Prion Disease

Prion DiseaseProteins from Hell Gissel ArandaJose VarelaJeremiah RamosKristina Barron

IntroductionTransmissible spongiform encephalopathies, called so because the brains of infected patients have prominent holes that resemble a sponge, are a collection of rare but deadly neurodegenerative disorders that occur in humans and animals and in some cases can be transmitted from animals to humans. The hypothesized mechanism that causes this disease is an unusual one. For one, unlike viruses or bacteria, it does not contain any nucleic acids in the form of DNA or RNA. Two, the incubation period, the period between exposure and neurological consequence, is a substantially long one taking up to 50 years to act. Researchers believe the cause of the disease is a specific protein called a prion. Prion proteins are found in a normal non-toxic form in the hosts cells and in pathogenic toxic forms, which is the cause of the disease. The pathogenic proteins contain the exact same amino-acid sequence as the healthy prions but are misfolded. When the pathogenic proteins come in contact with the host they signal healthy proteins to undergo conformational changes and misfold in the same manner as the pathogenic prion. This then leads to a chain reaction, as the newly host-derived misfolded prion will now signal other healthy prions to misfold. After the emergence of the misfolded prions the proteins aggregate in the cell and lead to neuronal cell death which lead to symptoms discussed later. The proposal of this disease mechanism has lead this disease to be coined prion disease. There have been several prion diseases identified in humans and animals including but not limited to; Creutzfeldt-Jakob disease (human), Variant Creutzfeldt-Jakob disease (human), Kuru (human), Bovine Spongiform Encephalopathies (animal), Chronic Wasting disease (animal), and Scrapie (animal). BackgroundCreutzfeldt-Jakob disease (CJD), first described by German neurologists Hans Gerhard Cruetzfeldt and Alfons Maria Jakob, is the most common occurring prion disease in humans with approximately 300 cases reported each year in the United States and in one person for every one million worldwide. This disease is extremely rare but its ramifications are severe as all cases are fatal. CJD is generally seen in individuals aged 60 and older and can emerge in one of three ways; 1) spontaneously (~90% of reported cases), here the disease emerges even though there is no family history of the disease, 2) inherited form (10% of reported cases), where there is family history or genetic predispostions, and 3) through transmission (less than 1% of cases) where an individual is exposed to nervous system tissue via medical procedures [1]. A separate category of Cruetzfeld-Jakob disease known as variant Cruetzfeld-Jakob (vCJD) disease is contracted through the ingestion of contaminated animal meats and will be discussed in detail later. Another human variant of the disease of historical importance is known as Kuru. Kuru was seen in epidemic levels during the 1950s and 1960s in the Fore tribes of New Guinea and was rampant due to ritualistic cannibalism in the population. The disease was transmitted through the consumption of infected human tissue. Like CJD the incubation period of the disease is long and initial symptoms did not appear for years or decades after consumption of the infected tissue. Education and intervention of cannibalistic practices by government authorities greatly reduced the levels of the disease and it has almost been completely eradicated. Chronic wasting disease (CWD) is an animal prion disease affecting deer and elk in the North American region. The infection spreads amongst free-roaming animals and has been seen in herds raised in captivity. The disease was first documented in 1967 but its precise location and time of origin remains unknown [2]. It was determined to be a spongiform encephalopathy soon afterwards by histological examinations of the affected animals who died from the disease. Incidences of CWD do not appear to have a single origin although some scientists believe the origin of the disease occurred decades prior to the first documented case and then spread to neighboring populations. [3]. While current documented cases of CWD are caused by prion infection the possibility of a familial or spontaneous form of CWD has yet to be ruled out [4]. Transmission of the disease has been suggested to occur by fecal shedding of infectious prions which then mix with animal feed. This transmission hypothesis is supported by immunohistochemistry of lymphoid tissues from the digestive tract as well as tonsils and mesenteric lymph nodes in infected animals [3]. It is further supported by the high incidence of infection and death from the disease that has been documented in captive herds when infected animals are not quarantined [2]. The asymptomatic phase of prion diseases in humans last anywhere from 5 to 50 years while the clinical symptomatic stage lasts approximately 14 months with death following, CWD however, has an incubation period of about 17 months [5]. Early symptoms in CJD and vCJD are prominent and consist of; gradual and progressive dementia, changes in behavior/personality, failure of motor skills, and deterioration in vision. In the more aggressive state of the disease, symptoms become exacerbated and include near complete mental failure, uncontrollable movements, loss of muscle cordination (ataxia), incontinence, complete loss of vision, muscle weakness, and comatose. The symptomology of Kuru disease is slightly different than that of CJD, as the prions