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Prion: toxic or infectious agent?
Medical Hypotheses (2003) 60(2), 209–214 ª 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-9877(02)00360-2
Prion: toxic or infectious agent? Andre´ Rico Veterinary School of Toulouse, Paris, France
Summary Prions are proteins that cause a number of invariably fatal neuro-degenerative diseases, which can be classified into two groups: genetic or sporadic diseases (GSD) and transmissible spongiform encephalopathies (TSE). Both types of disease require the development of both normal prion (PrP) and abnormal prion (PrPsc ) which differs from PrP in having a tertiary structure rich in b-sheets. In fact, PrPsc is a totally dehydrated protein with an anhydrous environment, probably a thin carbon dioxide gas gap, that is why it appears highly resistant to proteases, to chemical disinfectants in water phase except in certain conditions to sodium hydroxide and sodium hypochlorite, to heat and to radiation. GSD and TSE diseases differ in incubation time, primary symptoms, and nature of CNS lesions. This paper argues that diseases of the GSD type as inherited or hereditary metabolic disorders and diseases of the TSE type could be regarded as chemical poisonings. TSE is caused by a deficiency in the chemo-defense system (CDS), which is unable to destroy or eliminate PrPsc . As a result, the immune defense system (IDS) accommodates PrPsc as an inert particle if not a virus lure and routes it through to the nervous central system and the brain via the bodyÕs lymphoreticular system. In TSE PrPsc acts inside the cells as a toxic disruptor of post-translational phase of PrP biosynthesis. Unfortunately, CDS and IDS appear unable to neutralize PrPsc . ª 2003 Elsevier Science Ltd. All rights reserved.
INTRODUCTION Prions cause a number of invariably fatal neurodegenerative diseases, all of which involve transformation of the prion protein (PrP) into a modified isoform of PrP, designated PrPsc (1–3). Prion diseases can be broken down into two types. Genetic or sporadic diseases (GSD), including Creutzfeld–Jakob disease (CJD), sporadic CJD (sCJD), familial CJD (fCJD), fatal sporadic insomnia (FSI), fatal familial €ussler–Scheinker insomnia (FFI) and Gerstmann–Stra disease (GSS). Transmissible spongiform encephalopathies (TSE), including scrapie, kuru, bovine spongiform encephalopathy (BSE), variant Creutzfeld–Jakob disease (vCJD),
iatrogenic CJD (iCJD), transmissible mink encephalopathy (TME) and feline spongiform encephalopathy (FSE). These two disease types differ in pathological profile (4–6), but both seem requiring the combined presence of PrP and PrPsc . Why the title question? Because as veterinary biochemist and toxicologist, I am not convinced that PrPsc is an infectious agent. So this short review will be a challenging paper. It does not bring forward new factual data, except perhaps in term of structure. Rather, it takes a fresh interpretation of existing data at the light of structure and properties of PrP and PrPsc with a view to developing a new conceptual outlook. So in this paper I begin with an examination of critical aspects concerning the structure and properties of these two proteins, then go on to discuss GSD and TSE disease types. Some concluding remarks will be done.
Received 2 April 2002 Accepted 12 July 2002
PRIONS: STRUCTURE AND PROPERTIES Correspondence to: Professor Emeritus Andre´ Rico, Veterinary School of Toulouse (France), 24, rue Balard, F75015 Paris, France. Tel./Fax: +33-1-45-58-00-68. E-mail: [email protected]
Nuclear magnetic resonance (NMR) spectroscopy enables us to examine the three-dimensional structure of many recombinant prion proteins. All monomeric water-
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soluble cellular forms of PrP are very similar. For instance, normal human PrP is a sialoglycoprotein that is expressed specifically by the neuron and binds to the outer surface of the plasma membrane by means of a glycosyl-phosphotidyl-inositol (GPI) anchor. It presents two domains: a globular core containing three a-helices and two small b-sheets (three amino acids each), starting with a short –COOH tail that attaches to the membrane by means of the GPI anchor; and a highly flexible loosely structured NH2 terminus forming a kind of floating tail that can change conformation with the physico-chemical conditions of the medium (7). PrPsc is an isoform of PrP. Because it is not soluble in water, its structure cannot be studied using NMR, though spectroscopic techniques and genetic data do reveal that PrPsc resembles PrP in global structure (8,9), with a core including a short –COOH tail, plus a NH2 terminus sequence. However, PrPsc differs greatly from PrP in the tertiary structure of the core portion: in PrPsc , the globular core displays a four-stranded b-sheet, one side of which is covered by two a-helices. These differences in the tertiary structure are unable to explain the particular properties of PrPsc . So, something is missing in the structure discussed above. To my point of view, it is the implication of water. PrP is soluble in water, and it is important to realize the very varied and significant ways in which water molecules interact with all globular proteins, and, indeed, with macromolecules in general (10). Water that actively participates in the macromolecular structure is known as bound or fixed water (11), and it is essential for solubilization, stabilization and flexibility in sustaining the small conformational changes needed for performing biochemical functions. Water, which represents around 70% of the human being and more than 95% for medusa, is much more than simply an excipient for the body. So, it would be a serious oversight to attempt a structural description of a soluble macromolecule like a protein without making full allowance for water. It is not really a simple exercise, but water should be considered as lending a sort of fourth dimension, on top of the tertiary structure. A structural picture based solely on the peptide chain would be a static picture, a sort of snapshot, that failed to express system dynamics. In an aqueous medium, a soluble globular protein exists in a number of different closed transitional conformations, each of which is thermodynamically permissible. Small changes between these conformations may be harnessed to produce biological effects such as enzymatic regulation and metabolism stimulation or inhibition (12). This is true for PrP, and explains the following points: • Solubility in water, two consequences of which are that PrP is sensitive to proteases also soluble in water
