08 November 2017

Prions: diseases and treatment

Molecular biologist Byron Koi on the structure of prions, prion diseases, their causes and treatment

Post -science

Prions constitute a separate class of infectious agents; they have a protein basis and do not contain a genome consisting of nucleic acids. The concept of the existence of prions includes the idea of a new, protein heredity – altered forms of proteins in the host organism, which, when transferred to a new organism, can cause a phenotype change in the recipient. Most prions are pathogens, but compared to other classes of pathogens (e.g. viruses, bacteria, fungi, parasites) they are unique in that they spread within and between hosts without transferring or replicating their own DNA or RNA.

Prions, as a rule, are re–folded and aggregated proteins that spread by penetrating into the host organism and stimulating the refolding of the corresponding normal form of protein in it. The prion aggregate grows and then somehow fragments to generate more prion aggregates. In many ways, this is similar to Kurt Vonnegut's ice-nine: the growth of prions is similar to the growth of crystals, and prions themselves are often described as seed by analogy with seed crystals. Thus, prions do not need to transfer their genetic code, but the host organism must create a normal protein that pathogenic prions consist of. Many proteins (if not most) can regroup and/or assemble into ordered aggregates that can grow under certain natural or experimental conditions – in tissues or in vitro, however, not all such protein aggregates are pathogenic prions. The term "prion" means that at the biological level, the structure of a re-folded protein can spread between hosts or at least from cell to cell within a multicellular host organism. Recently, there was a proposal to collect under the umbrella term "prions" all the protein states that contribute to its growth in the form of multimeric clusters, but this expanded use of the term violates the underlying idea of transmittivity and does not allow prions to be isolated from many other cellular structures that can grow, but do not tend to spread to other cells and organisms..

Research history

The prototype of prion disease was a transmissible neurodegenerative disease of sheep – the mysterious deadly scrapie (or sheep scratching). Early studies have shown that the scrapy agent is unusually resistant to treatments that neutralize other pathogens, and can remain on pastures for years. The fact that the scrapy agent shows resistance, in particular, to radiation, led to the fact that in the 1960s J. S. Griffith and Tikvah Alper suggested that it represents a new class of pathogens that does not have its own nucleic genome and may be an abnormal self-reproducing form of protein or membrane. Meanwhile, the descriptions of brain pathology caused by human kuru disease in Papua New Guinea, which were performed by Carlton Gaydushek, led William Hadlow to the idea that kuru is similar to sheep scraping, and Hadlow recommended that kuru be tested for transmissibility from humans to other primates. Gaydushek successfully did this work and showed that the people of the Fore tribe fell ill with Kuru during ritual cannibalistic holidays. A striking feature of kuru and other prion diseases, which often hid their causes, is the long incubation period between infection and the appearance of clinical signs, which in humans can exceed four decades.


Lymph nodes of healthy (a) and infected (b) sheep – staining with antibodies clearly shows signs of prions of scrapy disease in the tissues of infected sheep / wikipedia.org

In the 1980s, Stanley Prusiner coined the term "prion" for these agents and was the first to identify a specific protein that is the main component of scrapie prions – prion protein (PrP). Homologues of the same protein have been found in many mammalian species, including humans, and abnormal PrP aggregates have been identified in other transmissible neurodegenerative diseases of humans and animals, similar to scrapie. These diseases are now known as prion diseases, or transmissible spongiform encephalopathies. Stanley Prusiner, Charles Weissman and other researchers have shown that PrP is an important factor in the susceptibility of prion diseases.

My laboratory, in collaboration with Peter Lansbury, has shown that disease-related forms of PrP can themselves cause the transformation of normal PrP molecules into abnormal forms. In these transformation reactions, we identified striking biochemical features that helped explain the characteristics of known prion strains and barriers to their transmission between different species. However, in order to prove unequivocally that prions consist of re-folded PrP aggregates and they do not need specific prion nucleic acids, it was necessary to develop methods for continuous cell-free amplification of prions or a de novo prion formation reaction. They were originally created by the laboratories of Soto, Supattapone and Prusiner in the 2000s; until then, it was difficult to completely rule out the possibility that these diseases were caused by unidentified viruses.

Although the word "prion" was first applied to the transmissible spongiform encephalopathies described above, the first unambiguous evidence that infectious proteins exist in biology was obtained when Reed Wickner realized in 1994 that some unexplained epigenetic elements in yeast are prions. These prions did not consist of a homologue of PrP, but of very special yeast proteins. The relative simplicity and power of yeast biology and genetics allowed Wickner and other researchers to clearly demonstrate a number of fundamental principles of prion biology and structure that were much more difficult to identify on mammalian prions.

