Prion protein isoforms in Gerstmann-Sträussler-Scheinker disease
Pedro Piccardo, Fabrizio Tagliavini, Orso Bugiani, Stephen R. Dlouhy and Bernardino Ghetti
Department of Pathology and Laboratory Medicine and Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA (PP, SRD, BG), Istituto Neurologico Carlo Besta, Milano, Italy (FT, OB).
Key words: Gerstmann-Sträussler-Scheinker disease (GSS), immunoblot, prion protein (PrP), prion protein gene (PRNP).
Abstract
Gerstmann-Sträussler-Scheinker disease, an autosomal dominant neurodegenerative disorder, is characterized clinically by ataxia and dementia and pathologically by the accumulation of prion protein amyloid. Gerstmann-Sträussler-Scheinker disease is caused by mutations in the coding region of the prion protein gene. Phenotypic variability in clinical and pathologic features has been observed in the described variants. This variability may be due to molecular mechanisms not completely understood. The aim of this study was to analyze prion protein isoforms in Gerstmann-Sträussler-Scheinker disease. In patients with spongiform changes we observed three major proteinase K resistant prion protein isoforms in the 21-30 kDa range, similar to those seen in Creutzfeldt Jakob disease, and variable amounts of distinct low molecular weight fragments of ca. 8-15 kDa. The 21-30 kDa isoforms were not prominent in Gerstmann-Sträussler-Scheinker variants without spongiform changes. In patients with no spongiform changes and severe neurofibrillary pathology, proteinase K resistant PrP species of 27-29, 18-19, and 8 kDa were consistently present. These studies suggest that Gerstmann-Sträussler-Scheinker disease is characterized by the presence of prion protein isoforms that can be partially cleaved to low molecular weight proteinase K resistant prion protein peptides, and that spongiform changes are associated with the presence of specific prion protein species.
Introduction
Gerstmann-Sträussler-Scheinker (GSS) disease, an autosomal dominant neurodegenerative condition, is characterized pathologically by the deposition of prion protein (PrP) amyloid in the brain (1). GSS disease is caused by mutations in the prion protein gene (PRNP) and the most common form is caused by a proline (P) to leucine (L) substitution at residue 102 (P102L). Other forms include the P105L, A117V, F198S, D202N, Q212P and Q217R variants.
The onset of GSS disease is generally insidious and the clinical evolution is that of a slowly progressing neurological disorder with an age at onset between the third and seventh decades. Presenting symptoms usually include cerebellar and pyramidal signs followed by progressive cognitive deterioration, the course of the disease is generally protracted over several years. However, in some patients with the P102L mutation, the disease may evolve rapidly with a clinical evolution indistinguishable from that of Creutzfeldt-Jakob disease (CJD). In these cases, electroencephalography (EEG) reveals pseudoperiodic synchronous discharges (PSDs) similar to those found in CJD. PSDs are not present in other forms of GSS disease. Thus, GSS disease variants may be heterogeneous for age of onset, symptoms accompanying ataxia, duration and EEG pattern. Clinical variability is not uncommon in patients with the same genetic mutation and even in patients within the same family. From the neuropathologic point of view, in addition to the amyloid deposits, GSS patients may present other features such as spongiform changes or neurofibrillary pathology. While spongiform changes are often found in patients with the P102L mutation and clinical features similar to those of CJD, lack of spongiform changes is characteristic of some patients with the P102L mutation. Neurofibrillary pathology is most prominent in patients with the F198S and Q217R mutations.
In addition to differences due to PRNP mutations, a variability in PrP degradation may underlie the clinical and pathologic variability of GSS disease. We hypothesize that the phenotypic variability among GSS disease variants may be related to the presence of distinct PrP species; therefore, the aim of this study was to determine the characteristics of PrP in various forms of GSS disease.
Materials and Methods
Patients
At total of twenty-four patients were studied. Nine patients with P102L; five with A117V; seven with F198S; one with D202N; one with Q212P and one with Q217R were analyzed. Seven subjects with the P102L mutations had spongiform changes, two subjects with the P102L mutation had no spongiform changes, ten subjects with the A117V [1], F198S [7], D202N [1] and Q217R [1] mutations had no spongiform changes but conspicuous neurofibrillary pathology, five subjects with the A117V [4] and Q212P [1] had neither spongiform changes nor neurofibrillary pathology. The clinical, pathologic and genetic information on these patients has been reported in detail (2-5).
Biochemical Analysis of PrP.
Brain tissue was homogenized in 9 volumes (10% w/v) of lysis buffer (100mM NaCl, 10mM EDTA, 0.5% Nonidet P-40, 0.5% Na deoxycholate in 10mM Tris-HCl [pH 7.4]) treated with proteinase K (PK) and fractionated in SDS-PAGE minigels as previously described (3,4). Non-PK-treated samples were also analyzed. Immunoblots were probed with monoclonal antibody 3F4 (Mab 3F4) recognizing an epitope corresponding to residues 109-112 of human PrP (6) or antibody AS 6800 raised to synthetic peptides representing human PrP residues 89-104 (provided by Dr. H. Diringer). Blots were visualized using an ECL chemiluminescence kit (Amersham, Arlington-Heights, IL). For selected samples, N-lauryl sarcosine sodium salt (Sarkosyl) insoluble and partially PK-resistant PrP was purified, as previously described (7,8).
