1Lecture 34: Prions.PRION = Proteinaceous Infectious particlePrion diseases can occur through:Infection: InfectiousAs a dominantly inherited genetic disorder: FamilialConsequent to a spontaneous mutation: Sporadic
2Mammalian Prion diseases. Characterized as Spongiform Encephalopathies.Invariably fatal.Characterized by amyloid plaques or spongy (spongiform) appearance of the affected areas of the brain, due to accumulation of vesicular structures in the brains of infected organisms.Vacuoles are seen in one neuronand in the neuropiles. Astrocyteswith small nucleus proliferate. Noinflammatory cells infiltrae in the brain.
3For many years, prion diseases were thought to be caused by slow-acting viruses, and were referred to as ‘slow virus diseases’, ‘transmissible spongiform encephalopathies’, or ‘unconventional viral diseases’.First discovered by showing that injection of a brain extract from a patient into a chimpanzee could result in infection. Incubation times to clinical disease are long (years).Inherited forms tend to appear in mid-life.
4Human Prion Diseases Creutzfeldt-Jakob disease (CJD). Human prion disease should be considered in any patient who develops a progressive subacute or chronic decline in cognitive or motor function. Typically adults between 40 and 80 years of age.Creutzfeldt-Jakob disease (CJD).Characterized by a progressive dementiaiCJD (infectious)injection of brain matter from a CJD patient into chimps suggested that CJD was a result of an infectious agent.Patients have been infected iatrogenically from injections of human growth hormone derived from human pituitary gland extracts.fCJD: (familial).Dominantly inherited trait, i.e. heterozygous individuals develop fCJD.Penetrance is 100%, i.e. if the carriers live long enough, they will all eventually develop prion disease.Inherited forms have been demonstrated in a number of families. Genealogic investigations from 4 southern English families show that they are related and argue for the existence of a single founder born more than 200 years ago.Lybian jews. Had been thought to be due to the consumption of lightly cooked/raw sheep brain and eyeballs. Later shown to be due to a specific mutation.Similar mutation found in people originating from Orava in North Central Slovakia, in a cluster of families from Chile, and a large German family living in the US.sCJD (spontaneous)CJD can occur in the absence of a familial history or infection. Can be due to a spontaneous mutation on the PrP gene, or even in the presence of only wild-type genes: a hint into the nature of the process.
5Human Prion Diseases Gerstmann-Steussler-Scheinker disease (GSS) The first of the spongiform encephalopathies that was described as a genetically inherited trait.Linkage analysis from GSS families demonstrated that PrP was the responsible gene.GSS may also arise spontaneously.Fatal Familial InsommniaFFI: characterized by adults generally over age 50 who present with a progressive sleep disorder who die within about a year of onset.More than 30 families worldwide have been identified.FSI (fatal sporadic insomnia). The sporadic form of FFI.KuruAffects the Fore people of New Guniea. Transmitted through ritual cannabalism.
6Regional distribution of PrPSc in transgenic mice inoculated with brain extracts from humans who died of prion diseaseMouse inoculated with brain extract from FFI patientMouse inoculated with brain extract from fCJD (E200K) patientHippocampusVentral posterior lateral thalamic nucleusNeocortexHabenulaThalamusHypothalamusAmygdala
7Animal Prion Diseases Scrapie The classic spongiform encephalopathy of sheepCharacterized by progressive loss of motor controlPropensity of infected animals to obsessively rub or scrape themselves against things (fenceposts, sides of enclosures, etc) to the point of scraping off all their hair and rubbing their skin raw.Horizontally transmissible through the herd.
8Animal Prion DiseasesMad Cow disease (Bovine Spongiform Encephalopathy or BSE)Characterized by progressive loss of motor control.Transmissible.Long incubation period is dose dependent: 2-10 years with a mean period of 5 years.Route of infection has been linked infective sheep and cattle tissue in meat and bone meal, a component of feed for cattle and other domestic livestock.A change in the traditional feed-rendering process was linked to the rise in BSE. The traditional includes an extraction step with organic solvents, which is thought to have extracted out the prion protein. In Great Britian in the early 1980's a new cost effective method was implemented that omitted the organic extraction step. The result was the BSE epidemic of the mid- to late-80's (thanks to Maggie Thacher).
9Animal Prion Diseases BSE (Con’t) Following the introduction of the ruminant feed ban in July1988, and the wholesale slaughter of infected herds, BSE is declining in Great Britian.Small epidemic in France linked to human growth hormone derived from cadaversBSE cow found in Canada in 2003BSE cow found in USA in 2004 – came from Canadian herd.Risks to human healthNew variant of CJD (vCJD) began to appear in humans 8 years after the first known case of BSE in cattle. Although there have only been 21 confirmed cases of vCJD to date, the young ages of the patients and the lack of mutant PrP alleles suggests that they developed disease via an infectious route.