Research methods

Unfortunately, many of the standard methods on which studies of common pathogens have been based for a long time – pathogen genetics, serology, X–ray diffraction analysis, nuclear magnetic resonance (NMR) spectroscopy - are extremely difficult to apply to prions. Without any specific pathogenic genes that could be sequenced or mutated, many standard genetic and reverse genetic approaches to identifying the structure and function of pathogens do not work. Since prions consist of host proteins, the host's immune response to the pathogen is very small; thus, it is very difficult to carry out a simple serological detection of prion infections based on interaction with antibodies. In addition, mammalian prions are usually densely packed, highly glycosylated and bound to other host molecules, and therefore even specific prion conformational epitopes (surfaces recognized by antibodies) on PrP aggregates are difficult to detect and use. All attempts to determine the three-dimensional structures of prions have been stalled for a long time, since the purified prions have an aggregated, but non-crystalline character.

For many years, the only way to detect and analyze mammalian prions was animal bioanalysis, which even on the fastest models – rodents – lasted from several months to one year. In a particular organism, different strains can usually be distinguished by incubation periods, neuropathological patterns, and biochemical signs of disease-related deposits of PrP or prions.

Fortunately, powerful cell-free amplification assays of prions have recently been developed, such as cyclic amplification of the prion form of protein (PMCA), vibration-induced real-time conversion analysis (RT-QuIC) and scrapy cell analysis. These methods are based on the replication mechanism inherent in prions. Both PMCA and RT-QuIC are extremely sensitive: they can amplify the presence of prions by a trillion times, almost to the point of detecting several prion particles. PMCA reactions spread prion infection, thereby reflecting and illuminating many aspects of prion biology, while RT-QuIC assays, as a rule, do not completely spread infectious prions, but provide faster, more practical and more high-performance methods for their detection, and thus they have become the most modern diagnostic tools prion diseases. Both PMCA and RT-QuIC in some cases help to distinguish important prion strains in certain host species.

There is slow progress in identifying the basic structure of prions. With the help of semiconductor NMR studies, the molecular architecture of some fungal prions and prion-like fibrillar structures of mammalian PrP was discovered. Electron crystallography, fiber diffraction, and cryoelectronic microscopic studies have helped to describe the key structural limitations of mammalian prions, but the application of these and possibly other structural biological methods still needs to be improved.

Structure and reproduction of prions

It is extremely difficult to understand the structure and mechanisms of mammalian prion replication, at least at the molecular level. First you need to explain how improperly folded proteins can spread as pathogens without transferring their own nucleic genome. Then it should also be explained how proteins with a single sequence of amino acids, such as the PrP of a particular host animal, can form different strains of prions that regularly spread and cause various disease phenotypes without genetic mutations that explain strain variations in common pathogens.

Many studies indicate that mammalian prions are ordered clusters of several PrP molecules, densely packed and often fibrillar or filamentous. The PrP molecules (monomers) in prions are almost completely inverted in comparison with normal free PrP molecules. When the right PrP molecules are incorporated into growing prion aggregates, these aggregates cause them to refold, with prions acting as strain-specific templates or seedings that somehow give their own aberrant forms to each incoming molecule, controlling the stable replication of their strain.

Beyond this rough description, the details of the structure and distribution of prions at the molecular level remain unclear. Also unresolved is the question of how prions spread beyond the initial site of infection in the host organism. Existing data suggest that the most effective intercellular prion transmission is associated with membranous structures such as exosomes or tunneling nanotubes – most likely because prions are usually bound to membranes by lipid anchors; however, the possibility of these membrane structures to promote the spread of prions in vivo has yet to be determined. It is very important to understand the mechanisms of prion propagation, since the ability of various improperly folded protein aggregates to spread inside and between cells, tissues and individuals determines whether they act as infectious pathogens or are relatively harmless failures of protein metabolism.

Prion diseases

Many mammalian species, including humans, lower primates, cattle, sheep, goats, deer, elk, cats, minks, rodents and various exotic ungulates, are susceptible to prion diseases of PrP. But not all species are like that: dogs and horses seem to be notable exceptions. Different species usually express slightly different normal PrP molecules, and differences in the amino acid sequence of PrP can greatly affect the host's susceptibility to incoming prion infections. For example, humans are known to be susceptible to bovine spongiform encephalopathy (bovine encephalopathy) to some extent, but apparently resistant to sheep scraping and, as far as we know, chronic debilitating deer disease. For some reason, forest voles and squirrel monkeys are unusually susceptible to a wide range of prion infections of other species.

The mechanisms by which prion infections cause neurodegenerative diseases are still unknown to us. Aggregates of various prion strains in host organisms of different species can accumulate mainly in different areas of the central nervous system and cause a number of neuropathological disorders. It is obvious that the final effect of at least partial damage is a malfunction of neurons and their loss, which causes many clinical symptoms and leads to a fatal outcome. It is known that a number of neurophysiological processes and pathways are disrupted, but much remains to be determined as to whether such disorders are associated with direct or indirect toxicity of prions and to what extent one or another insufficiency or combination of insufficiencies is most responsible for the death of the patient.

