Prion Explained

Prion Diseases (TSEs)
Icd10:A81

A prion is an infectious agent composed of protein in a misfolded form.[1] This is in contrast to all other known infectious agents (virus/bacteria/fungus/parasite) which must contain nucleic acids (either DNA, RNA, or both). The word prion, coined in 1982 by Stanley B. Prusiner, is a portmanteau derived from the words protein and infection.[2] Prions are responsible for the transmissible spongiform encephalopathies in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as "mad cow disease") in cattle and Creutzfeldt–Jakob disease (CJD) in humans. All known prion diseases affect the structure of the brain or other neural tissue and all are currently untreatable and universally fatal.[3]

Prions propagate by transmitting a misfolded protein state. When a prion enters a healthy organism, it induces existing, properly folded proteins to convert into the disease-associated, prion form; the prion acts as a template to guide the misfolding of more protein into prion form. These newly formed prions can then go on to convert more proteins themselves; this triggers a chain reaction that produces large amounts of the prion form.[4] All known prions induce the formation of an amyloid fold, in which the protein polymerises into an aggregate consisting of tightly packed beta sheets. Amyloid aggregates are fibrils, growing at their ends, and replicating when breakage causes two growing ends to become four growing ends. The incubation period of prion diseases is determined by the exponential growth rate associated with prion replication, which is a balance between the linear growth and the breakage of aggregates.[5] (Note that the propagation of the prion depends on the presence of normally folded protein in which the prion can induce misfolding; animals which do not express the normal form of the prion protein cannot develop or transmit the disease.)

This altered structure is extremely stable and accumulates in infected tissue, causing tissue damage and cell death.[6] This structural stability means that prions are resistant to denaturation by chemical and physical agents, making disposal and containment of these particles difficult. Prions come in different strains, each with a slightly different structure, and most of the time, strains breed true. Prion replication is nevertheless subject to occasional epimutation and then natural selection just like other forms of replication.[7] However, the number of possible distinct prion strains is likely far smaller than the number of possible DNA sequences, so evolution takes place within a limited space.

All known mammalian prion diseases are caused by the so-called prion protein, PrP. The endogenous, properly folded, form is denoted PrPC (for Common or Cellular) while the disease-linked, misfolded form is denoted PrPSc (for Scrapie, after one of the diseases first linked to prions and neurodegeneration.)[8] The precise structure of the prion is not known, though they can be formed by combining PrPC, polyadenylic acid, and lipids in a Protein Misfolding Cyclic Amplification (PMCA) reaction.

Proteins showing prion-type behavior are also found in some fungi, which has been useful in helping to understand mammalian prions. Interestingly, fungal prions do not appear to cause disease in their hosts.[9]

Discovery

Radiation biologist Tikvah Alper and mathematician John Stanley Griffith developed the hypothesis during the 1960s that some transmissible spongiform encephalopathies are caused by an infectious agent consisting solely of proteins.[10] [11] Their theory was developed to explain the discovery that the mysterious infectious agent causing the diseases scrapie and Creutzfeldt–Jakob disease resisted ionizing radiation. A single ionizing "hit" normally destroys an entire infectious particle, and the dose needed to hit half the particles depends on the size of the particles. The data suggested that the infectious agent was too small to be a virus.

Francis Crick recognized the potential importance of the Griffith protein-only hypothesis for scrapie propagation in the second edition of his "Central dogma of molecular biology": while asserting that the flow of sequence information from protein to protein, or from protein to RNA and DNA was "precluded", he noted that Griffith's hypothesis was a potential contradiction (although it was not so promoted by Griffith).[12] The revised hypothesis was later formulated, in part, to accommodate discovery of reverse transcription by Howard Temin and David Baltimore.

