Brain Advance Access originally published online on April 5, 2006
Brain 2006 129(6):1557-1569; doi:10.1093/brain/awl076
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Phenotypic heterogeneity in inherited prion disease (P102L) is associated with differential propagation of protease-resistant wild-type and mutant prion protein
1 MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London, National Hospital for Neurology and Neurosurgery Queen Square, London, UK 2 Institute of Neurology, Medical University Vienna Wien, Austria
Correspondence to: Prof. John Collinge, MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London, National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK E-mail: j.collinge{at}prion.ucl.ac.uk
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Summary |
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Inherited prion diseases are caused by PRNP coding mutations and display marked phenotypic heterogeneity within families segregating the same pathogenic mutation. A proline-to-leucine substitution at prion protein (PrP) residue 102 (P102L), classically associated with the Gerstmann–Sträussler–Scheinker (GSS) phenotype, also shows marked clinical and pathological heterogeneity, including patients with a Creutzfeldt–Jakob disease (CJD) phenotype. To date, this heterogeneity has been attributed to temporal and spatial variance in the propagation of distinct protease-resistant (PrPSc) isoforms of mutant PrP. Here, using a monoclonal antibody that recognizes wild-type PrP, but not PrP 102L, we reveal a spectrum of involvement of wild-type PrPSc in P102L individuals. PrPSc isoforms derived from wild-type and mutant PrP are distinct both from each other and from those seen in sporadic and acquired CJD. Such differential propagation of disease-related isoforms of wild-type PrP and PrP 102L provides a molecular mechanism for generation of the multiple clinicopathological phenotypes seen in inherited prion disease.
Key Words: Creutzfeldt–Jakob disease; Gerstmann–Sträussler–Scheinker disease; prion protein; prion disease
Abbreviations: CJD, Creutzfeldt–Jakob disease; ELISA, enzyme-linked immunosorbent assay; GSS, Gerstmann–Sträussler–Scheinker disease; PBS, phosphate buffered saline; PrP, prion protein
Received November 24, 2005. Revised February 3, 2006. Accepted March 8, 2006.
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Introduction |
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Prion diseases are fatal neurodegenerative disorders that include Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker disease (GSS), fatal familial insomnia (FFI), kuru and variant CJD (vCJD) in humans (Collinge, 2001
). Their central feature is the post-translational conversion of host-encoded, cellular prion protein (PrPC) to an abnormal isoform, designated PrPSc (Prusiner, 1998; Collinge, 2001). The propagation of transmissible prions can be acquired (by inoculation, or in some cases by dietary exposure) with infected tissue, arise sporadically or be inherited via germline mutations in the human prion protein gene (PRNP) (Prusiner, 1998; Collinge, 2001; Wadsworth et al., 2003). According to the protein-only hypothesis (Griffith, 1967), an abnormal PrP isoform is the principal, if not the sole, component of the transmissible prion, with prion propagation occurring through PrPSc acting to replicate itself with high fidelity by recruiting endogenous PrPC (Prusiner, 1998; Collinge, 2001). Distinct strains of prions are recognized that produce characteristic phenotypes when propagated in defined inbred mice, and which are associated with biochemically distinct PrPSc types that can be serially propagated in suitable experimental hosts (Bessen and Marsh, 1994; Collinge et al., 1996; Telling et al., 1996). Multiple human PrPSc types have been described in diverse human prion diseases (Collinge et al., 1996; Parchi et al., 1996; Wadsworth et al., 1999; Hill et al., 2003, 2006), and these human molecular strain types can be faithfully propagated in transgenic mice expressing only human PrP (Collinge et al., 1996; Hill et al., 1997; Asante et al., 2002; Wadsworth et al., 2004). Phenotypic diversity in sporadic and acquired human prion disease is significant, partly because of the propagation of distinct prion strains.
About 15% of human prion disease is associated with autosomal dominant pathogenic mutations in PRNP (Collinge, 2001; Kovacs et al., 2002; Wadsworth et al., 2003): to date more than 30 pathogenic PRNP mutations have been identified. While patients with these familial forms of prion disease have traditionally been classified by the clinical or pathological syndromes of GSS, CJD or FFI, the advent of molecular genetic diagnosis led to recognition of considerable phenotypic heterogeneity even within families with the same PRNP mutation (Collinge et al., 1989, 1990, 1992; Mallucci et al., 1999; Kovacs et al., 2002) and sub-classification of inherited prion disease by pathogenic mutation was proposed (Collinge and Prusiner, 1992; Collinge et al., 1992). The classical presentation of GSS is as a progressive cerebellar ataxia with dementia occurring much later in the relatively prolonged clinical course in distinction to CJD in which a rapidly progressive dementia is the typical dominant feature. GSS is diagnosed neuropathologically by the demonstration of characteristic multicentric amyloid plaques showing positive PrP immunoreactivity (Gerstmann et al., 1936; Kretzschmar et al., 1991; Hainfellner et al., 1995; Budka, 2003).