Results
Biochemical studies of cases with spongiform degeneration.
GSS P102L
PrPres isoforms in the ca 21-30 kDa range were observed. They showed an electrophoretic mobility like that of type 1 PrPres (9) from patients with CJD. In addition, all cases showed a low molecular weight band of ca.8 kDa. This PrPres fragment reacts with antibody (AS6800) directed to the midregion (residues 89-104) of PrP, in addition to reacting with Mab 3F4. The high and low molecular weight species can be seen in partially purified PrP preparations.
Biochemical studies of cases without spongiform degeneration
GSS P102L
The 21-30 kDa isoforms were not prominent. PrPres was seen (upon longer exposure) as a smear in the ca 15-30 kDa range and distinct bands of lower molecular weight (ca 8-15 kDa) were observed. As described above, the low molecular weight fragments reacted with antibody AS-6800 and Mab 3F4. This supports the concept that these peptides represent internal fragments of PrP.
In cases with and without spongiform degeneration small amounts of low molecular weight PrPres can be seen in some samples that have not been treated with PK. The amount of this fragment is increased substantially by PK treatment. Thus, quantitatively, the majority of low molecular weight PrP fragment(s) that we observe appears to derive by in vitro PK cleavage of preexisting, higher molecular weight intermediates, rather than from postmortem degradation. In addition, the high and low molecular weight PrP species can both be seen in partially purified PrP preparations.
GSS A117V
One patient presented neurofibrillary tangles (NFTs), while four patients did not. The case with NFTs presented PrPres bands of ca. 27-29, 18-19 and 8 kDa. This pattern differed from that seen in patients without NFTs. The latter contained major bands of ca. 14 and 7 kDa.
GSS F198S
Prominent bands of ca. 27-29, 18-19, and 8 kDa were seen. PK-resistant PrP fragments of similar electrophoretic mobility were seen in brain areas with small (caudate nucleus and putamen), moderate (frontal cortex), and severe (cerebellum) amounts of thioflavin S fluorescent deposits.
GSS D202N
Prominent bands of ca. 27-29, 18-19, and 8 kDa were seen and were similar to those seen in GSS A117V with NFTs as well as in GSS F198S and GSS Q217R.
GSS Q212P
Prominent bands of ca. 18-20 and 10 kDa were seen, in addition to a poorly resolved smear in the 25-35 kDa range.
GSS Q217R
Major bands of ca. 27-29, 18-19 and 8 kDa were seen, a pattern similar to that seen in patients with PrP A117V and NFTs, F198S and Q217R.
Discussion
GSS patients can be neuropathologically divided into two categories, based on the presence or absence of spongiform degeneration. For this study, the group of patients with spongiform degeneration comprised seven subjects, all with the P102L mutation and the group without spongiform degeneration comprised 17 subjects, two with the P102L and 15 with the remaining, A117V, F198S, D202N, Q212P and Q217R, variants.
The data obtained show heterogeneity of PrPres isoforms in brain extracts of patients with GSS. PrPres peptides ranged from ca. 8 kDa to over 30 kDa and differed in their relative distribution among patients. In GSS disease with the P102L mutation and severe spongiform changes, PrPres peptides with an electrophoretic mobility of ca. 21-30 kDa were the most abundant, while smaller isoforms (ca. 8-15 kDa) were less abundant and varied in amount among patients. The 21-30 kDa isoforms were identical in mobility to that of type 1 PrPres described in CJD (8). In patients without spongiform changes and with the P102L, A117V, F198S, D202N, Q212P and Q217R mutations, distinct 21-30 kDa isoforms were not present.
A distinctive biochemical feature of GSS is the presence of low molecular weight of PrP species (ca. 8-15 kDa). These peptides correspond to amino- and carboxy-truncated fragments. Their presence can be detected in all GSS variants and in all anatomical regions analyzed regardless of the presence or absence of spongiform degeneration. Similar-sized fragments were also found in GSS tissue prior to PK digestion, suggesting that truncated PrP is generated in vivo and that a significant amount can be produced in vitro by hydrolysis of preexisting intermediates. The presence of these fragments may be indicative of the existence of specific metabolic pathways associated with GSS amyloidosis. This concept is supported by the finding of truncated PrP peptides (ca. 7-11 kDa) in isolated amyloid cores of the GSS A117V, F198S, and Q217R variants (10-12).
PrP may undergo different conformational changes or processing, or both, in patients with the P102L mutation, even in distinct brain areas of the same patient. In patients with the F198S mutation, PrP species of 27-29, 18-19, and 8 kDa are present in all anatomical regions. It is also noteworthy that the F198S and Q217R variants have similarities in PrP patterns and are both neuropathologically characterized by presence of severe neurofibrillary pathology.
In conclusion, these studies indicate that presence of spongiform changes in GSS P102L is associated with a significant amount of high molecular weight (21-30 kDa) detergent insoluble PrPres similar to that seen in CJD. The presence of low molecular weight peptides (i.e., ca. 8-15 kDa) consistently observed in GSS, in the presence or absence of spongiform degeneration, suggests that in this disease, PrP intermediates can be partially cleaved to smaller protease resistant peptides.
Acknowledgments. Supported in part by PHS grants NS 29822 and AG 10133, and by the Project Development Program, Research and Sponsored Programs, Indiana University at Indianapolis.
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