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Prions and protein misfolding disorders

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Prions and protein-folding diseases
E. Norrby
J Intern Med 2011; 270: 1–14. http://dx.doi.org:/10.1111/j.1365-2796.2011.02387.x

Prions represent a group of proteins with a unique capacity to fold into different conformations. One isoform is rich in beta-pleated sheets and can aggregate into amyloid that may be pathogenic. This abnormal form propagates itself by imposing its confirmation on the homologous normal host cell protein. Pathogenic prions have been shown to cause lethal neurodegenerative diseases in humans and animals. These diseases are sometimes infectious and hence referred to as transmissible spongiform encephalopathies. In the present review, the remarkable evolution of the heterodox prion concept is summarized. The origin of this phenomenon is based on information transfer between homologous proteins, without the involvement of nucleic acid-encoded mechanisms. Historically, kuru and Creutzfeldt-Jakob disease (CJD) were the first infectious prion diseases to be identified in man. It was their relationship to scrapie in sheep and experimental rodents that allowed an unravelling of the particular molecular mechanism that underlie the disease process. Transmission between humans has been documented to have occurred in particular contexts, including ritual cannibalism, iatrogenic transmission because of pituitary gland-derived growth hormone or the use in neurosurgical procedures of dura mater from cadavers, and the temporary use of a prion-contaminated protein-rich feed for cows. The latter caused a major outbreak of bovine spongiform encephalopathy, which spread to man by human consumption of contaminated meat, causing approximately 200 cases of variant CJD. All these epidemics now appear to be over because of measures taken to curtail further spread of prions. Recent studies have shown that the mechanism of protein aggregation may apply to a wider range of diseases in and possibly also outside the brain, some of which are relatively common such as Alzheimer’s and Parkinson’s diseases. Furthermore, it has become apparent that the phenomenon of prion aggregation may have a wider physiological importance, but a full understanding of this remains to be defined. It may involve maintaining neuronal functions and possibly contributing to the establishment of long-term memory.

The history of the identification of the infectious nature of prion diseases and the discovery of the chemical nature of this type of infectious agent is remarkable. The great advances in this field of research have been recognized by two Nobel Prizes in Physiology or Medicine: one in 1976 to D. Carleton Gajdusek (a prize shared with Baruch S. Blumberg, for the discovery of hepatitis B virus) and the other in 1997 to Stanley B. Prusiner. Infectious prion diseases represent relatively rare phenomena mostly observed in the context of the spread of the agent by human intervention. However, the principal molecular mechanisms that lead to disease may have applications for a number of much more common noncontagious diseases. Two fundamental observations are relevant to the understanding of the molecular mechanisms involved. The first is that the same polypeptide chain, depending on the environmental conditions, including the possible presence of homologous proteins with a predetermined folding pattern, may take on dramatically altered folding. In certain cases, major aggregates of homologous proteins may be formed and such aggregates in turn may display cytopathogenic effects. A degenerative disease may ensue. The second relevant observation is that much still remains to be learnt about protein-folding phenomena and the role of information transfer systems engaging only proteins. Many proteins have sequences of amino acids that make them potentially prionogenic. Such sequences that under certain circumstances may be the cause of disease in mammals can, in another context, play a central role in physiological functions, for example, as the source of epigenetic mechanisms of protein signalling of importance for the survival of yeast cells.

In this review, our emerging understanding of the mechanisms of prion diseases will first be discussed, in particular their unique mechanisms of spread will be considered. The original belief in relatively firm barriers preventing the spread of prions between species was questioned when it became clear that bovine spongiform encephalopathy (BSE), also known as ‘mad cow disease’, could be transmitted to man. It was then shown, surprisingly, that the disease contracted from infected cattle could spread from man to man by blood transfusion. Next, the possibility of a much wider application of the pathogenic mechanism of cell destruction by protein aggregation, including degenerative processes causing for example Alzheimer’s and Parkinson’s diseases, will be discussed. The rapidly growing appreciation of the significance of signaling between proteins outside the canonical steps of the central dogma ofmolecular biology will finally be considered.  ….

Unique samples of brain collected by Gajdusek allowed identification of the histopathology of kuru. It showed many similarities to the changes in brains from patients with sporadic sCJD, but there were also similarities to the changes in brains from sheep with scrapie as noted by Hadlow [2]. His observation encouraged Gajdusek to attempt to transmit these non-inflammatory diseases to chimpanzees. He and his collaborators were successful in transmitting first kuru [3] and then CJD [4] by intracerebral inoculation of chimpanzees. The term transmissible spongiform encephalitis (TSE) was commonly used to refer to this kind of infection.

Next, after determining that kuru was infectious, the route of transmission was found to be the ritual cannibalism practiced by the Fore people [5]. The body of the deceased relative was prepared for the funeral meal, and the central nervous system containing the largest concentration of the infectious agent was consumed mainly by the women and children; thus, they were primarily affected by the disease (Fig. 1). However, no child born after 1960, the year in which the practice of ritual cannibalism ceased, has contracted kuru. The incubation time of the disease can be long, potentially longer than the life span of the individual, and cases of kuru have occurred in the present century [6]. The infectious nature of sCJD had been demonstrated, but it was initially unclear how it might spread. In fact, under normal conditions, CJD is not infectious. To date, careful studies of transfusion of blood from people incubating or even displaying early signs of CJD have not revealed any spread of the agent between individuals by this route [7]. Only under conditions of medical interventions involving brain material could an iatrogenic spread of the spontaneous form of the disease be demonstrated.

 

Throughout the 1970s, Gajdusek continued to refer to the infectious agents that cause kuru⁄CJD as slow viruses, and this situation did not change until Prusiner began to gain insights into the chemical nature of the agents using hamster scrapie as amodel to isolate and characterize them. He continued to purify the infectious material from hamster brains until he produced a relatively pure protein preparation [8]. Because of the lack of any evidence of participating nucleic acid in this preparation, he named it prion (proteinaceous and infectious particle) [9]. The protein preparation – referred to as PrP 27–30 (prion protein with a molecular weight 27–30 kD) – was pure enough to determine a short section of its amino terminal amino acid sequence. This information made it possible to determine that the gene responsible for the synthesis of the prion protein was not foreign, but a normal host cell gene [10]. Thus, it became possible to explain the absence of any inflammatory response in the tissues in the presence of this infectious disease; under healthy conditions, we do not develop immunological reactions to our own tissues.

Following the identification of the PrP gene, it was possible to produce mice deficient in PrP using ‘knockout’ technology [11]. At this time, it was known that the PrP protein appears in the brain early during embryonic development. It was found, unexpectedly, that development and life span appeared to be normal in animals without PrP protein. The PrP-knockout mice could be used for two types of critical follow-up experiments.

First, after infection with different doses of infectious prion material, the knockout mice were found to be completely refractory to development of disease [12, 13]. Furthermore, they could mount an immune response to the PrP protein, as this no longer represented an endogenous product. For the first time, antibodies could be generated against different parts of the protein. The protein that Prusiner et al. had identified was indeed critical to the pathogenic process in prion diseases.

