Linkage of the Indiana kindred of Gerstmann-Sträussler-Scheinker disease to the prion protein gene.

The Indiana kindred variant of Gerstmann-Sträussler-Scheinker disease has amyloid plaques that contain prion protein (PrP), but is atypical because neurofibrillary tangles like those of Alzheimer disease are present. To map the position of the disease causing gene, we used three markers for linkage analyses. A missense mutation at codon 198 of the PrP gene (PRNP) is found in all definitely affected individuals and yields a maximum lod score of 6.37 (theta = 0). The disease also is concordant with the two other PRNP-region markers. These results demonstrate tight linkage of the disease-causing gene to PRNP and support the hypothesis that the codon 198 mutation is the cause of IK-GSS. Our studies also suggest that methionine/valine heterozygotes at PRNP codon 129 have a later age of onset of the disease than codon 129 valine/valine homozygotes.

Mutant prion proteins in Gerstmann-Sträussler-Scheinker disease with neurofibrillary tangles.

Two families with Gerstmann-Sträussler-Scheinker disease (GSS) are atypical in possessing neocortical neurofibrillary tangles (NFTs), which are few or absent in other kindreds with GSS, in addition to amyloid plaques that react with prion protein (PrP) antibodies and protease-resistant PrP accumulation in the brain. A leucine substitution at PrP codon 102 has been genetically linked to GSS in some families. We examined the PrP gene in these families. A serine for phenylalanine substitution was found at codon 198 in the Indiana patients; arginine for glutamine substitution at codon 217 in the Swedish patients. These mutations in PrP are the first to be associated with the appearance of both PrP amyloid plaques and neocortical NFTs in GSS patients.

Insoluble wild-type and protease-resistant mutant prion protein in brains of patients with inherited prion disease.

We studied prion proteins (PrP) in skin and brains of Libyan Jews carrying the E200K mutation who died of familial Creutzfeldt-Jakob disease (CJD). Unexpectedly, studies with brain showed that PrP molecules encoded both by the wild-type (wt) and mutant alleles exhibit altered properties characteristic of the prion protein associated with prion diseases (PrPSc). Using monospecific antisera, we found that wtPrP was insoluble in the brains of three patients who were heterozygous for the E200K mutation, whereas mutant PrP was both insoluble and protease-resistant. Our results argue that both wild-type and mutant PrP undergo conformational changes and are particularly intriguing, because the normal isoform PrPc is soluble in nondenaturing detergents and is readily digested by proteases, whereas PrPSc is insoluble and resistant to proteolytic digestion. Our findings indicate that insoluble wtPrP represents a conformational intermediate, the first to be identified, within a pathway in which PrPc is converted to PrPSc.

Heritable disorder resembling neuronal storage disease in mice expressing prion protein with deletion of an alpha-helix.

Mice were constructed carrying prion protein (PrP) transgenes with individual regions of putative secondary structure deleted. Transgenic mice with amino-terminal regions deleted remained healthy at >400 days of age, whereas those with either of carboxy-terminal alpha-helices deleted spontaneously developed fatal CNS illnesses similar to neuronal storage diseases. Deletion of either C-terminal helix resulted in PrP accumulation within cytoplasmic inclusions in enlarged neurons. Deletion of the penultimate C-terminal helix resulted in proliferation of rough endoplasmic reticulum. Mice with the C-terminal helix deleted were affected with nerve cell loss in the hippocampus and proliferation of smooth endoplasmic reticulum. Whether children with the human counterpart of this malady will be found remains to be determined.

Eight prion strains have PrP(Sc) molecules with different conformations.

Variations in prions, which cause different incubation times and deposition patterns of the prion protein isoform called PrP(Sc), are often referred to as 'strains'. We report here a highly sensitive, conformation-dependent immunoassay that discriminates PrP(Sc) molecules among eight different prion strains propagated in Syrian hamsters. This immunoassay quantifies PrP isoforms by simultaneously following antibody binding to the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrP(Sc), each strain occupies a unique position, indicative of a particular PrP(Sc) conformation. This conclusion is supported by a unique pattern of equilibrium unfolding of PrP(Sc) found with each strain. Our findings indicate that each of the eight prion strains has a PrP(Sc) molecule with a unique conformation and, in accordance with earlier results, indicate the biological properties of prion strains are 'enciphered' in the conformation of PrP(Sc) and that the variation in incubation times is related to the relative protease sensitivity of PrP(Sc) in each strain.

Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells.

The cellular prion protein (PrPC) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrPSc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrPSc to proteolysis and to digestion with phosphatidylinositol-specific phospholipase C as well as the enhancement of PrPSc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrPSc in cultured cells. Using these immunoassays, we identified the sites of PrPSc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrPSc-producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrPSc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrPSc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrPSc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrPSc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrPSc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases.

Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells.

Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.

Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform.