A proline-to-leucine substitution at codon 102 (P102L) of human PrP is the most common mutation associated with the GSS phenotype and was first reported in a UK and a US family in 1989 (Hsiao et al., 1989). Many other kindreds have now been documented worldwide (Kovacs et al., 2002), including the original Austrian family reported by Gerstmann, Sträussler and Scheinker in 1936 (Kretzschmar et al., 1991; Hainfellner et al., 1995). Progressive ataxia is the dominant clinical feature, with dementia and pyramidal features occurring later in a disease course typically much longer than that of classical CJD. However, marked variability at both the clinical and neuropathological levels is apparent, with some patients developing a classical CJD-like phenotype with early and rapidly progressive dementia (Hainfellner et al., 1995; Barbanti et al., 1996; Majtenyi et al., 2000), and another phenotypic variation including marked amyotrophic features is reported (Kretzschmar et al., 1992). Such clinical heterogeneity has yet to be explained; however, in agreement with the hypothesis that divergent physicochemical properties of PrPSc encode and determine clinical and neuropathological phenotype, distinct forms of abnormal prion protein have been identified in P102L patients (Furukawa et al., 1998; Parchi et al., 1998; Piccardo et al., 1998; Hill et al., 2006).
Although proteinase K-resistant PrP fragments of 
21–30 kDa seen in P102L GSS have molecular masses similar in size to those seen in classical CJD (Furukawa et al., 1998; Parchi et al., 1998; Piccardo et al., 1998; Hill et al., 2006), the glycoform ratio is distinct from PrPSc fragments seen in both classical CJD (Furukawa et al., 1998; Parchi et al., 1998; Piccardo et al., 1998; Hill et al., 2006) and vCJD (Hill et al., 2006). Notably, however, abnormal PrP conformers generating high molecular mass protease-resistant PrP fragments are not uniformly detected throughout the brain in P102L GSS and appear to be restricted to areas of brain showing synaptic PrP deposition and spongiform vacuolation (Parchi et al., 1998; Piccardo et al., 1998). In brain regions in which amyloid plaques are a prominent feature, an alternate abnormal PrP isoform appears to predominate that generates smaller protease-resistant PrP fragments of 8 kDa derived from the central portion of PrP (Parchi et al., 1998; Piccardo et al., 1998; Hill et al., 2006), which in the longest form comprise PrP residues 74–153 (Parchi et al., 1998). To date, all abnormal PrP isoforms seen in P102L GSS have been thought to originate from mutant PrP (Parchi et al., 1998; Muramoto et al., 2000). Here, however, using a monoclonal antibody that recognizes wild-type human PrP, but not human PrP 102L, we show a spectrum of involvement of protease-resistant wild-type PrP that may contribute to phenotypic variability in P102L GSS.
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Methods |
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Use of human tissues
Human tissues were obtained at autopsy with consent for use in this research. This study was approved by the Institute of Neurology/National Hospital for Neurology and Neurosurgery Local Research Ethics Committee. Human prion disease tissue was handled according to strict biosafety protocols in a microbiological containment level 3 facility.
Transgenic mice
Transgenic mice homozygous for a human PrP 129M transgene array and murine PrP null alleles (Prnpo/o) designated Tg(HuPrP 129M+/+ Prnpo/o)-35 mice (Tg35 mice), or homozygous for a human PrP E200K 129M transgene array and murine PrP null alleles (Prnpo/o) designated Tg(HuPrP E200K 129M+/+ Prnpo/o)-49 mice (Tg49 mice) or homozygous for a human PrP P102L 129M transgene array and murine PrP null alleles (Prnpo/o) designated Tg(HuPrP P102L 129M+/+ Prnpo/o)-27 mice (Tg27 mice) were generated as described previously (Asante et al., 2002, 2004). Inbred FVB/NHsd mice were supplied by Harlan UK Ltd. Transmission of human prion isolates to transgenic mice was performed as described previously (Asante et al., 2002). Full details of these transmissions will be reported elsewhere. Care of mice was according to institutional guidelines.