Stanley B. Prusiner of the University of California, San Francisco announced in 1982 that his team had purified the hypothetical infectious prion, and that the infectious agent consisted mainly of a specific protein – though they did not manage to isolate the protein until two years after Prusiner's announcement.[13] Prusiner coined the word "prion" as a name for the infectious agent. While the infectious agent was named a prion, the specific protein that the prion was composed of is also known as the Prion Protein (PrP), though this protein may occur both in infectious and non-infectious forms. Prusiner was awarded the Nobel Prize in Physiology or Medicine in 1997 for his research into prions,[14] albeit not without controversy.[15]

Structure

See also: PrP structure.

Isoforms

The protein that prions are made of (PrP) is found throughout the body, even in healthy people and animals. However, PrP found in infectious material has a different structure and is resistant to proteases, the enzymes in the body that can normally break down proteins. The normal form of the protein is called PrPC, while the infectious form is called PrPSc — the C refers to 'cellular' or 'common' PrP, while the Sc refers to 'scrapie', a prion disease occurring in sheep.[16] While PrPC is structurally well-defined, PrPSc is certainly polydisperse and defined at a relatively poor level. PrP can be induced to fold into other more-or-less well-defined isoforms in vitro, and their relationship to the form(s) that are pathogenic in vivo is not yet clear.

PrPC

PrPC is a normal protein found on the membranes of cells. It has 209 amino acids (in humans), one disulfide bond, a molecular mass of 35-36 kDa and a mainly alpha-helical structure. Several topological forms exist; one cell surface form anchored via glycolipid and two transmembrane forms.[17] The normal protein is not sedimentable; meaning it cannot be separated by centrifuging techniques.[18] Its function is a complex issue that continues to be investigated. PrPC binds copper (II) ions with high affinity.[19] The significance of this finding is not clear, but it presumably relates to PrP structure or function. PrPC is readily digested by proteinase K and can be liberated from the cell surface in vitro by the enzyme phosphoinositide phospholipase C (PI-PLC), which cleaves the glycophosphatidylinositol (GPI) glycolipid anchor.[20] PrP has been reported to play important roles in cell-cell adhesion and intracellular signaling in vivo, and may therefore be involved in cell-cell communication in the brain.[21]

PrPSc

The infectious isoform of PrP, known as PrPSc, is able to convert normal PrPC proteins into the infectious isoform by changing their conformation, or shape; this, in turn, alters the way the proteins interconnect. Although the exact 3D structure of PrPSc is not known, it has a higher proportion of β-sheet structure in place of the normal α-helix structure.[22] Aggregations of these abnormal isoforms form highly structured amyloid fibers, which accumulate to form plaques. It is unclear if these aggregates are the cause of cell damage or are simply a side effect of the underlying disease process.[23] The end of each fiber acts as a template onto which free protein molecules may attach, allowing the fiber to grow. Under most circumstances, only PrP molecules with an identical amino acid sequence to the infectious PrPSc are incorporated into the growing fiber [18] . However, rare cross-species transmission is also possible. In a different prion, sup35p was shown to be able to be incorporated into existing aggregations even when three of the five oligopeptide repeats normally present were deleted. [24]

Prion replication mechanism

The first hypothesis that tried to explain how prions replicate in a protein-only manner was the heterodimer model.[25] This model assumed that a single PrPSc molecule binds to a single PrPC molecule and catalyzes its conversion into PrPSc. The two PrPSc molecules then come apart and can go on to convert more PrPC. However, a model of prion replication must explain both how prions propagate, and why their spontaneous appearance is so rare. Manfred Eigen showed that the heterodimer model requires PrPSc to be an extraordinarily effective catalyst, increasing the rate of the conversion reaction by a factor of around 1015.[26] This problem does not arise if PrPSc exists only in aggregated forms such as amyloid, where cooperativity may act as a barrier to spontaneous conversion. What is more, despite considerable effort, infectious monomeric PrPSc has never been isolated.

