WP1-3: Mutant PrPs Induce Distinct Neurodegenerative Diseases and the Spontaneous Generation of Prion Infectivity
Walker S. Jackson and Susan Lindquist
Whitehead Institute for Biomedical research; Massachusetts Institute of Technology; Howard Hughes Medical Institute; Cambridge, MA USA
Key words: prion protein mutation, genetic prion disease, transgenic model
Familial forms of prion diseases are a particularly fascinating subset in which multiple, strikingly different disease types can be caused by several mutations of the same protein, PrP. In order to investigate the cause of these differences we have developed mouse lines modeling two very different prion diseases, Creutzfeldt-Jakob disease and fatal familial insomnia. These lines differed from a third line expressing a non-mutated control version, by one amino acid, and all three were engineered by gene targeting at the endogenous PrP gene locus. Animals were subsequently phenotyped using an automated mouse behavioral analysis system and an in vivo magnetic resonance imaging procedure. We found that both of the mutant lines developed specific, neurodegenerative diseases spontaneously. The diseases were strikingly different from each other but very similar to the diseases of humans carrying the corresponding mutations. Since the mutations were “knocked-in” to the native PrP gene locus, phenotypic differences were not caused by differences in gene expression patterns. Furthermore, each disease could be transmitted to mice expressing non-mutated prion protein via injection of brain homogenates from each of the mutant lines, but not from the control line. Thus, various mutations in PrP are sufficient for the spontaneous generation of distinct prion diseases.
WP2-1: Animal TSEs – Epidemiological Surveillance of Potentially Zoonotic Diseases
Giuseppe Ru
Reference Centre for Animal Encephalopathies; Institute for Zooprophylaxis; Torino, Italy
Key words: epidemiology, risk assessment, surveillance, animal TSEs, atypical, BSE, scrapie
Introduction. The onset of some cases of Creutzfeldt-Jakob disease (CJD) in 1994–1995 with an atypical clinical and pathological presentation suggested the emerging of a new variant (vCJD) and dreadful disease. Since then, strong scientific evidence linking vCJD and bovine spongiform encephalopathy (BSE) in cattle has been cumulated. Moreover new concern came from the discovery that BSE may circulate within the sheep population and the detection of atypical strains of both scrapie and BSE. As a consequence a huge effort has been put internationally in surveillance on ruminants. Aim of this presentation is to provide a global description the international results of the testing for animal spongiform diseases.
Results and conclusion. Scrapie has a heterogeneous geographical distribution compared with the NOR98 atypical strain which doesn’t show any evidence of between animals contagiousness. BSE is keeping to fade out; however the differences in size and peaking of the national epidemics may have consequences in the human spongiform risk. With regards to the atypical BSE forms identified so far (L- and H-type), the small number of cases prevents any interpretation although they don’t look to be related each other or to BSE.
Methods. Data were obtained through a review of the relevant literature (epidemiology and risk assessment of spongiform diseases) and from a number of official web sources. The worldwide temporal and spatial distribution of the classical and atypical animal spongiform diseases has been compared calculating rates and relative risks and integrating them with the results of risk assessment exercises.
WP3-3: Update on Decontamination: Prions as an Informative Paradigm for Broad-range Disinfection of Surgical Instruments
Michael Beekes
Transmissible spongiform encephalopathies; Robert Koch-Institut; Berlin, Germany
Key words: decontamination, disinfection, surgical instruments, prions, prion protein
Effective disinfection of surgical instruments is of utmost importance for the prevention of nosocomial infections in hospitals and medical practices. However, pathogens such as mycobacteria, non-enveloped viruses, spores or prions have a high tolerance to inactivation. Recent studies identified new formulations for the simultaneous broad-range disinfection of such pathogens, and showed that prions can provide an informative paradigm for the decontamination of surgical instruments (Lehmann et al. J Hosp Infect 2009; 72:342−50; Beekes et al. J Gen Virol 2010; 91:580−9). Taking advantage of this appropach we have examined further candidate formulations for a rapid and effective decontamination of heat-sensitive instruments, aiming at the identification of material-friendly mixtures that are active against PrPTSE, the biochemical marker of prion infectivity, at pH values of >11 In these studies steel grids and/or steel wires were examined by direct western blotting or protein misfolding cyclic amplification (PMCA) in order to quantify both the residual presence and the residual misfolding activity of PrPTSE which may have “suvived” exposure to the tested disinfectant formulations. The decontamination efficacy was monitored for PrPTSE of model prions such as 263K scrapie as well as for PrPTSE from patients with sporadic or variant Creutzfeldt-Jakob disease. Addressing the disinfection of prions not only in its own right but in combination with that of conventional pathogens may open new avenues for the effective broad-range decontamination of surgical instruments and heat-sensitive medical devices.
