Cruetzfeldt-Jakob disease. Neuroscz Lett 183, 127-130.
lronside
56
44 GaJdusek, D. C and Zlgas, V (1957) Degenerative disease of the central nervous
system m New Gumea The endemtc occurrence of “km-u” m the native popula-
tton N Engl J Med 257,97&978
4.5 Neumann, M A , GaJdusek, D C , and Zigas, V (1965) Nemopathologic find-
mgs m exotic neurologic disorders among natives of the highlands of New Gumea
J Neuropathol Exp Neurol 18,48&507.
46 Gerstmann, J , Straussler, E , and Schemker, I (1936) Uber eine etgenarttge
heredrtar-famihare Erkrankung des Zentralnervensystems, zugletch em Benrag
zur Frage des vorzetttgen lokalen Alterns Z ges Neurol Psychzat 154,736-762
47 Ghetto, B , Dlouhy, S R., Glaccone, G., Buglam, 0 , Franglone, B , Farlow, M.
R , et al (1995) Gerstmann-Straussler-Schemker disease and the Indiana kindred
Bram Pathol 5,61-75
48 Gutroy, D C , Yanagihara, R , and GaJdusek, D C (1991) Localisatron of
amyloidogemc proteins and sulfated glycosammoglycans m nontransmissible and
transmissible cerebral amylotdoses. Acta Neuropathol 82,87-92
49 Brown, P , Gibbs. C J , Jr, Rogers-Johnson, P., Asher, D M , Suhma, M P ,
Bacote, A , et al (1994) Human spongtform encephalopathy the National Instt-
tutes of Health series of 300 experimentally transmitted dtsease Ann Neurof 35,
5 13-529
50 Ghetto, B , Taghavma, F , Masters, C L , Beyreuther, K , Giaccone, G , Verga, L ,
et al. (1989) Gerstmann-Straussler-Schemker disease II. Neurofibrdlary tangles and
plaques with PrP amylotd coexist m an affected family Neurology 39, 1453-146 1
51 Medort, R., Trttschler, J H., LeBlanc, A., Vtllare, F , Manetto, V , Ghen, H Y , et
al (1992) Fatal familial msomma, a priori disease with a mutation at codon 178 of
the prlon protein gene N Engl J Med 7,444-449
52 Collmge, J , Owen, F , Poulter, M , Leach, M , Crow, T. J., Rossor, M. N , et al
(1990) Priori dementia without characteristtc pathology. Lancet 336, 7-9
53 Manetto, V , Medort, R , Cortelh, P , Montagna, P , Tinuper, P , Baruzzt, A , et al
(1992) Fatal familial msomma. clmtcal and pathological study of five new cases
Neurology 42,3 12-3 19
54 Gambettt, P , Parchr, P , Petersen, R. B , Chen, S G., and Lugaresi, E (1995)
Fatal familial msomma and familial Creutzfeldt-Jakob dtsease: clmtcal, patho-
logical and molecular features Brazn Path01 5,43-5 1
55. Collmge, J , Palmer, M S , Sidle, K C., Gowland, I , Medort, R , Ironstde, J , and
Lantos, P. (1995) Transmission of fatal familial msomma to laboratory animals
Lancet 346,569,570
56 Lantos, P L., McGill, I S , Janota, I , Doey, L. J , Collinge, J., Bruce, M , et al
(1992) Prton protein tmmunocytochemistry helps to estabhsh the true incidence
of priori diseases. Neuroscl. Lett 147,67-7 1.
57 Edttortal (1990) Priori dlsease+-spongtform encephalopathres unveiled Lancet
336,21-22
Nakazato, Y , Htrato, J., Ishida, Y , Hoshi, S , Hasegawa, M., and Fukuda, T
58.
(1990) Swollen corttcal neurons m Creutzfeldt-Jakob disease contain a phospho-
rylated neurotilament epttope J Neuropathol Exp Neural 49, 197-205
Diagnosis of Human Pnon Disease 57