An alternative model assumes that PrPSc exists only as fibrils, and that fibril ends bind PrPC and convert it into PrPSc. If this were all, then the quantity of prions would increase linearly, forming ever longer fibrils. But exponential growth of both PrPSc and of the quantity of infectious particles is observed during prion disease.[27] [28] [29] This can be explained by taking into account fibril breakage.[30] A mathematical solution for the exponential growth rate resulting from the combination of fibril growth and fibril breakage has been found.[5] The exponential growth rate depends largely on the square root of the PrPC concentration.[5] The incubation period is determined by the exponential growth rate, and in vivo data on prion diseases in transgenic mice match this prediction.[5] The same square root dependence is also seen in vitro in experiments with a variety of different amyloid proteins.[31]

The mechanism of prion replication has implications for designing drugs. Since the incubation period of prion diseases is so long, an effective drug does not need to eliminate all prions, but simply needs to slow down the rate of exponential growth. Models predict that the most effective way to achieve this, using a drug with the lowest possible dose, is to find a drug that binds to fibril ends and blocks them from growing any further.[32]

PrP function

It has been proposed that neurodegeneration caused by prions may be related to abnormal function of PrP. However, the physiological function of the prion protein remains a controversial matter. While data from in vitro experiments suggest many dissimilar roles, studies on PrP knockout mice have provided only limited information because these animals exhibit only minor abnormalities. In recent research done in mice, it was found that the cleavage of prions in peripheral nerves causes the activation of myelin repair in Schwann Cells and that the lack of prions caused demyelination in those cells.[33]

PrP and long-term memory

A review of evidence in 2005 suggested that PrP may have a normal function in maintenance of long-term memory.[34] As well, a 2004 study found that mice lacking genes for normal cellular PrP protein show altered hippocampal long-term potentiation.[35]

PrP and stem cell renewal

A 2006 article from the Whitehead Institute for Biomedical Research indicates that PrP expression on stem cells is necessary for an organism's self-renewal of bone marrow. The study showed that all long-term hematopoietic stem cells expressed PrP on their cell membrane and that hematopoietic tissues with PrP-null stem cells exhibited increased sensitivity to cell depletion.[36]

Prion disease

See main article: Transmissible spongiform encephalopathy.

Diseases caused by prions
Affected animal(s)Disease
sheep, goatScrapie[37]
cattleBovine spongiform encephalopathy (BSE), mad cow disease
minkTransmissible mink encephalopathy (TME)
white-tailed deer, elk, mule deer, mooseChronic wasting disease (CWD)
catFeline spongiform encephalopathy (FSE)
nyala, oryx, greater kuduExotic ungulate encephalopathy (EUE)
ostrich[38] Spongiform encephalopathy
(Not been shown to be transmissible.)
rowspan=8humanCreutzfeldt–Jakob disease (CJD)
Iatrogenic Creutzfeldt–Jakob disease (iCJD)
Variant Creutzfeldt–Jakob disease (vCJD)
Familial Creutzfeldt–Jakob disease (fCJD)
Sporadic Creutzfeldt–Jakob disease (sCJD)
Gerstmann–Sträussler–Scheinker syndrome (GSS)
Fatal familial insomnia (FFI)[39]
Kuru

Prions cause neurodegenerative disease by aggregating extracellularly within the central nervous system to form plaques known as amyloid, which disrupt the normal tissue structure. This disruption is characterized by "holes" in the tissue with resultant spongy architecture due to the vacuole formation in the neurons.[40] Other histological changes include astrogliosis and the absence of an inflammatory reaction.[41] While the incubation period for prion diseases is generally quite long, once symptoms appear the disease progresses rapidly, leading to brain damage and death.[42] Neurodegenerative symptoms can include convulsions, dementia, ataxia (balance and coordination dysfunction), and behavioural or personality changes.

