Prion disease, Creutzfeldt- Jakob and other neurodegenerative diseases.

Cortical vacuolation obtained by scanning electron microscopy x 3500

Astrocytes labelling (astrogliosis) x 100

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Summarize

Neurodegenerative diseases (or amyloidosis) that primarily affect the central nervous system include among other Alzheimer’s disease (agent involved Aß), Parkinson’s disease (agent involved α-synuclein), Huntington’s disease (polyQ agent involved) and Creutzfeldt-Jakob disease (classic and variant involved agent Prion). All these diseases are characterized by an extracellular amyloid deposition no longer degraded by the cellular machinery and accumulates in amyloid plaques. Creutzfeldt-Jakob is the only transmissible amyloidosis. Taking account the current data, inductibility, not yet transmissibility, is the feature for other proteinopathies. Health french authorities have listed products as total inactivating prions (‘Protocol Standard Prions’). As a precaution and new consistent recent data generated, we propose to evaluate not only the total inactivation feature but also amyloicidal. Amyloicid is defined as the ability of a chemical formulation or physical process to disaggregate (depolymerize or defragment) amyloid plaques formed by the agents involved in neurodegenerative diseases; this is called amyloicidal activity.

1. PRION DISEASES

1.1. Characteristics and tissue distribution

Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative diseases that affect both humans and animals. The infectious agent responsible for these diseases, prion, would be purely protein. TSEs are not new diseases. Scrapie has been known since the 18th century and Creutzfeldt-Jakob disease (CJD) since the last century. However, the outbreak of bovine spongiform encephalopathy (BSE) began in 1986 and the passage of the bovine agent in man causing variant Creutzfeldt-Jakob (vCJD) has been reported in 1996. Between one and two million cattle would have entered into the human food chain. vCJD has been described in 1996 in UK after finding 10 atypical cases of CJD in patients under 40 years, of which nine were under 30 years (Will et al., 1996). The first case detected in France was reported in April 1996 in young patient (Chazot et al., 1996). Aside from their young age, these patients had an atypical profile to psychiatric beginning and an evolution of the unusually long illness (14 months on average, against 6 weeks to 6 months in the usual forms of CJD). In particular, the presence of amyloid plaques surrounded by vacuoles (florid plaques) is always deteted in histological sections of brain (Figures 1 and 2).

plaques-amyloide

Figure 1: Observation of a typical brain section from a vCJD patient with the detection of amyloid plaques surrounded by vacuoles or florid plaques (Will et al., 1996).

visuels

Figure 2: Histopathological lesions observed in a CJD patient (Hauw et al., 1998)
A: cortical vacuolation obtained by scanning electron microscopy x 3500.
B:astrocytes labelling (astrogliosis) x 100.

This variant of classic CJD has quickly led researchers to consider a link with the BSE crisis. The first experimental evidence of BSE causing vCJD in humans were established in 1996. The macaque inoculated with the BSE agent showed the same histopathological lesions characteristic as those described in patients with vCJD (florid plaques), the same electroencephalograms and the same biochemical pattern (Lasmezas et al., 1996; Collinge et al., 1996). In 1997, Bruce et al. confirmed this hypothesis by analyzing the lesion profiles (Bruce et al., 1997). The agent responsible for the appearance of vCJD in humans is the same as the origin of BSE. The most likely hypothesis of human infection is the transmission of BSE through highly infectious bovine tissues through oral food. The number of cases of vCJD has increased rapidly since 1996, especially in the UK. After three years without any new cases, two new ones were reported in France in June 2012 and were not still alive Interestingly, in april 2016, the last case of vCJD was reported in UK and he was heterozygous at codon129 of the PRNP gene (http://www.cjd.ed.ac.uk/index.html).
If the primary risk is controlled with routine screening of all animals at slaughter, in the context of secondary human contamination via blood transfusion and through the medical and surgical equipment (Gill et al., 2013; Chen et al., 2014), the risk still exists.

The distribution of infectious prions is not the same between classical forms (mainly in the central nervous system (CNS)) and the variant form (CNS and periphery) where the risk of the last is increased (figure 3).

distribution-tissulaire-en

Figure 3: Tissue distribution of classical and variant forms of prions (figure from Wadsworth et al., 2010).

