Prions Microbial Pathogenicity 2013 2014 Stephanie Boothphacaspc gc
Prions Microbial Pathogenicity 2013 -2014 Stephanie. Booth@phac-aspc. gc. ca
Topics to be Covered What are prions? n How are prions propagated? n How do prions enter the body? n The prion diseases n ¨ Animal diseases ¨ Human diseases n How can the prion problem be resolved?
What are prions? n Proteinaceous infectious agents n Prion diseases are a group of fatal neurodegenerative diseases affecting humans and diverse animal species n Prion diseases are unique in that they can be inherited, they can occur sporadically, or they can be infectious. n The infectious agent is different from conventional microorganisms n a misfolded protein is the main (and perhaps the sole) component of this unorthodox infectious agent
What differentiates Pr. PSc from Pr. PC -helix B -helix A -helix C Pr. PC -helix C Model of Pr. PSc (Pathologic Prion)
Another name for prion diseases is Transmissible Spongiform Encephalopathies (TSE) n n n This describes the main features of the disease – they are all transmissible They all show unique histopathology in the brain – small holes reminiscent of a sponge They all affect the brain
Spectrum of prion diseases of humans and animals
Background n n n The infectious nature of prion diseases was first evident more than 70 years ago when accidental transmission of scrapie (prototypic prion disease) occurred in sheep. Inoculation against a common virus with a formalin extract of tissue unknowingly derived from an animal with scrapie infected nearly 10% of the flock. Scrapie was subsequently transmitted experimentally to sheep and later to mice.
Prions are very small!! n The same group also reported that the minimum molecular weight to maintain infectivity (around 2 × 105 Da) was too small to possibly be a virus or other type of micro-organism. n Interestingly, this estimate is in the range of the size of the most infectious prion particles obtained with modern sophisticated techniques. This study identified clusters of 14 to 28 prion proteins as being the most infectious, and that clusters of less than six protein molecules have virtually no infectivity. n
No virus particles, bacteria, or fungi have been conclusively associated with prion diseases n n All but a few scientists agree Laura Manuelidis still asserts there is a virus present Electron micrographs of 120 S infectious material fractionated from hamster CJD brain. A – dark arrows point at the 25 nm particles. Open arrows show Pr. P associated gold particles. B – Higher magnification of A. Bars are 100 nm (Manuelidis, 2007).
Prions are Resistant to Radiation n Alper and coworkers demonstrated that procedures that obliterate nucleic acids did not destroy the infectious Alper T, et al. Does the agent of scrapie replicate without nucleic acid? Nature 1967; 214: 764– 766. Ø Irradiate UV light at increasing doses Ø One of the controls was the highly resistant organism Micorcoccus radiodurans Ø UV light forms cyclobutadiene thymine dimers in DNA whenever two T's are adjacent preventing the DNA from being replicated. ØScrapie titer was measured by serial dilution, using intra-cranial injection of 7 -8 mice at every dilution. Ø Scrapie resistance was complete at dosages that knocked the control viability down 3 logs in activity Ø Earlier studies had found the same result with ionizing radiation.
The Prion Hypothesis Prior to the discovery of prions, it was thought that all pathogens used nucleic acids to direct their replication. The "protein-only hypothesis" states that a protein structure can replicate without the use of nucleic acid. The idea was first proposed by Griffith in 1967. n This was initially controversial as it contradicts the so -called "central dogma of molecular biology", which describes nucleic acid as the central form of replicative information. The Nobel Prize in Physiology or Medicine 1997 was awarded to n Now much evidence Stanley B. Prusiner "for his discovery exists to support this of Prions - a new biological principle of hypothesis infection" n
The Prion Protein
Infectivity is associated with Pr. PSc protein and is reduced by agents that destroy protein structure n n Brain microsomes isolated from scrapie-infected hamsters contained Pr. PSc enriched approximately 5700 -fold with respect to total brain protein Na. Dod. SO 4 (SDS) reduces scrapie infectivity Polyclonal rabbit prion protein antiserum raised against Na. Dod. SO 4/PAGE-purified scrapie prion protein of 27 -30 k. Da reduced scrapie infectivity dispersed into detergent-lipid-protein complexes by a factor of 100. These results represent direct immunologic and chromatographic demonstrations of a relationship between Pr. PSc and prion infectivity and provide support for the contention that Pr. PSc is a major component of the infectious scrapie particle.