The second type of experiment was to investigate the effects of reintroducing into the PrP-knockout mice either a modified homologous PrP gene (carrying a substitution or deletion of a single or a stretch of amino acids) or a PrP gene from a different species. The former experiment enabled attempts to dissect the role of different parts of the PrP protein in the pathogenic process (as discussed below). The latter enabled the evaluation of the species specificity of PrP proteins. Mice are normally infected only by prions from othermice or rodents, such as hamsters, but not from more distant species. However, by generation of transgenic mice carrying for example a human or a bovine PrP gene, this species barrier can be overcome providing important opportunities for studies of different kinds of pathogenic PrP proteins of relevance for human disease. Inappropriate folding and aggregation of proteins can cause disease

Diseases caused by protein aggregation have been known for a long time. They are collectively referred to as amyloid diseases, a term reflecting the original misconception that the observed stainable deposits contained starch (amylum in Latin). Later, it was demonstrated that they were in fact composed of various kinds of proteins. The amyloid deposits have characteristic staining properties and show birefringence under polarized light. Protein aggregates showing these characteristics were demonstrated in the brains of patients with CJD. However, amyloid formation is not always a feature of prion disease [14]. It is in fact found in only about 10% of the brains of patients with sCJD, but at a much higher rate in patients with other forms of the disease.

It then became possible to determine the structure of purified PrP using nuclear magnetic resonance analysis [15, 16]. It was shown that PrP can appear in two completely different forms, albeit with an identical amino acid sequence. The ‘healthy’ protein, referred to as PrP-C (control), has a structure involving predominantly two large alpha-helix structures, whereas there is a predominance of beta-pleated sheets in the pathological PrP-TSE protein (Fig. 2). It is the PrP-TSE protein that may form amyloid protein aggregates. The reason for the two completely different configurations of the same protein is not known, but a critical observation is that if a small amount of PrP-TSE is added to a larger amount of PrP-C, the ‘healthy’ protein is converted to the TSE form by an as yet undefined contagious ‘snowball’ effect. Two theoretical models, nucleation-polymerization and template assistance, have been proposed to explain this ([17], Fig. 3). However, as discussed in the next section, only certain kinds of proteins are capable of forming amyloid and are truly infectious, and the term prion is reserved for them; the term prionogenic has been introduced to include noninfectious amyloid-generating proteins.

The awareness that CJD was a disease that could be transmitted to experimental animals immediately raised the question of to what extent itmight be transmissible between humans. There was no epidemiological evidence of connections between cases of CJD, except for an increased frequency of occurrence in certain families and ethnic groups. In the light of understanding the seminal importance of PrP in the disease, it could be deduced that familial cases must be because of inherited mutations in different sites of the PrP gene, whereas sporadic cases were likely to be caused by mutation(s) accumulated in somatic (possibly brain) cells or alternatively a spontaneous emergence of amisfolded PrP protein (the nucleation– polymerization model) during the lifetime of the individual. As already mentioned, to date it has not been possible to find evidence for transmission of disease from individuals with sCJD by blood transmission [7], but relatively recent data provide evidence for a possible transmission of scrapie in sheep by experimental blood transfusion [18].

Gajdusek and collaborators at an early stage began to search for evidence of the spread of CJD through medical intervention. They found that in CJD, as in scrapie, the majority of infectious prions were located in the brain; indeed, brain tissue is about 100 000 times more infectious than peripheral tissues, such as blood [19]. The first case of iatrogenic spread of CJD between two individuals was found in connection with a corneal transplant [20], and later the similar spread to two relatively young individuals was demonstrated as a result of using electrodes for intracerebral recording [21].

Three epidemics of strikingly different origins have been documented, and all comprise slightly in excess of two hundred cases with the maximum number of cases at the end of the 1990s (Fig. 4). The first of the three epidemics was caused by the use of human growth hormone prepared from pools of many hundreds of pituitary glands from cadavers [see 19 for references]. Because these preparations were used in growing individuals, many of the victims of the disease were relatively young. When this iatrogenic spread of CJD prions was discovered, the product was rapidly withdrawn, and it was progressively replaced by growth hormone prepared by recombinant DNA technology. The average incubation time of this parenterally injected material was estimated to be 15 years (range 4–36 years). The total number of cases to date is 206, and the epidemic seems to have just reached an end. Most cases occurred in France, 109 of 1700 treated individuals. The corresponding figures are 56 of 1848 treated individuals in the UK and 28 of 7700, in the USA. In the USA, an additional step of purification was introduced in 1977, which may have reduced the risk of transmission of infectious prions.

The second epidemic has a distinctly different iatrogenic background. It relates to the previous use of heterologous cadaveric dura mater material to improve the healing process after neurosurgical interventions [see 19 for references]. The total number of cases registered to date is 196 (only 142 shown in Fig. 4), the majority (63%) of which have been in Japan. The estimated average incubation time was 11 years (range 16 months – 23 years). The use of this type of graft was banned in the UK in 1989 and in Japan in 1997. In 1987 a disinfection step with NaOH was introduced, but eventually this was not considered safe. The alternatives used today are synthetic dura mater material, connective tissue (fascia lata orfascia temporalis) from the patient or material of animal origin (bovine pericardium)’. Overall, it seems that the threat of iatrogenic spread of CJD is now minimal [19]. With the present awareness of the situation, any potential occupational risk of disease, for example, for surgeons and nurses involved in brain surgical procedures, can in practical terms be eliminated.

The main focus of interest during the last 15 years has been the third epidemic, the unexpected spread of prions from cattle to man. …..

Fig. 2 Fundamentally different structures of normal and inappropriately folded PrP protein. The latter has a predominance of beta-pleated sheets, which gives it a propensity to aggregate with other homologous proteins potentially causing destruction of tissues. Figure kindly provided by Paul Brown.

Fig. 3 Schematic model of conversion of PrP-C to PrP-TSE. In the nucleation-polymerization model, there is a rapid conversion of PrP protein between the PrP-C (circles) and PrP-TSE forms (squares), but the former is more stable. In the presence of an aggregate large enough to act as a stable nucleus, illustrated by the collection of PrP-TSE squares, a change from PrP-C to PrP-TSE is favored. In the template-assistance model, the conversion of PrP-C or a modified conformation, PrP-INT (intermediate), to Prp-TSE is extremely slow in the absence of PrP-TSE, but the process of conversion is essentially irreversible. PrP-TSE is able to propagate itself by catalysing the conversion of other PrP-INT molecules to the PrP-TSE confirmation. The final product of the two models is amyloid,which is potentially responsible for the disease process. Modified from ref. 17

In 1989 mandatory changes in slaughtering techniques were introduced. These changes ensured that the brain and spinal cord, the main sources of prions, were excluded from products used for human consumption. The precaution was taken even though at the time it was not anticipated that prions could spread from cattle directly to man, as there had never been any evidence that the scrapie agent could spread from sheep to man. In principle, the same species barrier that had prevented such a spread for hundreds of years was expected to exist also between cows andman. However, in 1994 the first case of CJD of bovine origin was identified inman [22]. For several reasons, it was concluded that this case was caused by transmission of prions from cows. ….