10Animal Prion Diseases Other mammalian prion diseases Transmissible Mink Encephalopathy (TME): Mink are carnivores, raised on farms, infection results from being fed prion-containing meat and bone meal.Feline Spongiform Encephalopathy (FSE): Infection results from being fed prion-containing beef.Exotic Ungulate Encephalopathy (EUE): occurs in the greater kudu, nyala, oryx, i.e. ungulates housed in zoos. Infection results from being fed prion-containing meat and bone meal.Chronic Wasting Disease (CWD). Occurs in mule deer and elk. Unknown etiology, perhaps due to contact with infected farm animals. Concern in Wyoming that it will spread to cattle.
11Etiology, molecular biology & biochemistry The Prion Protein (PrP).Discovery of the prion protein used a mouse scrapie model.Pruisner and co-workers infected mice by intracerebellar injection with brain extracts from Scrapie infected sheep.Provided a bioassay that could be used to identify the prion-causing agent.The infectious fraction is:Nuclease resistant.Resistant to UV irradiation at 254 nm.Sensitive to proteases. Suggests that the agent is a protein.Strongly suggests that the agent is not a nucleic acid.
12The Prion Protein (PrP). Amyloid plaque stained with Congo RedBifefringence under polarized light
13The Prion ProteinInfectious agent purified to a single protein speciesNamed the Prion Protein (PrP).Infectious dose: ~105 PrPSc molecules correspond to one ID50 unit of prions.This leaves open the formal possibility of some nucleic acid contamination, and that the infectious agent is a small protein-associated nucleic acid.No difference in the quantity of PrP between normal and diseased brains.Constitutively expressed in brains of adult animals.Highly regulated during development.Cellular functionsLong term survival of Purkinje neurons??Circadian activity rhythms and patterns??
14Molecular characterization of the PrP gene. Protein sequence analysis was used to clone the PrP gene.Highly conserved gene among all vertebrates.Prion protein gene structure.PrP is encoded by a single open reading frame (ORF) encoding 253 amino acids in all known mammalian and avian PrP genes. Rules out the possibility that the infective form arises from alternative RNA splicing.
15Molecular characterization of the PrP gene. Alignment of the 44 known PrP sequences shows a striking degree of consevation between the mammalian sequences, suggestion the retention of an important function through evolution.However, PrP-null mice are fine. Therefore, PrP is not an essential protein.
16PrP protein structure PrP post-translationally processed to remove: a 22 amino acid NH2-terminal signal peptideat the COOH-terminus, 23 residues are removed during the addition of a glycophosphatidyl inositol (GPI) moiety that anchors the protein to the cell membrane.NH2-Terminal sequence repeatsAmino terminal domain of mammalian PrP contains 5 copies of a P(H/Q)GGG(G)WGQ octarepeat sequenceInsertions of octarepeats have been linked to Human diseaseDeletions of octarepeats do not appear to cause disease, however, deletions do not prevent disease.
17PrP protein structure Conserved Ala-Gly region Between A113 to Y128 is a highly conserved Gly- and Ala-rich region.Structurally, the sequence appears to have been selected for its properties of flexibility, and perhaps for the ability to undergo conformational change.Closest protein sequence is spider dragline silk, a highly β-sheet rich protein that is capable of forming filamentous polymers.A single point mutation A117V is linked to GSS.Two β-sheet (S12 and S2) and three α-helix (A, B & C) forming regions are conserved
19Human mutations fCJD sCJD fGSS FFI extra copies of the octarepeat D178NE200KsCJDHomozygosity for M129VfGSSP102LA117V (the sequence polymorphism that was used to demonstrate the inherited nature of GSS and prion disease).FFIRequires double mutation: D178N + M129VHuman mutations
20Structural studies PrPc is protease-sensitive; PrPsc is protease-resistantPrPc is α-helix rich: 40% a-helix, little β-sheet;PrPsc is β-sheet rich: 30% a-helix, 45% β-sheetNo differences in postranslational chemical modifications were found between the two forms of the protein.Rules out this as a possible cause of prion diseases.
21Structural studies PrPc is soluble PrPsc forms insoluble filaments 45,000X magnification electron micrograph of yeast prion proteinfibers formed in the test tube. The rigid fibers are similar to thoseobserved in amyloid diseases of mammals.