Scrapie-associated fibrils (SAFs) Incubation time bioassay Endpoint titrations Accelerated studies nearly 100 -fold 1981 scientists purified rodshaped structures – SAF’s – from concentrated suspensions of hamster infected scrapie brain Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain.
Prion Protein Isolated Proteinase K n n ~30 KD n S S N N 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 (PK) distinguished it from proteins of similar molecular weight found in normal hamster brain. The amount of this PK resistant protein correlates with the titer of the agent.
Identification of the Gene Coding for Prion Protein Amino acid sequence used to produce oligo probes to screen libraries from infected and uninfected cells + - scrapie infected uninfected
Identification of the Gene Coding for Prion Protein Amino acid sequence used to produce oligo probes to screen libraries from infected and uninfected cells Single copy host gene Exons are shown in light blue and the coding region (entirely contained in exon 3) is shown in dark blue The m. RNA for Pr. P found in both scrapie–infected and uninfected mice The Prnp gene codes for a normal host gene
Animals Lacking Pr. PC do not Contract Prion Disease. n n Prnp 0/0 mice devoid of Pr. PC show normal development and behavior. When inoculated with mouse scrapie prions, they remained free of scrapie symptoms while wild-type controls all died within 6 months. After introduction of Syrian hamster Pr. P transgenes, Prnp 0/0 mice became highly susceptible to hamster but not to mouse prions. These experiments show that Pr. PC is required for susceptibility to scrapie and that lack of homology between incoming prions and the host's Pr. P genes retards disease.
Prion protein n n n n Branched-chain Carbohydrate (attached to Asn residues) Normal host protein Expression enriched in CNS Expression is regulated during development GPI linked glycoprotein Disulphide bridge Enriched in lipid rafts linking two internal (detergent-resistant cystine residues membrane islands) Highly conserved (throughout mammals & perhaps amphibians) 3 alpha helices 2 stranded anti-parallel beta sheet
Cellular model of Pr. PSc formation and deposition • Pr. PC is produced in ER, processed in golgi, and transported to cell surface • Rapidly cycled by endocytosis • Conversion occurs on cell surface or in endosomes • (acid environment? Accessory molecules? ) • Accumulations may be neurotoxic directly or by changes induced in accessory cells
What does the normal form of Pr. PC do? Pr. PC has many implied functions although knock out mice have very subtle characteristics. n If we had a greater understanding of what Pr. PC does this may provide clues for the design of therapies n
Copper Binding: transport, carrier, enzyme? §Pr. PC is a metal ion-binding protein as it binds copper with low micromolar affinity §Mice devoid of Pr. P harbour 50% lower copper concentrations in synaptosomal fractions §Pr. Pc has been shown to harbour activity of a copper dependent superoxide dismutase Copper may play a structural role in Pr. P or influence binding to other proteins May play a roles in copper regulation at the synapse May have a role in re-uptake of copper across the plasma membrane
Cellular localization of Pr. PC § Pr. PC found at the cell membrane in cholesterol-rich lipid rafts § In epithelial cells (polarized) associated with basolateral location (unusual for a GPI-anchored protein) § In stomach epithelial cells - Pr. PC in secretion granules § Pr. PC localized in secretory mammary gland tissues - an association with exocytosis?
Pr. PC interacting molecules Prion interacting proteins: - §GFAP §Bcl-2 §Hsp 60 § 37 k. Da laminin receptor precusor §Protein disulphide isomerase §Calnexin §Calreticulin §Laminin §Heparan sulphate
Synaptic Function Pr. PC is associated with the synaptic membrane and synaptic vesicles; probably cycling between the two Synapsin Ib - Pr. Pc interacting protein • associated with small synaptic vesicles • synapsin and Grb 2 copurify with Pr. Pc in neuronal microsomal vesicles • some laboratories have noticed anomalies in neuronal excitability and synaptic transmission in Pr. PC null mice
Other Putative Functions Role in signaling interaction with fyn Role in cell survival and differentiation - Pr. Pc protected cells from Bax mediated cell death - Pr. Pc overexpression made cells more sensitive to staurosporine
Differences between Pr. PC and Pr. PSc
Prion rods composed of Pr. P 27 -30 100 nm
Trimeric left-handed -helical model of Pr. P 27 -30 2 D crystals Processed Image (Uranyl acetate (crystallographic staining) average) 1 THJ (bacterial carbonic anhydrase) Indication for a trimeric arrangement. Left-handed -helices readily form trimers
A trimeric left-handed -helical model of Pr. P 27 -30
Size of infectious agent 14 -28 prion molecules
How are prions propagated? n n Prion protein is absolutely required for infection No nucleic acid is involved Animals can be infected by inoculation with Pr. PSc from brain Prions consist of a self-propagating protein How does this work?
