American Journal of Pathology, Vol. 141, No. 2, August 1992 Copyright C American Association of Pathologists
Short Communication The Primary Structure of the Prion Protein Influences the Distribution of Abnormal Prion Protein in the Central Nervous System
Tetsuyuki Kitamoto, Katsumi Doh-ura, Tamaki Muramoto, Masayuki Miyazono, and Jun Tateishi From the Department of Neuropatholog, Neurological Institute, Kyushu University, Fukuoka,Japan
We immunohistochemically examined tissue sections from patients with prion protein (PrP) polymorphism using hydrolytic autoclaving enhancement. Abnormal PrP accumulations could be classified into plaque formations (plaque-type) and the diffuse gray matter stainings including synaptic structures (synaptic-type). Insertional polymorphism, a point mutation in codon 102 or 11 7/129, and a polymorphism in codon 129 (Val'29) result in plaque-type PrP accumulations. The patients with codon 102 mutation also have synaptic-type PrP accumulations. However, a point mutation in codon 200 did not show plaque-type accumulations, and only showed synaptic-type PrP accumulations. Likewise, sporadic Creutzfeldt-Jakob disease patients without any known mutations only have synaptic type accumulations. These results imply that the primary structures of PrP influence the phenotype of prion diseases, especially in abnormal PrP distributions of the central nervous system. (Am J Pathol 1992, 141:271-277)
Creutzfeldt-Jakob disease (CJD), kuru, and GerstmannStraussler syndrome (GSS) are transmissible neurodegenerative diseases in humans, whereas scrapie is a similar disorder in animals. These diseases are caused by a slow infectious pathogen-designated prion.1 Molecular biologic studies have revealed polymorphisms of the
prion protein (PrP) gene, especially in the PrP open reading frame.2 In animals, these polymorphisms of the PrP coding region influence the duration of the incubation period, susceptibility to the disease, or neuropathologic features.' In humans, codon 102 or codon 1 17 point mutation was reported to be linked to GSS.6 7 The results in codon 102 transgenic mice also strengthen the idea that the prion mutation is one of the essential events that cause GSS.8 Several polymorphisms were also reported to be linked to familial CJD and familial dementia.2>12 We developed a new pretreatment, designated as hydrolytic autoclaving, to enhance the immunoreactivity of the abnormal isoform of PrP (PrPCJD).13 This pretreatment revealed diffuse gray matter stainings including synaptic structures in the central nervous system of both sporadic CJD patients14 and CJD-infected mice,15 as well as follicular dendritic cell stainings in the lymphoid tissues of CJD-infected mice.13,15 With hydrolytic autoclaving, we could confirm the diagnosis of CJD immunohistochemically. Previous immunohistochemistry using formic acid pretreatment did not reveal either diffuse gray matter stainings or follicular dendritic cell stainings in paraffin sections.16 Therefore, it is interesting to examine the tissue sections from a patient with PrP polymorphisms using the hydrolytic autoclaving method. We herein report that PrP polymorphisms may influence the distribution of abnormal PrP depositions in the central nervous system.
Supported by grants (02454245, 03454171) from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, the Science and Technology Agency, and the Foundation for Advancement of Clinical Medicine. Accepted for publication May 22, 1992. Address reprint requests to Dr. Tetsuyuki Kitamoto, Department of Neuropathology, Neurological Institute, Kyushu University, Fukuoka 812, Japan.
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AJP August 1992, Vol. 141, No. 2
Materials and Methods
primers (5'-CATGAGCAGGCCCATCATAC-3', and 5'ACACATCTGCTCAACCACGC-3') to get a 244 bp frag-
PrP Gene Analysis
ment from codon 134 to codon 214 for analysis of Hph RFLP. The typical RFLP patterns in each polymorphism are shown in Figure lb.
