PrionsFirst identified with Spongiform encephalopathiesCharacteristics of infection:Loss of motor controlDementiaParalysisEncephalitisWidespread neuronal lossWays of infection:Infectious (including diet, after surgical procedures, corneal transplants etc.)Hereditary (autosomal and dominant)

vacuoleSource: UC Davis School of Veterinary MedicineBrain Damage from Spongiform Encephalopathy

Transmissible spongiform encephalopathiesAnimalsBovine spongiform encephalopathy (BSE)Scrapie in sheep and goatsTransmissible mink encephalopathyChronic wasting disease of deer, elkHumansKuruCreutzfeldt-Jacob disease (CJD)Fatal familial insomnia (FFI)Gerstmann-Straussler syndrome (GSS)TSEs are always fatal

Types of TSEsInfectiouse.g., kuru, BSE (mad cow disease), scrapieSpread by consumption of infected materialIatrogenic spread (organ transplant, esp. cornea)transfusionSporadic1-2 million infected worldwide, late in lifeEvidence mounting that some sporadic TSE is really result of infectionFamilialDue to autosomal dominant mutation of PrPInherited at least 10-15% of total human TSE casesEach of these can be transmitted experimentally

KuruIdentified by epidemiology in New Guinea base on anthropological research by Robert and Louise Glasse in 1950s1% of the Fore tribe was afflicted; mostly women, some children, few adult malesSymptoms: headache, joint pain, then 6-12 weeks later, difficulty walking, then death usually within 12 months, always within 2 yearsDisease was of recent origin: ~1910-1920Epidemiological evidence led the Glasses to suggest that endocannibalism was associated with diseaseThis hypothesis was not well accepted among medical community

South Fore

KuruAustralian government suppressed cannibalism among North Fore in early 1950sSouth Fore were convinced to discontinue the practice in 1959Incidence of kuru among North Fore ceased ~ 5 years before South Fore; no child born since then has died of kuruCarlton Gadjusek, a medical research scientist with NIH, inoculated chimps with brain extracts of kuru victims; all chimps died after 50 monthsNo unique antibodies were associated with disease, no virus particles or aberrant nucleic acids were identifiedGadjusek got Nobel Prize; Glasses didnt

ScrapieAn animal model was needed to study TSEsScrapie disease of sheep had many similarities to kuru in terms of symptomatology and etiologyCould be transmitted to hamsters and mice, kuru could notScrapie was used as first good animal model TSE2 month incubation in rodentsInfectious agent purified 5000 foldNuclease resistantUV and heat resistantSensitive to protease (only at high levels) & protein denaturants

+ = inactivated; - = no change in infectivity

Glasses research in1950s and 60sIn 1967 Tikvah Alper at Hammersmith Hospital found particles responsible for transmittable spongioform encephalitis contained no nucleic acids. Following characterization of viroids in 1971, many pursued the viroid hypothesis for prions.In 1982 Stanley Prusiner at UCSF concluded no NA, first named proteinaceous infectious particles that resist inactivation by procedures that modify nucleic acids as PRIONS-received Nobel Prize in 1997.Carlton Gajdusek receive Nobel Prize in 1976

Major Contributors to the History of Prions

Bovine spongiform encephalopathy (BSE)mad cow disease

In Britain in the 1970s, hydrocarbon-solvent extraction of meat and bone meal (MBM) for cattle feed was abandonedIn 1987, BSE emergedIn 1988, BSE became a reportable disease Epidemiology suggested a prion disease, and MBM use was abandonedBSE incubation period is ~5 yearsEstimated that over 1,000,000 cattle were infectedIn 1989, human consumption of bovine CNS tissue (thought to have the highest prion concentration) banned based on fears of transmission to humansIn 1996, a new type of CJD appeared in Britain and France; young patients (

Evidence that BSE gave rise to vCJD in humans

Disease was found in younger cohortCourse of vCJD disease was 14 months rather than 4-6 month for CJD, suggesting more distantly related sourceProteolytic degradation pattern suggests variant CJD (vCJD) closer to BSE than other CJD strainsMouse inoculations showed identical reactions with BSE and vCJD, different from classical CJD; sporadic CJD and all scrapie variants also different from BSE and vCJDWhen transgenic mice expressing bovine PrPc gene were inoculated with vCJD or BSE, course of disease was identical and different from inoculations with CJD or scrapie

Time course of epidemic BSE in the UK 1986-2000, with dates of major precautionary interventions. Mammalian ban on meat and bone meal in March 1996 extended a 1994 ban for farmed food animal species to include all mammalian species. SBO = specified bovine offals (brain, spinal cord, thymus, tonsil, spleen, and intestines from cattle >6 months of age); MBM = meat and bone meal (protein residue produced by rendering).

BSE continues to spread to other areas, but has not become epidemic as it was in Great Britain. It is a major concern because finding it may result in quarantines against beef from the country in which it is found.U.S. - 2003

Cost of Mad Cow Disease3 BSE-infected cows identified in Canada in May, 2003BSE identified in a cow, originally from Canada, in Washington state in Dec., 2003; another in Texas in 2005Embargoes against U.S. and Canadian beef brought immediately by most importersLoss to U.S. and Canadian beef industries so far due to embargoes: approximately $10 billionCanada and U.S. test only a small proportion (

After BSE was found in Japan in 2001, U.S stopped importing Japanese beef; Japanese consumption of beef also plunged

THE FIGURES: THE U.S. STORY (EXPORTS)Year Total beef exports to Japan (in 1,000 USD) Total beef exports to Canada 1998 1,296,265 242,802 1999 1,358,431 225,895 2000 1,449,734 245,003 2001 1,235,392 217,527 2002 831,489 217,690 Jan-May 2003 454,026 111,893