All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal.[43] A vaccine has been developed in mice, however, that may provide insight into providing a vaccine in humans to resist prion infections.[44] Additionally, in 2006 scientists announced that they had genetically engineered cattle lacking a necessary gene for prion production – thus theoretically making them immune to BSE,[45] building on research indicating that mice lacking normally occurring prion protein are resistant to infection by scrapie prion protein.[46]

Many different mammalian species can be affected by prion diseases, as the prion protein (PrP) is very similar in all mammals.[47] Due to small differences in PrP between different species it is unusual for a prion disease to be transmitted from one species to another. The human prion disease variant Creutzfeldt-Jakob disease, however, is believed to be caused by a prion which typically infects cattle, causing Bovine spongiform encephalopathy and is transmitted through infected meat.[48]

Transmission

It has been recognized that prion diseases can arise in three different ways: acquired, familial, or sporadic.[49] It is often assumed that the diseased form directly interacts with the normal form to make it rearrange its structure. One idea, the "Protein X" hypothesis, is that an as-yet unidentified cellular protein (Protein X) enables the conversion of PrPC to PrPSc by bringing a molecule of each of the two together into a complex.[50]

Current research suggests that the primary method of infection in animals is through ingestion. It is thought that prions may be deposited in the environment through the remains of dead animals and via urine, saliva, and other body fluids. They may then linger in the soil by binding to clay and other minerals.[51]

A University of California research team, led by Nobel Prize winner Stanley Prusiner, has proven that infection can occur from prions in manure.[52] And since manure is present in many areas surrounding water reservoirs, as well as used on many crop fields, it raises the possibility of widespread transmission. It was reported in January 2011 that researchers had discovered prions spreading through airborne transmission on aerosol particles, in an animal testing experiment focusing on scrapie infection in laboratory mice. Preliminary evidence supporting the notion that prions can be transmitted through use of urine-derived human menopausal gonadotropin, administered for the treatment of infertility, was published in 2011.

Sterilization

Infectious particles possessing nucleic acid are dependent upon it to direct their continued replication. Prions, however, are infectious by their effect on normal versions of the protein. Sterilizing prions therefore involves the denaturation of the protein to a state where the molecule is no longer able to induce the abnormal folding of normal proteins. Prions are generally quite resistant to proteases, heat, radiation, and formalin treatments,[53] although their infectivity can be reduced by such treatments. Effective prion decontamination relies upon protein hydrolysis or reduction or destruction of protein tertiary structure. Examples include bleach, caustic soda, and strongly acidic detergents such as LpH.[54] 134°C (274°F) for 18 minutes in a pressurized steam autoclave may not be enough to deactivate the agent of disease.[55] [56] Ozone sterilization is currently being studied as a potential method for prion denature and deactivation.[57] Renaturation of a completely denatured prion to infectious status has not yet been achieved; however, partially denatured prions can be renatured to an infective status under certain artificial conditions.[58]

The World Health Organization recommends any of the following three procedures for the sterilization of all heat-resistant surgical instruments to ensure that they are not contaminated with prions:

  1. Immerse in a pan containing 1N NaOH and heat in a gravity-displacement autoclave at 121°C for 30 minutes; clean; rinse in water; and then perform routine sterilization processes.
  2. Immerse in 1N NaOH or sodium hypochlorite (20,000 parts per million available chlorine) for 1 hour; transfer instruments to water; heat in a gravity-displacement autoclave at 121°C for 1 hour; clean; and then perform routine sterilization processes.
  3. Immerse in 1N NaOH or sodium hypochlorite (20,000 parts per million available chlorine) for 1 hour; remove and rinse in water, then transfer to an open pan and heat in a gravity-displacement (121°C) or in a porous-load (134°C) autoclave for 1 hour; clean; and then perform routine sterilization processes.[59]

Debate

Whether prions are the agent which causes disease or merely a symptom caused by a different agent is still debated by a minority of researchers. The following sections describe several hypotheses: some pertain to the composition of the infectious agent (protein-only, protein with other components, virus, or other), while others pertain to its mechanism of reproduction.

Protein-only 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. 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.