High molecular weight DNA was prepared from the peripheral blood lymphocytes or frozen brain tissues as described by Sambrook et al.17 The PrP coding region was amplified by the polymerase chain reaction (PCR) method as reported previously.18 The substitutions in codons 102, 117, 129, 178, 198, and 200 were detected by dot differential hybridization using allele specific oligonucleotides,7 18 and by restriction fragment length polymorphism (RFLP) using Dde 1, Pvu II, Nsp 1, Tth 111 1, Hph 1, or Bsm Al, respectively (Figure 1 a) 6,7,1011'19'20 For the analysils in patients with a codon 129 polymorphism (Val129), we sequenced the PrP coding region to detect any other mutations,21 and prepared a pair of PCR
a P G repetitive
102
'-
-l
117
129
'78
-FT
5'
~~~~~~~~~~1'Dde
t t llvul11
NsplI
198 200
11
t Tili1ll
i
3'
t\ Hpti BsmA
168bp ieisertion
b
l 2 3 4 5 6 6581489~ 267mm 0
Figure 1. a: Schematic presentation for RFLP to detect a point mutation. Codon 102 mutation creates a new Dde I site. Codon 117, 129, 178, 198, or 200 mutation abolishes a PvuII, NspI, Tthl 1l, HphI, or BsmAI site, respectively. Insertional polymorphisms are recognized in the proline-glycine rich repetitive sequence (P-G repetitive). b: RFLP of amplified PrP gene. Lane 1; healthy control, Lane 2; GSS'02, Lane 3; healthy control, Lane 4; CJD129, Lane 5; healthy control, Lane 6; CJIY'0. PCR-amplified PrP gene was digested with Ddel in Lane 1 and 2, with NspI in Lane 3 and 4, and with BsmnAl in Lane 5 and 6 Codon 102 mutation shows a new 361bp band in Lane 2. Codon 129 polymorphism represents an uncut 438bp band in Lane 4. Codon 200 mutation shows a whole-sized product of PCR (739bp) in Lane 6
Immunohistochemistry We studied formalin-fixed, paraffin-embedded tissue sections from 8 patients with codon 102 mutation (GSS102), 8 with a codon 129 polymorphism (Val129) (CJD129; 1 was homozygous, whereas the others were heterozygous), 2 with codon 200 mutation (CJD21), 1 with codon 117/129 mutations (GSS1 17'129), 1 with 168 bp insertions (Dementiainsert), and 15 without any known mutations (CJDWIld). All patients were Japanese, except for GSS117'129.22 All available paraffin blocks were cut into 7-pum sections. To enhance PrP immunoreactivity, we performed the hydrolytic autoclaving for pretreatment.13 The tissue sections were deparaffinized and blocked with 0.3% H202 in absolute methanol. Then, the sections were autoclaved at 1210C for 10 minutes in a stainless-steel vessel filled with 1.5, 3, 6, or 9 mmol HCI (final concentration) in distilled water to immerse the section completely. After the temperature in the autoclave had fallen to under 600C, the sections were taken out and washed with tap water and Tris buffer. The sections were then incubated overnight with PrP antiserum23'24 at 40C. The following steps were performed by the PAP method (DAKO, Denmark) according to the manufacturer's instruction.
Results
GSS102 As previously reported,25'26 kuru plaques are the site of major PrP accumulations. Numerous Congophilic kuru plaques were widely distributed in the central nervous system. In cerebral cortices with GSS102 (Leu102), hydrolytic autoclaving documented diffuse gray matter stainings including synaptic structures, in addition to the plaque-type stainings. Almost all patients with GSS102 have these synaptic-type diffuse gray matter stainings. In cerebellar cortices, we found plaque-type stainings (Figure 2a). In one of the seven GSS102 patients, we found another polymorphism at codon 129 (Val129). However, this patient had a similar clinical course and pathologic findings to other GSS102 patients (Miyazono et al, in preparation).