Prion growth appears to be akin to a crystal growth and fragmentation model n nucleation-dependent polymerization reaction (where the formation of growth nuclei from soluble proteins is slower than the elongation of preformed nuclei. Secondary nucleation mechanisms lead to the formation of additional nuclei from preexisting filaments. Secondary nucleation likely results via filament fragmentation
Cell-free conversion of Pr. PSc n n n If no factors other than prion protein are required for infectivity conversion of Pr. PC to Pr. PSc should be possible in vitro. Caughey and co-workers proved this principle by purifying Pr. PC and mixing it with stochiometric amounts of purified Pr. PSc. They produced a low yield of Pr. PRES, proteinase K resistant Pr. P reminiscent of Pr. PSc. This reaction was performed under nonphysiological conditions that precluded the generation infectivity. The next step, crucial to proving this was the invention of protein misfolding cyclic amplification (PMCA) technology
PMCA n n n PMCA is based on the assumption that prion replication occurs by a seeding/nucleation model Pr. PSc seeds bind and convert Pr. PC by incorporating the protein into the polymer. In PMCA, Pr. PSc and Pr. PC are mixed and incubated, allowing the misfolding of Pr. PC, which permits the incorporation and growth of Pr. PSc aggregates. After incubation, samples are submitted to sonication in order to fragment Pr. PSc polymers, thereby generating new free ends suitable for continued prion replication. This process is cyclically repeated in order to produce an exponential amplification of Pr. PSc conversion.
Is the prion hypothesis proven? n n n Despite the compelling evidence for the prion hypothesis, skeptics argued that definitive proof required the production of infectious material in vitro from pure, normal prion protein. In 2004 the production of “synthetic prions” via the in vitro induction of misfolding and aggregation of bacterially expressed recombinant prion protein (r. P) was reported. Recombinant mouse prion protein produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of β sheet–rich structures. Fibrils consisting of were inoculated intracerebrally and these mice were sick after 330 days and had protease resistant prion deposits in the brain. Unfortunately these prions were only transmissible to transgenic animals overexpressing Pr. P, and not to wild-type animals. This was a matter of concern, given the well known propensity for such animals to spontaneously develop a prionlike disease
n n n Use of a different protocol to induce misfolding to a beta-sheet enriched amyloid form of recombinant Pr. P produced in Escherichia coli, generated infectious prions that induced disease in wild type animals. Serial transmission in hamsters gave rise to a disease phenotype with highly unique clinical and neuropathological features Disease was characterized by prion like lesions and lots of Pr. PSc accumulation but slow clinical disease
Still a caveat? n n n A couple of caveats were still raised from this data ¨ 1. The disease was very slow long incubation period, and incomplete attack rate ¨ 2. Were the prions already present in the normal brain at very low levels; so that they do not cause disease within the normal life-time of the animal, however, this process is somehow accelerated on inoculation. One study showed infectivity was detectable in cells at low frequency following incubation of uninfected mouse brain homogenate on metal wires that presumably concentrated prions (Prions stick to metal!) These may be models of spontaneously occurring prion diseases? (those diseases that have no known cause)
Final Proof…. . n n n infected 8 -week-old female CD-1 mice by intracerebral injection. The r. P-res (inoculum 4) was prepared by propagating r. P-res through 24 rounds of PMCA. Inoculum 1, consisted of all the components used for r. P-res propagation except for rec. Pr. P and r. P-res seed. Inoculum 2, consisted of all the components of r. P -res propagation except for r. P-res seed, was incubated at 37°C for 24 days without sonication, and subjected to the same pelleting and washing treatments. Omitting the sonication step prevented the de novo r. P-res formation in this control sample. Inoculum 3 was prepared by directly mixing rec. Pr. P, POPG, and RNA in the inoculum diluent. The amount of each component was equal to the total amount in the final pool of inoculum 4, ensuring that the result was not influenced by insufficient dosage of rec. Pr. P or any other component. 150 dpi incubation period
Generation of a recombinant Pr. P seed Perform PMCA with recombinant protein, plus a mixture of lipid and polyanion (RNA) to simulate components of a cellular environment
From: Claudio Soto, Prion hypothesis: the end of the controversy? Trends in Biochemical Sciences Volume 36, 151– 158, 2011
How do Infectious Prions Enter the Body? n Most human prion disease occurs for unknown regions and germ line mutations. n Most infectious prions enter the body by extraneural pathways. n How can a protein or protein aggregate cross mucosal barriers, bypass innate and adaptive immunity and cross the blood brain barrier to cause disease?
