Prionoses--neurodegenerative diseases caused by prions, offectious proteinaceous molecules

Prionoses are a group of human and animal neurodegenerative diseases caused by prions, infectious pathogens that differ from bacteria, fungi, parasites, viroids, and viruses. Despite intensive searches over the past three decades, no nucleic acid has been found within prions and considerable experimental data argue that prions are composed exclusively of proteins (glycoproteins). Normal prion protein (PrPC) is encoded by a gene present in all nuclear cells of humans and other mammals but is constitutively expressed mainly in neurons. PrPC is protease sensitive and nonpathogenic but it can be modified to the pathological and protease resistant form designated PrPSC which is essential for infectivity. Prion diseases are manifested as infectious, genetic, or sporadic disorders and are also named as transmissible spongiform encephalopathies (TSE). TSE culminate with a progressive and fatal degeneration of the CNS. The human prionoses include Creutzfeldt-Jakob disease (CJD), kuru, Gerstman-Str?ussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). In mammals, more than 15 different species have been described to suffer from prion disorders till now. Scrapie of sheep and goats is the oldest and the most studied of the prion diseases. Bovine spongiform encephalopathy (BSE) and transmissible mink encephalopathy are thought to result from the feeding of scrapie-infected animal products, whereas BSE has been identified in transmission to mice, domestic cats, two exotic species of ruminant, and monkey. More than 20 cases of clinically and pathologically atypical form of CJD, referred to as "new variant" CJD (vCJD) have been recognized in unusually young people in the United Kingdom. There is a strong evidence that the same prion strain is involved in both BSE and vCJD. It suggest the breaking of species barrier which results by spreading of BSE to humans, putatively by dietary exposure. Understanding the function of prion proteins and their modification to the pathological form may give new insight into the etiologic and pathogenic mechanisms also other diseases caused by aberrant proteins, including Alzheimer' disease, amyotrophic lateral sclerosis, and Parkinson's disease. (Tab. 4, Fig. 3, Ref. 76.)     

Chicken prion tandem repeats form a stable, protease-resistant domain

Prion-linked diseases, such as mad cow disease, scrapie, and the human genetic disorder Creutzfeldt-Jakob disease, are fatal neurodegenerative diseases correlated with changes in the secondary structure of neural prion protein. We expressed recombinant chicken prion protein in Escherichia coli and purified the protein to homogeneity. Circular dichroism spectra of the 26 kDa recombinant protein closely resemble those of prion protein purified directly from healthy hamster brain. The chicken prion protein exists as a soluble, monodisperse monomer but can be forced to multimerize following lyophilization and resuspension. We analyzed the chicken prion protein domain structure by proteolysis and show that, unlike the mammalian homologues, the chicken prion protein N-terminal tandem amino acid repeats form a stable, protease-resistant domain. This domain probably represents a physiologically functional unit. As tested by both mass spectrometry and circular dichroism, the mature chicken prion protein does not bind copper, unlike synthetic peptides from the chicken prion N-terminus, suggesting that binding copper is not the physiological activity of the chicken prion. However, copper strongly destabilizes the prion protein and depresses the melting temperature by 30 degreesC, presumably by binding to the unfolded form of the prion protein. The chicken prion N-terminus may have evolved to fold without a cofactor, unlike mammalian prion proteins, whose N-termini are disordered without cofactors such as copper present. Chicken prion offers an alternative to intractable mammalian prions for structural studies of the amino-terminal domain.     

The use of transgenic mice in the investigation of transmissible spongiform encephalopathies

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and to be identical to PrPSc (prion protein: scrapie form), a modified form of the normal host protein PrPC (prion protein: cellular form) which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. The authors generated homozygous Prnp(o/o) ('PrP knockout') mice and showed that, after inoculation with prions, these mice remained free from scrapie for at least two years while wild-type controls all died within six months. There was no propagation of prions in the Prnp(o/o) animals. Surprisingly, heterozygous Prnp(o/+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie despite high levels of infectious agent and PrPSc in the brain at an early stage. After introduction of murine PrP transgenes, Prnp(o/o) mice became highly susceptible to mouse--but not to hamster--prions, while the insertion of Syrian hamster PrP transgenes rendered the mice susceptible to hamster prions but much less susceptible to mouse prions. These complementation experiments enabled the application of reverse genetics. The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.     

Generation of monoclonal antibodies against human prion proteins in PrP0/0 mice

BACKGROUND: Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available. MATERIALS AND METHODS: We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared. RESULTS: Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. CONCLUSIONS: These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.     