Medical Hypotheses (2003) 60(2), 209–214
and that PrP can be in contacts with other water-soluble tertiary or more simple structures; these contacts are essential to permit to PrP to express its biological activities (i.e., anti-oxidant) (13). • Vulnerability to heat, which breaks water-based links and contacts, thereby disrupting the tertiary structure and unwinding the peptide chain. With its four b-sheets, PrPsc is highly stable and rather inflexible. And since PrPsc is strongly hydrophobic, it would be reasonable to assume that water molecules do not participate in its structure, as they do with PrP, and that PrPsc is surrounded by a microanhydrous environment, perhaps as it has been reported recently for hydrophobic surface immersed in water an ultra thin gas gap with thickness of the order of 1 nm formed spontaneously (14). This gas gap could be considered as an insulating structure. According with the composition and the slightly acid pH of the cells, the only gas which could participate to the structure of PrPsc is carbon dioxide. This assertion is supported by the following structural chemical fact: Carbon dioxide is a symmetrical molecule, perfectly apolar, so doing able to have intimate contacts with hydrophobic structures as PrPsc and why not ‘‘prion proteins like (PPL)’’ existing in various neurodegenerative diseases such as AlzheimerÕs, ParkinsonÕs and HuntingdonÕs diseases. In terms of properties, insolubility in water explains why PrPsc appears highly resistant to digestive and cellular proteases, lysosomal proteases (15) in particular but also ubiquitin–proteasome system (16). It is resistant to various chemical deactivators all acting in an aqueous medium except for sodium hydroxide and sodium hypochlorite (17). The activities of these two compounds could be due to their alcaline properties able to transform carbon dioxide into carbonate or hydrocarbonate water soluble, so doing uncovering PrPsc , rendering it sensible to the strong alcaline pH of the middle (NaOH) or to the oxidative action (ClNaO) and so sustaining the role of carbon dioxide in the structure of PrPsc . We also note that PrPsc is not soluble in fat and not extractible by detergent, excluding its possible emulsion in water middle (i.e., blood and milk) (18). The absence of structural water also explains resistance to heat (19) and radiation (20,21). It is widely known that two different mechanisms are involved in the way radiation acts on tissue (22): a directimpact mechanism, which explains why sensitivity is lower for smaller targets; and an indirect mechanism involving ionization of water molecules to produce HOHþ þ e and other reactive species. The second, indirect, mechanism is the most important in living organisms that is why a dry product resists much more to
ª 2003 Elsevier Science Ltd. All rights reserved.
Prion: toxic or infectious agent?
radiation than the same wet product. Because PrPsc is hydrophobic and contains no structural water, the indirect mechanism is greatly inhibited, which means that only the direct mechanism remains operative. As PrPsc is a small protein, the direct effect of radiation is almost negligible. Working backwards, observation of PrPsc radiation resistance leads us to deduce the lack of water molecules in PrPsc structure. The particular properties of PrPsc explain also why it is persistent in the soils. Much more, structural rigidity and hydrophobicity might also impede biological recognition of PrPsc . We know that PrPsc is not recognized by B or T lymphocytes but also by macrophages, and that this explains the absence of immunological reaction. Non-recognition could be due to the fact that PrPsc is an isoform of a normal body protein. But it might be due to the fact that PrPsc is insoluble in water, inflexible, and surrounded by a microanydrous molecular environment; these factors could prevent effective contact with lymphocytes, thus leading to the absence of specific reactions. This assumption is supported by the fact that in conformation-dependent immunoassay (CDI), epitopes of protease-sensitive sPrPsc and protease-resistant rPrPsc are buried (23). So the active biochemical mechanisms that normally act specifically against viruses and bacteria are not working. This last point is crucial, because it explains largely the great participation of macrophages in the TSE pathology. This important point will be discussed later. However, this special structure of PrPsc does not exclude its association as dimmers, particles and perhaps with the primary structure of PrP at the end of the translational phase of its biosynthesis, all that realised without water implication. To sum up, we can say that PrPsc is a protein with a special strongly hydrophobic tertiary structure. It can be considered as a totally dehydrated pre-denatured globular protein with microanhydrous environment, and this explains its high resistance to various physical, chemical, biological agents and the absence of recognition by the specific part of immune defense system (IDS). GENETIC OR SPORADIC DISEASES Existing data on this category (which includes CJD, sCJD, fCJD, FFI, FSI, and GSS) can be summarized as follows: the prions involved in these diseases are endogenous prions, which never come from exogenous sources; except for FFI, initial symptoms are signs of dementia, followed by signs of ataxia; the prion is found only in the brain and never at least up to now in the RES as spleen, tonsils and lymph nodes; the patient age, especially in CJD, sCJD, fCJD is often fairly high; some of ª 2003 Elsevier Science Ltd. All rights reserved.