After the cellular prion protein (PrPC) transits to the cell surface where it is bound by a glycophosphatidyl inositol (GPI) anchor, PrPC is either metabolized or converted into the scrapie isoform (PrPSc). Because most GPI-anchored proteins are associated with cholesterol-rich membranous microdomains, we asked whether such structures participate in the metabolism of PrPC or the formation of PrPSc. The initial degradation of PrPC involves removal of the NH2 terminus of PrPC to produce a 17-kD polypeptide which was found in a Triton X-100 insoluble fraction. Both the formation of PrPSc and the initial degradation of PrPC were diminished by lovastatin-mediated depletion of cellular cholesterol but were insensitive to NH4Cl. Further degradation of the 17-kD polypeptide did occur within an NH4Cl-sensitive, acidic compartment. Replacing the GPI addition signal with the transmembrane and cytoplasmic domains of mouse CD4 rendered chimeric CD4PrPC soluble in cold Triton X-100. Both CD4PrPC and truncated PrPC without the GPI addition signal (Rogers, M., F. Yehieley, M. Scott, and S. B. Prusiner. 1993. Proc. Natl. Acad. Sci. USA. 90:3182-3186) were poor substrates for PrPSc formation. Thus, it seems likely that both the initial degradation of PrPC to the 17-kD polypeptide and the formation of PrPSc occur within a non-acidic compartment bound by cholesterol-rich membranes, possibly glycolipid-rich microdomains, where the metabolic fate of PrPC is determined. The pathway remains to be identified by which the 17-kD polypeptide and PrPSc are transported to an acidic compartment, presumably endosomes, where the 17-kD polypeptide is hydrolyzed and limited proteolysis of PrPSc produces PrP 27-30.

Molecular biology of prion diseases.

Prions cause transmissible and genetic neurodegenerative diseases, including scrapie and bovine spongiform encephalopathy of animals and Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases of humans. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein, which is encoded by a chromosomal gene. A posttranslational process, as yet unidentified, converts the cellular prion protein into an abnormal isoform. Scrapie incubation times, neuropathology, and prion synthesis in transgenic mice are controlled by the prion protein gene. Point mutations in the prion protein genes of animals and humans are genetically linked to development of neuro-degeneration. Transgenic mice expressing mutant prion proteins spontaneously develop neurologic dysfunction and spongiform neuropathology. Understanding prion diseases may advance investigations of other neurodegenerative disorders and of the processes by which neurons differentiate, function for decades, and then grow senescent.

Novel proteinaceous infectious particles cause scrapie.

After infection and a prolonged incubation period, the scrapie agent causes a degenerative disease of the central nervous system in sheep and goats. Six lines of evidence including sensitivity to proteases demonstrate that this agent contains a protein that is required for infectivity. Although the scrapie agent is irreversibly inactivated by alkali, five procedures with more specificity for modifying nucleic acids failed to cause inactivation. The agent shows heterogeneity with respect to size, apparently a result of its hydrophobicity; the smallest form may have a molecular weight of 50,000 or less. Because the novel properties of the scrapie agent distinguish it from viruses, plasmids, and viroids, a new term "prion" is proposed to denote a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids. Knowledge of the scrapie agent structure may have significance for understanding the causes of several degenerative diseases.

Prion diseases and the BSE crisis.

Bovine spongiform encephalopathy (BSE) and human Creutzfeldt-Jakob disease (CJD) are among the most notable central nervous system degenerative disorders caused by prions. CJD may present as a sporadic, genetic, or infectious illness. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular prion protein (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. It is thought that BSE is a result of cannibalism in which faulty industrial practices produced prion-contaminated feed for cattle. There is now considerable concern that bovine prions may have been passed to humans, resulting in a new form of CJD.

Reversible chemical modification of the scrapie agent.

The scrapie agent causes a degenerative nervous system disease in sheep and goats. Studies with extensively purified preparations demonstrated that the agent contains a protein that is required for infectivity. Chemical modification of the scrapie agent by diethyl pyrocarbonate reduced the titer 1000-fold. Exposure of the inactivated agent to hydroxylamine, a strong nucleophile, resulted in complete restoration of infectivity. Presumably, nucleophilic residues within a scrapie agent protein undergo carbethoxylation on reaction with diethyl pyrocarbonate, and subsequent addition of hydroxylamine displaces these carbethoxy groups.

Identification of a protein that purifies with the scrapie prion.

Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain. The protein migrated with an apparent molecular size of 27,000 to 30,000 daltons in sodium dodecyl sulfate polyacrylamide gels. The resistance of this protein to digestion by proteinase K distinguished it from proteins of similar molecular weight found in normal hamster brain. Initial results suggest that the amount of this protein correlates with the titer of the agent.

Unusual topogenic sequence directs prion protein biogenesis.