The prion diseases

The human prion diseases are fatal neurodegenerative maladies that may present as sporadic, genetic, or infectious illnesses. The sporadic form is called Creutzfeldt-Jakob disease (CJD) while the inherited disorders are called familial (f) CJD, Gerstmann-Straussler-Scheinker (GSS) disease and fatal familial insomnia (FFI). Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. In fCJD, GSS, and FFI, mutations in the PrP gene located on the short arm of chromosome 20 are the cause of disease. Considerable evidence argues that the prion diseases are disorders of protein conformation.     

A review of the current research on prions. The evidence suggests the possibility of transmission of the mad cow disease to humans

Further evidence of the transmissibility of bovine spongiform encephalopathy (BSE) across the species barrier from cow to man has been derived from epidemiological analysis and the characterisation of prion strains. Recent research has shown the persistence of prions after experimental transmission to resistant murine species, and subclinical persistence in cows. The accumulation of pathological prion proteins in tonsils and appendix has been demonstrated prior to clinical confirmation of the presence of the new variant of Creutzfeldt-Jakob disease. Current prion research is focused on the involvement of B lymphocytes as carriers, on the species barrier and cellular receptors, and on macromolecules involved in the conformational change from normal to pathological prion proteins.     

Prion protein amyloidosis

The prion protein (PrP) plays an essential role in the pathogenesis of a group of sporadic, genetically determined and infectious fatal degenerative diseases, referred to as "prion diseases", affecting the central nervous system of humans and other mammals. The cellular PrP is encoded by a single copy gene, highly conserved across mammalian species. In prion diseases, PrP undergoes conformational changes involving a shift from alpha-helix to beta-sheet structure. This conversion is important for PrP amyloidogenesis, which occurs to the highest degree in the genetically determined Gerstmann-Str?ussler-Scheinker disease (GSS) and prion protein cerebral amyloid angiopathy (PrP-CAA), while it is less frequently seen in other prion diseases. GSS and PrP-CAA are associated with point mutations of the prion protein gene (PRNP); these conditions show a broad spectrum of clinical presentation, the main signs being ataxia, spastic paraparesis, extrapyramidal signs and dementia. In GSS, parenchymal amyloid may be associated with spongiform changes or neurofibrillary lesions; in PrP-CAA, vascular amyloid is associated with neurofibrillary lesions. A major component of the amyloid fibrils in the two diseases is a 7 kDa peptide, spanning residues 81-150 of PrP.     

Prions

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.     

The prionoses and other conformational disorders

The basic pathogenesis of numerous neurodegenerative disorders is now thought to be related to abnormal protein conformation. The common theme in all these diseases is the conversion of a normal cellular and/or circulating protein into an insoluble, aggregated, beta-sheet rich form which is deposited in the brain, sometimes in the form of amyloid. These deposits are toxic and produce neuronal dysfunction and death. The most common of these illnesses is Alzheimer's disease (AD), in which a central event is the conversion of the normal soluble amyloid beta (sA beta) peptide to amyloid beta (A beta) within neuritic plaques and cerebral vessels. A unique category of the conformational conditions are prion related diseases (or prionoses), where the etiology is thought to be related to conversion of the normal prion protein, PrPC, into an infectious and pathogenic form, PrPSc. In the case of AD and the prionoses, the conformational change can be influenced by the presence of mutations in various gene products, as well as by chaperone proteins. Apolipoprotein E is thought to act as such a chaperone protein in AD; however, among the prionoses such a protein has been hypothesized to exist only by indirect evidence and is called "protein X". Our growing understanding of the mechanisms involved in this category of diseases, raises the possibility of therapeutic approaches based directly on the prevention and reversal of pathologic protein conformation.     

Hereditary amyloid cardiomyopathy caused by a variant apolipoprotein A1

Autosomal dominant hereditary amyloidosis with a unique cutaneous and cardiac presentation and death from heart failure by the sixth or seventh decade was found to be associated with a previously unreported point mutation (thymine to cytosine, nt 1389) in exon 4 of the apolipoprotein A1 (apoA1) gene. The predicted substitution of proline for leucine at amino acid position 90 was confirmed by structural analysis of amyloid protein isolated from cardiac deposits of amyloid. The subunit protein is composed exclusively of NH2-terminal fragments of the variant apoA1 with the longest ending at residue 94 in the wild-type sequence. Amyloid fibrils derived from four previously described apoA1 variants are composed of similar fragments with carboxyl-terminal heterogeneity, but contrary to those variants, which all carry one extra positive charge, the substitution Leu90Pro does not result in any charge modification. It is unlikely, therefore, that amyloid fibril formation is related to change of charge for a specific residue of the precursor protein. This is in agreement with studies on transthyretin amyloidosis in which no unifying factor such as change of charge for amino acid residues has been noted.     

