Allelic origin of protease-sensitive and protease-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease with the P102L mutation

Gerstmann-Str?ussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP(Sc), consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrP(Sc) isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrP(Sc) has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrP(Sc) allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrP(Sc) molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrP(Sc) quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.     

A synthetic peptide initiates Gerstmann-Sträussler-Scheinker (GSS) disease in transgenic mice

The molecular basis of the infectious, inherited and sporadic forms of prion diseases is best explained by a conformationally dimorphic protein that can exist in distinct normal and disease-causing isoforms. We identified a 55-residue peptide of a mutant prion protein that can be refolded into at least two distinct conformations. When inoculated intracerebrally into the appropriate transgenic mouse host, 20 of 20 mice receiving the beta-form of this peptide developed signs of central nervous system dysfunction at approximately 360 days, with neurohistologic changes that are pathognomonic of Gerstmann-Str?ussler-Scheinker disease. By contrast, eight of eight mice receiving a non-beta-form of the peptide failed to develop any neuropathologic changes more than 600 days after the peptide injections. We conclude that a chemically synthesized peptide refolded into the appropriate conformation can accelerate or possibly initiate prion disease.     

Specific amyloid-β42 deposition in the brain of a Gerstmann-Sträussler-Scheinker disease patient with a P105L mutation on the prion protein gene

ABSTRACT

Although colocalization of amyloid β (Aβ) with prion protein (PrP) in the kuru plaque has previously been observed in the brain of prion diseases patients, the participating Aβ species has not been identified. Here, we present an immunohistochemical assessment of the brain and spinal cord of a 69-year-old Japanese female patient with Gerstmann-Sträussler-Scheinker disease with a P105L mutation on the PRNP gene (GSS-P105L). Immunohistochemical assessment of serial brain sections was performed using anti-PrP and -Aβ antibodies in the hippocampus, frontal and occipital lobes. She died 69 years after a 21-year clinical course. Immunohistochemistorical examination revealed that ~50% of the kuru plaques in the cerebrum were colocalized with Aβ, and Aβ42 was predominantly observed to be colocalized with PrP-plaques. The Aβ deposition patterns were unique, and distinct from diffuse plaques observed in the normal aging brain or Alzheimer’s disease brain. The spinal cord exhibited degeneration in the lateral corticospinal tract, posterior horn, and fasciculus gracilis. We have demonstrated for the first time that Aβ42, rather than Aβ40, is the main Aβ component associated with PrP-plaques, and also the degeneration of the fasciculus gracilis in the spinal cord in GSS-P105L, which could be associated with specific clinical features of GSS-P105L.     

Gerstmann-Sträussler-Scheinker disease with P102L-V129 mutation: a case with psychiatric manifestations at onset

Gerstmann-Str?ussler-Scheinker disease (GSS) is an adult onset, rare, genetically determined autosomal dominant prion disease. Clinically, it is characterized predominantly by slowly progressive spino-cerebellar dysfunction with ataxia, absent reflexes in the legs and cognitive impairment. Onset is usually in the fifth decade and in the early phase, ataxia is predominant. Mutations in the prion protein gene (PRNP) had been identified and the most important of these is at codon 129. A genotype-phenotype relationship with genetic polymorphism at residue 129 between methionine and valine has been supposed. We describe a patient with GSS and P102L-V129 mutation in which the onset with prominent psychiatric features characterized by apathy and depression and not with cerebellar sign and the clinical course with seizures, nor observed in P102L-V129 cases, allow us to confirm observations that the GSS caused by the 102 mutation is influenced by the codon 129 polymorphism with a specific genotype-phenotype influence, but probably other additional factors might be considered as background for phenotypic variability.     

Allelic Origin of Protease-Sensitive and Protease-Resistant Prion Protein Isoforms in Gerstmann-Str?ussler-Scheinker Disease with the P102L Mutation

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrP^Sc, consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrP^Sc isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrP^Sc has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrP^Sc allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrP^Sc molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrP^Sc quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.     