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the diseases appear to be inherited, if not totally hereditary. As PrPsc exists only in the brain, its formation is certainly endogenous to the brain neurons, leading over a long period to deposition of rod-shaped particles in the cell, usually at the beginning in the mental cortex, at least in CJD, sCJD, fCJD, which explains the predominance of signs of dementia as initial symptoms. Coming back to the structures, we note that the primary structure of PrP and PrPsc are identical. The differences appear in the tertiary structure. In biosynthesis of proteins, to go from the primary structure which finalizes the transmission of genetic information, to the tertiary structure, mechanisms are involved described as post-translation phase (24). So it is logical to suggest that the apparition of PrPsc in the neuron is a metabolic disorder affecting the post-translational mechanisms and involving defective folding of normal PrP, a process that correlates with dysfunctioning of the chaperons involved in PrP synthesis (25–28). It is also linked to polymorphism in codon 129 of the prion gene (29). The disorder comes belatedly because, as is well known in biology, regulation processes become less efficient with age. For example, defective regulation of DNA replication is a well-known phenomenon, with replication errors occurring more frequently in older than in younger subjects. In summary, we can say that GSD should be considered as metabolic disorders, which are largely inherited if not truly hereditary and coming from a perturbation of the post-translational PrP biosynthesis process occurring most easily in old people. Note. In the process of biosynthesis of PrPsc , PrPsc itself can catalyse the transformation. It is the same for PrPsc dimer and the rod-shaped particles. The formation of these particles can act simply by shifting the chemical equilibrium of the reaction (30). TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES The TSE category includes naturally and experimentally transmissible spongiform encephalopathies: scrapie, kuru, BSE, vCJD, iCJD, TME, FSE. Existing data can be summarized as follows: all TSE diseases are induced by exogenous PrPsc ; incubation time depends on penetration route (oral, cutaneous, general or intracerebral) and on the amount and strains of PrPsc entering the body. It will be shorter than for genetic or sporadic disorders (CJD, sCJD, fCJD, FFI); primary symptoms usually correlated with signs of ataxia, signs of dementia appear later; brain lesions with vCJD differ from those observed with CJD. For example, they are well located in the cerebellum; and patients tend to be younger than with Medical Hypotheses (2003) 60(2), 209–214
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classic CJD; PrPsc is found in the reticulo-endothelial system (RES), and specifically in the spleen, lymph nodes, tonsils and gut-associated lymphoid tissue (GALT), though it does not induce febrile response, leucocytosis, pleocytosis or humoral immune response (i.e., no immunoglobulins). These diseases clearly have different pathological profiles from GSD diseases. PrPsc is not destroyed in the gut by the digestive proteolytic enzymes. The absorption of PrPsc is located in GALT and taken up by the lymphoepithelial M cells. CD68 cells may transport the agent to dendritic cells without any direct involvement of bone-marrow-derivated cells such as lymphocytes. All the cells appertaining to macrophage populations (31) are involved in PrPsc phagocytosis, especially in the RES and nervous system. This takes the form of non-specific action of the immune defense system (32). Owing to its peculiar properties, PrPsc escapes to the chemo-defense system of the host (33). As it is not reactive, it is seen by the body as an inert particle if not a virus lure, and is treated as such by the immune defense system. The phagocytosis of inert particle by macrophages has been demonstrated € pfer in 1876 and Metchnikoff in 1901. Macroby Ku phages are in fact the body dustmen. The B and T lymphocytes, which have a specific action, are not involved in the process. The macrophages are unable to identify and destroy PrPsc . From macrophages PrPsc is going into the cells by a process of internalisation. Today it is suggested that this internalisation involves interlacing of PrPsc with PrP prior to penetration. PrPsc much more stable and deformable, acting as a template for duplication (25,30). In term of biochemistry, structural characteristics and properties of PrP and PrPsc , this hypothesis is unclear. So, I suggest another mechanism. The half life of PrP on the surface of the cell is short 3–6 h. Over time more PrP appear to be degraded, liberating the site of fixation on the GPI anchor (34,35). This free site should be occupied by the COOH short tail of PrPsc as it is done by PrP. PrPsc is very resistant with an half life of more than 48 h (35) and finally penetrates into the cell by endocytosis. Inside the cell, PrPsc acts as a disruptor of the post-translational process of PrP biosynthesis disturbing this phase and conducing to the synthesis of PrPsc from the primary structure of PrP. But how could it work? Recently it has been stated that chaperons to act begin by sticking themselves on hydrophobic clusters (36) and it is known that these types of cluster exist in PrP (37) As PrPsc is strongly hydrophobic, it also could make the same, disturbing the activity of chaperons and acting at this stage as a template for the folding of PrP primary structure. This altered biosynthesis is similar to this appearing de novo in GSD. Medical Hypotheses (2003) 60(2), 209–214