Once it became clear that the presence of the PrP gene was absolutely essential to the development of PrPTSE-derived diseases and that animals without PrP, unexpectedly, seemed to develop normally, it was important to determine the physiological role of the PrP protein. However, despite many studies, its fundamental function(s) still remains to be definitively identified. Different studies have highlighted a wide range of different functions [37–42]. The chromosomal gene denoted Prnp encodes PrP. It is a member of the Prn gene family, which also genes encoding two other proteins. The PrP open reading frame is encoded within a single exon directing the synthesis of a protein with 254 amino acids. This protein is post-translationally modified by removal of a 22-amino acid, amino terminal signal peptide and a 23-amino acid carboxy terminal. The latter directs the addition of a glycosylphosphatidyl inositol membrane anchor. Under normal conditions, PrP is a membrane-bound protein, but it can also show biological activity and cause infectious amyloid disease in a nonmembrane-bound form [43]. The protein has two glycosylation sites and an internal disulphide bond. All these properties are shared between molecules exerting their normal physiological function(s) and proteins causing prion diseases. The majority, but not all, PrP proteins are relatively resistant to protease digestion. This property was used in the early attempts to purify the protein. Proteinase digestion cleaves about 67 amino acids from the amino terminal of the 209-amino acid final protein product. This produces PrP 27–30, a truncated protein, which can still form amyloid. This was the protein used by Prusiner et al. to identify the nature of PrP. Alignment of PrP sequences of different mammalian origin shows a striking degree of conservation, highlighting a crucial biological function preserved through evolution [42]. However, there are also differences, which explain the species barrier to disease transmission mentioned earlier.

A number of different functions have been proposed for the normal protein: modulation of signal pathways of importance for the survival of cells, protection against oxidative stress and binding of copper. It was recently reported that membrane-bound PrP represents the major cellular receptor for the oligomeric beta-amyloid involved in Alzheimer’s disease [44]. Whether there is any significance to this possible connection between mechanisms of development of these two neurodegenerative diseases that both depend on transmission of inappropriately folded proteins remains to be seen. A number of recent studies points towards the particular importance of the PrP protein for long-term maintenance of neuronal functions. One study involving four independently targeted mouse strains depleted of PrP-C demonstrated a role of the gene product for peripheral myelin maintenance [45]. Ablation of the protein triggered chronic demyelinating neuropathy. Other recent results suggest that the seminal role of normal PrP is to maintain brain cells in good condition and protect them from overexcitement.   ….

The early studies of transmissible prion brain disease inmice and hamsters revealed that agents of different origin and⁄ or passage history could cause disease after different incubation times and with different histopathologies [51]. Originally, this was interpreted to mean that nucleic acids must play a role in prion inheritance because it was believed at the time that only this type ofmolecule could be the source of stably inherited properties. However, the more the system was analysed, the more it became clear that proteins alone could be a source of diversity, the expression of which to a considerable extent was dependent on the environmental conditions. To date, studies of hereditable human prion diseases have demonstrated correlations with more than 40 different mutations in the PrP gene [see ref 42]. These genetic variants include single-nucleotide base changes, deletions and occurrence of a varying number of segmental repeats. The effects of many of these types of genetic changes have now been mimicked by the use of transgenic mice. It has been shown that prions exist as conformationally diverse populations and that amongst these there are different strains that can replicate with independent reproducibility. Prion transformation may occur by competition and selection [52]. Other studies have focused on the effect of deletion of the part of the PrP-TSE protein that is responsible for anchoring to the cytoplasmic membrane [43]. The soluble form of PrP-TSE can still cause disease, but there is a major change in the incubation time and in histopathological changes in the infected brain [53, 54].

The propensity of certain proteins to form potentially pathogenic aggregates can be examined currently by four different approaches.

  1. Synthetic peptides exploring amino acid-dependent conformational differences that determine the emergence of polymorphic amyloid fibrils structurallymimicking prion strains.
  2. Performance of bioinformatic proteome-wide surveys for prionogenic proteins in certain species.
  3. Examination of the product(s) of replication of prions of different molecular characteristics in transgenic mice with a PrP gene construct of a preselected, potentially different species origin (possibly a chimera), with different molecular characteristics and displaying varying levels of expression.
  4. Treatment of prion inocula prior to inoculation by different procedures to attempt to increase the infectiousness of the preparation. This is referred to as in vitro generation of prions, but it should be kept in mind that the read-out of prion ‘replication’ is always anin vivo system. ….

Prion replication in mammalian systems requires the presence of both PrP-C and PrP-TSE. The latter serves as a seeding nucleus or a template onto which the physiological form of the protein is refolded into the infectious conformation (see Fig. 3). To undergo conversion, it is likely that PrP-C must develop an intermediate state.

Experimentally induced increase in the infectiousness of a prion-containing material can be achieved in vivo or in vitro. Many different experiments have demonstrated that it is the characteristics of the seeding nucleus or template that decides the nature of the final product.  ….

In further experiments, including denaturation by guanidine hydrochloride at varying concentrations, it was demonstrated that the conformational stability of the prions (either native or synthetic) correlated with the incubation period of disease [65–68]. Even protease-sensitive forms of PrP have been found to be capable of inducing disease [69].

In vitro replication of infectious PrP using a mixture in which all reagents are defined and employing a cell culture read out system as not yet been demonstrated. Nevertheless, there is general agreement that the successful generation of new infectious material that has been achieved both in vivo and in vitro rules out the possibility that prion replication is dependent on information stored in nucleic acids. ….

It has become increasingly realized that there is an extensive flow of information, or cross-talk, between proteins. Many proteins do not have a firm three-dimensional form in their native state, but represent a random coil; on coming into contact with a specific part of another protein or another chemical structure that they take on a fixed three-dimensional structure. Others can, under certain conditions, spontaneously move from secondary to tertiary and even quaternary structures. Still, protein folding as a general phenomenon has only been incompletely explained, and it is known that in many cases assisting proteins, like the chaperones, need to be present. It has been clearly demonstrated that the same polypeptide chain may take on very different conformations and that this occurs under various environmental conditions, in particular in the presence of homologous proteins already folded into one form or another. Epigenetics, i.e. the transfer of resilient genetic information not stored in nucleic acid sequences, is a rapidly expanding field and there is room for still more surprises from the study of prions.

Infectious prion diseases are rare, but the mechanism of tissue destruction by aggregation of proteins via their beta-pleated sheets seems to also apply to many other diseases, some of which are common [71, 72]. Several examples are given in Table 1. One interesting case is the beta-amyloid protein, which plays a central role in Alzheimer’s disease. …

Table 1 Prions and potential prionoids

Brain extracts from transgenic mice expressing mutant forms of tau protein have been injected into brains of other transgenic mice expressing human wild-type tau, leading to development of aggregates of the human tau [75]. Thus ‘tauopathies’ may be the result of a prion-like process in which hyperphosphorylation of the protein leads to polymerization and subsequently produces filamentous protein aggregates. There is also evidence for prion-like transmission of polyglutamine protein aggregates, characteristic of Huntington’s disease [76]. Studies have shown that amyloid protein A, the critical protein in secondary amyloidosis, injected into mouse brain can lead to degenerative disease [77]. Additional studies of material from patients with Parkinson’s disease have revealed that the occurrence of inappropriate protein folding can be transmitted from the cells of the host to transplanted cells (see [78]). Also, diseases outside the central nervous system can involve cells subjected to degenerative processes induced by inappropriately folded proteins; one example of this is diabetes type 2 [79]. Although these different diseases appear to have their origin in self-sustained aggregation of prionoid proteins, it should be noted that there is no evidence that they may be transmitted by an infectious process.