Antibodies
Anti-PrP monoclonal antibody 3F4 (Kascsak et al., 1987) was supplied by Signet Laboratories Inc, Dedham, MA, USA. ICSM 18, ICSM 35 and ICSM 37 were supplied by D-Gen Ltd, London, UK. ICSM antibodies were raised in Prnpo/o mice against recombinant 
- or ß-PrP (Jackson et al., 1999) as described elsewhere (Khalili-Shirazi et al., 2005a, b). 3F4 is an IgG2a whose epitope spans residues 104–113 of human, hamster or feline PrP (Kascsak et al., 1987; Kanyo et al., 1999). ICSM 18 is an IgG1 whose epitope spans residues 142–153 of human PrP (Khalili-Shirazi et al., 2005b). ICSM 37 and ICSM 35 are IgG2a and IgG2b immunoglobulins, respectively, both with epitopes spanning residues 93–105 of human PrP (Khalili-Shirazi et al., 2005a, b).
Immunoblotting
Ten per cent (w/v) brain homogenates were prepared in Dulbeccos phosphate buffered saline (PBS) lacking Ca2+, Mg2+ or lysis buffer [10 mM Tris, 10 mM EDTA, pH 7.4, containing 100 mM NaCl, 0.5% (w/v) NP-40 and 0.5% (w/v) sodium deoxycholate] by serial passage through needles of decreasing diameter (Collinge et al., 1996; Hill et al., 2006). Aliquots were analysed with or without proteinase K digestion (50 or 100 µg/ml final protease concentration, 1 h, 37°C) by electrophoresis and immunoblotting as described previously (Wadsworth et al., 2001; Hill et al., 2006). Blots were blocked in PBS containing 0.05% v/v Tween-20 (PBST) and 5% non-fat milk powder and probed with anti-PrP monoclonal antibodies (0.2 µg/ml final concentration in PBST) in conjunction with anti-mouse IgG-alkaline phosphatase conjugated secondary antibody and chemiluminescent substrate CDP-Star (Tropix Inc, Bedford, MA, USA), and visualized on Biomax MR film (Kodak; Anachem Ltd, Luton, UK) as described (Wadsworth et al., 2001). For quantification and analysis of PrP glycoforms, blots were developed in chemifluorescent substrate (AttoPhos; Promega, Southampton, UK) and visualized on a Storm 840 phosphoimager (Molecular Dynamics; GE Healthcare UK Ltd, Chalfont St.Giles, UK). Quantification of PrPSc glycoforms was performed using ImageQuaNT software (Molecular Dynamics).
Detergent solubility studies
Fifty microlitre aliquots of 10% brain homogenate were adjusted with 200 µl PBS and 250 µl PBS containing 4% (w/v) sodium lauroylsarcosine (Calbiochem; Merck Biosciences Ltd, Nottingham, UK). Following addition of 1 µl benzonase (Benzon nuclease, purity 1; Merck KGaA, Darmstadt, Germany) samples were incubated for 30 min at 37°C with constant agitation. Samples were then centrifuged at 13 200 r.p.m. (16 100 g) for 30 min in a microfuge. After careful isolation of the supernatant, detergent-insoluble pellets were re-suspended to the original starting volume of 50 µl with PBS containing 0.1% (w/v) sodium lauroylsarcosine. Aliquots of supernatant and re-suspended pellet were analysed with or without proteinase K digestion (50 µg/ml final protease concentration, 1 h, 37°C) by electrophoresis and immunoblotting as described above.
Analysis of brain homogenate by enzyme-linked immunosorbent assay (ELISA)
Ten microlitre aliquots of 10% brain homogenate were treated with 1 µl benzonase (Benzon nuclease, purity 1; Merck) for 5 min at 37°C with constant agitation. Subsequently, samples were adjusted with 2 µl of 500 mM 4-(2-aminoethyl)-benzene sulphonyl fluoride and 10 µl 2% (w/v) sodium dodecyl sulphate (SDS) and heated at 100°C for 5 min. Samples were centrifuged at 800 r.p.m. (100 g) for 30 s before adjustment with 600 µl PBS containing 2% (v/v) Tween-20 and 2.5% (v/v) soya milk. Twenty-five microlitre aliquots were transferred into the wells of microtitre plates (Greiner, microlon 96W, Greiner Bio-One Ltd, Stonehouse, UK) containing immobilized anti-PrP monoclonal antibody ICSM 18 (250 ng per well). After incubation with constant agitation for 1 h at 37°C, wells were washed in 90 s with 3 x 300 µl PBS containing 0.05% v/v Tween-20 using an automated microplate washer followed by the addition of 100 µl PBS containing 1% (v/v) Tween-20 and 1 µg/ml biotinylated anti-PrP monoclonal antibody ICSM 35 or ICSM 37. Following incubation with constant agitation for 1 h at 37°C, wells were washed (as above) followed by the addition of 100 µl PBS containing 1% (v/v) Tween-20 and a 1 : 10 000 fold dilution of streptavidin-horseradish peroxidase conjugate (Dako UK Ltd, Ely, UK). After incubation with constant agitation for 30 min at 37°C, wells were washed in 120 s with 4 x 300 µl PBS containing 0.05% v/v Tween-20. Plates were developed with 100 µl per well 3,3',5,5' tetramethylbenzidine and quenched with 50 µl 3 M sulphuric acid before determining absorbance at 450 nm using a Tecan spectra image microplate reader.