Oral uptake n n Natural transmission of prions has taken place via the oral route in a number of animal and human infections. This has caused large epidemics or epizootics. Prions, despite being made up only of protein, are incredibly resistant and can pass through the gut environment of low p. H and enzymatic attack. They can also survive procedures such as irradiation and cooking
Resistance of Prions n Prions are resistant to heat, chemicals and enzymes n autoclaving ineffective UV and ionizing radiation have no effect on infectivity Infectivity of agent resistant to ribonuclease digestion, psoralen photoadduct formation, Zn 2+-catalyzed hydrolysis, and chemical modification with NH 20 H Difficult to remove prions from food/medical products n n n
Intracerebral Administration n n Most efficient means of transmission Mainly occurs experimentally…. Unfortunately several iatrogenic CJD (i. CJD) transmissions have occurred via neurosurgery and dura mater grafts. Electrodes for stereotactic electroencephalographic (EEG) used for a CJD patient and reused after sterilization with ethanol and formaldehyde vapor (neither of which are capable of decontaminating prions)
Peripheral exposure n n Intraperitoneal inoculation is a widely used route for experimental studies A second route for iatrogenic CJD infections Growth hormone, to treat dwarfism, and fertility hormones used to be prepared from cadaveric pituitary glands. This led to over 160 prion deaths. v. CJD can be transmitted from person to person via blood transfusion
Aerosols n n Prions are not considered airborne pathogens However, recently (albeit in rather artificial experimental conditions) prions have been transmitted to mice through aerosols Did they really inhale these prions where they crossed the nasal mucosa, or did the aerosol pass into the throat where the inoculum was ingested? No evidence of this type of transmission in a natural situation
How do Prions Reach the Central Nervous System? n Excellent review for more information: Mabbott NA & Mac. Pherson G. 2006. Prions and their lethal journey to the brain. Nature Reviews Microbiology 4: 201 -211
Tissues involved in TSE pathogenesis Infectivity is often first detectable in the spleen and other lymphoreticular tissues prior to neuroinvasion CNS Peripheral nerves Gut
Pr. P can be detected in the gut following ingestion TSE diseases are usually acquired by peripheral exposure, eg: ingestion. Route of entry may follow invasion of Peyers patches and other gut lymphoid tissues. Pr. PSC’s resistance to proteases presumably allows it to survive fairly well in the gut
Peyers Patches §When lymphoid tissue involvement was assessed, the earliest Pr. PSc deposition occurred in Peyer’s patches and enteric ganglia. §Pr. PSc was associated with follicular dendritic cells, ‘dome’ macrophages, M cells Enteric ganglia and Peyer's patches are early reservoirs of infection and that they may be important sites for subsequent neuroinvasion
Potential mechanisms of transmissible spongiform encephalopathy (TSE)agent translocation across the intestinal epithelium.
Are FDCs potential targets for intervention? § Cytokines produced by B cells provide important stimuli that induce follicular dendritic cell maturation and maintain them in their differentiated state. §Lymphotoxin-beta signalling blockade temporarily inactivates FDCs
Innervation of the spleen and lymph nodes • The spleen is innervated by sympathetic nerve fibers closely associated with the splenic artery. Some fibers extend into the white pulp. • May be the route of neuroinvasion although few, if any, nerve fibers associate with follicular dendritic cell in the B-cell follicles.