THE FIGURES: THE U.S. STORY (IMPORTS) Year Total beef imports from Japan Total beef imports from Canada 1997 678 603,022 1998 870 722,828 1999 1,435 918,940 2000 248 962,732 2001 0 1,083,866 2002 0 1,096,238 Source: Foreign Agriculture Service USDA (Figures in U.S. dollars)

Different prions affect different parts of the brain

Cerebral cortex When the cerebral cortex is affected, the symptoms include loss of memory and mental acuity, and sometimes also visual imparement (CJD).Thalamus Damage to the thalamus may result in insomnia (FFI). Cerebellum Damage to the cerebellum results in problems to coordinate body movements and difficulties to walk (kuru, GSS). Brain stem In the mad cow disease (BSE), the brain stem is affected.

Convert PrPc into PrScPrSc has identical primary structure but different beta structures leading to resistance of protease cleavage.Brain tissue collects PrSc causing too much protein accumulation.Distinguished by nerve cell death causing large vacuoles and plaques in brain tissue

Effect of prions on neural tissue

How do prions function?PrPScPrPc

Prion biologyFor a prion (PrPSc) to infect a host, the host must have a recognizable cellular form (PrPc) of that prionGenerally, the closer the phylogenetic relationship between the donor host and the recipient, the greater the chance for infection, and the more rapidly symptoms occurLevel of accumulation of prion does not necessarily correspond to level of diseaseMice in which PrPc copy is knocked out have altered sleep/wake cycles and circadian rhythm

Species barrierInfectous dose between species is usually higher than between animals of the same species (possibly a million fold), but it is sometimes the same (e.g. between scrapie doses for mink) When a species has been infected with a TSE of a different species it can then go on to infect a range of animals that the original species could not, and with a different dose. When a species has been infected, it can infect additional animals of the same species with much lower doses of agent. The histopathology of the disease in an animal infected from another species is not the same as if it had been infected from one of the same species. The incubation period of an animal infected from another species is much longer than that of an animal from one of the same species.

Criteria for prion demonstrationTransmissible and associated with phenotypeReversible curability from cured individual, phenomenon can arise again because the same event may reoccur in the same genotypeOverproduction of normal protein increases frequency of prion formation more normal molecules will be converted to prion formPhenotype relationship of prion and mutation of the normal gene for its protein in the host

Sequence of prion protein

Cellular trafficking of PrPC and PrPSc. PrPC (yellow dots) follows the secretory pathway of the cell through the endoplasmic reticulum (ER) and the Golgi. Mature PrPC is inserted via its GPI anchor into plasma membrane lipid rafts. The conversion of PrPC to PrPSc (orange ovals) occurs either on the cell surface or, following endocytosis, in a cellular compartment such as the endosome. PrPSc formed at the surface and released into the extracellular space may cause the plaques seen in TSE diseases such as human vCJD. The diffuse PrPSc deposits and neuronal vacuolation common to many sheep scrapie strains may be due to PrPSc formation in endocytic compartments or to endocytosed surface PrPSc accumulating inside the cell. Misfolded PrPC (squiggle) accumulating in the cytosol may also trigger PrPSc formation. (Inset) Structure of PrPC showing the GPI anchor, the glycan chains, the copper-binding octapeptide repeats, and the regions where the helices and loop structure of PrPC (red, blue) may be converted to the sheets of PrPSc. ERAD, endoplasmic reticulum associated degradation.Cellular trafficking of PrPC and PrPSc

Advances in prion controlBSE-resistant cattleBovine PrPc gene cloned, modified by site-directed mutagenesis to produce BSE-resistance form Cattle were transformed with modified form of the gene, targeted to replace natural PrPc geneTransgenic animals homozygous for mutant gene express mutant copy and are resistant to BSE, but do not show altered sleep/wake cycles as seen in knockout miceDepleting neuronal PrPc in prion infection prevents disease and reverses spongiosisUsing transgenic mice, first demonstration that prion infection and pathology can be reversed by ceasing expression of endogenous PrPc copy

Prions of yeast and fungiYeast and filamentous fungi make great experimental tools because they are eukaryotes that normally grow as haploids with small genome sizes and powerful geneticsPrions in yeast first identified by Wickner as non-Mendelian elements associated with nitrogen metabolism [URE3], then as a component of a suppressor tRNA activity [PSI].The first prion in filamentous fungi was identified in association with heterokaryon (vegetative) incompatibility in the ascomycete Podospora anserina This is the only prion identified to date that is not associated with a diseased state

Identity of alleles at the het-s locus is required for hyphae of different Podospora colonies to fuse. However, an encounter of het-s and het-S colonies will only result in the lethal reaction that comprises the incompatibility reaction if the Het-s protein is in its prion form (called [Het-s]).From Wickner, 1999, J.Biol. Chem. 274: 555The [Het-s] prion results in heterokaryon incompatibility in the filamentous fungus Podospora anserina.

Structural model of the SHaPrPC molecule. The model depict relative sizes of and locations of the Asn-linked oligosaccharides relative to the published structure of SHaPrP fragments inferred from NMR spectroscopy. SHaPrPC is shown attached to the plasma membrane by its GPI anchor to indicate how the range of movement of the N-terminal half of the molecule might be constrained in vivo. The putative protein X binding sites are indicated with an X with lines pointing to the discontinuous epitope on helices C and B with which it interacts (Kaneko et al., 1997). Adapted from DeArmond et al. (DeArmond et al., 1999).