Peptide ELISA
Synthetic peptides corresponding to human PrP residues 92–111 with either proline or leucine at residue 102 and biotin covalently coupled at the N-terminus were immobilized (100 ng peptide per well) on streptawell microtitre plates (Roche, Diagnostics Ltd, Lewes, UK). One hundred microlitres of 1 µg/ml anti-PrP monoclonal antibody (ICSM 35, 37 or 18) in PBS were added to each well and incubated for 1 h at 37°C with constant agitation. After aspiration of the antibody, wells were washed four times (5 min at 37°C) with 200 µl PBS containing 0.2% (v/v) Tween-20, followed by incubation with 100 µl of goat anti-mouse IgG horseradish peroxidase conjugate (Sigma, Poole, UK) (1 : 2000 dilution in PBS) for 30 min at 37°C with constant agitation. Wells were washed in 90 s with 3 x 200 µl PBS containing 0.2% v/v Tween-20 and developed with 100 µl per well 3,3',5,5' tetramethylbenzidine before quenching with 50 µl 3 M sulphuric acid and determination of absorbance at 450 nm.
Immunohistochemistry
Brain were fixed in 10% buffered formalin, immersed in 98% formic acid for 1 h, formalin post-fixed and paraffin wax-embedded. Serial sections of 4 µm nominal thickness were pre-treated with Tris-citrate EDTA buffer for antigen retrieval. PrP deposition was visualized using ICSM 35 or ICSM 18 as the primary antibody (2 µg/ml final concentration), on a Ventana automated immunohistochemical staining machine (Ventana Medical Systems Inc., Tucson, Arizona) using proprietary secondary detection reagents (Ventana Medical Systems Inc.) before development with 3'3 diaminobenzedine tetrachloride as the chromogen. Sections were counterstained with haematoxylin. Bright field photographs were taken on an ImageView digital camera (www.soft-imaging.de) and composed with Adobe Photoshop. For double-labelling experiments, Alexa 488-labelled anti-mouse IgG1 antibody (Molecular Probes A-21121, Invitrogen Ltd, Paisley, UK) and Alexa 546-labelled anti-mouse IgG2b antibody (Molecular Probes A-21143) were used to detect ICSM 18 and ICSM 35, respectively. Fluorescence confocal microscopy was performed with a confocal laser scanning microscope (Zeiss LSM510 META, mounted on Zeiss Axiovert 200M) using a Plan-Neofluar 40x/1.30 oil DIC or Plan-Apochromat 20x/0.75 objective. Image analysis was performed using Zeiss LSM5 software.
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Results |
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Antibody ICSM 35 detects wild-type but not P102L human PrP
Anti-PrP monoclonal antibody ICSM 35 is an IgG2b mouse immunoglobulin that binds to human PrP (Asante et al., 2002
; Wadsworth et al., 2004) (Fig. 1) via an epitope spanning amino acid residues 93–105 (Khalili-Shirazi et al., 2005b). This sequence is highly conserved in mammals (Collinge and Palmer, 1997; Wopfner et al., 1999) and a single coding change at residue 97 of human PrP in the corresponding sequence of mouse or bovine PrP does not appear to influence recognition by ICSM 35 (Asante et al., 2002; Lloyd et al., 2004; Wadsworth et al., 2004).
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However, during the course of characterizing transgenic models of inherited prion disease, we discovered that ICSM 35 failed to recognize human PrP 102L on immunoblots of brain tissue from Prnpo/o transgenic mice expressing human PrP 102L [designated Tg(HuPrP P102L 129M+/+ Prnpo/o)-27 mice] (Fig. 1A). In contrast, human PrP 102L was efficiently detected by anti-PrP monoclonal antibodies 3F4 (Kascsak et al., 1987
) or ICSM 18 (Khalili-Shirazi et al., 2005b) (Fig. 1A), and both wild-type human PrP and human PrP E200K from Tg(HuPrP129M+/+ Prnpo/o)-35 (Asante et al., 2002) and Tg(HuPrP E200K 129M+/+ Prnpo/o)-49 transgenic mice, respectively, were efficiently recognized by all three antibodies (Fig. 1A). Further studies showed that ICSM 35 similarly failed to recognize 3F4-reactive truncated fragments of PrPSc derived from human PrP 102L in Tg27 mice inoculated with human prions from a patient with iatrogenic CJD (Fig. 1B). Indeed, exhaustive overexposure of immunoblots showed no residual non-specific interaction of ICSM 35 with any form of human PrP P102L (data not shown).