So the persistency of PrPsc leads to multiplication and accumulation in cells, which present normal PrP on their membranes. Contaminated cells with non-negligible turnover, like spleen cells, liberate PrPsc when they die by apoptosis and they are replaced. They thus of contribute actively to the multiplication and dispersion of PrPsc . Finally by mechanism of neuron invasion (38) through peripheral nerves and spinal cord PrPsc go into the brain. The brain – a sort of cul-de-sac in the labyrinth of the body – is the final destination, from which there is no way out. This assumption is supported by two facts: neural progression of PrPsc by reverse transport (39); and quasi inexistent turnover of neuron cells, which are never replaced. After internalisation and multiplication in the neuron, PrPsc dimers accumulate and polymerize to form rodshaped particles, which act as crystallization nuclei, a well-known phenomenon in classical chemistry, first described by Pasteur regarding tartrate crystals in the nineteenth century, and accelerate the process. The PrPsc concentration thus increases until it kills the neuron, with the voluminous rod-shaped particles staying in place. Since neuron turnover is very low or non-existent, the neuron will not be replaced, and this gives rise to the characteristic microscopic spongiform lesions. Penetration into the peripheral nervous system, involving migration to the spinal cord and cerebellum before other parts of the brain are reached, explains the early symptoms observed, with signs of ataxia preceding signs of dementia. The distribution and multiplication of exogenous PrPsc by RES also explains why young people where the anabolic process largely exceed catabolic process ingesting sufficient quantities can be affected by the disease. To sum up, we can say that PrPsc mimes an inert particle or a virus lure, thereby triggering non-specific IDS action. Because it is disseminated by the macrophage population and multiplies in cells able to synthesize PrP (including neurons) as a result of a purely biochemical mechanism, we cannot reasonably consider it as an infectious agent. Rather, TSE should be regarded as poisoning by a chemical poison, PrPsc , having some very special properties and acting as disruptor and template for post-translational phase of PrP biosynthesis, so inducing the disease start Notes. (1) Because it is not soluble in water or lipids, and not extractible by detergents (i.e., no affinity for amphiphilic structures), PrPsc is not found in the blood plasma. Presence in blood, if found in non-experimental diseases, might be associated with mononuclear cells (40). And the same applies to milk. (2) The species barrier might be explained by the structure of the host PrP and exogenous PrPsc : the greater the closure in these two structures, the less effective the species barrier (1,35). ª 2003 Elsevier Science Ltd. All rights reserved.
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CONCLUDING REMARKS: GSD VERSUS TSE
ACKNOWLEDGEMENTS
The table below sets out the main characteristics of the two types of disease.
Many thanks to Professors J.P. Braun, D. Dormont, P.L. Toutain and M. Tubiana for their support and advice, and also to Mrs. M. Rico for preparing and typing this manuscript.
Characteristics
GSD
TSE
Endogenous PrPsc Exogenous PrPsc Only brain, particularly RSE (spleen, lymph neurons nodes, tonsils) peripheral and central NS. In the brain, the cerebellum affected first. Multiplication De novo synthesis in In RSE (spleen, cell brain neuron turnover)and NS at least intracell synthesis Early symptoms Dementia usually Ataxia (cerebellum precedes ataxia CJD attack), usually precedes dementia. vCJD iCJD Age Usually in old people. Often in young people Nature Metabolic disorder, Poisoning by chemical often inherited or contaminant (PrPsc ) (dehydrated protein) even hereditary Origin Age-linked Deficiency of the deregulation of chemo-defense system rigorous protein exo. PrPsc : disruptor of the post-translational folding process. synthesis phase of PrP Chaperon dysfunctions? Agent Tissue distribution
GSD and TSE, therefore, appear to be different disease types, having different pathological profiles. They are both induced by PrPsc , but though this chemical agent is similar in both cases, it is not always structurally identical, and it appears in the body in different circumstances (endogenous or exogenous origin). A GSD is a metabolic disorder caused by defective biosynthesis of PrP in the neurons, as a result of defective regulation of the protein folding process. It appears with age and it seems related to chaperon dysfunction. Deregulation is facilitated by small inherited or hereditary structural modifications to PrP (i.e., codon 129). In other words, GSD is basically an inherited or hereditary metabolic disorder, and thus a strictly biological anomaly. A TSE involves poisoning by the rather special chemical agent PrPsc , which escapes destruction by the hostÕs chemo-defense system so it is treated as an inert particle if not a virus lure by non-specific action of the immune defense system, which is unable to identify and destroy it. In other words, TSE are chemical toxicological events. In any case the CDS and IDS are unable to destroy PrPsc . This paper try to introduce new concepts in the prion debate. Certain details in this conceptual paper may be criticized and some basic aspects may be controversial. This is a good thing, because it will open up a new area of discussion and should initiate new scientific investigations. ª 2003 Elsevier Science Ltd. All rights reserved.
