General intro
Prion proteins are a common part of cell surface proteins in the mammalian nervous system, and they can, upon change of its conformation, become a highly infectious and pathogenic agent. The physiological form (PrPC) is encoded by the PRNP gene on chromosome 20 and when misfolded and aggregated, the pathological form (PrPSc) occurs, lacking any specific nucleic acid and its primary structure being determined by the PrPC form. When accumulated in the central nervous system (CNS) of mammals, PrPSc is known to be responsible for uprise of several untreatable progressive neurodegenerative diseases, generally called as transmissible spongiform encephalopathies (TSEs). These include kuru, fatal familial insomnia (FFI) and Creutzfeld-Jacob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, transmissible mink encephalopathy (TME) in mink, feline spongiform encephalopathy (FSE) in cat or chronic wasting disease (CWD) in deer and elk (Imran et al., 2011; Prusiner, 1997; Sigurdson et al., 2019).
Table of contents
1. Protein structure
2. Misfolding
3. Pathogenesis
4. Prion diseases
5. Diagnosis and treatment
Protein structure
The PrPSc differs from PrPC solely in conformation and is its isoform. The mature PrPC consists of approx. 208 amino acids, arranged as a disordered N-terminus and a globular C-terminal domain consisting of three α-helices and a short, antiparallel β-pleated sheet. (Riek et al., 1996; Zahn et al., 2002). There is a GPI membrane anchor at the C-terminus that tethers the protein to cell membranes and proteins that are secreted and lacking the anchor component has been proven to be unaffected by the infectious isoform (Chesebro et al., 2005). In contrast to the natural form of prion protein with only about 3 % of β-sheet secondary structure, the PrPSc form has about 47 % of the secondary structure in β-sheets (Pan et al., 1993) that create a core of four-rung β-solenoid fold (Wille and Requena, 2018). Accordingly, they also differ in their properties. PrPC is soluble, has a life-span between 2 and 4 hours, and is sensitive to proteolytic cleavage – when exposed to proteases, the protein is degraded completely (Pan et al., 1993). The two most important cleavage events are the α cleavage which removes the unstructured N-terminal tail and leaves the globular domain attached to the cell membrane, and the cleavage on the C-terminal end (termed PrPC shedding) which releases PrPC into the extracellular space (Sigurdson et al. 2019). Under the same conditions, PrPSc is hydrolysed by proteases only partially by forming resistant core fragment PrP 27-30 (Pan et al., 1993). In addition, it is insoluble in detergents and has a very long half-life, therefore accumulates in tissues easily. It has a tendency to form aggregates and fibrillar structures and is generally susceptible to oligomerization, whereas the PrPC form mainly exist as a monomer (Cohen and Prusiner 2002). Monomeric PrPSc has never been isolated.
Misfolding
The fundamental event in propagation of the infectious form lies in the PrPSc template-directed misfolding of the natural form into the pathogenic, β-sheet-rich version of itself (Pan et al. 1993). This process is now widely accepted as a current prion theory, and the most striking fact is that this action lacks any nucleic acid template (Sigurdson et al. 2019). However, the replication cycle of PrPSc does need the PRNP gene to direct PrPC synthesis. Also, the interaction between the pathogenic and physiological form must be quite specific to propagate the conversion. The replication process itself can be explained by stochastic fluctuations in the PrPC structure, that would create the intermediate form, PrP*. This partially unfolded monomer can then switch back to the natural conformation, adopt a PrPSc one, or be degraded. Normally, there is an equilibrium between the PrPC and PrP* states which favors the physiological one. Depending on the specific cause of the disease, the PrP* state can adopt a PrPSc conformation either upon contant with a dimer of this infectious form by forming a heteromultimer which is further converted into a homomultimer of PrPSc, or through encounter with another PrP* molecule. This PrP*/PrP* dimer can then form an infectious dimer and initiate the replication cycle (Cohen et al., 1994; Cohen and Prusiner, 2002). It has been found that certain host-specific RNAs can assist with the conversion into the pathogenic form (Deleault et al. 2003).
Pathogenesis
In the case of infectious/iatrogenic diseases, pathogenic prion proteins enter the body alimentary via ingestion of affected neural tissues, or via infected materials and tissues such as during blood transfusions, corneal transplants or dura mater grafts. In this case, the exogenous PrPSc form would serve as a template to promote the conversion of PrP* and, due to its insolubility, make this exponential conversion process irreversible (Cohen et al. 1994).
Genetic, or inherited cause comprise the familial TSEs and comes from DNA mutations in the PRNP gene, which then produces mutant, unstable forms of PrPC with higher tendency of folding into the PrP* form. This further increases the chance of PrPSc forming (Cohen and Prusiner, 2002). The mutations in PRNP are autosomal dominant, highly penetrant, and consist of missense mutations, insertions and deletions (Sigurdson et al., 2019). With higher probability of protein misfolding in the old age, they usually incite disease onset in the late decades of life and ultimately lead to accumulation of prion proteins and rapid development of neurodegenerative disease (Khanam et al. 2016).
Concerning sporadic form of prion disease, the concentration of PrPSc may eventually reach a threshold level upon which a positive feedback loop would stimulate the formation of PrPSc. It requires solely a rare molecular event of formation of the PrP*/PrP* complex, or a somatic cell mutation followed by the mechanism of the initiation of inherited disease. Once formed, the replication cycle is primed for subsequent conversion (Cohen et al., 1994, Cohen and Prusiner, 2002).
Ultimately, in all cases this leads to PrPSc polymerization, forming a rod-like structures and amyloid plaques.