PrP Influences Distribution of PrPCJD in the CNS
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:
r i; S i ze vr4 J~~~~~~/ Al
Short Communication The Primary Structure of the Prion Protein Influences the Distribution of Abnormal Prion Protein in the Central Nervous System
Tetsuyuki Kitamoto, Katsumi Doh-ura, Tamaki Muramoto, Masayuki Miyazono, and Jun Tateishi From the Department of Neuropatholog, Neurological Institute, Kyushu University, Fukuoka,Japan
We immunohistochemically examined tissue sections from patients with prion protein (PrP) polymorphism using hydrolytic autoclaving enhancement. Abnormal PrP accumulations could be classified into plaque formations (plaque-type) and the diffuse gray matter stainings including synaptic structures (synaptic-type). Insertional polymorphism, a point mutation in codon 102 or 11 7/129, and a polymorphism in codon 129 (Val'29) result in plaque-type PrP accumulations. The patients with codon 102 mutation also have synaptic-type PrP accumulations. However, a point mutation in codon 200 did not show plaque-type accumulations, and only showed synaptic-type PrP accumulations. Likewise, sporadic Creutzfeldt-Jakob disease patients without any known mutations only have synaptic type accumulations. These results imply that the primary structures of PrP influence the phenotype of prion diseases, especially in abnormal PrP distributions of the central nervous system. (Am J Pathol 1992, 141:271-277)
Creutzfeldt-Jakob disease (CJD), kuru, and GerstmannStraussler syndrome (GSS) are transmissible neurodegenerative diseases in humans, whereas scrapie is a similar disorder in animals. These diseases are caused by a slow infectious pathogen-designated prion.1 Molecular biologic studies have revealed polymorphisms of the
prion protein (PrP) gene, especially in the PrP open reading frame.2 In animals, these polymorphisms of the PrP coding region influence the duration of the incubation period, susceptibility to the disease, or neuropathologic features.' In humans, codon 102 or codon 1 17 point mutation was reported to be linked to GSS.6 7 The results in codon 102 transgenic mice also strengthen the idea that the prion mutation is one of the essential events that cause GSS.8 Several polymorphisms were also reported to be linked to familial CJD and familial dementia.2>12 We developed a new pretreatment, designated as hydrolytic autoclaving, to enhance the immunoreactivity of the abnormal isoform of PrP (PrPCJD).13 This pretreatment revealed diffuse gray matter stainings including synaptic structures in the central nervous system of both sporadic CJD patients14 and CJD-infected mice,15 as well as follicular dendritic cell stainings in the lymphoid tissues of CJD-infected mice.13,15 With hydrolytic autoclaving, we could confirm the diagnosis of CJD immunohistochemically. Previous immunohistochemistry using formic acid pretreatment did not reveal either diffuse gray matter stainings or follicular dendritic cell stainings in paraffin sections.16 Therefore, it is interesting to examine the tissue sections from a patient with PrP polymorphisms using the hydrolytic autoclaving method. We herein report that PrP polymorphisms may influence the distribution of abnormal PrP depositions in the central nervous system.
Supported by grants (02454245, 03454171) from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, the Science and Technology Agency, and the Foundation for Advancement of Clinical Medicine. Accepted for publication May 22, 1992. Address reprint requests to Dr. Tetsuyuki Kitamoto, Department of Neuropathology, Neurological Institute, Kyushu University, Fukuoka 812, Japan.
271
Kitamoto et al
272
AJP August 1992, Vol. 141, No. 2
Materials and Methods
primers (5'-CATGAGCAGGCCCATCATAC-3', and 5'ACACATCTGCTCAACCACGC-3') to get a 244 bp frag-
PrP Gene Analysis
ment from codon 134 to codon 214 for analysis of Hph RFLP. The typical RFLP patterns in each polymorphism are shown in Figure lb.