To date, the focus in studies of mammalian prions and prionogenic proteins has been on their potential for development of disease. Whether this category of proteins may also, as in the case of fungi, carry important physiological functions remains to be determined. It was recently demonstrated that the cytoplasmic polyadenylation element binding protein can form prion-like multimers in sensory neurons in the nervous system of the giant marine snail Aplysia [80]. This modification has been proposed to serve a function in long-term memory. Thus, for readers who have followed this review to the end, recollection of the salient facts and speculations presented – if stored for the future – may be due to aggregation of prionogenic proteins in the brain, provided of course that the fundamental long-term memory mechanisms of the human brain are similar to those of Aplysia.

 

Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution
Massimo Stefani · Christopher M. Dobson
J Mol Med 2003; 81:678–699      http://dx.doi.org:/10.1007/s00109-003-0464-5

The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of more than 20 degenerative conditions affecting either the central nervous system or a variety of peripheral tissues. As these conditions include Alzheimer’s, Parkinson’s and the prion diseases, several forms of fatal systemic amyloidosis, and at least one condition associated with medical intervention (haemodialysis), they are of enormous importance in the context of present-day human health and welfare. Much remains to be learned about the mechanism by which the proteins associated with these diseases aggregate and form amyloid structures, and how the latter affect the functions of the organs with which they are associated. A great deal of information concerning these diseases has emerged, however, during the past 5 years, much of it causing a number of fundamental assumptions about the amyloid diseases to be reexamined. For example, it is now apparent that the ability to form amyloid structures is not an unusual feature of the small number of proteins associated with these diseases but is instead a general property of polypeptide chains. It has also been found recently that aggregates of proteins not associated with amyloid diseases can impair the ability of cells to function to a similar extent as aggregates of proteins linked with specific neurodegenerative conditions. Moreover, the mature amyloid fibrils or plaques appear to be substantially less toxic than the prefibrillar aggregates that are their precursors. The toxicity of these early aggregates appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increases in free Ca2+ that eventually lead to apoptotic or necrotic cell death. The ‘new view’ of these diseases also suggests that other degenerative conditions could have similar underlying origins to those of the amyloidoses. In addition, cellular protection mechanisms, such as molecular chaperones and the protein degradation machinery, appear to be crucial in the prevention of disease in normally functioning living organisms. It also suggests some intriguing new factors that could be of great significance in the evolution of biological molecules and the mechanisms that regulate their behavior.

The genetic information within a cell encodes not only the specific structures and functions of proteins but also the way these structures are attained through the process known as protein folding. In recent years many of the underlying features of the fundamental mechanism of this complex process and the manner in which it is regulated in living systems have emerged from a combination of experimental and theoretical studies [1]. The knowledge gained from these studies has also raised a host of interesting issues. It has become apparent, for example, that the folding and unfolding of proteins is associated with a whole range of cellular processes from the trafficking of molecules to specific organelles to the regulation of the cell cycle and the immune response. Such observations led to the inevitable conclusion that the failure to fold correctly, or to remain correctly folded, gives rise to many different types of biological malfunctions and hence to many different forms of disease [2]. In addition, it has been recognised recently that a large number of eukaryotic genes code for proteins that appear to be ‘natively unfolded’, and that proteins can adopt, under certain circumstances, highly organised multi-molecular assemblies whose structures are not specifically encoded in the amino acid sequence. Both these observations have raised challenging questions about one of the most fundamental principles of biology: the close relationship between the sequence, structure and function of proteins, as we discuss below [3]. It is well established that proteins that are ‘misfolded’, i.e. that are not in their functionally relevant conformation, are devoid of normal biological activity. In addition, they often aggregate and/or interact inappropriately with other cellular components leading to impairment of cell viability and eventually to cell death. Many diseases, often known as misfolding or conformational diseases, ultimately result from the presence in a living system of protein molecules with structures that are ‘incorrect’, i.e. that differ from those in normally functioning organisms [4]. Such diseases include conditions in which a specific protein, or protein complex, fails to fold correctly (e.g. cystic fibrosis, Marfan syndrome, amyotonic lateral sclerosis) or is not sufficiently stable to perform its normal function (e.g. many forms of cancer). They also include conditions in which aberrant folding behaviour results in the failure of a protein to be correctly trafficked (e.g. familial hypercholesterolaemia, α1-antitrypsin deficiency, and some forms of retinitis pigmentosa) [4]. The tendency of proteins to aggregate, often to give species extremely intractable to dissolution and refolding, is of course also well known in other circumstances. Examples include the formation of inclusion bodies during overexpression of heterologous proteins in bacteria and the precipitation of proteins during laboratory purification procedures. Indeed, protein aggregation is well established as one of the major difficulties associated with the production and handling of proteins in the biotechnology and pharmaceutical industries [5].

Considerable attention is presently focused on a group of protein folding diseases known as amyloidoses. In these diseases specific peptides or proteins fail to fold or to remain correctly folded and then aggregate (often with other components) so as to give rise to ‘amyloid’ deposits in tissue. Amyloid structures can be recognised because they possess a series of specific tinctorial and biophysical characteristics that reflect a common core structure based on the presence of highly organised β- sheets [6]. The deposits in strictly defined amyloidoses are extracellular and can often be observed as thread-like fibrillar structures, sometimes assembled further into larger aggregates or plaques. These diseases include a range of sporadic, familial or transmissible degenerative diseases, some of which affect the brain and the central nervous system (e.g. Alzheimer’s and Creutzfeldt-Jakob diseases), while others involve peripheral tissues and organs such as the liver, heart and spleen (e.g. systemic amyloidoses and type II diabetes) [7, 8]. In other forms of amyloidosis, such as primary or secondary systemic amyloidoses, proteinaceous deposits are found in skeletal tissue and joints (e.g. haemodialysis-related amyloidosis) as well as in several organs (e.g. heart and kidney). Yet other components such as collagen, glycosaminoglycans and proteins (e.g. serum amyloid protein) are often present in the deposits protecting them against degradation [9, 10, 11]. Similar deposits to those in the amyloidoses are, however, found intracellularly in other diseases; these can be localised either in the cytoplasm, in the form of specialised aggregates known as aggresomes or as Lewy or Russell bodies or in the nucleus (see below).