59. Kato, S , Htrano, A , Umahara, T , Llena, J F., Herz, F , and Ohama, E (1992)
Ultrastructural and immunohistological studies of ballooned cortical neurons m
Creutzfeldt-Jakob diseases expression of aB-crystallm, ubtqumn and stress-
response protein 27 Acta Neuropathol 84,443-448
60 Tateishi, J , Kitamoto, T , Doh-Ura, K , Boellaard, J W , and Petffer, J (1992)
Creutzfeldt-Jakob disease with amyloid angiopathy: diagnosis by immunologic
analysis and transmission experiments Acta Neuropathol 83, 559-563
61 Gray, F , Chretien, F., Cesaro, P., Chatelam, J , Beaudry, P , Laplanche, J. L , et
al (1994) Creutzfeldt-Jakob disease and cerebral amyloid angiopathy Acta
Neuropathol 88, 106-l 11
62 Powers, J M., Lm, Y , Hair, L S , Kascsack, R J , Lewis, L D , and Levy, L A (1991)
Concomitant Creutzfeldt and Alzheimer diseases Acta Neuropathol 83, 95-98
63 Ironside, J W , McCardle, L , Hayward, P. A. R , and Bell, J E (1993) Ubtquttm
immunocytochemistry in human spongtform encephalopathies Neuropathol
Appl Neuroblol 19, 134-140
64 Laszlo, L , Lowe, J., Self, T , Kenward, N , Landon, M , McBride, T . et al (1992)
Lysosomes as key organelles m the pathogenesis of priori encephalopthies J
Path01 166,333-341
65 Hansen, L. A., Mashah, E , Terry, R D., and Mirra, S S. (1989) A neuropatho-
logical subset of Alzheimer™s disease with concomttant Lewy body disease and
spongiform change Acta Neuropathol 78, 194-20 1
66 Collmge, J., Palmer, M S., and Dryden, A J. (1991) Genetic predisposition to
iatrogetuc Creutzfeldt-Jakob disease Lancet 337, 1441,1442
67 de Silva, R , Ironside, J W , McCardle, L , Esmonde, T., Bell, J E , Will, R G., et
al. (1994) Neuropathological phenotype and “priori protein” genotype correlation
m sporadic Creutzfeldt-Jakob disease Neuroscz Lett 179, 50-52
68 Amouyel, P , Vidal, 0 , Launay, J M , and Laplanche, J L (1994) The
apoltpoprotem E alleles as maJor suscepttbthty factors for Creuztfeldt-Jakob dts-
ease Lancet 344, 13 15-l 3 18.
69 Hayward, P. A R , Bell, J E , and Ironside, J W (1994) Prion protein tmmuno-
cytochemistry the development of reliable protocols for the mvestigation of
Creutzfeldt-Jakob disease Neuropathol Appl Neuroblol 20, 375-383
70 Sutherland, K , Bame, C., and Ironside, J W (1994) Automattc quantification of amyloid
plaque formation in human spongifonn encephalopathy Neurodegeneratron 3,293-300
71 Turner, C., Bell, J. E , and Ironstde, J. W. (1993) Localisation of microglia m
CNS amyloid plaques an mrmunocytochemical and confocal microscopic study.
J Path01 170,401.
72 Libersh, P P., Budka, H , Sluga, E., and Barcikowska Kwiecmski, H. (1992) Tubulo-
vesicular structures m Creutzfeldt-Jakob disease. Acta Neuropathol S4,238-243.
73 Liberskt, P P , Yanagihara, R , Gibbs, C J., and Gajdusek, D. C (1990) Appear-
ance of tubulovestcular structures in experimental Creutzfeldt-Jakob disease and
scrapie precedes the onset of clmtcal disease Acta Neuropathol 79, 349-354.
74 DeArmond, S J (1993) Alzheimer™s disease and Creutzfeldt-Jakob disease over-
lap of pathogenic mechanisms Curr Open Neural 6,872-88 1
Neuropathological Diagnosis
of Human Prion Disease
PrP Immunocytochemical Techniques