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21. Fraser H., Farquhar C. F., Mc Connel J., Davies D. The scrapie disease process is unaffected by ionising radiation. Prog Clin Biol Res 1989; 317: 653–658. 22. Hobbs C. H., Mc Cellan R. O. Toxic effects of radioactive materials. In: C. D. Klaasen, M. C. Amdus, J. Dull (eds). Toxicology – The Basic Science of Poisons. New York: Macmillan Publishing Company, 1986: 668–705. 23. Peretz D., Willamson R. A. A conformational transition at the N terminus of the prion protein features in formation of the scrapie isoform. J Mol Biol 1997; 273: 614–622. se et remaniement des prote ines; 24. Coopers G. M. Synthe gulation de leur fonction. In: G. M. Coopers (ed). La cellule re , 1999: une approche moleculaire. Paris: De Boeck Universite 290–300. 25. Harrison P. M., Bamborough P., Dagget V., Prusiner S. B., Cohen F. E. The prion folding problem. Curr Opin Struct Biol 1997; 7(1): 53–59. culaire et physiopathologie des 26. Lasmedas C. T. Biologie mole phalopathies subaigue €s spongiformes transmissibles. C ence R Acad Agric F 1999; 25(4): 71–78. 27. Tatzelt J., Prusiner S. B., Welch W. J. Chemical chaperones interfere with the formation of scrapie prion protein. EMBO J 1996; 15(23): 6363–6373. 28. Ranson N. A., White H. E., Saibil H. R. Chaperones. Biochem J 1998; 333: 233–242. 29. Ironside J. W. Neuropathology of Variant CJD, Abstract. Paris: Symp. Acad. Sciences, 2001. 30. Caughey B. Interactions between prion protein isoforms: the kiss of death? Trends Biochem Sci 2001; 26(4): 235–242.
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ª 2003 Elsevier Science Ltd. All rights reserved.
Prion: toxic or infectious agent? Andre´ Rico Veterinary School of Toulouse, Paris, France
Summary Prions are proteins that cause a number of invariably fatal neuro-degenerative diseases, which can be classified into two groups: genetic or sporadic diseases (GSD) and transmissible spongiform encephalopathies (TSE). Both types of disease require the development of both normal prion (PrP) and abnormal prion (PrPsc ) which differs from PrP in having a tertiary structure rich in b-sheets. In fact, PrPsc is a totally dehydrated protein with an anhydrous environment, probably a thin carbon dioxide gas gap, that is why it appears highly resistant to proteases, to chemical disinfectants in water phase except in certain conditions to sodium hydroxide and sodium hypochlorite, to heat and to radiation. GSD and TSE diseases differ in incubation time, primary symptoms, and nature of CNS lesions. This paper argues that diseases of the GSD type as inherited or hereditary metabolic disorders and diseases of the TSE type could be regarded as chemical poisonings. TSE is caused by a deficiency in the chemo-defense system (CDS), which is unable to destroy or eliminate PrPsc . As a result, the immune defense system (IDS) accommodates PrPsc as an inert particle if not a virus lure and routes it through to the nervous central system and the brain via the bodyÕs lymphoreticular system. In TSE PrPsc acts inside the cells as a toxic disruptor of post-translational phase of PrP biosynthesis. Unfortunately, CDS and IDS appear unable to neutralize PrPsc . ª 2003 Elsevier Science Ltd. All rights reserved.
INTRODUCTION Prions cause a number of invariably fatal neurodegenerative diseases, all of which involve transformation of the prion protein (PrP) into a modified isoform of PrP, designated PrPsc (1–3). Prion diseases can be broken down into two types. Genetic or sporadic diseases (GSD), including Creutzfeld–Jakob disease (CJD), sporadic CJD (sCJD), familial CJD (fCJD), fatal sporadic insomnia (FSI), fatal familial €ussler–Scheinker insomnia (FFI) and Gerstmann–Stra disease (GSS). Transmissible spongiform encephalopathies (TSE), including scrapie, kuru, bovine spongiform encephalopathy (BSE), variant Creutzfeld–Jakob disease (vCJD),
iatrogenic CJD (iCJD), transmissible mink encephalopathy (TME) and feline spongiform encephalopathy (FSE). These two disease types differ in pathological profile (4–6), but both seem requiring the combined presence of PrP and PrPsc . Why the title question? Because as veterinary biochemist and toxicologist, I am not convinced that PrPsc is an infectious agent. So this short review will be a challenging paper. It does not bring forward new factual data, except perhaps in term of structure. Rather, it takes a fresh interpretation of existing data at the light of structure and properties of PrP and PrPsc with a view to developing a new conceptual outlook. So in this paper I begin with an examination of critical aspects concerning the structure and properties of these two proteins, then go on to discuss GSD and TSE disease types. Some concluding remarks will be done.