Creutzfeldt-Jakob disease(CJD) and Gerstmann-Str?ussler-Scheinker syndrome(GSS)

Prion diseases are now a focus of attention since the diseases are known to disseminate beyond expectation. They are various types of Gerstmann-Str?ussler-Scheinker syndrome(GSS), fatal familial insomnia, iatrogenic Creutzfeldt-Jakob disease(CJD), and variant CJD which suspected to have disseminated from bovine spongiform encephalopathy. Abnormal prion protein deposits as amyloid structure in the brain of the patients with these diseases. Mutation of the prion protein gene exists in a part of these diseases. Depending on their kind, patients represent a variety of clinical and neuropathological manifestations. They can be clinically divided into two general groups of the CJD type and the GSS type. For clinical diagnosis of the GSS type prion diseases, analysis of prion protein gene is useful.     

Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations

We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies.     

BSE: can we predict the future?

Prion diseases are transmissible neurodegenerative disorders of humans and animals. The prion protein (PrPc) gene is expressed to some extent in many cell types but principally in neurons. Normal PrPc may contribute in the protection of neurons and are protease sensitive. Abnormal prions consist of a post-translationally modified form of PrP, PrPsc, which is partly protease resistant. PrPsc is a protein with high resistance to inactivation by irradiation, heat and harsh chemical treatments. It is currently proposed that PrPsc is an infectious protein that propagates by inducing the normal PrPc to become the abnormal PrPsc. PrPsc cause transmissible spongiform encephalopathies (TSE), an unusual group of degenerative brain diseases that can be transmitted by inoculation or ingestion of diseased brain or other tissues. The human diseases occur in an inherited, acquired and sporadic forms. Transmission of prion diseases between species is limited by a species barrier, determined in part by the degree of sequence homology between the host PrP and inoculated PrPsc. The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom is a new disease that has affected over 160,000 cattle and has presumably arisen from dietary exposure to PrPsc from sheep with scrapie. Until shown otherwise we must assume that oral consumption of infectious BSE protein is a new factor for Creutzfeldt-Jakob (CJD) disease in man. This disease is a new variant of CJD (nvCJD) and has a different clinical picture. Early diagnostic markers to facilitate the diagnosis and screen blood and organ donors are not available. The control of the disease relies on the measures to eradicate the spread of BSE by banning the use of ruminant tissues in ruminant feed and slaughter and rendering procedures to ensure inactivation of prions of all infected animals. The control of nvCJD is based on reduction of exposure to BSE by banning a variety of tissues for consumption. A surveillance worldwide is increased for both BSE and nv-CJD and the WHO update regularly recommendations to limit the risk of transmitting the disease. The extent and size of the nvCJD will depend on different factors among others the dose of infectious material, the route of infection, the genetic susceptibility of the person. Therefore is not possible to predict how important the disease will be.     

The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation

BACKGROUND: A conformational change seems to represent the major difference between the scrapie prion protein (PrPSc) and its normal cellular isoform (PrPC). We recently proposed a set of four helix bundle models for the three-dimensional structure of PrPC that are consistent with a variety of spectroscopic and genetic data. RESULTS: We report a plausible model for the three-dimensional structure of a biologically important fragment of PrPSc. The model of residues 108-218 was constructed by an approach that combines computational techniques and experimental data. The proposed structures of this fragment of PrPSc display a four-stranded beta-sheet covered on one face by two alpha-helices. Residues implicated in the prion species barrier are found to cluster on the solvent-accessible surface of the beta-sheet of one of the models. This interface could provide a structural template that would assist the conversion of PrPC to PrPSc and hence direct prion propagation. CONCLUSIONS: Molecular models of the PrP isoforms should prove very useful in developing structural hypotheses about the process by which PrPC is transformed into PrPSc, the mechanisms by which PrP gene mutations give rise to the inherited human prion diseases, and the species barrier that seems to protect humans from animal prions. It seems likely that PrPC represents a kinetically trapped intermediate in PrP folding.     