Jeanne E. Bell


1. Introduction
Routme histologtcal exammation of cases of Creutzfeldt-Jakob disease
(CJD) reveals a spectrum of pathological involvement, particularly with regard
to the extent and locabzatton of sponglform change. Although there ISno dtffi-
culty m confirmmg a dlagnosrs of CJD m cases with widespread and severe
spongiform change mvolvmg the corttcal gray matter or cerebellar molecular
layer, all too often the cortex may be only mmrmally or focally affected, and m
some casespathological changes are largely confined to the basal ganglia or
cerebellum. Unless wide-ranging pathological examination IS undertaken, the
diagnosis of CJD may be mtssed in such cases.Thus is a particular problem m
the interpretation of cortical bropsres in cases of suspected CJD and IS one
reason why biopsy in such cases is now discouraged (I). In addition, there are
other conditions that give rise to spongy appearances in the gray matter (2) and
the difficulties of achieving a firm diagnosis of CJD, based on conventional
histological appearances, have been outlined m Chapter 3. Occasional reports
of the coexistence of CJD with other dementmg illnesses, particularly
Alzhelmer disease, also add to the dragnostrc difficulties (3). CJD is a rare
disease (0.5 cases/million of the population/yr) and it 1slikely that mdtvidual
experience of the possible range of CJD pathological changes will be limited.
For all of these reasons, it would be useful to develop some additional method
of confirming the diagnosis of CJD in tissue sections. Since it IS now known
that CJD and related diseasesare associated with accumulation in the brain of
a protease-resistant protein known as prton protein (4), the demonstration of
From Methods In Mo/ecu/ar M8d/Cin8 Pnon DlS8aS8S
Edlted by H Baker and R M Rldley Humana Press Inc , Totowa. NJ
60 Bell

prton presence m suspected cases would represent such a development.
Recently, mununocytochemical techniques have been devised that accurately
locahze disease-related priori protein (PrPCJD) m human cases (5-13) The
potenttal value of such additional techmques m clarifying the diagnosis in dif-
ficult and subtle cases of CJD 1s obvtous. However, the implementatron of
PrPCJD immunocytochemrstry may have other benefits. Since PrPCJD closely
1s
associated, if not identical, with the transmissible agent known to be present in
the central nervous system (CNS), its localization clearly 1sa matter of scten-
trfic interest and may help to throw light on the spread of the agent from the
point of entry, at least m those cases m which the disease has an exogenous
origm, such as patients treated with human pmutary-derived hormones (14).
All the maJor human forms of prton disease, including sporadic (2,15) and
familial CJD (2,16), Gerstmann-Straussler-Schemker syndrome (GSS) (16),
and iatrogemc CJD (dura mater and human pmutary hormone cases) (17) show
deposits of PrPCJDm different CNS locattons. Stmllar accumulations occur m
animal priori diseases, notably scraple (IS) and bovine spongiform encepha-
lopathy (BSE) (19). This chapter is concerned with the methodology for
immunolocalization of PrPCJD withm the human CNS, and with the dtlemmas
of interpretation associated with this technique
2. PrPCJD
PrPCJDis an msoluble, protease-resistant protein that accumulates m the
brains of patients with CJD and other related diseases (20). It is deposited
within the brain parenchyma m part as amylord that is detectable by periodic
acid Schrff and congo red stammg if present m sufficient quantity to form the
plaques that are seen m some casesof CJD and more commonly m other priori
diseases, particularly GSS (2,20,21). It 1sbelieved that PrPCJDis a perverted
form of a normal soluble cellular protein, PrPC,which is encoded by a gene on
chromosome 20 and is expressed m many cells (mcludmg non-CNS cells) but
parttcularly on neuronal membranes. The function of PrPC is not known. In
CJD and other related diseases, PrPC 1sposttranslatronally altered to PrPCJD,
which, because it is protease resistant, 1scapable of accumulatmg m tissues m
which it is generated (20). It is known that a number of different mutations in
the PrP gene predispose to this posttranslattonal modrficatron with consequent
accumulation of PrPCJD(22). The assoctatton of PrP mutations with different
forms of prton disease 1sreviewed elsewhere m this volume. The relationship
between PrP genotype and phenotypic expression of priori disease 1scomplex,
and currently is the focus of intense research activity.
PrPC and PrPCJDare closely similar and antibodies raised against one will
crossreact against the other (2) The gene mvolved 1shighly conserved across
species (20) and is expressed m mammals and m some birds Antibodies have
61
Diagnosis of Human Priori Disease
Table 1
Pretreatments Used
in PrP lmmunostaining Protocols
Protemase K 10 ug/mL 15 mitt 37°C (6)
Pepsin 10% 30 mm 37°C (8)
80-I 00% fornnc acrd 5-60 mm (5,7,9, IO, 13)*
30% formic acid 1 mm m microwave oven (1 I)
4M guamdine thtocyanate 2 h (13,23)
Hydrated autoclavmg drstilled water (13)O
Hydrolytic autoclaving 2 5 n&I HCI 10 mm 12 1“C (24)
aIncIudmg those currently used III Edmburgh