Received 2 April 2002 Accepted 12 July 2002
PRIONS: STRUCTURE AND PROPERTIES Correspondence to: Professor Emeritus Andre´ Rico, Veterinary School of Toulouse (France), 24, rue Balard, F75015 Paris, France. Tel./Fax: +33-1-45-58-00-68. E-mail: [email protected]
Nuclear magnetic resonance (NMR) spectroscopy enables us to examine the three-dimensional structure of many recombinant prion proteins. All monomeric water-
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soluble cellular forms of PrP are very similar. For instance, normal human PrP is a sialoglycoprotein that is expressed specifically by the neuron and binds to the outer surface of the plasma membrane by means of a glycosyl-phosphotidyl-inositol (GPI) anchor. It presents two domains: a globular core containing three a-helices and two small b-sheets (three amino acids each), starting with a short –COOH tail that attaches to the membrane by means of the GPI anchor; and a highly flexible loosely structured NH2 terminus forming a kind of floating tail that can change conformation with the physico-chemical conditions of the medium (7). PrPsc is an isoform of PrP. Because it is not soluble in water, its structure cannot be studied using NMR, though spectroscopic techniques and genetic data do reveal that PrPsc resembles PrP in global structure (8,9), with a core including a short –COOH tail, plus a NH2 terminus sequence. However, PrPsc differs greatly from PrP in the tertiary structure of the core portion: in PrPsc , the globular core displays a four-stranded b-sheet, one side of which is covered by two a-helices. These differences in the tertiary structure are unable to explain the particular properties of PrPsc . So, something is missing in the structure discussed above. To my point of view, it is the implication of water. PrP is soluble in water, and it is important to realize the very varied and significant ways in which water molecules interact with all globular proteins, and, indeed, with macromolecules in general (10). Water that actively participates in the macromolecular structure is known as bound or fixed water (11), and it is essential for solubilization, stabilization and flexibility in sustaining the small conformational changes needed for performing biochemical functions. Water, which represents around 70% of the human being and more than 95% for medusa, is much more than simply an excipient for the body. So, it would be a serious oversight to attempt a structural description of a soluble macromolecule like a protein without making full allowance for water. It is not really a simple exercise, but water should be considered as lending a sort of fourth dimension, on top of the tertiary structure. A structural picture based solely on the peptide chain would be a static picture, a sort of snapshot, that failed to express system dynamics. In an aqueous medium, a soluble globular protein exists in a number of different closed transitional conformations, each of which is thermodynamically permissible. Small changes between these conformations may be harnessed to produce biological effects such as enzymatic regulation and metabolism stimulation or inhibition (12). This is true for PrP, and explains the following points: • Solubility in water, two consequences of which are that PrP is sensitive to proteases also soluble in water
Medical Hypotheses (2003) 60(2), 209–214
and that PrP can be in contacts with other water-soluble tertiary or more simple structures; these contacts are essential to permit to PrP to express its biological activities (i.e., anti-oxidant) (13). • Vulnerability to heat, which breaks water-based links and contacts, thereby disrupting the tertiary structure and unwinding the peptide chain. With its four b-sheets, PrPsc is highly stable and rather inflexible. And since PrPsc is strongly hydrophobic, it would be reasonable to assume that water molecules do not participate in its structure, as they do with PrP, and that PrPsc is surrounded by a microanhydrous environment, perhaps as it has been reported recently for hydrophobic surface immersed in water an ultra thin gas gap with thickness of the order of 1 nm formed spontaneously (14). This gas gap could be considered as an insulating structure. According with the composition and the slightly acid pH of the cells, the only gas which could participate to the structure of PrPsc is carbon dioxide. This assertion is supported by the following structural chemical fact: Carbon dioxide is a symmetrical molecule, perfectly apolar, so doing able to have intimate contacts with hydrophobic structures as PrPsc and why not ‘‘prion proteins like (PPL)’’ existing in various neurodegenerative diseases such as AlzheimerÕs, ParkinsonÕs and HuntingdonÕs diseases. In terms of properties, insolubility in water explains why PrPsc appears highly resistant to digestive and cellular proteases, lysosomal proteases (15) in particular but also ubiquitin–proteasome system (16). It is resistant to various chemical deactivators all acting in an aqueous medium except for sodium hydroxide and sodium hypochlorite (17). The activities of these two compounds could be due to their alcaline properties able to transform carbon dioxide into carbonate or hydrocarbonate water soluble, so doing uncovering PrPsc , rendering it sensible to the strong alcaline pH of the middle (NaOH) or to the oxidative action (ClNaO) and so sustaining the role of carbon dioxide in the structure of PrPsc . We also note that PrPsc is not soluble in fat and not extractible by detergent, excluding its possible emulsion in water middle (i.e., blood and milk) (18). The absence of structural water also explains resistance to heat (19) and radiation (20,21). It is widely known that two different mechanisms are involved in the way radiation acts on tissue (22): a directimpact mechanism, which explains why sensitivity is lower for smaller targets; and an indirect mechanism involving ionization of water molecules to produce HOHþ þ e and other reactive species. The second, indirect, mechanism is the most important in living organisms that is why a dry product resists much more to
ª 2003 Elsevier Science Ltd. All rights reserved.
Prion: toxic or infectious agent?
radiation than the same wet product. Because PrPsc is hydrophobic and contains no structural water, the indirect mechanism is greatly inhibited, which means that only the direct mechanism remains operative. As PrPsc is a small protein, the direct effect of radiation is almost negligible. Working backwards, observation of PrPsc radiation resistance leads us to deduce the lack of water molecules in PrPsc structure. The particular properties of PrPsc explain also why it is persistent in the soils. Much more, structural rigidity and hydrophobicity might also impede biological recognition of PrPsc . We know that PrPsc is not recognized by B or T lymphocytes but also by macrophages, and that this explains the absence of immunological reaction. Non-recognition could be due to the fact that PrPsc is an isoform of a normal body protein. But it might be due to the fact that PrPsc is insoluble in water, inflexible, and surrounded by a microanydrous molecular environment; these factors could prevent effective contact with lymphocytes, thus leading to the absence of specific reactions. This assumption is supported by the fact that in conformation-dependent immunoassay (CDI), epitopes of protease-sensitive sPrPsc and protease-resistant rPrPsc are buried (23). So the active biochemical mechanisms that normally act specifically against viruses and bacteria are not working. This last point is crucial, because it explains largely the great participation of macrophages in the TSE pathology. This important point will be discussed later. However, this special structure of PrPsc does not exclude its association as dimmers, particles and perhaps with the primary structure of PrP at the end of the translational phase of its biosynthesis, all that realised without water implication. To sum up, we can say that PrPsc is a protein with a special strongly hydrophobic tertiary structure. It can be considered as a totally dehydrated pre-denatured globular protein with microanhydrous environment, and this explains its high resistance to various physical, chemical, biological agents and the absence of recognition by the specific part of immune defense system (IDS). GENETIC OR SPORADIC DISEASES Existing data on this category (which includes CJD, sCJD, fCJD, FFI, FSI, and GSS) can be summarized as follows: the prions involved in these diseases are endogenous prions, which never come from exogenous sources; except for FFI, initial symptoms are signs of dementia, followed by signs of ataxia; the prion is found only in the brain and never at least up to now in the RES as spleen, tonsils and lymph nodes; the patient age, especially in CJD, sCJD, fCJD is often fairly high; some of ª 2003 Elsevier Science Ltd. All rights reserved.