1.2. Nature of prion agents

Prions are rather hydrophobic agents. This characteristic gives them a high adsorption capacity on various solid surfaces. Surface contamination is known in human health and some iatrogenic CJD cases are related to a bad cleaning and disinfection of medical and surgical equipment. Indeed, a key feature of prions is their resistance to almost all conventional inactivation methods. Only drastic treatments (sodium hydroxide, sodium hypochloride, autoclaving 134°C) as recommended by the World Health Organization (WHO) are effective but incompatible with thermosensitive devices. It becomes crucial from a public health point of view to be able to propose effective inactivation processes compatible with the medical and surgical equipment.

1.3. Transmissibility of prion agents and secondary risk of human transmission

As previously shown, there is a proven risk of human to human secondary transmission of CJD via the medical and surgical equipment to the hospital if it has not undergone the specific procedures (Gibbs et al., 1994). Indeed, a surface or material contaminated by the causative agent of the prion (PrP) can only be addressed in three specific recommendations issued by the WHO (2000): either two chemical (20,000 ppm at least 1 hour or 1M sodium hydroxide solution at least 1 hour) or a physical procedures (autoclaving at 134°C, 3 bars, 18 minutes). These are drastic and incompatible with medical devices (MD) and they are incinirated if CJD is subsequently confirmed on a patient. Many countries edited specific guidelines related to CJD (Fichet et al., 2007). Interestingly, France is the only country who published ‘Protocol Standard Prions’ to recommend specific physical processes and chemical formulations with total inactivating prion activity (a new version is under discussion, ANSM 2011).

2. OTHER NEURODEGENERATIVE DISEASES AND LINKS WITH PRION DISEASES

2.1. Features and mechanistic

CJD or prion diseases (more than 16,000 references to date) fits into the broader context of neurodegenerative CNS diseases like mainly Alzheimer’s (over 85,000 references), Parkinson’s disease (more 70000 references) and Huntington’s disease (more than 18000 references). They seem to have a similar mechanism in the spread of the disease by circulating in the periphery and accumulating under the deposition of misfolded proteins, called amyloid plaques.

Amyloidosis is one of the term used for all diseases forming aggregates. Amyloicidal is the term used to describe the action of a chemical formulation or a physical process by depolymerization and / or defragmentation aggregates. While currently prionicidal is only dedicated to prion inactivation, definition would be extended to others depending on the evolution of knowledge.

For many years, research teams have studied the kinetics of the conversion reaction of the prion protein and have proposed a model of polymerization from a nucleation core.

figure-4

Figure 4: model for the conformational conversion of PrPc into PrPSc (figure from Aguzzi et al., 2004)

This theory postulates that the infectious protein (PrPSc) and the normal protein (PrPc) exist in solution in a thermodynamic equilibrium. PrPc monomer is a natural component of the cells while the infectious agent is multimeric, highly ordered PrPSc aggregates that grow by recruiting surrounding monomeric PrPc. The infectivity increases when PrPSc aggregates become so big that they would eventually break into small nuclei, each of them being able to recruit new PrP molecules and act as an infectious unit. These features of prion proteins can extend to other proteins involved in neurodegenerative diseases. For example, these proteins fit together to become insoluble protein aggregates that are beyond the cellular machinery of protein degradation (Renner et al., 2014).

2.2. In vitro modelization

An in vitro technique was then developed from this hypothesis: since its initial publication in 2001, the PMCA (“Protein Misfolding Cyclic Amplification” (Saborio et al., 2001)) has emerged as a technique to effectively amplify prions test tube. In principle, it amplifies small amounts of pathological prion proteins to make them detectable by conventional techniques immunoblotting, alternating sonication cycles and incubation in a substrate containing the normal prion protein.

figurine-5-pmca-en

Figure 5: diagrammatic representation of the PMCA procedure (figure from Saborio et al., 2001).

Amplification is based on multiple cycles of PrPSc incubation in the presence of excess PrPC followed by sonication. During the incubation periods, the size of oligomeric PrPSc is increased by incorporation of PrPC into the growing aggregate, while during sonication the aggregates are disrupted, producing an expanded population of converting units (legend from Saborio et al., 2001).

This technique widely developed for prions tried to be adapted to other proteins without success before two publications come almost simultaneously provide very encouraging results for both of beta amyloid (Aß) and alpha synuclein fibers (Herva et al., 2014 ; Salvadores et al., 2014).