been raised both to the disease-related forms of PrP, such as scrapte-associated
frbrtls, and more recently to synthetic peptides followmg the successful
sequencing of the PrP gene. A number of different polyclonal and monoclonal
antibodies are available (but not yet commercially) and many of these have
been used m umnunocytochemmal studies (5-13). Antibodies raised agamst
scrapie fibrrls readily produce positive staining m human CJD cases,thus dem-
onstrating crossreacttvlty between different species (2,23)
The fact that these antrbodtes cannot clearly distinguish between PrPC and
PrPCJD raises potential problems of interpretation. In order to cu-cumvent these
dtfficulttes, strategres have been developed that arm to eliminate PrPC from
trssue sections before exposure to the primary antibody, with the arm of vrsual-
rzing only disease-related, protease-resistant PrPCJD(Table 1). A number of
different enhancing treatments are also currently in use that allow the primary
anttbody to be used at much higher drlution (5I3,23,24). As a result, tt now
seems unlikely that the vrsualizatton of PrPc is a significant confounding fac-
tor m sections stained for PrPCJD. Nevertheless, tt IS even more important than
usual to include immunocytochemtcal checks and controls m any protocol for
the demonstration of PrPCJD The use of negative and positive control case
material, and negative secttons m which the primary antibody is omitted, are
mandatory in each test run Other checks mclude comparrson of results using a
panel of different PrP antrbodres, including anttbodres preabsorbed against the
specific antigen.
Protocols for PrPCJDunmunocytochemistry (Table 2) are now reasonably
well validated for CNS tissue sections. In our laboratory, PrP tmmunostainmg
is part of the dtagnosttc procedure, used in conjunction with routine histo-
pathology of the CNS, and to demonstrate the topographtcal cellular localiza-
non of PrPCJD. Western blotting and immunoblottmg (25) represent alternative
mvestrgatory procedures for the presence of PrPCJD neither allows locahza-
but
non at the cellular level m the way that rmmunocytochemistry does.
Bell
62

Table 2
Protocol for PrP lmmunocytochemistry
1 5-pm sectlons floated on to shdes coated with Vectabond
2 Sectlons to water
3 Picrlc acid 15 mm (to remove formalm pigment)
4 Water
5 Hydrogen peroxlde 30 mm (to block endogenous peroxide)
6 Water
7 Autoclave m dlstllled water 12 1°C for 10 mm
8 Water
9 96% Formic acid 5 mm
10 Water
11 4M guamdme thlocyanate 2 h at 4™C
12 Water followed by Trls buffered salme
13 Blocking serum 10 mm
14 Primary PrP antibody most are used overnight (Table 3)
15 Wash m Trls buffered salme
16 Secondary antlbody (1.200) for 30 mm
17 Wash m Trls buffered salme
18 Avldm blotm for 30 mm
19 Wash m Trls buffered salme
20 Visualize with chammobenzldme
21 Wash in water
22 Counterstain lightly with hemotoxylm
23 Sections dehydrated, cleared, and mounted m Pertex
24 Mounted shdes (after drymg) further decontaminated by immersion m 96% formic
acid for 5 mm


Just as the correlation between PrP genotype, clinical phenotype, and classl-
cal pathology is under intense mvestlgation at present, so also is the dlstnbu-
tion of PrPCJD different manifestations of prion disease. It remains to be seen
in
whether tightly consistent patterns of PrPCJDdeposition will be demonstrated
but considerable progress 1sbeing made in this field.
3. Protocols for PrP lmmunocytochemistry
Based on the published experience of PrP immunostaining, we introduced
this procedure in the UK National CJD Surveillance Umt m Edinburgh m 1992,
experimentally at first in order to explore its potential role m diagnostic confir-
mation of CJD. Since that time we have modified the protocol m accordance
with recently published work and with our own experience m order to further
enhance PrPCJDlocahzatlon and to reduce the risk of PrPC detection. In 1993,
our Unit hosted an MRC-funded workshop to which all the UK centers under-
63
Diagnosis of Human Priori Disease

<<

. 9
( 45 .)



>>