WP4-1: Neuroinvasion Revisited: Scrapie
Sílvia Sisó, Martin Jeffrey, Stuart Martin and Lorenzo González
Veterinary Laboratories Agency
Key words: TSEs, scrapie, neuroinvasion
Other than by direct invasion following traumatic and iatrogenic incidents, infectious agents can gain access to the brain by two mechanisms: using peripheral nerves as physical conduits (neural neuroinvasion) or via the blood (haematogenous neuroinvasion). Documented examples of the first include infections by lyssaviruses, herpesviruses and listeriosis. Examples of the second are infections by lentiviruses or flaviviruses, and septicemic or thromboembolic bacterial meningoencephalitides.
The precise nature of the infectious agent of the transmissible spongiform encephalopathies (TSEs) or prion diseases is still to be determined, although the most widely accepted theory is that they are caused by an infectious proteinaceous agent. In natural TSE infections, this agent, usually termed “prion” and operationally designated as PrPd, is believed to be acquired by the alimentary route, so that the first barrier that it encounters is the gastrointestinal epithelium. Once this barrier has been crossed, TSE agents could theoretically reach the brain by the same two pathways as any other infectious agent. The prevailing hypothesis of neuroinvasion in animal TSEs is that it occurs through the neural route, after replication of the agent in the enteric nervous system and retrograde transport through autonomic nerves. However, recent evidence from natural and experimental infections in different species suggests that the haematogenous route can also play an alternative or complementary role. In such case, TSE agents would appear to reach the brain through the circumventricular organs which, having fenestrated capillaries, lack a blood-brain barrier and have been shown to accumulate PrPd in early stages of infection.
WP6-2: De Novo Prion Formation from Recombinant Mouse PrP
Gregory J. Raymond, Danielle K. Offerdahl, Ravindra Kodali, Brent Race, Roger A. Moore, Andrew G. Hughson and Gerald S. Baron
Laboratory of Persistent Viral Diseases; Rocky Mountain Laboratories; National Institute of Allergy and Infectious Diseases; NIH; Hamilton, MT USA
Key words: prion protein, synthetic prion, amyloid fibril
Prions are thought to consist of misfolded aggregates of the prion protein (PrP), suggesting that prions could be generated spontaneously from appropriately refolded, purified PrP. Failures to induce prion disease in wild-type animals inoculated with amyloid fibrils derived solely from bacterially-expressed recombinant PrP (rPrP) have raised the possibility that other accessory components may be required and recent data provide support for this hypothesis. Nevertheless, it is important to determine whether purified rPrP by itself can spontaneously misfold into an infectious agent that can be propagated in animals expressing wild-type levels of PrP. To investigate methods to create prions de novo, we developed conditions for mouse rPrP amyloid fibril formation that direct the spontaneous formation of fibrils with enhanced levels of a PrPSc-like protease-resistant conformer without addition of any exogenous biomolecules. These fibrils induced the formation of protease-resistant, amyloid plaque-like deposits of PrP after inoculation into mice co-expressing PrP with and without a glycosylphosphatidyl inositol (GPI) anchor (TgPrPGPIpos/neg). Secondary passage into wild-type and TgPrPGPIpos/negmice induced prion disease with a novel clinical phenotype accompanied by classical histopathological and biochemical markers. Our data strongly suggest that we have generated a novel prion agent solely from purified mouse rPrP that was detected after amplification in mice expressing a molecularly similar PrP and with wild-type levels of total PrP. These observations provide new insights into the minimal elements required to create prions in vitro.