Aggregation/fibrillogenesis of recombinant human prion protein and Gerstmann-Sträussler-Scheinker disease peptides in the presence of metal ions

In this study we investigated the role of Cu(2+), Mn(2+), Zn(2+), and Al(3+) in inducing defective conformational rearrangements of the recombinant human prion protein (hPrP), which trigger aggregation and fibrillogenesis. The research was extended to the fragment of hPrP spanning residues 82-146, which was identified as a major component of the amyloid deposits in the brain of patients affected by Gerstmann-Str?ussler-Scheinker (GSS) disease. Variants of the 82-146 wild-type subunit [PrP-(82-146)(wt)] were also examined, including entirely, [PrP-(82-146)(scr)], and partially scrambled, [PrP-(82-146)(106)(-)(126scr)] and [PrP-(82-146)(127)(-)(146scr)], peptides. Al(3+) strongly stimulated the conversion of native hPrP into the altered conformation, and its potency in inducing aggregation was very high. Despite a lower rate and extent of prion protein conversion into altered isoforms, however, Zn(2+) was more efficient than Al(3+) in promoting organization of hPrP aggregates into well-structured, amyloid-like fibrillar filaments, whereas Mn(2+) delayed and Cu(2+) prevented the process. GSS peptides underwent the fibrillogenesis process much faster than the full-length protein. The intrinsic ability of PrP-(82-146)(wt) to form fibrillar aggregates was exalted in the presence of Zn(2+) and, to a lesser extent, of Al(3+), whereas Cu(2+) and Mn(2+) inhibited the conversion of the peptide into amyloid fibrils. Amino acid substitution in the neurotoxic core (sequence 106-126) of the 82-146 fragment reduced its amyloidogenic potential. In this case, the stimulatory effect of Zn(2+) was lower as compared to the wild-type peptide; on the contrary Al(3+) and Mn(2+) induced a higher propensity to fibrillation, which was ascribed to different binding modalities to GSS peptides. In all cases, alteration of the 127-146 sequence strongly inhibited the fibrillogenesis process, thus suggesting that integrity of the C-terminal region was essential both to confer amyloidogenic properties on GSS peptides and to activate the stimulatory potential of the metal ions.     

Conformational plasticity of the Gerstmann-Sträussler-Scheinker disease peptide as indicated by its multiple aggregation pathways

The existence of several prion strains and their capacity of overcoming species barriers seem to point to a high conformational adaptability of the prion protein. To investigate this structural plasticity, we studied here the aggregation pathways of the human prion peptide PrP82-146, a major component of the Gerstmann-Sträussler-Scheinker amyloid disease.By Fourier transform infrared (FT-IR) spectroscopy, electron microscopy, and atomic force microscopy (AFM), we monitored the time course of PrP82-146 fibril formation. After incubation at 37 °C, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands. At a critical oligomer concentration, the emergence of a new FT-IR band allowed to detect fibril formation. A different intermolecular β-sheet interaction of the peptides in oligomers and in fibrils is, therefore, detected by FT-IR spectroscopy, which, in addition, suggests a parallel orientation of the cross β-sheet structures of PrP82-146 fibrils. By AFM, a wide distribution of PrP82-146 oligomer volumes—the smallest ones containing from 5 to 30 peptides—was observed. Interestingly, the statistical analysis of AFM data enabled us to detect a quantization in the oligomer height values differing by steps of ∼ 0.5 nm that could reflect an orientation of oligomer β-strands parallel with the sample surface. Different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly.Thermal aggregation of PrP82-146 was also investigated by FT-IR spectroscopy, which indicated for these aggregates an intermolecular β-sheet interaction different from that observed for oligomers and fibrils. Unexpectedly, random aggregates, induced by solvent evaporation, were found to display a significant α-helical structure as well as several β-sheet components.All these results clearly point to a high plasticity of the PrP82-146 peptide, which was found to be capable of undergoing several aggregation pathways, with end products displaying different secondary structures and intermolecular interactions.     

How to assess the pathogenicity of mutations in Charcot-Marie-Tooth disease and other diseases?

Knowledge whether a certain DNA variant is a pathogenic mutation or a harmless polymorphism is a critical issue in medical genetics, in which results of a molecular analysis may serve as a basis for diagnosis and genetic counseling. Due to its genetic heterogeneity expressed at the levels of loci, genes and mutations, Charcot-Marie-Tooth (CMT) disease can serve as a model group of clinically homogenous diseases for studying the pathogenicity of mutations. Close to a 17p11.2-p12 duplication occurring in 70% of patients with the demyelinating form of CMT disease, numerous mutations have been identified in poorly characterized genes coding for proteins of an unknown function. Functional analyses, segregation analyses of large pedigrees, and inclusion of large control groups are required to assess the potential pathogenicity of CMT mutations. Hence, the pathogenicity of numerous CMT mutations remains unclear. Some variants detected in the CMT genes and originally described as pathogenic mutations have been shown to have a polymorphic character. In contrast, polymorphisms initially considered harmless were later reclassified as pathogenic mutations. However, the process of assessing the pathogenicity of mutations, as presented in this study for CMT disorders, is a more general issue concerning all disorders with a genetic background. Since the number of DNA variants is still growing, in the near future geneticists will increasingly have to cope with the problem of pathogenicity of identified genetic variants.     