22Allosteric model of Prion propagation Prion form is “enciphered” thru protein conformationNormalformHeterodimerHomodimerPrion+
23Subunit interaction model for prion formation +NormalformHeterodimerHomodimer
28Overcoming the species barrier…implications for hBSE
29Advantages of the prion model Self-encoded protein explains the lack of lymphocytic infiltrationEpidemiology is best explained by the prion model:Spontaneous forms are due to the rare, stochastic conversion of native, wild-type protein to prion form.Inherited form is due to the PrP gene mutations that make the conformational conversion more energetically favorable.Infectious because infectious form also makes the conformational conversion of endogenous PrPc more energetically favorable.Templating/EncipheringIncubation times. Once the species barrier has been crossed, of the host species PrPc to PrPsc becomes more energetically favorable.Strain differences, e.g. FFI vs CJD, and deposition patterns are explained by the prion hypothesis.
30Cannot make or isolate completely pure PrPsc . Problems with the prion modelCannot make or isolate completely pure PrPsc .Therefore, the argument can be made that infectious innoculum contains a small amount of an infectious nucleic acid.
31Opens up new possibilities, e.g. Altzheimers, Sickle Cell disease Q: Does requirement for transmissability limit our conceptual understanding of prion disease?By the allosteric model, any protein that can template a conformational change should also fit the definition.Opens up new possibilities, e.g.Altzheimers, Sickle Cell diseaseEarly-Onset Familial Alzheimer Disease With Coexisting-Amyloid and Prion Pathology (JAMA. 2000;283: )Figure. Double Immunostaining for -Amyloid and Prion Protein (PrP) in the Frontal CortexSenile plaques immunopositive for -amyloid40 (arrowheads in panels A and C), PrP (arrowheads in B), and for -amyloid40 plus PrP (double arrowheads in C) are shown. Twodifferent chromogens were used, first diaminobenzidine dihydrochloride to reveal the -amyloid peptide (reddish-brown) and second, benzidine dihydrochloride to reveal the PrP (blue). When both signals are superposed, the blue signal appears dark and is localized in the center of the plaque.Scale bar is 50 µm for panels A and B, and 75 µm for panel C.
32Fundamental significance of viroids Molecular signals that induce host polymerase to accept viroids as templates fortranscription (missing from cellular RNAs?)Molecular mechanisms of viroid replication (operative in uninfected cells?)How do viroids induce disease?Why are viroids restricted to higher plants?How did viroids originate?T.O. Diener (1987)
33First report of viroid disease - Schultz and Folsom (1923)
35Viroids Smallest known agents of infectious disease Discovered by T.O. Diener (1971)Small, covalently closed circular, single stranded RNAsNo protein coding capacity ( nt)Autonomous replication (no helper virus)29 species (8 genera, 2 families)
36Viroid classification Host range may be broad(HSVd) or narrow (CCCVd)Mechanically transmissableNot insect transmittedMost important vector = man
37Viroid pathogenicity Host contribution Induction of “pathogenesis related” proteinsProtein kinases / signaling cascadesViroid contributionRole of pathogenicity domainInduction of RNA silencing
38Viroid Movement, i.e. spread within infected plant Nuclear / chloroplast importCell-to-cell movementLong distance via phloem
39Movement of viroids and viruses Infected leafDevelopmental controls on movement as wellSource-to-sink movementMoves from infected leaf to roots, up phloem to actively photosynthesizing leaves, then back down the plant
40Origin of viroidsThree pieces of evidence that viroids are relics of a precellular RNA world:Structural periodicity / polyploid genomes: Suggests that they are constructed from very ancient functional RNA domains.Minimal ribozymes / genomic tags: represent ancient enzymesViroids / introns / transposons: all ancient and phylogenetically related to one another
41Phylogeny of subviral RNAs: They’re genetically related to one another ASBVdViroidsLTSVHDV: hepatitis delta virusASSVdAGVdSCMoVGYSVdPlant SatellitevirusesSNMVGVd1BVTMoVHLVdCTiVdSatTRSVCCCVdHSVdCLVdCEVdPSTVdTASVdTPMVd100 ntSatArMVCSVdElena et al Proc. Natl. Acad. Sci. USA 88,
42Hepatitis delta virus: a human viroid? Rod-like secondary structureReplicates in nucleusRNA polymerase IIRibozyme-mediated cleavageProtein coding region /RNA editing
43Some interesting musings… Viroids can only be propagated horizontally/vegetatively.Cannot be propagated vertically, do not make it through meiosis.Origins of sex?Implications for animal cloning?