WP7-1: A Transgenic Mice Model of E200K fCJD Presents Fatal Neurodegenerative Disease, PK Resistant PrP and Intraneuronal Accumulation of PrPSc
Yael Friedman-Levy,1 Zeev Meiner,1 Gabor G. Kovacs,2 Herbert Budka,2 Dana Avrahami1 and Ruth Gabizon1,†
1Department of Neurology; The Agnes Ginges Center for Human Neurogenetics; Jerusalem, Israel; 2Institute of Neurology; Medical University; Vienna; Austria
†Presenting author
Key words: genetic prion disease, prion protein mutation, transgenic model
Genetic prion diseases are late onset fatal neurodegenerative disorders linked to pathogenic mutations in the prion protein-encoding gene. The most prevalent of these is the substitution of Glutamate for Lysine at codon 200 (E200K), causing familial CJD in several clusters. To investigate the mechanism of prion disease linked to the E200K mutation, we characterized a line of transgenic mice expressing a chimeric human-mouse PrP harboring the E200K mutation on either a null or wt C57BL background. Both lines, carrying either PrP E200K alone or also the wt allele, presented clinical neurological signs as early as 5–6 month of age, which aggravated gradually until their death at about 1 year of age. Clinical signs included hind limb atrophy, asymmetric weakness or paraplegia, and ended with dystonic limb posture, blindness and kyphosis. Occasionally, animals showed tail plasticity, resembling the posture observed in infectious prion disease mice models. There was great variability both in score and clinical symptoms between littermates of the same age and genetic background. Histopathological examination of the brain and spinal cord reveled late and minor spongiform change, early gliosis and prominent and widespread intraneuronal PrPSc deposition. Concomitantly, we found significant levels of protease resistance PrP in brain samples by immunobloting. We believe this line represents an excellent model of a genetic prion disease that shows substantial similarities to the corresponding human disease. This model may play an important role in elucidating central neuropathogenesis and developing prophylactic treatment for at risk carriers of genetic prion diseases.
WP8-4: Prion-like Induction of Alzheimer-type Proteopathy in Transgenic Mice
Lary C. Walker
Yerkes Center; Emory University; Atlanta, GA USA
Key words: abeta, Alzheimer, amyloid, prion, seeding, strains, transgenic, transmission
Alzheimer’s disease and prion disease both involve the accumulation of disease-specific proteins in the brain, suggesting pathogenic commonalities. In Alzheimer’s disease, the aggregation of the protein fragment Aβ is a seminal event. Similar to the templated corruption of prion protein, cerebral Aβ deposition can be induced in β-amyloid precursor protein (APP)-transgenic mice and rats by the intracerebral injection of Aβ-rich brain extracts from patients with Alzheimer’s disease or APP-transgenic mice. Our studies indicate that the characteristics of the seeded deposits depend on the source of the seeding extract, the type of host, and the seeded brain region. We are using the amyloid-binding agent Pittsburgh Compound B (PIB) as a marker of a potential AD-specific form of multimeric Aβ, and are attempting to induce alternative conformations in the transgenic mouse Aβ-seeding model. In addition, we are investigating non-intracerebral routes of administration and the ability of heterologous agents to induce Aβ deposition. Analysis of Aβ-seeding in vivo could yield fresh insights into the origins of idiopathic Alzheimer’s disease.
Acknowledgements
Key collaborators on these studies are Mathias Jucker (U. Tübingen), Rebecca Rosen (Emory U.) and Harry LeVine III (U. Kentucky). Supported by NIH RR-00165 and the CART Foundation.