The failure of ICSM 35 to recognize human PrP 102L on immunoblots was further investigated by the analysis of transgenic mouse brain by ELISA. Again, ICSM 35 reacted efficiently with human PrP 200K but not human PrP 102L (Fig. 2A). In contrast, anti-PrP monoclonal antibody ICSM 37, whose epitope also lies within residues 93–105 of human PrP (Khalili-Shirazi et al., 2005a), reacted efficiently with both forms of mutant PrP as determined by both ELISA (Fig. 2A) and immunoblotting (Fig. 1). ICSM 35 and ICSM 37 thus appear to bind to distinct epitopes within residues 93–105 of human PrP, with the P102L mutation specifically perturbing the binding of ICSM 35. To investigate this directly, we synthesized peptides corresponding to residues 92–111 of human PrP with either proline or leucine at residue 102. In ELISA, ICSM 37 bound efficiently to both forms of the peptide, whereas ICSM 35 only recognized the peptide with wild-type PrP sequence (Fig. 2B). Collectively, these findings show that the presence of leucine at residue 102 of human PrP effectively abolishes high-affinity recognition by ICSM 35. At present, we have not determined whether proline-102 is a major determinant of the binding affinity of ICSM 35 or whether leucine-102 has a considerable negative affect on the strength of the antibody–antigen interaction. Importantly, however, the inability of ICSM 35 to recognize human PrP 102L provides an opportunity to directly investigate the involvement of wild-type PrP in human P102L inherited prion disease.
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Immunoblot detection of protease-resistant wild-type PrP in P102L inherited prion disease
Previously we have reported the analysis of brain samples (
1 g frontal cortex) from seven P102L inherited prion disease patients by immunoblotting using anti-PrP monoclonal antibody 3F4 (Hill et al., 2006). Analysis of multiple homogenates prepared from each specimen identified varying concentrations of proteinase K-resistant PrP fragments of 21–30 and 8 kDa in four cases, whereas only the 8 kDa PrP fragment was detected in the other three cases (Hill et al., 2006). These data are consistent with those of other researchers (Parchi et al., 1998; Piccardo et al., 1998), and typify both the spectrum and spatial variability of proteinase K-resistant PrP species seen in P102L inherited prion disease. We have now re-examined brain homogenates from three of the patients that generate both 21–30 and 8 kDa protease-resistant PrP fragments, and have used antibody 3F4 to detect total PrP and antibody ICSM 35 to detect wild-type PrP only. The clinical and neuropathological features of these patients are summarized in Table 1. Immunoblotting with antibody 3F4 showed that P102L Cases 1 and 3 propagated PrPSc with a fragment size corresponding to type 1 PrPSc seen in sporadic CJD, while Case 2 propagated PrPSc with a fragment size corresponding to type 2 PrPSc seen in sporadic CJD (Hill et al., 2006).
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In the absence of proteinase K digestion, we used normal human brain and brain from a patient with E200K inherited prion disease (Hill et al., 2006
) to calibrate PrP immunoblots to allow direct comparison of 3F4 and ICSM 35 immunostaining in tissue from P102L patients. We observed that wild-type PrP labelled by ICSM 35 in P102L patients represented 30–40% of total PrP labelled by 3F4 (Fig. 3). ICSM 35 detected only full-length di-, mono- and non-glycosylated wild-type PrP bands, whereas 3F4 also labelled higher and lower molecular mass PrP species (Fig. 3). Although an 8 kDa PrP species can sometimes be detected in non-proteinase K-digested P102L brain homogenate (Parchi et al., 1998; Piccardo et al., 1998; Zou et al., 2003), PrP fragments of this molecular mass were not detected in our samples with either 3F4 or ICSM 35 in the absence of proteinase K digestion.
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Next we analysed proteinase K-digested brain homogenate again using tissue from an E200K patient to calibrate immunoblots to facilitate direct comparison of PrP immunostaining by 3F4 or ICSM 35 in tissue from P102L patients. Remarkably, all samples from P102L patients with 3F4-reactive proteinase K-resistant fragments of
21–30 kDa also had similar molecular mass PrP fragments labelled with ICSM 35, indicating that they were generated from wild-type PrP (Fig. 4).
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In sharp contrast, however,
8 kDa protease-resistant PrP fragments detected with 3F4 were not detected by ICSM 35 (Fig. 4, and data not shown). Reported amino acid sequencing (Parchi et al., 1998) shows that the 8 kDa protease-resistant PrP fragment in its shortest form comprises PrP residues 82–147 (that encompasses the ICSM 35 epitope); thus the lack of recognition by ICSM 35 suggests an origin solely from mutant PrPSc.
