ABSTRACT
Although there is considerable evidence that PrP(Sc) is the infectious form of the prion protein, it has recently been proposed that a transmembrane variant called (Ctm)PrP is the direct cause of prion-associated neurodegeneration. We report here, using a mutant form of PrP that is synthesized exclusively with the (Ctm)PrP topology, that (Ctm)PrP is retained in the endoplasmic reticulum and is degraded by the proteasome. We also demonstrate that (Ctm)PrP contains an uncleaved, N-terminal signal peptide as well as a C-terminal glycolipid anchor. These results provide insight into general mechanisms that control the topology of membrane proteins during their synthesis in the endoplasmic reticulum, and they also suggest possible cellular pathways by which (Ctm)PrP may cause disease.
Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Prions/metabolism , Protein Sorting Signals , Animals , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Cricetinae , Cysteine Endopeptidases/metabolism , Glycosylphosphatidylinositols/metabolism , Membrane Proteins/genetics , Mice , Multienzyme Complexes/metabolism , Mutagenesis , Prions/genetics , Proteasome Endopeptidase Complex , Tumor Cells, CulturedABSTRACT
We have generated lines of transgenic mice that express a mutant prion protein (PrP) containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop a neurological illness with prominent ataxia at 65 or 240 days of age, depending on whether the transgene array is, respectively, homozygous or hemizygous. Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles the scrapie isoform of PrP, and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of granule cells in the cerebellum. Our analysis provides important insights into the molecular pathogenesis of inherited prion disorders in humans.
Subject(s)
Apoptosis , Cerebellum/pathology , Neurons/pathology , Prions/genetics , Prions/metabolism , Animals , Endopeptidases/metabolism , Homozygote , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred Strains , Mice, Transgenic , Mutagenesis, Insertional , Prion Diseases/genetics , Recombinant Proteins/metabolism , Scrapie/pathologyABSTRACT
Prion diseases are neurodegenerative disorders that result from conformational transformation of a normal cell surface glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). Although the normal physiological function of PrP(C) has remained enigmatic, the recent observation that the protein binds copper ions with micromolar affinity suggests a possible role in brain copper metabolism. In this study, we have used mice that express 0, 1, and 10 times the normal level of PrP to assess the effect of PrP expression level on the amount of brain copper and on the properties of two brain cuproenzymes. Using mass spectrometry, we find that the amount of ionic copper in subcellular fractions from brain is similar in all three lines of mice. In addition, the enzymatic activities of Cu-Zn superoxide dismutase and cytochrome c oxidase in brain extracts are similar in these groups of animals, as is the incorporation of (64)Cu into Cu-Zn superoxide dismutase both in cultured cerebellar neurons and in vivo. Our results differ from those of another set of published studies, and they require a re-evaluation of the role of PrP(C) in copper metabolism.
Subject(s)
Brain/metabolism , Copper/metabolism , Electron Transport Complex IV/metabolism , Prions/metabolism , Superoxide Dismutase/metabolism , Animals , Mice , Mice, Transgenic , PrPC Proteins/genetics , PrPC Proteins/metabolism , PrPSc Proteins/genetics , PrPSc Proteins/metabolism , Prions/genetics , Subcellular Fractions/chemistry , Superoxide Dismutase-1ABSTRACT
We have generated lines of transgenic mice that express a mutant prion protein containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop an ataxic illness that begins at 65 days of age when the transgene array is homozygous, and results in death by 115-138 days. Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles PrP(Sc), and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of cerebellar granule cells, as well as astrocytosis and deposition of PrP in a punctate pattern. These results establish a new transgenic animal model of an inherited human prion disease, and provide important insights into the molecular pathogenesis of these disorders.
Subject(s)
Disease Models, Animal , Mice, Transgenic , Prion Diseases , Prions/genetics , Animals , Brain/metabolism , Brain/pathology , Humans , Mice , Mutation , PrPSc Proteins/chemistry , PrPSc Proteins/metabolism , Prion Diseases/genetics , Prion Diseases/pathology , Prion Diseases/physiopathology , Prions/chemistry , Prions/metabolism , TransgenesABSTRACT
Exon trapping was employed to identify coding sequences from a collection of 46 bovine cosmids, previously characterized for the presence of microsatellite markers and physically mapped to chromosomes by FISH. The sequence analysis of 104 clones revealed 18 putative exons, 10 of which showed near identity to known sequences. Among these were the human (cytosine-5)-methyltransferase (DNMT), ATP-citrate lyase (ACLY), the mouse Lbcl1 oncogene, the bovine mitochondrial aconitase (ACO2) and beta-arrestin 1 (ARR1). The chromosomal localization of the cloned exons was inferred from the localization of the parent cosmids. DNMT and ACLY were not previously known in cattle, but the physical localization of the cloned bovine exons is in agreement with the published comparative human and bovine maps. The trapping of exons for bovine ACO2 and ARR1 confirms the available mapping information based on synteny and provides a physical assignment for the genes.