responsible for Kuru preferentially affect the cerebellum of infected individuals [6]. Patients with Kuru exhibit a drastic deterioration in motor function, and eventually enter a comatose state before death. Symptoms of CWD that are most prevalent are loss of body condition (from where the name of the disease originates) and changes in behavior. These two categories can include decreased or increased socialization with herd members or human handlers, repetitive behaviors such as walking in specific patterns, depression, and decreased food intake leading to wasting. As the disease becomes more severe polydipsia and polyuria may occur as well as increased salivation, problems with coordination, posterior ataxia, and/or head tremors. Like all other forms of infectious prion disease, it is fatal [5].As deadly and debilitating as human cases of prion disease are the most impactful on society are the animal cases with bovine spongiform encephalopathy (BSE), more commonly known as mad cow disease, being the most serious. Mad cow disease is a progressive neurological disorder, in cows, that is caused by the same mechanisms seen in CJD; a pathogenic prion is introduced into a healthy host and begins to misfold healthy prion proteins. Interestingly, BSE can be transmitted to humans, where it is known as Variant CJD. This fact has caused major ramifications in the cattle industry, disease control, and world trade. The country most affected by BSE is the United Kingdom with more than 180,000 known cattle infected and 4.4 million others killed as a precaution [7]. The lapse in action cost the lives of 80 individuals, who contracted the disease after consuming contaminated meats, and over $5 billion dollars in costs to added consumer safety measures, law suits, and aid for the beef industry. While it is not known how BSE originated, researchers have concluded that the disease was spread when animal remains, in the form of meat and bone meal, were processed for cattle food. The disease, being contagious, was spread further when more and more infected animals entered the livestock food chain. As a result of the diseases capability of cross species transmission, a ban on British beef was enacted by the European Union, a major importer of UK beef. The ban would last ten years and was finally lifted in May 2006 as cases of BSE in the UK dropped dramatically with improvements and measures taken to eradicate the presence of the disease [8].TherapiesPrion disease is hypothesized to be a result of normal host prion proteins (PrPc) misfolding into protease resistant isoforms (PrPsc), which then aggregate and lead to cell death [9]. Multiple attempts have been made that aim to inhibit the transformation of PrPc to PrPsc. One such drug is pentosan polysulfate (PPS), a large polyglycoside molecule, that when administered in the ventricles (PPS cannot cross the bloodbrain barrier) of PrPsc mice, significantly lengthened the incubation period of the disease and attenuates the formation of PrPsc, thereby prolonging the clinical phase of the disease [9]. As promising as animal trials have been, human trials have been mostly disappointing. Worldwide clinical trials have been mixed, with one study in Japan having seven patients shown no improvement from PPS treatment and four patients experience longer than average survival periods, 26 months compared to 14 months untreated patients, with no patients showing improvement in symptomology [10]. Another study conducted in the UK showed minimal improvement in survival time, 16-month average survival compared to 14 months, also with no improvement in symptoms [11].Another drug that possesses PrPsc inhibitory characteristics is quinacrine. Quinacrine, which is in human use as an anti-malarial drug, was shown to prevent aggregation of PrPsc in TSE infected mice [12]. As with PPS, quinacrine showed promise in animal and cell culture studies, but crossover of benefits in human cases has been absent. Due to the very limited to no effectiveness of treatment of prion diseases, most effort is given in alleviating diseases symptoms. Drugs such opioids are administered to alleviate pain and clonazepam is given to assist with involuntary muscle movement. Researchers are exploring many other interventions in prion disease, with the most exciting one being the use of antibodies. Antibody use has shown to be successful in prion-infected cell cultures and in-vivo in mice [13]. The potential for antibody use is so promising the British Medical Research Council has begun probing the possibility of human clinical trials of prion antibodies.Antibody treatments currently under investigation would alter one or all of the proposed mechanisms for conversion of PrPc to PrPsc. Anti-PrP antibodies would interfere with the interaction of PrP on cell membrane lipid rafts where conversion is said to initiate, disrupt co-factors associated with the conversion, and/or interfere with the internalization of infectious PrP [14]. Investigation into immunizations with heterologous PrP was shown to slow disease onset in mice [15]. Homologous PrP immunizations did not have this effect, however, and it is speculated that heterologous allows for better vaccination because of its higher immunogenicity [16]. However, some scientists claim PrP vaccines could cause an autoimmune response as PrP is a host protein. Furthermore, recombinant PrP has been shown to convert to the infectious form in vitro complicating the possibility of it ever being used as a prospect for immunizations [17].Mechanisms of ActionTransmissible Spongiform Encephalopathies are characterized by