Parkinson's disease-associated mutations in α-synuclein and UCH-L1 inhibit the unconventional secretion of UCH-L1

Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is an intracellular protein abundantly expressed in neurons, and a mutation in UCH-L1 has been identified in familial Parkinson’s disease. UCH-L1 has been detected in human cerebrospinal fluid, raising the possibility that UCH-L1 is secreted from neurons. In the present study, we showed that a portion of UCH-L1 is secreted from cultured cells. The secretion of D30K UCH-L1, which lacks ubiquitin binding activity, was decreased compared with that of wild-type UCH-L1, while the secretion of C90S UCH-L1, which lacks hydrolase activity, was not. Treatment with Brefeldin A, an inhibitor of vesicle transport from the endoplasmic reticulum to the Golgi, did not block the secretion of UCH-L1, indicating that UCH-L1 is secreted by an unconventional pathway. The UCH-L1 sequence from Leu-32 to Leu-39 is similar to the unconventional secretory signal sequence of engrailed 2, and substitution of the leucines within this region (L32S/L32A/L34S/L34A/L39S/L39A) reduced the secretion of UCH-L1. We found that the Parkinson’s disease-associated mutation I93M in UCH-L1 decreased the secretion of I93M UCH-L1. In addition, Parkinson’s disease-linked α-synuclein mutants reduced the secretion of endogenous UCH-L1. Our results indicate that the hydrolase activity is not necessary for the unconventional secretion of UCH-L1, and suggest that the ubiquitin binding activity and the sequence between Leu-32 and Leu-39 are involved in the secretion. Moreover, the secretion of UCH-L1 could be involved in the pathology of Parkinson’s disease.     

A 7-kDa prion protein (PrP) fragment, an integral component of the PrP region required for infectivity, is the major amyloid protein in Gerstmann-Sträussler-Scheinker disease A117V

Gerstmann-Str?ussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Str?ussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.     

Biochemical correlation of activity of the α-dystroglycan-modifying glycosyltransferase POMGnT1 with mutations in muscle-eye-brain disease

Congenital muscular dystrophies have a broad spectrum of genotypes and phenotypes and there is a need for a better biochemical understanding of this group of diseases in order to aid diagnosis and treatment. Several mutations resulting in these diseases cause reduced O-mannosyl glycosylation of glycoproteins, including α-dystroglycan. The enzyme POMGnT1 (protein-O-mannose N-acetylglucosaminyltransferase 1; EC 2.4.1.-) catalyses the transfer of N-acetylglucosamine to O-linked mannose of α-dystroglycan. In the present paper we describe the biochemical characterization of 14 clinical mutants of the glycosyltransferase POMGnT1, which have been linked to muscle-eye-brain disease or similar conditions. Truncated mutant variants of the human enzyme (recombinant POMGnT1) were expressed in Escherichia coli and screened for catalytic activity. We find that three mutants show some activity towards mannosylated peptide substrates mimicking α-dystroglycan; the residues affected by these mutants are predicted by homology modelling to be on the periphery of the POMGnT1 surface. Only in part does the location of a previously described mutated residue on the periphery of the protein structure correlate with a less severe disease mutant.     

A role for NOX NADPH oxidases in Alzheimer's disease and other types of dementia?

Because of population ageing, dementias are likely to become a major scourge of the 21st century. Causes of dementia include Alzheimer's disease, cerebrovascular disease, and lesser known entities such as frontotemporal dementia or dementia with Lewy bodies. Neuroinflammation is likely to play an important role in the pathogenesis of dementia by the killing of neurons through inflammatory mechanisms. Such a role of neuroinflammation is well documented for Alzheimer's disease, and it is likely to play a role in other types of dementia as well. Reactive oxygen species (ROS) play a key role in inflammatory tissue destruction. The phagocyte NADPH oxidase NOX2 is the best studied ROS-generating system. In the central nervous system, it is expressed in microglia and--to a lesser extent--in neurons. Indeed, there is emerging experimental evidence for a role of NOX2 in Alzheimer's and cerebrovascular disease. Recently, six novel ROS-generating NADPH oxidases with homology to NOX2 have been discovered. Several of them are also expressed in the central nervous system. In this article, we hypothesize a role of NOX-type NADPH oxidases in inflammatory neuronal loss. We review presently available evidence and suggest that NOX-type NADPH oxidases may become promising pharmacological targets for the treatment and prevention of dementia.     