In humans, the causes of prion diseases can be genetic (due to specific mutations of the PrP gene), acquired (caused by infection - for example, exposure to kuru, GECRS or other prion–containing material) or sporadic (of unknown origin; it is usually assumed that they are due to spontaneous formation of prions in a particular individual). The vast majority of human prion diseases are sporadic, and among them the most common is sporadic Creutzfeldt–Jakob disease (sCJD), the incidence of which is approximately one case per million people worldwide per year. A number of different mutations in the PrP gene can cause many familial prion diseases, while some mutations are completely penetrant (always causing disease in mutation carriers), and others are less penetrant. The clinical symptoms and progression of the disease may vary markedly in different host organisms and in different types of prion diseases, but may include dementia, coordination disorder, insomnia, hallucinations, muscle stiffness, confusion, fatigue and difficulty speaking.

There are also important prion diseases of animals. GECRS emerged as a large-scale epidemic of cattle in connection with what could be called "agricultural cannibalism" in the 1990s. Consumption of GECRS – infected beef – then caused almost two hundred cases of a variant of Creutzfeldt–Jakob disease in humans, but preventive measures almost eliminated GECRS and prevented the emergence of new cases. Chronic debilitating deer disease is sweeping across North America at an alarming rate, with cases also emerging in South Korea and Norway. Scraping is a constant problem with sheep and goats in many parts of the world.

Diagnosis and treatment of prion diseases

Recently, significant progress has been made in accurately and relatively noninvasively diagnosing human prion diseases in living patients based on new prion-specific tests of smears from the nose, cerebrospinal fluid, blood, urine or skin. For example, RT-QuIC testing of cerebrospinal fluid and/or nasal brush biopsy materials can achieve 100% accuracy in the diagnosis of sporadic Creutzfeldt–Jakob disease. These tests are beneficial because they measure the pathogens of prion disease, but they have not yet been fully tested and are not officially recommended by organizations such as WHO. Otherwise, the diagnosis of sporadic prion diseases in humans depends primarily on a combination of clinical signs, brain scan results, electroencephalograms and other biomarkers, which together may have high diagnostic sensitivity, but are not completely specific for prion diseases.

Despite the recent successes described above in the development of new prion tests, the current guidelines are as follows: for the final diagnosis of sporadic or acquired prion disease, neuropathological examination of brain tissues obtained as a result of biopsy (which is rare) or autopsy is necessary. I believe that these recommendations will be changed soon, they will include new, less invasive lifetime tests for detecting prions. Unfortunately, despite the fact that this progress in the early diagnosis of prion diseases should improve the prospects for the development and use of therapeutic agents, there are currently no available treatment methods that would prove their effectiveness in clinical trials.

Open questions and future research directions

In the field of mammalian prion diseases, the following key questions remain open: "What is the self-propagating structure of prions and how does it vary depending on the prion strain?", "How does prion activity lead to brain damage?", "How can we prevent these damages or restore them in the treatment of prion diseases?", "What are the most relevant mechanisms of transmission of prion diseases in humans and animals?", "What prion diseases of animals (in addition to GECRS), if such exist, have zoonotic potential, that is, can cause disease in humans?", "To what extent do other pathogenic, improperly folded proteins, which can also act as a seed, behave how are PrP-based prions in their ability to spread within or between humans, causing disease?"

Indeed, the latter question represents an important milestone in the study of many diseases associated with improper protein formation, especially those associated with the pathogenic accumulation of abnormal fibrillary protein deposits (for example, amyloid fibrils and plaques). These diseases include Alzheimer's, Parkinson's and Huntington's diseases, as well as amyotrophic lateral sclerosis, frontotemporal dementia, chronic traumatic encephalopathy and type II diabetes. Various host proteins form clusters in these and many other diseases, but, like prions, such clusters usually grow due to the inclusion of normal soluble protein molecules in the seed. Thus, the potential for prion-like protein propagation exists at the molecular level. There is also growing evidence that many different disease-related protein deposits can grow and spread in the same way as prions, causing pathologies after inoculation into localized areas in experimental animals.

The results of these studies raise pressing questions about whether numerous diseases based on repeated protein folding – and they are often much more common than prion diseases based on PrP – can be transmitted to humans or animals in real conditions. Creutzfeldt–Jakob disease is transmitted between people through tissue transplantation, hormone injections obtained from corpses, blood transfusions and infected medical instruments. The second factor in such iatrogenic transmissions is the fact that prions are often not completely inactivated by standard clinical disinfection procedures.

It remains to be determined whether other types of potentially prion-like, disease-associated protein aggregates can be identified that may also be resistant to inactivation and at the same time capable of initiating or accelerating pathogenic processes in humans. I do not know of any epidemiological indications that this is the case, but further careful study of this issue seems justified.

About the author:
Byron W. Caughey – Ph.D., Chief of the TSE/Prion Biochemistry Section of the Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases.

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