The presence in tissue of proteinaceous deposits is a hallmark of all these diseases, suggesting a causative link between aggregate formation and pathological symptoms (often known as the amyloid hypothesis) [7, 8, 12]. At the present time the link between amyloid formation and disease is widely accepted on the basis of a large number of biochemical and genetic studies. The specific nature of the pathogenic species, and the molecular basis of their ability to damage cells, are however, the subject of intense debate [13, 14, 15, 16, 17, 18, 19, 20]. In neurodegenerative disorders it is very likely that the impairment of cellular function follows directly from the interactions of the aggregated proteins with cellular components [21, 22]. In the systemic non-neurological diseases, however, it is widely believed that the accumulation in vital organs of large amounts of amyloid deposits can by itself cause at least some of the clinical symptoms [23]. It is quite possible, however, that there are other more specific effects of aggregates on biochemical processes even in these diseases. The presence of extracellular or intracellular aggregates of a specific polypep tide molecule is a characteristic of all the 20 or so recognised amyloid diseases. The polypeptides involved include full length proteins (e.g. lysozyme or immunoglobulin light chains), biological peptides (amylin, atrial natriuretic factor) and fragments of larger proteins produced as a result of specific processing (e.g. the Alzheimer β- peptide) or of more general degradation [e.g. poly(Q) stretches cleaved from proteins with poly(Q) extensions such as huntingtin, ataxins and the androgen receptor]. The peptides and proteins associated with known amyloid diseases are listed in Table 1. In some cases the proteins involved have wild type sequences, as in sporadic forms of the diseases, but in other cases these are variants resulting from genetic mutations associated with familial forms of the diseases. In some cases both sporadic and familial diseases are associated with a given protein; in this case the mutational variants are usually associated with early-onset forms of the disease. In the case of the neurodegenerative diseases associated with the prion protein some forms of the diseases are transmissible. The existence of familial forms of a number of amyloid diseases has provided significant clues to the origins of the pathologies. For example, there are increasingly strong links between the age at onset of familial forms of disease and the effects of the mutations involved on the propensity of the affected proteins to aggregate in vitro. Such findings also support the link between the process of aggregation and the clinical manifestations of disease [24, 25].

The presence in cells of misfolded or aggregated proteins triggers a complex biological response. In the cytosol, this is referred to as the ‘heat shock response’ and in the endoplasmic reticulum (ER) it is known as the ‘unfolded protein response’. These responses lead to the expression, among others, of the genes for heat shock proteins (Hsp, or molecular chaperone proteins) and proteins involved in the ubiquitin-proteasome pathway [26]. The evolution of such complex biochemical machinery testifies to the fact that it is necessary for cells to isolate and clear rapidly and efficiently any unfolded or incorrectly folded protein as soon as it appears. In itself this fact suggests that these species could have a generally adverse effect on cellular components and cell viability. Indeed, it was a major step forward in understanding many aspects of cell biology when it was recognised that proteins previously associated only with stress, such as heat shock, are in fact crucial in the normal functioning of living systems. This advance, for example, led to the discovery of the role of molecular chaperones in protein folding and in the normal ‘housekeeping’ processes that are inherent in healthy cells [27, 28]. More recently a number of degenerative diseases, both neurological and systemic, have been linked to, or shown to be affected by, impairment of the ubiquitin-proteasome pathway (Table 2). The diseases are primarily associated with a reduction in either the expression or the biological activity of Hsps, ubiquitin, ubiquitinating or deubiquitinating enzymes and the proteasome itself, as we show below [29, 30, 31, 32], or even to the failure of the quality control mechanisms that ensure proper maturation of proteins in the ER. The latter normally leads to degradation of a significant proportion of polypeptide chains before they have attained their native conformations through retrograde translocation to the cytosol [33, 34]. For example, the most common mutation of the CFTR chloride channel associated with cystic fibrosis interferes with the cor rect folding of the polypeptide chain; as a consequence, much of the mutated protein is not secreted but is retained in the ER and rapidly degraded even though, when properly folded, it could still function as ion channel at the cell surface ([35] and references therein).

Table 1 A summary of the main amyloidoses and the proteins or peptides involved

Table 2 Neurodegenerative diseases with inclusion bodies shown to be linked to deficits of the ubiquitin-proteasome pathway (modified from [26])

It is now well established that the molecular basis of protein aggregation into amyloid structures involves the existence of ‘misfolded’ forms of proteins, i.e. proteins that are not in the structures in which they normally function in vivo or of fragments of proteins resulting from degradation processes that are inherently unable to fold [4, 7, 8, 36]. Aggregation is one of the common consequences of a polypeptide chain failing to reach or maintain its functional three-dimensional structure. Such events can be associated with specific mutations, misprocessing phenomena, aberrant interactions with metal ions, changes in environmental conditions, such as pH or temperature, or chemical modification (oxidation, proteolysis). Perturbations in the conformational properties of the polypeptide chain resulting from such phenomena may affect equilibrium 1 in Fig. 1 increasing the population of partially unfolded, or misfolded, species that are much more aggregation-prone than the native state. …

Fig. 1 Overview of the possible fates of a newly synthesised polypeptide chain. The equilibrium ① between the partially folded molecules and the natively folded ones is usually strongly in favour of the latter except as a result of specific mutations, chemical modifications or partially destabilising solution conditions. The increased equilibrium populations of molecules in the partially or completely unfolded ensemble of structures are usually degraded by the proteasome; when this clearance mechanism is impaired, such species often form disordered aggregates or shift equilibrium ② towards the nucleation of pre-fibrillar assemblies that eventually grow into mature fibrils (equilibrium ③). DANGER! indicates that pre-fibrillar aggregates in most cases display much higher toxicity than mature fibrils. Heat shock proteins (Hsp) can suppress the appearance of pre-fibrillar assemblies by minimising the population of the partially folded molecules by assisting in the correct folding of the nascent chain and the unfolded protein response target incorrectly folded proteins for degradation.

The various peptides and proteins associated with amyloid diseases have no obvious similarities in size, amino acid composition, sequence or structure. Nevertheless, the amyloid fibrils into which they convert have marked similarities both in their external morphology (Fig. 2) and in their internal structure (Fig. 3). Circular dichroism and Fourier transform infra-red spectroscopy both indicate a high content of β-structure, even when the monomeric peptide or protein is substantially disordered or rich in α-helical structure. Although it has not yet proved possible to obtain a detailed definition of the molecular structure of any amyloid fibril, investigations by electron and atomic force microscopy show that they are typically long, straight and unbranched. The fibrils are typically 6–12 nm in diameter and usually consist of two to six ‘protofilaments’, each of diameter about 2 nm, that are often twisted around each other to form supercoiled rope-like structures [38, 39]. Each protofilament in such structures appears to have a highly ordered inner core that X-ray fibre diffraction data suggest consists of some or all of the polypeptide chain arranged in a characteristic cross-β structure. In this structural organisation, the β-strands run perpendicular to the protofilament axis, resulting in a series of β-sheets that propagate along the direction of the fibril (Fig. 3).

Little is known at present about the detailed arrangement of the polypeptide chains themselves within amyloid fibrils, either those parts involved in the core β- strands or in regions that connect the various β-strands. Recent data suggest that the sheets are relatively untwisted and may in some cases at least exist in quite specific supersecondary structure motifs such as β-helices [6, 40] or the recently proposed µ-helix [41]. It seems possible that there may be significant differences in the way the strands are assembled depending on characteristics of the polypeptide chain involved [6, 42]. Factors including length, sequence (and in some cases the presence of disulphide bonds or post-translational modifications such as glycosylation) may be important in determining details of the structures. Several recent papers report structural models for amyloid fibrils containing different polypeptide chains, including the Aβ40 peptide, insulin and fragments of the prion protein, based on data from such techniques as cryo-electron microscopy and solid-state magnetic resonance spectroscopy [43, 44]. These models have much in common and do indeed appear to reflect the fact that the structures of different fibrils are likely to be variations on a common theme [40]. It is also emerging that there may be some common and highly organised assemblies of amyloid protofilaments that are not simply extended threads or ribbons. It is clear, for example, that in some cases large closed loops can be formed [45, 46, 47], and there may be specific types of relatively small spherical or ‘doughnut’ shaped structures that can result in at least some circumstances.