Variations of sitting posture and physical activity in different types of school furniture

The development of neuro-degenerative diseases often involves amyloidosis, that is the formation of polymeric fibrillar structures from normal cellular proteins or peptides. For example, in Alzheimer's disease, a 42 amino acid peptide processed from the amyloid precursor protein forms filaments with a beta-sheet structure. Because of this, the structure and dynamics of polymeric peptide filaments is of considerable interest. We showed previously that a 23 amino acid peptide constituting a single leucine-rich repeat (LRRN) polymerises spontaneously in solution to form long filaments of a beta-sheet structure, a property similar to that of Alzheimer's beta-amyloid and prion peptides. Here we report that a variant of LRRN in which a highly conserved asparagine residue is replaced by aspartic acid does not form either filaments or beta structure. By contrast, a variant which replaces this asparagine residue with glutamine forms filaments ultrastructurally indistinguishable from those of LRRN. Electron micrographs of LRRN filaments show that many consist of two interleaved strands which appear to have a ribbon-like morphology. X-ray diffraction patterns from oriented LRRN fibres reveal that they are composed of long beta-sheet arrays, with the interstrand hydrogen bonding parallel to the filament axis. This 'cross-beta' structure is similar to that adopted by beta-amyloid and prion derived fibres. Taken together, these results indicate that the LRR filaments are stabilised by inter- or intra-strand hydrogen bonded interactions comparable to the asparagine ladders of beta-helix proteins or the 'glutamine zippers' of poly-glutamine peptides. We propose that similar stabilising interactions may underlie a number of characterised predispositions to neuro-degenerative diseases that are caused by mutations to amide residues. Our finding that amyloid-like filaments can form from a peptide motif not at present correlated with degenerative disease suggests that a propensity for beta-filament formation is a common feature of protein sub-domains.     

A prion disease with a novel 96-base pair insertional mutation in the prion protein gene

There are coding mutations in the prion protein gene in familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, and other phenotypes that make up the inherited prion diseases. Insertional mutations consisting of two, five, six, seven, eight, and nine additional octapeptide repeat elements are seen in the inherited prion diseases and usually present as atypical dementias with considerable intrafamilial phenotypic variability. A four-octarepeat insertion was reported previously in an individual without neurodegenerative disease who died of hepatic cirrhosis. Here we report a novel four-octarepeat insertional mutation in a case with classical clinical, electroencephalographic and histopathologic features of CJD with the unusual finding of pronounced prion protein immunoreactivity of the molecular layer of the cerebellum.     

Permeability and stability in buffer and in human serum of fluorinated phospholipid-based liposomes

Calsequestrin is the major Ca(2+)-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its alpha-helical content and the intrinsic fluorescence upon binding of Ca2+. Calsequestrin binds Ca2+ with high-capacity and with moderate affinity and it functions as a Ca2+ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca2+ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsequestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Traumatic myositis ossificans of the levator scapulae muscle

The Chou-Fasman method has been widely used for predicting protein secondary structure. It is based on knowledge of the potential of amino acid residues to form alpha-helical or beta-sheet regions in proteins. Our main interest in this study was to examine the reliability of these Chou-Fasman parameters. We calculated the Chou-Fasman parameters, with 95% confidence limits, of 144 non-homologous proteins consisting of 155 chains, and a total of 33 118 amino acid residues. All of the protein chains used were X-ray structures known at a resolution of at least 2.5 A. We compared the results of our calculations with those previously done by Chou and Fasman. Our results show that Chou and Fasman classified four amino acid residues wrongly in alpha-helical regions and one in a beta-sheet region. This is so, because the confidence limits we calculated did not include the values determined by Chou and Fasman. Moreover, the confidence limit calculations contradict most of the Chou-Fasman classification of amino acid residues.     

Molecular genetics of C1 inhibitor

More than 100 different C1 inhibitor gene mutations have been described in hereditary angioedema (HAE) patients. Sixty-nine mutations have been reported in patients with the quantitative C1 inhibitor defect (type 1 HAE) in two recent large-scale studies. These changes were found distributed over all exons and exon/intron boundaries. The molecular defects can be divided as follows: Alu-repeat-mediated deletions or duplications (accounting for 21% of all cases), missense mutations (> 36%), frameshifts (14%), Stop codon mutations (10%), promoter variants (4%), splice site mutations (7-10%), deletions of a few amino acids (less than 3%). Several recent studies indicate that up to 25% of these changes are found in patients without a family history of angioedema and represent de novo mutations. Pathogenic amino acid substitutions were found distributed over the entire length of the coding sequence, except for the 100 amino-acid-long glycosylated amino-terminal extension, whose sequence tolerates extensive variation, as indicated by comparisons across species. Functional studies have been carried out only on a fraction of these amino acid substitutions and indicate that defects affecting intracellular transport are often at the basis of type 1 hereditary angioedema. An interesting promoter variant (a C to T transition at position -103) was found in an exceptional family with recessive transmission of the disease. Regulatory elements in the promoter region and in intron 1 were revealed by their sequence conservation in mouse and man and by functional studies. C1 inhibitor "minigene" constructs directing correct mRNA and protein synthesis in transgenic mice have provided valuable information on hormonal control and cell-type specificity of gene expression.