Evidence in favor of a protein-only hypothesis includes:

Genetic factors

A gene for the normal protein has been identified: the PRNP gene.[60] In all inherited cases of prion disease, there is a mutation in the PRNP gene. Many different PRNP mutations have been identified and these proteins are more likely to fold into abnormal prion.[61] Although this discovery puts a hole in the general prion hypothesis, that prions can only aggregate proteins of identical amino acid make up. These mutations can occur throughout the gene. Some mutations involve expansion of the octapeptide repeat region at the N-terminal of PrP. Other mutations that have been identified as a cause of inherited prion disease occur at positions 102, 117 & 198 (GSS), 178, 200, 210 & 232 (CJD) and 178 (Fatal Familial Insomnia, FFI). The cause of prion disease can be sporadic, genetic, and infectious, or a combination of these factors.[62] For example, in order to have scrapie, both an infectious agent and a susceptible genotype need to be present.[61]

Multi-component hypothesis

In 2007, biochemist Surachai Supattapone and his colleagues at Dartmouth College produced purified infectious prions de novo from defined components (PrPC, co-purified lipids, and a synthetic polyanionic molecule).[63] These researchers also showed that the polyanionic molecule required for prion formation was selectively incorporated into high-affinity complexes with PrP molecules, leading them to hypothesize that infectious prions may be composed of multiple host components, including PrP, lipid, and polyanionic molecules, rather than PrPSc alone.[64]

In 2010, Jiyan Ma and colleagues at The Ohio State University produced infectious prions from a recipe of bacterially expressed recombinant PrP, POPG phospholipid, and RNA, further supporting the multi-component hypothesis.[65] This finding is in contrast to studies that found minimal infectious prions produced from recombinant PrP alone.[66] [67]

Heavy metal poisoning hypothesis

Recent reports suggest that imbalance of brain metal homeostasis is a significant cause of PrPSc-associated neurotoxicity, though the underlying mechanisms are difficult to explain based on existing information. Proposed hypotheses include a functional role for PrPC in metal metabolism, and loss of this function due to aggregation to the disease associated PrPSc form as the cause of brain metal imbalance. Other views suggest gain of toxic function by PrPSc due to sequestration of PrPC-associated metals within the aggregates, resulting in the generation of redox-active PrPSc complexes. The physiological implications of some PrPC-metal interactions are known, while others are still unclear. The pathological implications of PrPC-metal interaction include metal-induced oxidative damage, and in some instances conversion of PrPC to a PrPSc-like form.[68]

Viral hypothesis

The protein-only hypothesis has been criticised by those who feel that the simplest explanation of the evidence to date is viral.[69] For more than a decade, Yale University neuropathologist Laura Manuelidis has been proposing that prion diseases are caused instead by an unidentified slow virus. In January 2007, she and her colleagues published an article reporting to have found a virus in 10%, or less, of their scrapie-infected cells in culture.[70] [71]

The virion hypothesis states that TSEs are caused by a replicable informational molecule (which is likely to be a nucleic acid) bound to PrP. Many TSEs, including scrapie and BSE, show strains with specific and distinct biological properties, a feature which supporters of the virion hypothesis feel is not explained by prions.

Evidence in favor of a viral hypothesis includes:[72]

Recent studies propagating TSE infectivity in cell-free reactions[73] and in purified component chemical reactions[63] strongly suggest against TSE viral nature. More recently, using a similar defined recipe of multiple components (PrP, POPG lipid, RNA), Jiyan Ma and colleagues generated infectious prions from recombinant PrP expressed from E. coli,[65] casting further doubt on the viral hypothesis.

Fungi

See main article: Fungal prions. Fungal proteins exhibiting templated conformational change were discovered in the yeast Saccharomyces cerevisiae by Reed Wickner in the early 1990s. For their mechanistic similarity to the prion hypothesis, they were termed yeast prions. Subsequently, a prion has also been found in the fungus Podospora anserina. These prions behave similarly to PrP, but are generally nontoxic to their hosts. Susan Lindquist's group at the Whitehead Institute has argued some of the fungal prions are not associated with any disease state, but may have a useful role; however, researchers at the NIH have also provided arguments suggesting fungal prions could be considered a diseased state.[74] Thus, the issue of whether fungal proteins are diseases, or have evolved for some specific functions, still remains unresolved.[75]

As of 2010, there are eight known prion proteins in fungi, seven in Saccharomyces cerevisiae (Sup35, Rnq1, Ure2, Swi1, Mca1, Mot3, Cyc8) and one in Podospora anserina (HET-s).