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the diseases appear to be inherited, if not totally hereditary. As PrPsc exists only in the brain, its formation is certainly endogenous to the brain neurons, leading over a long period to deposition of rod-shaped particles in the cell, usually at the beginning in the mental cortex, at least in CJD, sCJD, fCJD, which explains the predominance of signs of dementia as initial symptoms. Coming back to the structures, we note that the primary structure of PrP and PrPsc are identical. The differences appear in the tertiary structure. In biosynthesis of proteins, to go from the primary structure which finalizes the transmission of genetic information, to the tertiary structure, mechanisms are involved described as post-translation phase (24). So it is logical to suggest that the apparition of PrPsc in the neuron is a metabolic disorder affecting the post-translational mechanisms and involving defective folding of normal PrP, a process that correlates with dysfunctioning of the chaperons involved in PrP synthesis (25–28). It is also linked to polymorphism in codon 129 of the prion gene (29). The disorder comes belatedly because, as is well known in biology, regulation processes become less efficient with age. For example, defective regulation of DNA replication is a well-known phenomenon, with replication errors occurring more frequently in older than in younger subjects. In summary, we can say that GSD should be considered as metabolic disorders, which are largely inherited if not truly hereditary and coming from a perturbation of the post-translational PrP biosynthesis process occurring most easily in old people. Note. In the process of biosynthesis of PrPsc , PrPsc itself can catalyse the transformation. It is the same for PrPsc dimer and the rod-shaped particles. The formation of these particles can act simply by shifting the chemical equilibrium of the reaction (30). TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES The TSE category includes naturally and experimentally transmissible spongiform encephalopathies: scrapie, kuru, BSE, vCJD, iCJD, TME, FSE. Existing data can be summarized as follows: all TSE diseases are induced by exogenous PrPsc ; incubation time depends on penetration route (oral, cutaneous, general or intracerebral) and on the amount and strains of PrPsc entering the body. It will be shorter than for genetic or sporadic disorders (CJD, sCJD, fCJD, FFI); primary symptoms usually correlated with signs of ataxia, signs of dementia appear later; brain lesions with vCJD differ from those observed with CJD. For example, they are well located in the cerebellum; and patients tend to be younger than with Medical Hypotheses (2003) 60(2), 209–214
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classic CJD; PrPsc is found in the reticulo-endothelial system (RES), and specifically in the spleen, lymph nodes, tonsils and gut-associated lymphoid tissue (GALT), though it does not induce febrile response, leucocytosis, pleocytosis or humoral immune response (i.e., no immunoglobulins). These diseases clearly have different pathological profiles from GSD diseases. PrPsc is not destroyed in the gut by the digestive proteolytic enzymes. The absorption of PrPsc is located in GALT and taken up by the lymphoepithelial M cells. CD68 cells may transport the agent to dendritic cells without any direct involvement of bone-marrow-derivated cells such as lymphocytes. All the cells appertaining to macrophage populations (31) are involved in PrPsc phagocytosis, especially in the RES and nervous system. This takes the form of non-specific action of the immune defense system (32). Owing to its peculiar properties, PrPsc escapes to the chemo-defense system of the host (33). As it is not reactive, it is seen by the body as an inert particle if not a virus lure, and is treated as such by the immune defense system. The phagocytosis of inert particle by macrophages has been demonstrated € pfer in 1876 and Metchnikoff in 1901. Macroby Ku phages are in fact the body dustmen. The B and T lymphocytes, which have a specific action, are not involved in the process. The macrophages are unable to identify and destroy PrPsc . From macrophages PrPsc is going into the cells by a process of internalisation. Today it is suggested that this internalisation involves interlacing of PrPsc with PrP prior to penetration. PrPsc much more stable and deformable, acting as a template for duplication (25,30). In term of biochemistry, structural characteristics and properties of PrP and PrPsc , this hypothesis is unclear. So, I suggest another mechanism. The half life of PrP on the surface of the cell is short 3–6 h. Over time more PrP appear to be degraded, liberating the site of fixation on the GPI anchor (34,35). This free site should be occupied by the COOH short tail of PrPsc as it is done by PrP. PrPsc is very resistant with an half life of more than 48 h (35) and finally penetrates into the cell by endocytosis. Inside the cell, PrPsc acts as a disruptor of the post-translational process of PrP biosynthesis disturbing this phase and conducing to the synthesis of PrPsc from the primary structure of PrP. But how could it work? Recently it has been stated that chaperons to act begin by sticking themselves on hydrophobic clusters (36) and it is known that these types of cluster exist in PrP (37) As PrPsc is strongly hydrophobic, it also could make the same, disturbing the activity of chaperons and acting at this stage as a template for the folding of PrP primary structure. This altered biosynthesis is similar to this appearing de novo in GSD. Medical Hypotheses (2003) 60(2), 209–214