As the proteins may also find be detected in periphery, and thus the risk of soiling of the DM during a surgical contamination can not be excluded. There are many in vitro and in vivo experimental models much more described for CJD. CJD is the disease only currently transmitted in standard conditions while others are in experimental conditions using transgenic animal models susceptible to the agent involved.

2.3. Risk assessment of inter-human transmission of the agents involved

A first publication showed that Alzheimer Aß protein deposits are found in conventional mice inoculated with human brain from an Alzheimer patient (Morales et al., 2011). Other recent article published by the team of Prof. Prusiner (prion discoverer and Nobel Prize) has shown that by means of bioluminescence technique following injection of synthetic peptides in a transgenic mouse model, it is possible to follow a self-production of the mutated protein; it then spreads in both hemispheres without any co-factor identified. This is one of the characteristics of the prion that does not recruit any cofactor to spread. Therefore, these new data would suggest a prion model… even if the limit is still the use of transgenic animals (Stöhra et al., 2012). Research is focused towards diagnostic of ante mortem detection of agents. There are already available methods applied to research prions in fluids by amplification of a weak signal and tried to be adapted for the others. An american team, the same who had described for the first time a method for amplifying prions in the early 2000s, has described a test with a specificity of 90% but the collection of cerebrospinal fluid remains very invasive for the patient (Salvadores et al., 2014).

Parkinson’s disease affects over 1% of the population aged over 65 years. The marker of this disease is a protein of 140 amino acids, alpha synuclein. It is excreted, it is possible to detect in different biological fluids both in central and peripheral regions such as cerebrospinal fluid, plasma, saliva and mononuclear cells of peripheral blood (El-Agnaf et al., 2003; Devic et al., 2011; Prigione et al., 2010). The disease was transmitted to transgenic animals but an american team recently reported that the transmission was now possible with conventional mice (Lee et al., 2012). This same team has just published the concept of strains named synucleinopathies (Lee et al., 2013). As mentioned earlier, the development of small-signal amplification technique (PMCA) has been recently reported for assessing quantitative measurements (Herva et al., 2014). But can already speak of a prion and therefore an non-conventional transmissible agent for the main agent involved in Parkinson’s disease? The answer is yes to Prof. Prusiner (Prusiner et al., 2015). Moreover, of Dr. Collinge’s team has shown that following treatment with growth hormone in the UK, in 50% of the patients analysed, deposits of amyloid aggregates distinct from prion aggregates were detected (Jaunmuktane et al., 2015). A similar study is underway among french patients treated with growth hormone: preliminary data also reported the detection of amyloid beta lesions without associated-tau pathology (Haik et al., 2016, unpublished data.). Finally, a swiss-austrian studied young patients died of CJD who received dura mater grafts. Amyloid beta deposits were detected in 70% of patients analyzed (Frontzek et al., 2016).

Taking into account all these data (detection in the periphery in addition to the central region, and for the moment the only inductibility feature), we propose by the precautionary principle to validate formulations with amyloicidal properties to inactivate agents implied in all neurodegenerative diseases.

3. PRECAUTIONARY PRINCIPLE: DEVELOPMENT OF AMYLOICIDAL FORMULATIONS

While prion inactivation followed specific guidelines in many countries, there is no recommended protocol for studying, for example, alpha-synuclein involved in Parkinson’s disease. Based on the literature, we have developed a new protocol. Screening of candidate formulations have been initiated using in vitro models before validation in recipient animals. First in vitro results are already available (see PCEA_2nd part report) underlining the characteristics of an amyloicidal formulation that could be used as a pre-disinfectant. Those agents involved in neurodegenerative diseases forming amyloid plaques have a great affinity for surfaces and not just about medical devices. For example, the pharmaceutical industry also requires a high level of quality and safety but zero risk does not exist: stainless steel tanks (surface with high affinity for proteins) are most often used for the production of recombinant proteins and in case of bad cleaning between production batches, contamination of subsequent batches can not be excluded. More generally, it is therefore necessary to develop amyloicidal formulations allowing depolymerizing and / or defragmentation of amyloid plaques.

4. REFERENCES

ANSM 2011 : http://ansm.sante.fr/Dossiers/Creutzfeldt-Jakob-et-produits-de-sante/Protocole-Standard-Prion-lutte-contre-les-infections-liees-aux-soins/(offset)/0

Aguzzi A, Polymenidou M. Mammalian prion biology: one century of evolving concepts. Cell. 2004 Jan 23;116(2):313-27.