several factors that contribute to the development of its pathological features and the transmission of its distinctive proteinaceous infectious particle. The cellular prion protein is an essential component of the disease development and is present in all animals and humans. This protein is encoded by the PRNP gene and is found in a wide array of cells including skeletal muscle, heart, kidney, lymphoid tissue and cells within the CNS [18]. Within the CNS, PrPc is found on the cell surface of neurons, glial cells and peripheral cells. Furthermore, the structure of the mature PrPc non-pathogenic protein is shared among species and is characterized by abundant alpha helices and a flexible N-terminal tail; which makes it easily soluble and digested by proteases. Its broad expression pattern suggests a wide array of physiological functions within the organism, including immunoregulation, synaptic transmission and neurogenesis; however, its role is still unclear [19]. Despite of the role of PrPc in mammals, non-pathogenic protein conversion to PrPsc alters not only the structure but might also alter the function of the normal protein, which is believed to be a critical factor and cause of neurodegeneration. The abnormal form of PrPc, PrPsc, has the same amino acid sequence and primary structure as the non-pathogenic protein. However, the secondary structure of this protein is dominated by Beta sheets; which make the protein insoluble, very stable and resistant to digestion by proteases, heat and sterilization [18]. The unusual properties of the pathogenic protein led researchers to conclude that it might be composed of protein only (protein only hypothesis), which is as of today the most accepted idea among the field. Different experimental models have been used in order to describe the nature and mechanism for the formation of PrPsc and its method of replication. Knockout mice with deletion of the PRNP gene (which encodes for the cellular prion protein PrPc) are resistant to the development of the pathogenesis after inoculation of the infectious protein [20]. Furthermore, familial cases of the disease are caused by mutations of the PRNP gene. All of this suggesting that the cellular non-pathogenic PrPc needs to be present in order for the disease to develop. Taking this as a basis to investigate the main mechanism for PrPsc formation and replication, two main models for the molecular conversion of PrPc to PrPsc have been proposed. The first mechanism is the template-directed refolding suggests that PrPc needs to be unfolded and then refolded, using PrPsc as template. In other words, hanging with the bad guys makes it likely to convert into a bad guy as well. After infection occurs, the incoming PrPsc starts a catalytic cascade using partially unfolded PrPc as a substrate; converting the cellular non-pathogenic protein into a pathogenic Beta rich protein. However, a high energy barrier prevents a spontaneous conversion of PrPc into PrPsc; chaperone activity (protein X) and energy are required for PrPsc formation. In addition, continuous formation of PrPc-PrPsc heterodimers lowers the high energy barrier, increasing the number of PrPc being recruited and unfolded; thus, leading to the formation of PrPsc oligomers and fibril aggregates [18].

The second model, seeded nucleation model, states that PrPc and PrPsc are in reversible monomeric equilibrium, and that an unknown trigger stimulates seed formation. Once the seed is present in the system, in this case, PrPsc, further monomer addition is accelerated; in other words, the presence of several seeds or PrPsc monomers (harmless) lead to the formation of a stable nucleus or seed which stimulates the rapid autocatalytic growth of a PrPsc polymer. This polymers later lead to infectious amyloid entities [18].

There is supporting evidence for the seeded nucleation model due to the conversion of recombinant PrPc into protease resistant PrPsc through the presence of a PrPsc seed in transgenic mice models. In addition, recombinant PrPc is converted reversibly between the two secondary structures (alpha to beta) [19]. However, the molecular mechanisms of the conversion of the pathogenic protein are still unclear. The molecular mechanisms that work behind Prion pathogenesis vary among different species and are observed in the appearance of different prion strains. Its believed that this phenotypic variance is due to the conformational flexibility of the pathogenic protein. Prion pathogenesis and PrPsc structure are determined by both the conformation of the entering PrPsc and the characteristics and limitations that are presented by the infected host [18]. The growth and development of different prion strains can also be observed in how they express themselves in different organ systems. Some strains such as classical BSE are more abundant in the CNS and less visible or absent in secondary lymphoid organs. Furthermore, strains such as vCJD, CWD and scrapie are highly abundant in secondary lymphoid organs even though theyre also detected highly in the CNS. This might be due to the different cellular interactions that occur within systems and how the flexibility of PrPscs structure affects its compatibility with different receptors; also, different cofactors might be playing a role in how the pathogenic protein is replicated only in particular cells [18]. After exposure to infection, prion reaches the CNS through different cellular and molecular mechanisms that facilitate its transport and replication. PrPc is the main key player in carrying infection to several peripheral sites and reaching the central nervous