So the persistency of PrPsc leads to multiplication and accumulation in cells, which present normal PrP on their membranes. Contaminated cells with non-negligible turnover, like spleen cells, liberate PrPsc when they die by apoptosis and they are replaced. They thus of contribute actively to the multiplication and dispersion of PrPsc . Finally by mechanism of neuron invasion (38) through peripheral nerves and spinal cord PrPsc go into the brain. The brain – a sort of cul-de-sac in the labyrinth of the body – is the final destination, from which there is no way out. This assumption is supported by two facts: neural progression of PrPsc by reverse transport (39); and quasi inexistent turnover of neuron cells, which are never replaced. After internalisation and multiplication in the neuron, PrPsc dimers accumulate and polymerize to form rodshaped particles, which act as crystallization nuclei, a well-known phenomenon in classical chemistry, first described by Pasteur regarding tartrate crystals in the nineteenth century, and accelerate the process. The PrPsc concentration thus increases until it kills the neuron, with the voluminous rod-shaped particles staying in place. Since neuron turnover is very low or non-existent, the neuron will not be replaced, and this gives rise to the characteristic microscopic spongiform lesions. Penetration into the peripheral nervous system, involving migration to the spinal cord and cerebellum before other parts of the brain are reached, explains the early symptoms observed, with signs of ataxia preceding signs of dementia. The distribution and multiplication of exogenous PrPsc by RES also explains why young people where the anabolic process largely exceed catabolic process ingesting sufficient quantities can be affected by the disease. To sum up, we can say that PrPsc mimes an inert particle or a virus lure, thereby triggering non-specific IDS action. Because it is disseminated by the macrophage population and multiplies in cells able to synthesize PrP (including neurons) as a result of a purely biochemical mechanism, we cannot reasonably consider it as an infectious agent. Rather, TSE should be regarded as poisoning by a chemical poison, PrPsc , having some very special properties and acting as disruptor and template for post-translational phase of PrP biosynthesis, so inducing the disease start Notes. (1) Because it is not soluble in water or lipids, and not extractible by detergents (i.e., no affinity for amphiphilic structures), PrPsc is not found in the blood plasma. Presence in blood, if found in non-experimental diseases, might be associated with mononuclear cells (40). And the same applies to milk. (2) The species barrier might be explained by the structure of the host PrP and exogenous PrPsc : the greater the closure in these two structures, the less effective the species barrier (1,35). ª 2003 Elsevier Science Ltd. All rights reserved.
Prion: toxic or infectious agent?
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CONCLUDING REMARKS: GSD VERSUS TSE
ACKNOWLEDGEMENTS
The table below sets out the main characteristics of the two types of disease.
Many thanks to Professors J.P. Braun, D. Dormont, P.L. Toutain and M. Tubiana for their support and advice, and also to Mrs. M. Rico for preparing and typing this manuscript.
Characteristics
GSD
TSE
Endogenous PrPsc Exogenous PrPsc Only brain, particularly RSE (spleen, lymph neurons nodes, tonsils) peripheral and central NS. In the brain, the cerebellum affected first. Multiplication De novo synthesis in In RSE (spleen, cell brain neuron turnover)and NS at least intracell synthesis Early symptoms Dementia usually Ataxia (cerebellum precedes ataxia CJD attack), usually precedes dementia. vCJD iCJD Age Usually in old people. Often in young people Nature Metabolic disorder, Poisoning by chemical often inherited or contaminant (PrPsc ) (dehydrated protein) even hereditary Origin Age-linked Deficiency of the deregulation of chemo-defense system rigorous protein exo. PrPsc : disruptor of the post-translational folding process. synthesis phase of PrP Chaperon dysfunctions? Agent Tissue distribution
GSD and TSE, therefore, appear to be different disease types, having different pathological profiles. They are both induced by PrPsc , but though this chemical agent is similar in both cases, it is not always structurally identical, and it appears in the body in different circumstances (endogenous or exogenous origin). A GSD is a metabolic disorder caused by defective biosynthesis of PrP in the neurons, as a result of defective regulation of the protein folding process. It appears with age and it seems related to chaperon dysfunction. Deregulation is facilitated by small inherited or hereditary structural modifications to PrP (i.e., codon 129). In other words, GSD is basically an inherited or hereditary metabolic disorder, and thus a strictly biological anomaly. A TSE involves poisoning by the rather special chemical agent PrPsc , which escapes destruction by the hostÕs chemo-defense system so it is treated as an inert particle if not a virus lure by non-specific action of the immune defense system, which is unable to identify and destroy it. In other words, TSE are chemical toxicological events. In any case the CDS and IDS are unable to destroy PrPsc . This paper try to introduce new concepts in the prion debate. Certain details in this conceptual paper may be criticized and some basic aspects may be controversial. This is a good thing, because it will open up a new area of discussion and should initiate new scientific investigations. ª 2003 Elsevier Science Ltd. All rights reserved.
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