The patterns of labelling of 21–30 kDa species generated from wild-type PrP were equivalent in tissue from all three P102L patients examined; however, the relative concentration varied between and within cases (Fig. 4, and data not shown). In two of the patients, wild-type PrP contributed up to 1% of total protease-resistant PrP fragments, whereas in the other patient (Case 2, Fig. 4) levels of up to 10% were observed. Although direct comparison of the relative size of 21–30 kDa PrP fragments labelled with 3F4 or ICSM 35 was precluded by differences in signal strength, glycoform determination was possible in P102L Case 2 (Fig. 5). Proteolytic fragments generated from wild-type PrP labelled with ICSM 35 showed a marked predominance of non-glycosylated PrP, whereas those labelled with 3F4 (that originate principally from mutant PrP), showed a clear predominance of di-glycosylated PrP (Fig. 5). In sharp contrast, the glycoform ratios of 21–30 kDa PrP fragments labelled with 3F4 and ICSM 35 in the E200K case were indistinguishable (Fig. 5). These findings show that protease-resistant PrP conformers generated from wild-type PrP or mutant PrP in P102L inherited prion disease have distinct physicochemical properties. Indeed, 21–30 kDa proteolytic fragments generated from PrP 102L and wild-type PrP in P102L inherited prion disease are not only distinct from each other but are also distinct from those generated from wild-type PrP in sporadic or acquired CJD (Hill et al., 2003, 2006).
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In a further series of experiments, we examined the detergent solubility of abnormal isoforms of wild-type PrP and PrP 102L. Brain homogenates from P102L Case 2 or normal human brain were treated with 2% sodium lauroylsarcosine and then centrifuged to generate detergent-soluble supernatants and detergent-insoluble pellets. Immunoblot analysis of the supernatants (data not shown) or the re-suspended pellets (Fig. 6), before or after proteinase K digestion, showed that protease-resistant isoforms of both PrP 102L or wild-type PrP from P102L brain were efficiently recovered (>90%) into the detergent-insoluble pellet. In contrast, PrPC from normal human brain was not recovered into the pellet (Fig. 6). Immunoblotting of P102L brain samples, using 3F4 to detect total PrP or ICSM 35 to detect wild-type PrP only, showed that a similar relative proportion (
10%) of total PrP or wild-type PrP was insoluble and recovered in the pellet (Fig. 6). However, a difference was observed after proteinase K digestion of insoluble PrP. While only a slight reduction in the signal intensity of total PrP was observed with antibody 3F4, a much greater reduction in signal intensity of wild-type PrP was observed with ICSM 35 (Fig. 6). Collectively, these findings suggest either that abnormal isoforms of PrP 102L or wild-type PrP differ in their intrinsic resistance to proteolysis or that additional detergent-insoluble, protease-sensitive isoforms of wild-type PrP may exist in P102L brain. Further studies will now be required to distinguish between these possibilities. However, in this context, it should be noted that insoluble protease-sensitive PrP has been previously reported in P102L inherited prion disease (Parchi et al., 1998) and that an insoluble protease-sensitive isoform of wild-type PrP has been identified in E200K inherited prion disease (Gabizon et al., 1996). In addition, aggregated, protease-sensitive isoforms of mouse PrP 101L have been identified in transgenic models of GSS (Tremblay et al., 2004; Nazor et al., 2005).
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Immunohistochemical detection of wild-type PrP deposition in P102L inherited prion disease
Initially, we performed immunoblot analysis of P102L inherited prion disease brain samples blind to the clinical phenotype of the patient. However, after unblinding the samples we realized that Cases 2 and 3 presented with a rapidly progressive dementia consistent with a CJD phenotype rather than the GSS phenotype seen in Case 1 (Table 1). P102L Case 2, who consistently showed the highest levels of protease-resistant wild-type PrP (Fig. 4), is the grandniece of the original Gerstmann patient (Gerstmann et al., 1936
), and extensive neuropathology and PrP immunohistochemistry has been reported previously by Hainfellner et al. (1995). This atypical case [designated Case no. 19 in this study (Hainfellner et al., 1995)], showed histological signs of severe cortical damage and a severe burden of amyloid deposits throughout the brain, and was distinguished from historic ataxic patients in this pedigree by prominent fine granular synaptic PrP deposition in the middle and deep layers of the cerebral cortex accompanied by severe neuronal loss (Hainfellner et al., 1995).