Potential role of exosomes in Pr. P transport • DCs, lymphocytes, mast cells, platelets, reticulocytes, melanoma and epithelial cells secrete exosomes • Approximately 30 -100 nm • Shown to carry infectious prions
Human Prion Diseases Inherited/Sporadic – Creutzfeldt-Jakob disease (CJD) • Sporadic (s. CJD) • Inherited (f. CJD) – Gerstmann-Straussler. Sheinker (GSS) – Fatal Familial Insomnia (FFI) – Fatal Sporadic Insomnia (FSI) Transmissible – Kuru – Iatrogenic CJD (i. CJD) – New variant CJD (v. CJD) • Bovine Spongiform Encephalopathy (BSE)
s. CJD n Most common human Transmissible Spongiform Encephalopathy ¨ Approx 95% n Distributed worldwide n Incidence estimated at 1 per million ¨ US: n 0. 1/million in <55 yo n 5. 3/million in >55 yo ¨ Mean age of onset of 60 years
s. CJD symptoms n Typical Clinical Presentation ¨ Generally rapid progression ¨ Triad n Dementia n Myoclonus n Characteristic EEG ¨ No gender predilection ¨ No epidemiological factors identified ¨ Duration of illness: 4. 5 months
s. CJD diagnostic criteria n Diagnostic Criteria ¨ Definitive n n n Progressive dementia in < 2 years Periodic sharp wave EEG or 14 -3 -3 protein in CSF 2/4 of the following ¨ ¨ ¨ Myoclonus Visual and/or cerebellar symptoms Pyramidal and/or extrapyramidal disease Akinetic mutism Possible n Above criteria with no EEG or CSF protein
Typical Periodic Pattern on EEG Generalised bi- or triphasic periodic sharp wave complexes appear with a frequency of around 1 -2 per second.
CSF in s. CJD ¨ No inflammatory cells ¨ Total protein content may be elevated 14 -3 -3 Protein ¨ Normal neuronal protein released by multiple insults n n n n Herpes simplex encephalitis and other viral encephalitides Recent cerebral infarction or haemorrhage Subarachnoid haemorrhage Hypoxic brain damage Glioblastoma Carcinomatous meningitis Paraneoplastic encephalopathy Rule out these conditions – biomarker for CJD
• Gerstmann-Straussler-Scheinker Syndrome – Incidence: 2 per 100 million – Autosomal dominant (>50 families) – Age of onset: 43 -48 yr – Duration of illness: 2 -6 years – Clinical • Incoordination • Ataxia • Dementia and myoclonus (late) • Fatal Familial Insomnia – – – Approx 27 families Autosomal dominant Age of onset: 36 -62 yr Duration of illness: 8 mon-6 years Clinical • Disrupted sleep > intractable insomnia • Autonomic hyperactivation • Dementia • Myoclonus Chromosome 20
PRNP codon 129 genotype frequencies MM MV VV Normal population 37% 51% 12% Sporadic CJD 63% 19% 18%
Kuru n an infectious origin was suspected for kuru, a prion disease identified in some geographically isolated tribes of New Guinea in the 1950’s Fore tribe
Kuru Figures n Incidence 1 % (population ~ 15, 000) F : M = 10 : 1 n < 20 years of age F : M = 3 : 1 (20 % of cases) n 20 -40 years of age F : M = 15 : 1
Etiology of kuru n n Primary infectious: no fever, no CSF cell raise Genetic disease: Pro: in families, in patients who moved out of Fore region. ¨ Con: also in patients who came to live in Fore region, as well in young as in old patients. ¨ n n Toxic/deficiency: no toxic compound isolated, balanced diet. Endocanibalism In 1976 Carleton Gajdusek became corecipient of the Nobel Prize in Medicine for his "discoveries concerning new mechanisms for the origin and dissemination of infectious diseases. "
Similarities to previously described prion diseases n Scrapie n CJD (first described in 1920)
Kuru is transmissible n a formal demonstration came in 1966 with the transmission of kuru to monkeys
How BSE changed the face of prion research n New impetus for research in the prion field came when an outbreak of a prion disease in cattle called BSE (Bovine Spongiform Encephalopathy) in Great Britain was mediated by prion infection. n Between the discovery of this disease as a prion disorder and the implementation of measures to prevent possible transmission to humans, it is likely that the entire population of UK and a large part of Europe might have consumed infected meat. These initial concerns were confirmed when a new form of human disease, termed variant Creutzfeldt-Jakob disease, appeared and was convincingly linked to BSE. n
BSE epidemic in the UK n n n The clinical signs of BSE may include tremors, gait abnormalities particularly of hindlimb (ataxia), aggressive behavior, apprehension, and hyperreactivity to stimuli. Pr. PSc accumulation and spongiform vacuolation are usually found in the brain. Long incubation period, usually 2 -8 years Therefore most affected cattle are 3 to 6 years of age Disease not recognized until thousands of animals were already infected – nearly 200, 000 infected animals were identified
Geographical distribution of BSE
How was BSE caused n n n The main cause of the disease was the feeding of MBM (meat and bone meal) to previously uninfected cattle. Cattle are normally herbivores, and the MBM included the remains of other cattle, which, when fed to uninfected cattle, caused the disease to spread more rapidly. These included concentrates and milk replacers fed to calves under 6 months which appeared to be efficient disease transmitters. The BSE prion agent survived the rendering process (by which animal remains or byproducts are made into useful byproducts) Prior to the epidemic the rendering heating processing in was reduced. It is likely that this meant that the infectious agent remained active and therefore spread more rapidly. There are 2 possible origins of the very first BSE prion likely during the 1970’s: ¨ the disease may be a variation of Scrapie – a disease in sheep, which was transmitted to a cow and then subsequently amplified in feed. ¨ the disease could have evolved from a spontaneous (sporadic) case of the disease in a cow.