Fig. 4 Some amyloid-related peptides/proteins form early aggregates of globular appearance that further organise into beaded chains, globular annular ‘doughnut’ shaped assemblies eventually giving mature protofilaments and fibrils. Pre-fibrilar aggregates may interact with reconstituted phospholipid membranes and with cell membranes where they form aspecific channels (pores) disrupting cellular homeostasis. The latter possible mechanism of toxicity is similar to that displayed by antimicrobial peptides, pore-forming eukaryotic proteins and bacterial toxins and newly synthesised cyclic peptide antibiotics (see text). The electron micrographs of the globular and beaded chains of Aβ peptides are taken from Harper et al. [200]. The electron micrographs of the rings of the α-synuclein A53T (upper row) and A30P (middle row) mutants and of the Alzheimer precursor protein artic mutant (lower row) are from [201].

The similarity of some early amyloid aggregates with the pores resulting from oligomerisation of bacterial toxins and pore-forming eukaryotic proteins (see below) also suggest that the basic mechanism of protein aggregation into amyloid structures may not only be associated with diseases but in some cases could result in species with functional significance. Recent evidence indicates that a variety of micro-organisms may exploit the controlled aggregation of specific proteins (or their precursors) to generate functional structures. Examples include bacterial curli [52] and proteins of the interior fibre cells of mammalian ocular lenses, whose β-sheet arrays seem to be organised in an amyloid-like supramolecular order [53]. In this case the inherent stability of amyloid-like protein structure may contribute to the long-term structural integrity and transparency of the lens. Recently it has been hypothesised that amyloid-like aggregates of serum amyloid A found in secondary amyloidoses following chronic inflammatory diseases protect the host against bacterial infections by inducing lysis of bacterial cells [54]. One particularly interesting example is a ‘misfolded’ form of the milk protein α-lactalbumin that is formed at low pH and trapped by the presence of specific lipid molecules [55]. This form of the protein has been reported to trigger apoptosis selectively in tumour cells providing evidence for its importance in protecting infants from certain types of cancer [55]. ….

Until about 30 years ago proteolysis was considered to be the primary factor triggering the formation of amyloid aggregates in vivo, following the demonstration that lysosomal enzymes, at acidic pH values, are able to convert amyloidogenic proteins into amyloid fibrils [56]. This idea was challenged around 10 years ago when it was shown that transthyretin can be converted in vitro into amyloid fibrils following an acid-induced conformational change [57]. This finding demonstrated that a modification of the three-dimensional structure was sufficient to enable the production of an aggregation-prone species. This suggestion was not immediately accepted, at least in part as a consequence of the well established fact that the peptide found in the plaques characteristic of Alzheimer’s disease resulted from proteolysis of the Alzheimer’s precursor protein. Following these initial observations a large number of proteins known to aggregate in vivo were found to form fibrillar aggregates in vitro as a result of induced conformational changes; these data, however, reinforced the idea that the molecular basis of protein aggregation was an unusual feature of the few peptides and proteins found to be associated with the amyloid diseases, resulting from a specific conformational change related to the specific amino acid sequences. In 1998 two papers were published, each reporting the observation that a protein unrelated to any amyloid disease aggregated in vitro to form structures indistinguishable from the amyloid fibrils that could be produced from the disease-associated peptides and proteins [58, 59]. These observations were made by chance, but it was soon shown that a similar conversion could be achieved deliberately for other proteins by a rational choice of solution conditions [60, 61]. Since then a substantial number of similar studies have been reported ([61] and references therein; Table 3). In each case aggregation of a full-length protein to form amyloid fibrils was found to require solution conditions (such as low pH, lack of specific ligands, high temperature, moderate concentrations of salts or co-solvents such as trifluoroethanol) such that the native structure was partially or completely disrupted but under which interactions such as hydrogen-bonding were not completely inhibited. …..

It was generally assumed until recently that the proteinaceous aggregates most toxic to cells are likely to be mature amyloid fibrils, the form of aggregates that have been commonly detected in pathological deposits. It therefore appeared probable that the pathogenic features underlying amyloid diseases are a consequence of the interaction with cells of extracellular deposits of aggregated material. As well as forming the basis for understanding the fundamental causes of these diseases, this scenario stimulated the exploration of therapeutic approaches to amyloidoses that focused mainly on the search for molecules able to impair the growth and deposition of fibrillar forms of aggregated proteins. An increasing quantity of recent experimental data suggests, however, that in many cases at least the species that are most highly toxic to cells are the pre-fibrillar aggregates (sometimes referred to as amorphous aggregates, protein micelles or protofibrils) rather than the mature fibrils into which they often develop. In particular, a number of reports concerning Aβ peptides, α synuclein and transthyretin indicate that these early aggregates are the most toxic species [18, 76, 77, 78, 79, 80]; in addition, the presence of such species has also been reported for huntingtin [44], and possibly the androgen receptor [81] in diseased transgenic mice. The hypothesis that toxicity is exhibited primarily by early aggregates also provides an explanation for the lack of existence of a direct correlation between the density of fibrillar plaques in the brains of victims of Alzheimer’s disease and the severity of the clinical symptoms [82]. ….

The presence of toxic aggregates inside or outside cells can impair a number of cell functions that ultimately lead to cell death by an apoptotic mechanism [95, 96]. Recent research suggests, however, that in most cases initial perturbations to fundamental cellular processes underlie the impairment of cell function induced by aggregates of disease-associated polypeptides. Many pieces of data point to a central role of modifications to the intracellular redox status and free Ca2+ levels in cells exposed to toxic aggregates [45, 89, 97, 98, 99, 100, 101]. A modification of the intracellular redox status in such cells is associated with a sharp increase in the quantity of reactive oxygen species (ROS) that is reminiscent of the oxidative burst by which leukocytes destroy invading foreign cells after phagocytosis. In addition, changes have been observed in reactive nitrogen species, lipid peroxidation, deregulation of NO metabolism [97], protein nitrosylation [102] and upregulation of heme oxygenase-1, a specific marker of oxidative stress [103]. …..

It is not clear why protein aggregation is followed, even in vitro, by production of ROS. In the case of Aβ42, Met35, Gly29 and Gly33 have been suggested to be involved [109]; a role has also been proposed for metal ions such as Fe, Cu and Zn, for example, through the generation of hydroxide radicals from hydrogen peroxide [110, 111]. An upregulation of the activity of hydrogen peroxide-producing membrane enzymes, such as plasma membrane NADPH oxidase and ER cytochrome P450 reductase, has also been reported in Aβ-induced neurotoxicity in microglia and in cortical neurons [112, 113]. More generally, intracellular oxidative stress could be related to some form of destabilisation of cell membranes by toxic species leading to a failure to regulate appropriately plasma membrane proteins such as receptors and ion pumps [114] and/or to impairment of mitochondrial function. Mitochondria play a well recognised role in oxidative stress and apoptosis; in this regard, a key factor in Aβ peptide neurotoxicity could be the opening of mitochondrial permeability transition pores by Ca2+ entry in neuronal mitochondria [115] followed by release of cytochrome c, a strong inducer of apoptosis. …

Since 1993, a ‘channel hypothesis’ of the molecular basis of the cytotoxicity of amyloid aggregates has been put forward [131] by similarity with the proposed mechanism of toxicity of pore-forming peptides and proteins [90, 91]. As is pointed out above, this idea stems from a number of pieces of evidence leading to the proposal that unchaperoned, positively charged and misfolded proteins, or early aggregates of such species, can interact with lipid membranes ([90, 91] and references therein). Evidence for this proposal comes from the study of both artificial model systems, such as phospholipid bilayers, and cell membranes; in the latter the function of specific membrane proteins has been found to be impaired [78, 91]. In most cases interaction of a misfolded species with a membrane would occur via a two-step mechanism involving electrostatic interaction of the positively charged residues with negatively charged or polar lipid head groups followed by the insertion of hydrophobic regions into the membrane hydrophobic interior [91].