Research into fungal prions has given strong support to the protein-only concept, since purified protein extracted from cells with a prion state has been demonstrated to convert the normal form of the protein into a misfolded form in vitro, and in the process, preserve the information corresponding to different strains of the prion state. It has also shed some light on prion domains, which are regions in a protein that promote the conversion into a prion. Fungal prions have helped to suggest mechanisms of conversion that may apply to all prions, though fungal prions appear distinct from infectious mammalian prions in the lack of cofactor required for propagation. The characteristic prion domains may vary between species—e.g. characteristic fungal prion domains are not found in mammalian prions.

Fungal Prions
ProteinNatural hostNormal functionPrion statePrion phenotypeYear identified
Ure2pSaccharomyces cerevisiaeNitrogen catabolite repressor[URE3]Growth on poor nitrogen sources1994
Sup35pS. cerevisiaeTranslation termination factor[PSI+]Increased levels of nonsense suppression1994
HET-SPodospora anserinaRegulates heterokaryon incompatibility[Het-s]Heterokaryon formation between incompatible strains
Rnq1pS. cerevisiaeProtein template factor[RNQ+],[PIN+]Promotes aggregation of other prions
Mca1S. cerevisiaePutative yeast caspase[MCA+]Unknown2008
Swi1S. cerevisiaeChromatin remodeling[SWI+]Poor growth on some carbon sources2008
Cyc8S. cerevisiaeTranscriptional repressor[OCT+]Transcriptional derepression of multiple genes2009
Mot3S. cerevisiaeNuclear transcription factor[MOT3+]Transcriptional derepression of anaerobic genes2009

Potential treatments and diagnosis

Advancements in computer modeling have allowed for scientists to identify compounds which can serve as a treatment for prion-caused diseases, such as one compound found to bind a cavity in the PrPC and stabilize the conformation, reducing the amount of harmful PrPSc.[76]

Recently, antiprion antibodies capable of crossing the blood-brain-barrier and targeting cytosolic prion protein (an otherwise major obstacle in prion therapeutics) have been described.[77]

In 2011 it was discovered that prions could be degraded by lichens.[78] [79]

There continues to be a very practical problem with diagnosis of prion diseases, including BSE and CJD. They have an incubation period of months to decades during which there are no symptoms, even though the pathway of converting the normal brain PrP protein into the toxic, disease-related PrPSc form has started. At present, there is virtually no way to detect PrPSc reliably except by examining the brain using neuropathological and immunohistochemical methods after death. Accumulation of the abnormally folded PrPSc form of the PrP protein is a characteristic of the disease, but it is present at very low levels in easily accessible body fluids like blood or urine. Researchers have tried to develop methods to measure PrPSc, but there are still no fully accepted methods for use in materials such as blood.

In 2010, a team from New York described detection of PrPSc even when initially present at only one part in a hundred thousand million (10−11) in brain tissue. The method combines amplification with a novel technology called Surround Optical Fiber Immunoassay (SOFIA) and some specific antibodies against PrPSc. After amplifying and then concentrating any PrPSc, the samples are labelled with a fluorescent dye using an antibody for specificity and then finally loaded into a micro-capillary tube. This tube is placed in a specially constructed apparatus so that it is totally surrounded by optical fibres to capture all light emitted once the dye is excited using a laser. The technique allowed detection of PrPSc after many fewer cycles of conversion than others have achieved, substantially reducing the possibility of artefacts, as well as speeding up the assay. The researchers also tested their method on blood samples from apparently healthy sheep that went on to develop scrapie. The animals’ brains were analysed once any symptoms became apparent. The researchers could therefore compare results from brain tissue and blood taken once the animals exhibited symptoms of the diseases, with blood obtained earlier in the animals’ lives, and from uninfected animals. The results showed very clearly that PrPSc could be detected in the blood of animals long before the symptoms appeared.[80] [81]

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Notes and References

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