Rare V203I mutation in the PRNP gene of a Chinese patient with Creutzfeldt–Jakob disease

Here, we report a Chinese case of Creutzfeldt–Jakob disease (CJD) with a rare mutation in the prion protein gene (PRNP) leading to an exchange of amino acid from valine (Val) to isoleucine (I) at codon 203 (V203I). The 80-y-old male presented with sudden memory loss, rapid loss of vocabulary, inattention and slow responses, accompanied by dizziness, blurred vision and ataxia. Two weeks after admission, he exhibited tremor, myoclonus and bilateral Babinski signs. At the end of the clinical course, he developed severe akinetic mutism. The cerebrospinal fluid (CSF) was positive for 14-3-3 protein. Increased bilateral signal intensity in the frontal and parietal lobes was seen on diffusion-weighted imaging (DWI); periodic activity was recorded on an electroencephalogram (EEG). There was no family history of similar symptoms. The total clinical course was approximately two months.     

Point mutations in the upstream region of the α-galactosidase A gene exon 6 in an atypical variant of Fabry disease

Summary Single point mutations in the upstream region of exon 6 of the -galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; ³⁰¹ArgGln (⁹⁰²GA) in a case that has already been published and ²⁷⁹GlnGlu (⁸³⁵CG) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; ³²⁸GlyArg (⁹⁸²GA) in the downstream region of exon 6 in one case and two combined mutations, ⁶⁶GluGln (¹⁹⁶GC)/¹¹²ArgCys (³³⁴CT), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the -galactosidase A gene.     

The food and parasites of fish in some deep basins of northern L. Päijänne

The composition of the fish stock, food and macroparasites were studied in eleven basins (22–100 m) of Lake Jyväsjärvi and North Päijänne in August–September 1976. The fishing was done by means of a series of nets (meshes 15, 21 and 35 mm) laid on the bottom overnight. No fish were found in the two northernmost basins owing to bad oxygen conditions caused by waste waters. Smelt and burbot were the most abundant fish in the catches in other basins but the vendace was rare. Relict crustaceans and some deep water copepods (e.g. Heterocope borealis) played an important role in the food of fish in all basins.Parasites spreading via relict crustaceans were abundant in smelt and burbot, but absent in vendace and ruffe. The most common parasite in smelt was Cystidicola farionis, in burbot Echinorhynchus borealis, in ruffe Triaenophorus nodulosus and in vendace the gill parasite Ergasilus sieboldi. Females of a relatively rare copepod Salmincola lotae were found in the oral cavity of five burbot (at depths of 50 and 100 m).     

A cluster of familial Creutzfeldt-Jakob disease mutations recapitulate conserved residues in Doppel: a case of molecular mimicry?

Intrachromosomal deletions linking Dpl expression to the PrP promoter produce cerebellar degeneration that can be abrogated by the introduction of wild-type PrP transgenes. Since Dpl-like truncated forms of PrP are neuropathogenic in mice and likewise counterbalanced by expression of PrP(C) we asked whether naturally occurring mutant forms of human PrP have Dpl-like attributes. Five PRNP missense mutations causing familial Creutzfeldt-Jakob disease (F-CJD) map to a helical region found in both PrP(C) and Dpl and result in amino acids identical to conserved residues in Dpl. These F-CJD alleles may cause mutant PrP to become a weak mimetic of Dpl structure and/or function.     

The genetic basis of Cowden’s syndrome: three novel mutations in PTEN/MMAC1/TEP1

Cowden’s syndrome (CS) is an autosomal dominant disorder associated with an increased risk of developing benign and malignant tumors in a variety of tissues, including the skin, thyroid, breast and brain. Women with CS are felt to have an increased risk of developing breast cancer, and virtually all women with CS develop bilateral fibrocystic disease of the breast. Recently, a series of germline mutations have been identified from CS families in a gene known as PTEN/MMAC1/TEP1. In this study, we used heteroduplex analysis and direct sequencing analysis and identified three novel germline mutations in the PTEN/MMAC1/TEP1 coding sequence from unrelated individuals with CS. We report a de novo transition (T→C) at nucleotide 335 in exon 5. This missense mutation resulted in a leucine to proline (CTA to CCA) change at codon 112. We also describe a novel splice site mutation (801+2T→G) in intron 7 that caused exon skipping in PTEN/MMAC1/TEP1 mRNA. The third mutation we report is a missense mutation, consisting of a transition (T→C) at nucleotide 202 in exon 3, resulting in a tyrosine to histidine (TAC to CAC) change at codon 68. Finally, we also detected a rare polymorphism in exon 7 of the PTEN/MMAC1/TEP1 coding sequence. These data confirm the observation that mutations of the PTEN/MMAC1/ TEP1 coding sequence are responsible for at least some cases of CS, and further define the spectrum of mutations in this autosomal dominant disorder. Received: 21 October 1997 / Accepted: 15 December 1997