According to this hypothesis, misfolding of proteins, such as at least some of those involved in neurodegenerative diseases, would then induce cytotoxicity. Such cytotoxicity would be a direct consequence of the exposure of hydrophobic regions, favouring the interaction of the misfolded species with the plasma membrane and other cell membranes and leading to membrane damage via the formation of non-specific ion channels. These channels, or pores, have been described for a number of peptide and proteins associated with amyloid disease including Aβ peptides [19, 45, 78, 89] and their fragments [132], α-synuclein [133], islet-amyloid polypeptide [86], the 106–126 fragment of the prion protein [41], poly(Q) stretches [134, 135], the C-type natriuretic peptide [84], β2-microglobulin [48], transthyretin [136], murine serum amyloid A [137] and the N-terminal peptide of an acutephase isoform variant of human serum amyloid A1.1 (SAAp) [54]. The channels have been investigated primarily by recording ion currents across biological or reconstituted membranes, but ‘doughnuts’ of channel-like assemblies of pre-fibrillar aggregates of Aβ1–42, α-synuclein, transthyretin and serum amyloid A have also been observed by electron and atomic force microscopy [45, 46, 49, 137]. ….

In general, heterogeneity of amyloid intermediates, including globules, chains, doughnuts, protofilaments and fibrils, could result in increased potency of toxicity since the different types of intermediates may act in differing ways on membranes such as the suite of peptides in venoms. In the case of α-synuclein the ‘pores’ coexist with fibrils under conditions of molecular crowding [138], raising the possibility that the former are more stable under cytoplasmic conditions and leading to the proposal that they are the pathogenic species in Parkinson’s disease [46]. The size-dependent permeabilisation of artificial vesicles by protofibrillar α-synuclein suggests that permeabilisation occurs mainly as a result of a specific membrane perturbation via the formation of pores at least 2.5 nm in diameter [46]. If α-synuclein annular protofibrils are the pathogenic species in Parkinson’s disease and other amyloidoses, inhibition of their production should represent a suitable therapeutic strategy. However, it is difficult to imagine a drug molecule able to distinguish specifically among chain protofibrils, annular protofibrils and mature fibrils, when one considers that protofibril elongation into fibrils and protofibril annulation are likely to involve the same interactions leading to β-sheet extension [88]. ….

Fig. 6 Flow-chart of the main molecular steps leading misfolded polypeptide chains to induce cell death. In the panel, aggregation of proteins into fully formed, mature amyloid fibrils could be considered to be potentially beneficial in the light of recent findings indicating that, at least in most cases, the true toxic species are the early pre-fibrillar aggregates, whereas mature fibrils appear less toxic or devoid of toxicity. Degradation of misfolded proteins is carried out by the ubiquitin-proteasome machinery. The path leading to cell death occurs when the chaperone and clearing cellular machineries are overwhelmed by the presence of an excess of unfolded/malfolded proteins; the latter is followed by the appearance of unstable amyloid nuclei and pre-fibrillar assemblies further growing into mature fibrils; such assemblies may also interact with cell membranes destabilising them and modifying ion balance possibly by formation of aspecific membrane pores. The rise of the intracellular free Ca2+ and ROS is one of the earliest modification in the path of cell death following cell exposure to early amyloid aggregates of most peptides and proteins. (Modified from [91]).

The potential cytotoxicity of many aggregated proteins suggests that, in addition to providing cells with mechanisms to clear unfolded and misfolded proteins and to minimise their ability to induce toxicity, evolution must also have operated to eliminate protein sequences with a high intrinsic propensity to aggregate [8]. Thus mutations that are neutral with respect to protein function could be selected against because they enhance the tendency of proteins to aggregate under physiological conditions. It is interesting in this regard that most of the polypeptide chains associated with aggregation diseases are either intact, or fragments of, proteins that are secreted or membrane bound. It could be that such proteins are more easily able to escape the cellular mechanisms that protect against misfolding and aggregation. Moreover, it is possible that processing in the ER prior to secretion through the Golgi, or indeed the events involved in the retrograde translocation into the cytosol of polypeptide chains that have failed the quality-control tests in the ER [35], represent additional steps associated with folding in which errors could occur or accumulate. The recent studies of the ways in which structural adaptations of proteins can minimise their tendency to misfold and aggregate mentioned above show, however, that polypeptides are far from optimised in their ability to resist aggregation. One reason for this fact is that sequences must encode many features of proteins, such as their need to fold and to bind to other species. Another is that sequences selected by evolution are in general optimised only to an extent that allows a particular organism to function efficiently during its normal life span [148]. …

The data reported in the past few years strongly suggest that the conversion of normally soluble proteins into amyloid fibrils and the toxicity of small aggregates appearing during the early stages of the formation of the latter are common or generic features of polypeptide chains. Moreover, the molecular basis of this toxicity also appears to display common features between the different systems that have so far been studied. The ability of many, perhaps all, natural polypeptides to ‘misfold’ and convert into toxic aggregates under suitable conditions suggests that one of the most important driving forces in the evolution of proteins must have been the negative selection against sequence changes that increase the tendency of a polypeptide chain to aggregate. Nevertheless, as protein folding is a stochastic process, and no such process can be completely infallible, misfolded proteins or protein folding intermediates in equilibrium with the natively folded molecules must continuously form within cells. Thus mechanisms to deal with such species must have co-evolved with proteins. Indeed, it is clear that misfolding, and the associated tendency to aggregate, is kept under control by molecular chaperones, which render the resulting species harmless assisting in their refolding, or triggering their degradation by the cellular clearance machinery [166, 167, 168, 169, 170, 171, 172, 173, 175, 177, 178]. Misfolded and aggregated species are likely to owe their toxicity to the exposure on their surfaces of regions of proteins that are buried in the interior of the structures of the correctly folded native states. The exposure of large patches of hydrophobic groups is likely to be particularly significant as such patches favour the interaction of the misfolded species with cell membranes [44, 83, 89, 90, 91, 93]. Interactions of this type are likely to lead to the impairment of the function and integrity of the membranes involved, giving rise to a loss of regulation of the intracellular ion balance and redox status and eventually to cell death. In addition, misfolded proteins undoubtedly interact inappropriately with other cellular components, potentially giving rise to the impairment of a range of other biological processes. Under some conditions the intracellular content of aggregated species may increase directly, due to an enhanced propensity of incompletely folded or misfolded species to aggregate within the cell itself. This could occur as the result of the expression of mutational variants of proteins with decreased stability or cooperativity or with an intrinsically higher propensity to aggregate. It could also occur as a result of the overproduction of some types of protein, for example, because of other genetic factors or other disease conditions, or because of perturbations to the cellular environment that generate conditions favouring aggregation, such as heat shock or oxidative stress. Finally, the accumulation of misfolded or aggregated proteins could arise from the chaperone and clearance mechanisms becoming overwhelmed as a result of specific mutant phenotypes or of the general effects of ageing [173, 174].