Insufficient inactivation? Animal feed Meat and bone meal Rendering Sheep scrapie Nyala and Kudu Cat Experimental infection Mice Experimental infection Very similar pathology and incubation periods, clearly distinguishable from contemporary scrapie
What parts of a cow are infectious n n At the terminal stages of the disease, BSE prions may be detected in spinal cord, retina, ileum, adrenal glands, tonsils, bone marrow, peripheral nerves, dorsal root ganglia, trigeminal ganglion and thoracic ganglia. BSE infectivity is present in brain tissues as early as 2 years after post inoculation. Epidemiological and transmission studies have found no evidence of BSE prions in milk, semen or embryos. There is little to no evidence of its horizontal transmission (mother to fetus). BSE doesn’t seem to be shed in urine or feces and so we don’t see environmental contamination
The origins of v. CJD n Within weeks of the identification of BSE concern over the risk of transmission to humans began: ¨ 1990 setting up of CJD surveillance unit ¨ 1993 extension of this surveillance to Europe ¨ Transmission to domestic cats and zoo ungulates was found to be widespread
CJD surveillance n During first 10 years after BSE was identified the incidence of CJD did not increase in the general population and there were no increase in cases for high risk groups eg abattoir workers n May to October 1995 – three unusual cases of CJD were reported patients were 16, 19 and 29 years old all had amyloid plaques (usually only in 5 -10% of s. CJD) n December 1995 – 2 more suspected cases n These young patients all had a distinctive and extensive plaque formation the similar clinical syndrome distinct from s. CJD ¨ ¨ ¨ young age at onset early psychiatric symptoms prominent ataxia absence of periodic electroencephalographic activity comparatively prolonged illness no mutations identified
Differences between v. CJD and s. CJD §Large florid plaques §Prion deposits in lymphoid tissue, such as tonsils §Differing MRI profile §Differing EEG profile § 14 -3 -3 rare in CSF
Proving v. CJD is a new prion disease originating from BSE n Experiments were performed to look at the disease characteristics which are conserved between different prion strains: To determine whether BSE and v. CJD prions had the same transmission characteristics in rodents ¨ To determine whether Pr. PBSE and Pr. Pv. CJD were biochemically identical ¨ To determine whether the brain pathology is the same in BSE and v. CJD ¨
To determine whether BSE and v. CJD prions had the same transmission characteristics in rodents Scrapie BSE FSE Transmit scrapie, BSE, v. CJD to 3 strains of wild-type mice and measure the incubation periods v. CJD
v. CJD and BSE glycoform similarity a) b) Proportions of di- to mono- glycosylated prion protein in CJD, v. CJD and BSE Western blot after Protease K treatment 1 – v. CJD 2 – v. CJD inoculated mouse 3 – BSE 4 – BSE inoculated mouse
v. CJD and BSE brain pathology show very similar characteristics Scrapie BSE v. CJD
Cluster of 5 human nv. CJD cases in a village in Leicestershire A local “knacker’s yard” owner was convicted and jailed in 1982 for supplying meat “unfit for human consumption”
Comparison of sporadic and variant CJD s. CJD v. CJD Age at death (y) 66 28 Duration of illness (m) 4 13 Psychiatric features Rare Common Rapid dementia Common Rare Sensory symptoms Rare Common Myoclonus Common
How can the prion problem be resolved? n Prevention ¨ Many steps for prevention have been undertaken resulting in the virtual irradication of the BSE epidemic and scrapie ¨ CWD is an ongoing problem, rapidly spreading across North America n Cure ¨ No significant advancement towards a cure as yet!