Biosynthetic studies of the prion protein (PrP) have shown that two forms of different topology can be generated from the same pool of nascent chains in cell-free translation systems supplemented with microsomal membranes. A transmembrane form is the predominant product generated in wheat germ (WG) extracts, whereas a completely translocated (secretory) form is the major product synthesized in rabbit reticulocyte lysates (RRL). An unusual topogenic sequence within PrP is now shown to direct this system-dependent difference. The actions of this topogenic sequence were independent of on-going translation and could be conferred to heterologous proteins by the engineering of a discrete set of codons. System-dependent topology conferred by addition of RRL to WG translation products suggests that this sequence interacts with one or more cytosolic factors.

Spontaneous neurodegeneration in transgenic mice with mutant prion protein.

Transgenic mice were created to assess genetic linkage between Gerstmann-Sträussler-Scheinker syndrome and a leucine substitution at codon 102 of the human prion protein gene. Spontaneous neurologic disease with spongiform degeneration and gliosis similar to that in mouse scrapie developed at a mean age of 166 days in 35 mice expressing mouse prion protein with the leucine substitution. Thus, many of the clinical and pathological features of Gerstmann-Sträussler-Scheinker syndrome are reproduced in transgenic mice containing a prion protein with a single amino acid substitution, illustrating that a neurodegenerative process similar to a human disease can be genetically modeled in animals.

Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity.

The fundamental event in prion diseases seems to be a conformational change in cellular prion protein (PrPC) whereby it is converted into the pathologic isoform PrPSc. In fatal familial insomnia (FFI), the protease-resistant fragment of PrPSc after deglycosylation has a size of 19 kilodaltons, whereas that from other inherited and sporadic prion diseases is 21 kilodaltons. Extracts from the brains of FFI patients transmitted disease to transgenic mice expressing a chimeric human-mouse PrP gene about 200 days after inoculation and induced formation of the 19-kilodalton PrPSc fragment, whereas extracts from the brains of familial and sporadic Creutzfeldt-Jakob disease patients produced the 21-kilodalton PrPSc fragment in these mice. The results presented indicate that the conformation of PrPSc functions as a template in directing the formation of nascent PrPSc and suggest a mechanism to explain strains of prions where diversity is encrypted in the conformation of PrPSc.

A transmembrane form of the prion protein in neurodegenerative disease.

At the endoplasmic reticulum membrane, the prion protein (PrP) can be synthesized in several topological forms. The role of these different forms was explored with transgenic mice expressing PrP mutations that alter the relative ratios of the topological forms. Expression of a particular transmembrane form (termed CtmPrP) produced neurodegenerative changes in mice similar to those of some genetic prion diseases. Brains from these mice contained CtmPrP but not PrPSc, the PrP isoform responsible for transmission of prion diseases. Furthermore, in one heritable prion disease of humans, brain tissue contained CtmPrP but not PrPSc. Thus, aberrant regulation of protein biogenesis and topology at the endoplasmic reticulum can result in neurodegeneration.

Molecular biology and pathogenesis of prion diseases.

Prions cause a group of human and animal neurodegenerative diseases, which are now classified together because their etiology and pathogenesis, involve modification of the prion protein (PrP). Prion diseases are manifest as infectious, genetic and sporadic disorders. These diseases can be transmitted among mammals by the infectious particle designated 'prion'. Despite intensive searches over the past three decades, no nucleic acid has been found within prions, yet a modified isoform of the host-encoded PrP designated PrPSc is essential for infectivity. In fact, considerable experimental data argue that prions are composed exclusively of PrPSc. Earlier terms used to describe the prion diseases include transmissible encephalopathies, spongiform encephalopathies and slow virus diseases. The human prion disorders include kuru, Creutzfeldt-Jackob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI).

Selective neuronal targeting in prion disease.

The pattern of scrapie prion protein (PrP(Sc)) accumulation in the brain is different for each prion strain. We tested whether the PrP(Sc) deposition pattern is influenced by the Asn-linked oligosaccharides of PrP(C) in transgenic mice. Deletion of the first oligosaccharide altered PrP(C) trafficking and prevented infection with two prion strains. Deletion of the second did not alter PrP(C) trafficking, permitted infection with one prion strain, and had a profound effect on the PrP(Sc) deposition pattern. Our data raise the possibility that glycosylation can modify the conformation of PrP(C). Glycosylation could affect the affinity of PrP(C) for a particular conformer of PrP(Sc), thereby determining the rate of nascent PrP(Sc) formation and the specific patterns of PrP(Sc) deposition.

Genetics of prion infections.

Although the infectious prions causing scrapie and several human transmissible neurodegenerative diseases resemble viruses in many respects, molecular and genetic analyses indicate that prions are fundamentally different from viruses in their structure and the mechanisms by which they cause disease. The only macromolecule that has been identified in infectious prion preparations is a disease-specific isoform of the prion protein, which is encoded by a host gene. A growing body of data supports the contention that prion infections represent a novel host-pathogen interaction.