system. In addition, hematopoietic cells such as dendritic, macrophages, B and T cells help to transport prions to secondary lymphoid organs in which they replicate and accumulate and are transported to the brain through different routes. An in vitro study has shown that uptake by nerve endings in the intestine or spleen are important routes of transmission from the periphery to the CNS. Also, Peyers patches and lymphoid components such as follicular dendritic cells and B lymphocytes have shown to be key players for prion replication, accumulation and development in mice models. Infective prion proteins are prominent habitants of follicular dendritic cell membranes, follicles of the spleen, lymph nodes and Peyers patches in different prion strains such as variant CJD, scrapie and CWD [21]. Time course studies have provided evidence for the involvement of the vagal nerve and the sympathetic nervous systems in the transmission of prion to the CNS. Sympathetic innervation of lymphoid organs in mice indicated shorter incubation times after peripheral prion exposure; which indicates the importance of the above mentioned in prion infectivity and transmission [22]. Since lymphoid organs are such important players of prion pathogenesis, the involvement of pro inflammatory cytokines and immune cells was analyzed in correlation with inflammation and prion distribution. In vivo and in vitro studies indicated that inflammation is a key player in the distribution of prion infectivity among different organs in the periphery and suggests that other inflammatory disorders might influence the mode of distribution and replication of prions [23]. Thus, chronic inflammation and autoimmune diseases might lead to up regulation of lymphotoxins alpha and beta after pro-inflammatory cytokines attract B and T lymphocytes; leading to prion pathogen replication and accumulation in known as well other sites that were previously believed to be pathogen free [18].All TSE strains that follow horizontal transmission vary from one another in how they are expressed and do not affect everybody equally. Among these strains is kuru, and vCJD. Kuru was the first human disease related to scrapie and was transmitted from one individual to another by cannibalism; showing incubation periods of as long as 60 years. Most of the patients that suffered Kuru in geographic areas such as Papua Guinea were heterozygous at amino acid codon 129. On the other hand, different genetic factors are of great clinical importance for differential infectivity in variant CJD cases. Three compelling indications of transmission of BSE to humans (vCJD) are similar molecular markers, type 1 and 4 associated with Methionine 129. Four different strains of prion have been observed which have the same sequence of amino acids, but different conformation and pathology. The variance of amino acid 129 in PrPc influences the susceptibility and propagation of the four human PrPsc types. Also, due to different glycosylation profiles of PrPsc, different PrPsc conformations result in different digested fragments by protease K. Studies with transgenic mice expressing different variants of PrPc were analyzed for TSE development. Type 4 PrPsc infected mice carrying human PrPc Methionine 129 with BSE or vCJD (129 MM and type 4 prion strain) showed 100% infectivity and no species barrier. However, no infection was observed in 129 VV transgenic mice due to differential sequence and species barrier [24]. These studies showed what factors play important roles in the transmission of vCJD from BSE, and how Methionine 129 is key to influencing differential infectivity among individuals. Research FindingsPrions diseases have been found to be fatal on mouse models once infection occurs, and there are no current known treatments for the aggregation of the PrPsc proteins once this takes place[25]. Because of the PrPsc conformation of the prion is resistant to both chemical and physical sterilization, it makes it very difficult for the diseases caused by this prion to be treated [26]. This type of prion has been found to be resistant to proteases, heat, high pressure, radiation, and formalin treatments [27]; although their infectivity can be reduced by these treatments. The infectious state of prions depends on the tertiary structure conformation of the protein, so it could be assumed that denaturing the -sheet conformation of the prion would remove the aggregative properties [28]. Although this might be true the problem comes in when the protein refolds, it has been found that the prions would retain the PrPsc conformation according to the template-directed refolding model when refolding takes place after denaturing the prion [18]. A renowned professor in the field of prions, Giovanna Mallucci, suggested that all prion disorders could be prevented back in 2003 [25]. Her study focused on how PrPc deficient mice would not be affected by scrapie a form of prion disease characterized by scratching of the animal and peeling of the fur. Prior to this there has been no effective treatment for these animals after onset of clinical signs, and no agent prevented the disease progression either. Because of this it was thought that the disease should be treated prior to this happening; and because of the already existing knowledge that PrPsc is a change in conformation form PrPc, this would be their new target. Their hypothesis derived from this information was that targeting PrPc would remove the substrate for the conversion to PrPsc, making it a more effective therapy rather than reducing the already existing PrPsc accumulation. The effects