Bruce ME, Will RG, Ironside JW, et al. Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 1997;389(6650):498-501. Cell Rep. 2014 Apr 10;7(1):261-8.

Chazot G, Broussolle E, Lapras C, Blattler T, Aguzzi A, Kopp N. New variant of Creutzfeldt-Jakob disease in a 26-year-old French man. Lancet 1996;347(9009):1181.

Chen CC, Wang YH. (2014). Estimation of the exposure of the UK population to the BSE agent through dietary intake during the period 1980 to 1996. PLoS One.;9(4).

Collinge J, Rossor M. A new variant of prion disease. Lancet 1996;347(9006):916-7.

Devic et al. Salivary α-synuclein and DJ-1: potential biomarkers for PD’s. Brain, 134: e178, 2011.

El-Agnaf et al. Alpha-synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma. FASEB J, 17: 1945-1947, 2003.

Frontzek K, Lutz MI, Aguzzi A, Kovacs GG, Budka H. Amyloid-β pathology and cerebral amyloid angiopathy are frequent in iatrogenic Creutzfeldt-Jakob disease after dural grafting. Swiss Med Wkly. 2016 Jan 26;146:w14287.

Gibbs CJ, Jr., Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC. Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery. J Neurol Neurosurg Psychiatry 1994;57(6):757-8.

Gill ON, Spencer Y, Richard-Loendt A, et al. Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey. BMJ. 2013 15;347:f5675.

Hauw JJ, Seilhean D, Colle MA, Hogenhuys J, Duyckaerts C. Neuropathologic markers in degenerative dementias. Rev Neurol (Paris) 1998;154 Suppl 2:S50-64.

Herva ME, Zibaee S, Fraser G, Barker RA, Goedert M, Spillantini MG. Anti-amyloid compounds inhibit alpha-synuclein aggregation induced by Protein Misfolding Cyclic Amplification. J Biol Chem. 2014.

Jaunmuktane Z, Rudge P, Adlard P et al. Iatrogenic CJD due to pituitary-derived growth hormone with genetically determined incubation times of up to 40 years.Brain. 2015 Nov;138(Pt 11):3386-99.

Lasmezas CI, Deslys JP, Demaimay R, et al. BSE transmission to macaques. Nature 1996; 381 (6585): 743-4.

Lee et al. Pathological α-Synuclein Transmission Initiates Parkinson-like Neurodegeneration in Nontransgenic Mice. Science 338, 949 (2012).

Lee et al. Distinct α-Synuclein Strains Differentially Promote Tau Inclusions in Neurons., Cell 154, 103–117, July 3, 2013.

Morales R et al. De novo induction of amyloid-ß deposition in vivo., Molecular Psychiatry (2011), 1–7.

Prigione et al. Alpha-synuclein nitration and autophagy response are induced in peripheral blood cells from patients with Parkinson disease. Neurosci Lett, 477: 6-10, 2010.

Prusiner SB, Woerman AL, Mordes DA et al. Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism. Proc Natl Acad Sci U S A. 2015 Sep 22;112(38):E5308-17.

Renner M, Melki R. Protein aggregation and prionopathies. Pathol Biol (Paris). 2014 Mar 31.

Saborio G, Permanne B and Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 2001 411(6839):810-3.

Salvadores N, Shahnawaz M, Scarpini E, Tagliavini F, Soto C. Detection of misfolded aβ oligomers for sensitive biochemical diagnosis of Alzheimer’s disease. Cell Rep. 2014 Apr 10;7(1):261-8.

Stöhra et al. Purified and synthetic Alzheimer’s amyloid beta (Aβ) prions., Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):11025-30.

Wadsworth JD, Asante EA, Collinge J. Review: contribution of transgenic models to understanding human prion disease. Neuropathol Appl Neurobiol. 2010 Dec;36(7):576-97.

WHO Infection control guidelines for transmissible spongiform encephalopathies. World Health Organization report 23-26 march 1999, WHO/CDS/CSR/APH/2000.3, 1999.

Will RG, Ironside JW, Zeidler M, et al. A new variant of CJD in the UK. Lancet 1996;347(9006):921-5.