High molecular weight DNA was prepared from the peripheral blood lymphocytes or frozen brain tissues as described by Sambrook et al.17 The PrP coding region was amplified by the polymerase chain reaction (PCR) method as reported previously.18 The substitutions in codons 102, 117, 129, 178, 198, and 200 were detected by dot differential hybridization using allele specific oligonucleotides,7 18 and by restriction fragment length polymorphism (RFLP) using Dde 1, Pvu II, Nsp 1, Tth 111 1, Hph 1, or Bsm Al, respectively (Figure 1 a) 6,7,1011'19'20 For the analysils in patients with a codon 129 polymorphism (Val129), we sequenced the PrP coding region to detect any other mutations,21 and prepared a pair of PCR
a P G repetitive
102
'-
-l
117
129
'78
-FT
5'
~~~~~~~~~~1'Dde
t t llvul11
NsplI
198 200
11
t Tili1ll
i
3'
t\ Hpti BsmA
168bp ieisertion
b
l 2 3 4 5 6 6581489~ 267mm 0
Figure 1. a: Schematic presentation for RFLP to detect a point mutation. Codon 102 mutation creates a new Dde I site. Codon 117, 129, 178, 198, or 200 mutation abolishes a PvuII, NspI, Tthl 1l, HphI, or BsmAI site, respectively. Insertional polymorphisms are recognized in the proline-glycine rich repetitive sequence (P-G repetitive). b: RFLP of amplified PrP gene. Lane 1; healthy control, Lane 2; GSS'02, Lane 3; healthy control, Lane 4; CJD129, Lane 5; healthy control, Lane 6; CJIY'0. PCR-amplified PrP gene was digested with Ddel in Lane 1 and 2, with NspI in Lane 3 and 4, and with BsmnAl in Lane 5 and 6 Codon 102 mutation shows a new 361bp band in Lane 2. Codon 129 polymorphism represents an uncut 438bp band in Lane 4. Codon 200 mutation shows a whole-sized product of PCR (739bp) in Lane 6
Immunohistochemistry We studied formalin-fixed, paraffin-embedded tissue sections from 8 patients with codon 102 mutation (GSS102), 8 with a codon 129 polymorphism (Val129) (CJD129; 1 was homozygous, whereas the others were heterozygous), 2 with codon 200 mutation (CJD21), 1 with codon 117/129 mutations (GSS1 17'129), 1 with 168 bp insertions (Dementiainsert), and 15 without any known mutations (CJDWIld). All patients were Japanese, except for GSS117'129.22 All available paraffin blocks were cut into 7-pum sections. To enhance PrP immunoreactivity, we performed the hydrolytic autoclaving for pretreatment.13 The tissue sections were deparaffinized and blocked with 0.3% H202 in absolute methanol. Then, the sections were autoclaved at 1210C for 10 minutes in a stainless-steel vessel filled with 1.5, 3, 6, or 9 mmol HCI (final concentration) in distilled water to immerse the section completely. After the temperature in the autoclave had fallen to under 600C, the sections were taken out and washed with tap water and Tris buffer. The sections were then incubated overnight with PrP antiserum23'24 at 40C. The following steps were performed by the PAP method (DAKO, Denmark) according to the manufacturer's instruction.
Results
GSS102 As previously reported,25'26 kuru plaques are the site of major PrP accumulations. Numerous Congophilic kuru plaques were widely distributed in the central nervous system. In cerebral cortices with GSS102 (Leu102), hydrolytic autoclaving documented diffuse gray matter stainings including synaptic structures, in addition to the plaque-type stainings. Almost all patients with GSS102 have these synaptic-type diffuse gray matter stainings. In cerebellar cortices, we found plaque-type stainings (Figure 2a). In one of the seven GSS102 patients, we found another polymorphism at codon 129 (Val129). However, this patient had a similar clinical course and pathologic findings to other GSS102 patients (Miyazono et al, in preparation).
PrP Influences Distribution of PrPCJD in the CNS
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r i; S i ze vr4 J~~~~~~/ Al