of depletion of PrPc pre infection of scrapie showed no major detrimental effects. Two lines of transgenic MloxP mice were used, tg37 and tg46 that express PrPc from MloxP transgenes, these two mice lines would naturally succumb to scrapie at ~12 and ~18 weeks post infection (wpi) respectively. Cre-recombinase, an enzyme that excises the PrP-coding sequences in the MloxP transgenes can eliminate expression of PrPc in these mice [25]. Cre was introduced to both strains of mice using the neurofilament heavy chain promoter, the mice with the Cre-recombinase showed a neuronal depletion of PrPc at around 12 weeks of age. Both NFH-Cre/MloxP mice and MloxP mice were inoculated with scrapie at 3 to 4 weeks of weaning and the results were favorable to the NFH-Cre/MloxP mice. Both tg37 and tg46 mice tested positive for PrPsc, showed scrapie symptoms, neuronal loss, gliosis, HC spongiosis, and died at 12 wpi and 18 wpi respectively. NFH-Cre/tg37 and NFH-Cre/tg46 mice also tested positive for PrPsc and gliosis but were asymptomatic, there was no neuronal loss, no HC spongiosis and most importantly they survived for more than 58 wpi and eventually died due to natural causes [25].With these findings a possible treatment could be developed by means of either preventing expression of the PrPc protein with the conclusion that pathogenic prions require a precursor. Once aggregation of PrPsc takes place the disease can be attenuated, apparently the PrPsc concentration has to reach a certain level before the onset of symptoms, neuronal loss, and eventually death [25].Extensive research has also been done regarding the incidence of the prion related diseases, as they could be because of mutations as well as epigenetic factors it has been found that it could be transmitted via human-human infection as well as cross-species infections [18, 25, 26, 29]. A prime example of this route of infection can be related to both the variant Creutzfeldt-Jakob disease (vCJD) and kuru [18]. A high incidence rate for vCJD was noted in the United Kingdom starting in the mid-1980s and followed until the mid-1990s, while ten years earlier a high incidence rate for bovine spongiform encephalopathy (BSE) in cows also in the UK where the vCJD cases were noted [26]. What was known about BSE is that it caused death of the infected cattle and was knows as the mad-cow disease due to the symptoms shown by the infected. These cows became infected through the oral ingestion of meat-and-bone-meal (MBM), which consisted of carcasses of fallen stock, and poultry rejected for human consumption [26]. The processes for preparing this MBM is called rendering, this extracts water, separates fat, and leaves a protein rich material that would be ideal for cattle fodder BSE infected carcasses would often be used for MBM and because of the high resistance of the prions this rendering procedure would not neutralize the prion content [26]. This way BSE cases would rise yearly during the 1980s taking the number of infected cattle to the tens of thousands by the mid-1980s, once people figured out the source of infection BSE carcasses were no longer used for MBM [26, 29]. On the other hand BSE cattle was still being used for human consumption as a cross-species barrier was assumed, also at first BSE was thought to be a variant a of the agent of scrapie that affected sheep so it was thought that human transmission was unlikely. This was thought to be the case for the next couple of years where no cases of human BSE were noted, until the first cases of vCJD appeared in 1995 [29] upon post-mortem inspection the brain lesions appeared very similar to those of BSE cattle containing microscopic vacuoles and fibrils [30]. At this point BSE was related to being the cause for the human vCJD, although an individual suffering from vCJD could not be traced back to consuming BSE infected beef. Incidence trends between these two were compared, and there was a direct correlation between the numbers of BSE infected cattle and the numbers of vCJD deaths with an average of 10 years apart from one another [29, 31]. Although the incubation period for vCJD cannot be exactly determined post infection, epidemiologal data analysis provided the necessary data to reach this conclusion [29].A different approach for identifying prion disease is through the electrophysiological activity of affected neurons, it is believed that irregularities on the firing patters of affected neurons could lead to neuropahological events in the brain [32]. As it is known brain activity depends directly on the asynchrony of neuronal cells for proper function, and it was hypothesized that upon PrPsc infection and aggregation abnormal synaptic plasticity as well as irregular membrane depolarization and hyper-polarization are present. It is believed that this electrophysiological feature is due to neuronal malfunction but not due to neuronal loss [32]. This change in neuronal activity is what causes the behavioral changes in the affected mice as well as piloerection, weight loss, motor incoordination; eventually leading to, in more advanced cases, total impairment of synaptic plasticity vacuolation and ultimately neuronal loss. It is unsure if this membrane depolarization is a feature of the early prion disease or only seen at a later stage of the disease, but triggers inappropriate inactivation of NMDA receptors. This activation creates an ion imbalance increasing the intracellular Ca2+ concentration activating the potassium channels in the cell, as well as potentially triggering cytotoxic damage [32].

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