On the basis of preliminary experiments, showing that ICSM 18-reactive deposits of human PrP 102L in prion-inoculated Tg27 mouse brain were not reactive with ICSM 35 (data not shown), we performed PrP immunohistochemical analysis on tissue from human P102L inherited prion disease patients using antibody ICSM 18 to detect total PrP and antibody ICSM 35 to detect only wild-type PrP. We examined the atypical Case no. 19 from the Austrian pedigree together with a typical ataxic GSS case from the same pedigree [designated Case no. 11 (Hainfellner et al., 1995)] and three historic ataxic P102L GSS cases from the UK family (Adam et al., 1982) as described by Hsiao et al. [Cases III 22, IV 2, IV 12 (Hsiao et al., 1989); Case IV 12, corresponds to Case 1 in Fig. 4]. The clinical and neuropathological features of these patients are summarized in Table 1.
Conventional immunohistochemistry on available fixed sections of frontal cortex showed a spectrum of involvement of wild-type PrP. In two of the historic UK ataxic GSS cases [III 22 and IV 2 (Hsiao et al., 1989)], PrP plaques were labelled with ICSM 18 only (Fig. 7, and data not shown) and we saw no specific labelling of abnormal PrP deposition by ICSM 35 on any section (Fig. 7, and data not shown). However, in sections of frontal cortex from the other historic UK ataxic case [Case IV 12 (Hsiao et al., 1989); Case 1, Fig. 4], and both the typical and atypical Austrian GSS phenotypes [Cases no. 11 and no. 19, respectively (Hainfellner et al., 1995)], we observed some PrP plaques labelled with either ICSM 18 or ICSM 35 indicative of the presence of wild-type PrP (Fig. 7, and data not shown). The involvement of wild-type PrP in plaques was also demonstrated by fluorescence confocal microscopy double-labelling experiments using different secondary antibody fluorophores to detect ICSM 18 (IgG1) or ICSM 35 (IgG2b), respectively. Conditions for double-labelling PrP deposits by this method were optimized using tissue from human sporadic CJD patients (Fig. 8, and data not shown). In accordance with findings from conventional immunohistochemistry, in two of the UK classic ataxic GSS cases [III 22 and IV 2 (Hsiao et al., 1989)], PrP deposits were labelled by ICSM 18 only (Fig. 8, and data not shown), indicative of involvement of only mutant PrP. However, in the atypical Austrian GSS case [no. 19 (Hainfellner et al., 1995)], we observed prominent double labelling of PrP plaque deposits (Fig. 8), thereby establishing the presence of wild-type PrP in these plaques. At present we do not know whether plaques labelled with ICSM 35 are entirely composed of wild-type PrP.
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While all of the cases examined showed prominent PrP plaques, marked variability was observed in synaptic PrP deposition in these cases. Cases III 22, IV 2 and no. 11 showed no synaptic deposition of PrP with either ICSM 18 or ICSM 35 (data not shown). Case IV 12 showed synaptic PrP deposition with ICSM 18 only (data not shown) whereas the atypical Case no. 19 was distinguished from all the classic ataxic cases by prominent fine synaptic deposition of PrP labelled with both ICSM 18 and ICSM 35 (Fig. 7). Although immunoreactivity was stronger with ICSM 18 than ICSM 35 these data clearly indicate the involvement of wild-type PrP.
Collectively, our findings show that wild-type, PrP can be associated with both amyloid plaques and synaptic PrP deposits in P102L inherited prion disease. Previous studies of P102L GSS have shown that abnormal conformers of PrP 102L that generate protease-resistant fragments of either 8 or 21–30 kDa differ in their association with either amyloid plaques or synaptic PrP deposits, respectively (Parchi et al., 1998; Piccardo et al., 1998). Unfortunately, however, owing to the limited quantities of P102L brain we had available, we were unable to study the relative distribution of wild-type PrP between amyloid plaques or synaptic deposits. Further prospective studies will now be required to address this and to investigate whether wild-type and mutant PrP co-localize within amyloid plaques.
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Discussion |
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Within the framework of the protein-only hypothesis of prion propagation, clinical heterogeneity in P102L inherited prion disease has been attributed to temporal and spatial variance in the propagation of distinct abnormal PrP isoforms (Parchi et al., 1998
; Piccardo et al., 1998; Hill et al., 2006). To date, however, all abnormal PrP isoforms have been thought to originate from mutant PrP 102L, (Parchi et al., 1998; Muramoto et al., 2000). Here, using a monoclonal antibody that recognizes wild-type but not mutant PrP 102 L we reveal a spectrum of involvement of protease-resistant wild-type PrP in P102L inherited prion disease. The high sensitivity and specificity of our methods, coupled with the variable abundance and irregular distribution of wild-type PrPSc in the brain (both within and between P102L inherited prion disease patients), may explain why our findings contrast with those of previous work (Parchi et al., 1998; Muramoto et al., 2000). Our findings in P102L inherited prion disease complement previous studies of inherited prion diseases associated with other PRNP mutations that also demonstrated the presence of abnormal isoforms of wild-type PrP (Gabizon et al., 1996; Chen et al., 1997; Silvestrini et al., 1997). Collectively, these data argue that propagation of pathological isoforms of wild-type PrP may make a significant contribution to phenotypic variability in inherited prion disease. These data also have significant implications for interpreting the transmission properties of inherited prion disease isolates in both conventional and transgenic mice.