Prion diseases
Up to this date, many different types of TSEs are known (see General intro), affecting many animal species as well as humans and showing various symptoms. As was mentioned in previous chapters, all prion diseases promote their negative effects through accumulation of PrPSc in the CNS. However, since most of the TSEs are transmitted by peripheral routes, either orally or transcutaneously, events critical for their pathogenesis take place at peripheral parts of the organism, especially in peripheral lymph organs (Aucouturier et al., 2000). In the following text, probably the two most important prion diseases and facts known about their mechanism of infection are described.
Bovine spongiform encephalopathy
Commonly known as the mad cow disease, bovine spongiform encephalopathy (BSE) is a type of prion disease that affects cattle. Among major symptoms observed in affected animals are abnormal behavior, anxiety, ataxia, hypersensitivity to touch and noise and poor body condition – from movement and posture problems all the way down up to paralysis. Onset symptoms usually emerge after 4-4.5 years from the infection (Casalone C and Hope J., 2018). From that point, the disease is very progressive in degeneration of animal’s nervous system and leads to its death, generally within the time horizon of weeks to months (Konold et al., 2004).
Several types of the disease are distinguished: classic BSE (C-type BSE), L-type BSE and H-type BSE. Latter two types are considered to be sporadic, uncommon and classified as atypical since they arise spontaneously. H and L denotation has its origin in structural features of these two forms. The classic form, on the other hand, is classified as typical and arise most likely from ruminant-derived protein feed supplements (i.e. meat-and-bone meal) as epidemiological analyses of BSE-affected herds implied (Kimberlin and Wilesmith, 1994). After oral uptake of infected feed, it was found that PrPSc gather in some intestinal lymphatic tissues (mainly in Peyer’s patches of the distal ileum and also tonsils). Infectivity of BSE subsequently slowly spreads centripetally into the CNS, probably through the peripheral nervous system. However, it still is not clear, how the disease passes from intestinal mucosa to the lymphoid system of the cattle (Espinosa JC et. al., 2007).
Creutzfeld-Jacob disease
Creutzfeld-Jacob disease (CJD) is the most common human prion disease. It occurs in three distinct forms, based on the source of the disease: sporadic, acquired and inherited (Knight R, 2017). Sporadic form of CJD is denoted as sCJD and it predominantly affects middle aged and elderly. Its classical clinical symptoms are rapid cognitive decline, dementia, cerebellar ataxia and myoclonus terminating in an akinetic mute state (Mackenzie G, Will R, 2017). Due to a very rapid progress of the disease, mean survival of patients is merely six months and more than 90 % die within a year from onset of the first symptoms (Ladogana A et al., 2005). There are certain speculations about the cause of sCJD, e.g. stochastic protein folding or a somatic mutation in PRNP gene, but the true reasons remain unrevealed (Knight R, 2017).
Acquired forms of CJD are caused by infection from exogenous source and consist of variant CJD (vCJD) and iatrogenic CJD (iCJD). Latter is caused by accidental transmission of the disease through medical and surgical procedures, mainly by cadaveric-derived human dura mater grafts (e.g. in cases of corneal transplantation (Duffy P, 1974; Maddox RA, 2008)) or by treatment with human growth hormone (hGH) originating from sCJD affected pituitary glands (Peden AH, 2007). Additionally, few cases caused by treatment with infected human gonadotropin were also described (Cochius JI, 1992). Symptoms of iCJD are generally identical with those of sCJD. However, cases caused by infected hGH are more specific, i.e. progressive cerebellar ataxia and lower limb dysaesthesia with other features, including cognitive impairment (Rudge P et al., 2015).
The vCJD form was described for the first time during the BSE epidemic in the United Kingdom in 1996 and was termed as the “new variant CJD” (Will RG, 1996). It is primarily caused by ingestion of food with infectious contamination originating from BSE affected cattle (Ward HJ et al., 2006). Nonetheless, infection can also arise from transfusion by an infected blood (Urwin PJ et al., 2016) or a blood product, i.e. factor VIII important in the pathway of the blood coagulation (Peden A et al., 2010). The BSE epidemic is the main reason for which the vCJD still occurs with highest incidence in the UK and France. Since the 1996 till 25.04.2017, total count of 231 definite or probable cases of vCJD was reported (Knight R, 2017). Symptoms of vCJD are again mostly identical or very similar to those of other CJD forms, including painful sensory symptoms and involuntary movements (Heath CA et al., 2010). In compare with sCJD, mean survival of patients from the onset symptoms is significantly longer, approximately 14 months from the onset symptoms (Heath CA et al., 2011).
The susceptibility to infection and disease, incubation period and duration of survival in all CJD forms are dependent on several different factors including age of onset and genetic predispositions. So called PRNP-129 polymorphism is related to all CJD forms, but especially to iCJD. Its principle lies in genotype combinations of Met129 and Val129 alleles (MM, MV or VV) and in iCJD, it has substantial impact on susceptibility and incubation period of the disease caused by infection from previously mentioned treatment with hGH (Brandel JP et al., 2003). It appears that VV and mainly MM homozygotes are more susceptible to infection by CJD and have shorter incubation period than MV heterozygotes (Brandel JP et al., 2003; Parchi P et al., 1999). Furthermore, supporting this hypothesis almost all definite and probable vCJD and sCJD reported cases are of MM genotype (Parchi P et al., 1999; Kaski D et al., 2009)
Diagnosis and treatment
To this date, there are no known treatment approaches for prion diseases, therefore these remain 100% fatal. One approach might be increasing the stability of the natural PrPC protein, but the existing compounds (like quinacrine) have been proven far too toxic (Khanam et al. 2016).
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