Protecting the food supply from BSE n n Active post-mortem surveillance Ban on feeding ruminant’s to ruminants in feed. Ban the inclusion of tissue that could contain high levels of prions CNS tissue and ileum in cattle. In some countries - a ban on the inclusion of MBM from all mammalian species in feed
The course of the BSE epidemic
Protecting patients from iatrogenic transmission Use of recombinant products such as hormones n Use of disposable equipment in brain or lymph node surgeries if possible n No blood screening test available at present although a test for v. CJD in blood is in development in the UK n
v. CJD is transmissible in blood Brown et al. (1998): Rodent blood contains ~1 ID 50 unit/ml based on rodent bioassays. Hunter et al. (2002): Transfusion of 400 ml of blood from sheep with scrapie or BSE produced disease in many recipient sheep. Llewelyn et al. (2004): 69 y. o. male (MM 129) died of v. CJD in Britain. He received a blood transfusion 6. 5 years ago from a young donor, who subsequently died 3 years ago after developing v. CJD. This patient was first of 48 recipients who received blood or blood products from 15 donors who eventually developed v. CJD. Peden et al (2004): The second of these 48 recipients died of a nonneurological disease but had Pr. PSc in the spleen and a cervical lymph node. The patient (MV 129) received a blood transfusion 5 years ago from a young donor, who subsequently died 3. 5 years ago after developing v. CJD. Hilton et al (2004): 3 of 12, 674 appendixes taken at routine operation were found to have Pr. PSc arguing that >4, 000 people in the UK currently harbor v. CJD prions in lymphoid tissues. (Feb 2009) v. CJD transmission plasma-derived factor concentrates to a haemophilia patient
176 victims of v. CJD in UK We can estimate the average incubation period for the development of VCJD from this graph ~ distance between the peaks
s. CJD numbers stable in the population Mean age at death 67 (range 20 -95) Median age at death 68 Mean age at onset 66 (range 15 -94) Median age at onset 67 Median duration of illness 4 months (range 1 -74) Male/female illness 578 male/575 female Genotype 63% MM 19% MV 18% VV Monitoring numbers and characteristics of CJD cases enables any unusual cases or increased numbers that may indicate the emergence of a new disease
Curing prion disease n Many unknowns: ¨ ¨ ¨ ¨ ¨ Why do brain cells die when prions replicate in the brain? What is the toxic form of the agent? Does the replication of the agent to the toxic form interfere with the normal role of the cellular prion protein? Is the infectious agent a different species from the toxic agent? Animal experiments suggest that neurons may be rescued during early stages of the disease, is this true in humans? How do we diagnose the disease before the clinical signs are not severe? Can we identify biomarkers as a tool to identify patients that could be treated? If we block prion replication will we stop the progress of disease? Can we induce neurons to regenerate to repair damage? Could treatments be of general use to multiple neurodegenerative conditions?
Treatment by removal of Pr. P n BSE-resistant cattle Bovine Pr. Pc 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 Pr. Pc gene ¨ Transgenic 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 mice ¨ n Depleting neuronal Pr. Pc in prion infection prevents disease and reverses spongiosis ¨ Using transgenic mice, first demonstration that prion infection and pathology can be reversed by ceasing expression of endogenous Pr. Pc copy
Dementia – a burgeoning public health problem Disease Number of cases in US Alzheimer’s disease 4, 000 Parkinson’s disease 1, 500, 000 Dementia with Lewy bodies 350, 000 Frontotemporal dementias 200, 000 Progressive supranuclear palsy 35, 000 Multiple system atrophy 35, 000 Huntington’s disease 30, 000 Motor neuron disease 30, 000 Prion disease 300 Population 305 million
Research Priorities § Treatments and Early Diagnosis (go hand in hand) § Identification of genetic, epigenetic and environmental risk and protective factor
Neurological diseases associated with abnormal brain proteins Disease Abnormal protein Alzheimer’s disease Aβ, tau Parkinson’s disease α synuclein Creutzfeldt-Jakob disease Pr. PSc Huntington’s disease huntingtin Multiple system atrophy α synuclein, tau Lots of overlap with prion diseases : – • Early dendritic, synaptic alterations and loss – at this stage disease may be “curable” • Misfolding of associated proteins seems to be possible by a similar mechanism of template dependent refolding allowing cell-to-cell spread
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