At the molecular level, we show that three isoforms of protease-resistant PrP with divergent physicochemical properties can be propagated in P102L inherited prion disease brain. Two distinct abnormal conformers derived from PrP 102L generate protease-resistant fragments of either 21–30 or 8 kDa, while abnormal conformers of wild-type PrP appear to generate proteolytic fragments of only 21–30 kDa. Glycoform ratios of 21–30 kDa proteolytic fragments generated from PrP 102L and wild-type PrP are not only distinct from each other but are also distinct from those generated from wild-type PrP in sporadic or acquired CJD (Hill et al., 2003, 2006). Differences in neuropathological targeting of these distinct disease-related PrP species, together with differences in their abundance and potential neurotoxicity, provide a molecular mechanism for generation of multiple phenotypes in P102L inherited prion disease. That wild-type PrP can contribute up to 10% of total protease-resistant PrP species in P102L inherited prion disease clearly suggests that this may have significant impact on disease phenotype. However, while it is intriguing that the highest levels of protease-resistant wild-type PrP were seen in a highly atypical P102L patient, further prospective studies with a greater number of cases will be required in order to establish a clear correlation between disease phenotype and the regional distribution of protease-resistant wild-type PrP in the brain. In these studies it will be important to establish the relative contribution of protease-resistant wild-type PrP to amyloid plaques or synaptic PrP deposits and to also investigate the occurrence of abnormal, protease-sensitive isoforms of wild-type PrP.
The distinct nature of disease-related isoforms of wild-type and mutant PrP seen in P102L inherited prion disease provide a critical insight into the role of the pathogenic point mutation in specifying the physicochemical properties of disease-related PrP isoforms that can be understood within a conformational selection model of prion propagation (Collinge, 1999, 2001; Hill and Collinge, 2003), a model also recently supported by work with yeast prions (Tanaka et al., 2005). This model predicts that prion propagation involving distinct PrP sequences will depend upon the degree of overlap between thermodynamically permissible disease-related conformations for each PrP sequence. Where no favoured conformations are shared by distinct PrP sequences, a substantial transmission barrier to propagation would be expected and indeed propagation would necessitate a change in PrPSc conformation and prion strain type. While considerable evidence supports a critical role for PRNP codon 129 polymorphism in dictating the thermodynamic permissibility of human PrPSc conformation (Collinge, 2001; Hill and Collinge, 2003; Hill et al., 2003; Wadsworth et al., 2004), less evidence is available regarding the precise role of pathogenic PRNP point mutations. Our finding that protease-resistant isoforms of wild-type PrP M129 or mutant PrP 102L M129 are distinct from each other and from pathological isoforms of wild-type PrP M129 seen in sporadic or acquired CJD (Hill et al., 2003, 2006) clearly suggests that the PrP 102L point mutation can powerfully dictate thermodynamic preferences for disease-related PrP assembly states that cannot be adopted by wild-type PrP.
The aetiology of sporadic CJD remains unclear although its remarkably uniform worldwide incidence and apparently random distribution suggests involvement of a stochastic process such as somatic PRNP mutation (Brown et al., 1987; Collinge, 2001). In this context, an inherited prion disease patient in whom prion propagation, although initially triggered by the PRNP mutation, principally involves PrP expressed from the wild-type allele could be considered a model for sporadic CJD. However, since the abnormal isoform of wild-type PrP seen in P102L inherited prion disease is distinct from abnormal isoforms of wild-type PrP seen in sporadic CJD, these data suggest that somatic PRNP mutations underlying sporadic CJD are more likely to be octapeptide repeat insertion mutations, as the physicochemical properties of PrPSc seen in these cases closely resemble those seen in sporadic CJD (Hill et al., 2006).
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Acknowledgements |
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We are grateful to R. Young for preparation of figures and M. Poulter for helpful discussion. We especially thank all patients and their families for generously consenting to use of human tissues in this research, and the UK neuropathologists who have kindly helped in providing these tissues. This work was funded by the UK Medical Research Council and European Commission.
Conflict of interest statement. J.C. is a director, and J.C. and J.D.F.W. are shareholders and consultants of D-Gen Limited, an academic spin-out company working in the field of prion disease diagnosis, decontamination and therapeutics. D-Gen markets the ICSM antibodies used in this study.
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