The topics discussed in this review not only provide a great deal of evidence for the ‘new view’ that proteins have an intrinsic capability of misfolding and forming structures such as amyloid fibrils but also suggest that the role of molecular chaperones is even more important than was thought in the past. The role of these ubiquitous proteins in enhancing the efficiency of protein folding is well established [185]. It could well be that they are at least as important in controlling the harmful effects of misfolded or aggregated proteins as in enhancing the yield of functional molecules.
https://www.researchgate.net/profile/Massimo_Stefani/publication/10595052_Protein_Aggregation_and_Aggregate_Toxicity_New_Insights_into_Protein_Folding_Misfolding_Diseases_and_Biological_Evolution/links/0046352171a742f19a000000.pdf

 

Protein Misfolding, Evolution and Disease
Dobson C. M.
Trends in biochemical sciences 1999; 24(9): 329-332   0968-0004
http://www.ncbi.nlm.nih.gov/pubmed/10470028
http://dx.doi.org/10.1016/S0968-0004(99)01445-0

 

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Role of infectious agent in Alzheimer’s Disease?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Role of Infection in Alzheimer’s Ignored, Experts Say

Nancy A. Melville   http://www.medscape.com/viewarticle/860615

The potentially critical role of infection in the etiology of Alzheimer’s disease is largely neglected, despite decades of robust evidence from hundreds of human studies, as well as the possible therapeutic implications, experts say.

“Despite all the supportive evidence, the topic [of linking infections to Alzheimer’s disease] is often dismissed as ‘controversial,’ ” the authors of an editorial, signed by an international group of 33 researchers and clinicians, write.

The editorial was published online March 8 in theJournal of Alzheimer’s Disease.

Antiviral Treatment

“One recalls the widespread opposition initially to data showing that viruses cause some types of cancer, and that a bacterium causes stomach ulcers,” the authors write.

The implications could be just as important with regard to Alzheimer’s disease, coauthor Ruth F. Itzhaki, PhD, of the Faculty of Life Sciences at the University of Manchester, United Kingdom, toldMedscape Medical News.

“The implications are that patients could be treated with antiviral agents. These would not cure them, but might slow or even stop the progression of the disease,” she said.

The evidence points to herpes simplex virus type 1 (HSV1), Chlamydia pneumoniae, and several types of spirochetes, which make their way into the central nervous system (CNS), where they can remain in latent form indefinitely, the authors note.

The link with HSV1 is supported by as many as 100 studies. Only two studies oppose the association; both were published more than a decade ago, the authors state.

Under the prevailing theory, agents such as HSV1 undergo reactivation in the brain during aging and with the decline of the immune system, as well as when persons are under stress.

“The consequent neuronal damage ― caused by direct viral action and by virus-induced inflammation ― occurs recurrently, leading to (or acting as a cofactor for) progressive synaptic dysfunction, neuronal loss, and ultimately AD [Alzheimer’s disease],” the authors write

Importantly, that damage includes the induction of amyloid-β (Aβ) peptide deposits, considered a hallmark of Alzheimer’s disease, which initially appears to be only a defense mechanism, the authors add.

Causative Role?

In outlining some of the strongest evidence behind the theory, the authors note that although viruses and other microbes are common in the elderly brain and are usually dormant, influences such as stress and immunosuppression can cause reactivation.

“For example, HSV1 DNA is amplified in the brain of immunosuppressed patients,” they write.

In addition, herpes simplex encephalitis is known to damage regions of the CNS linked to the limbic system, and therefore to memory as well as cognitive and affective processes, the same regions affected in Alzheimer’s disease.

HSV infection is known to be significantly associated with the development of Alzheimer’s, and the disease is known to have a strong inflammatory component that is characteristic of infection, the authors say.

On a genetic level, research has shown that polymorphisms in the apolipoprotein E gene (APOE) that are linked to the risk for Alzheimer’s also control immune function and susceptibility to infectious disease.

In terms of evidence of a causative role of infection in Alzheimer’s disease, the authors cite studies indicating that brain infection, such as HIV or herpes virus, is linked to pathology similar to Alzheimer’s.

Notably, infection with HSV1 or bacteria in mice and cell culture studies has been shown to result in Aβ deposition and tau abnormalities typical of Alzheimer’s disease.

In addition, the olfactory dysfunction that is an early symptom of Alzheimer’s disease is consonant with a role of infection: The olfactory nerve leads to the lateral entorhinal cortex, where Alzheimer’s pathology spreads through the brain, and it is the likely portal of entry of HSV1 and other viruses into the brain, the authors note.

“Further, brainstem areas that harbor latent HSV directly irrigate these brain regions: brainstem virus reactivation would thus disrupt the same tissues as those affected in Alzheimer’s disease,” they write.

In terms of mechanisms, the authors cite mounting evidence that virus infection selectively upregulates the gene encoding cholesterol 25-hydroxylase (CH25H), and innate antiviral immunity is induced by its enzymatic product 25-hydroxycholesterol (25OHC).

The human CH25H polymorphisms control susceptibility to Alzheimer’s as well as Aβ deposition.

Consequently, “Aβ induction is likely to be among the targets of 25OHC, providing a potential mechanistic link between infection and Aβ production,” the authors write.

Considering the devastating toll Alzheimer’s disease takes on individual lives and society, the need to reconsider the collective evidence of a role for infection is pressing, the authors note.

“Alzheimer’s disease causes great emotional and physical harm to sufferers and their carers, as well as having enormously damaging economic consequences,” they write.

“Given the failure of the 413 trials of other types of therapy for Alzheimer’s disease carried out in the period 2002-2012, antiviral/antimicrobial treatment of Alzheimer’s disease patients, notably those who areAPOE ɛ4 carriers, could rectify the ‘no drug works’ impasse.

“We propose that further research on the role of infectious agents in Alzheimer’s disease causation, including prospective trials of antimicrobial therapy, is now justified.”

Chicken or the Egg?

Commenting on the editorial for Medscape Medical News, Richard B. Lipton, MD, Edwin S. Lowe Professor, vice chair of neurology, and director of the Division of Cognitive Aging and Dementia at Albert Einstein College of Medicine in New York City, applauded the effort to raise awareness of the issue.

“The authors are to be commended for reminding us of the hypothesis that infection may contribute to Alzheimer’s disease,” he told Medscape Medical News.

He noted the variety of genetic and environmental factors that can influence onset and progression of complex disorders such as Alzheimer’s disease.

“For Alzheimer’s disease, most people would agree that cardiovascular risk factors, traumatic brain injury, and stress increase risk of disease,” he said.

“It is entirely plausible that infectious agents may be one of many factors that contribute to the development of Alzheimer’s disease. Infectious agents could operate through several mechanisms.”

The evidence does not necessarily prove a causative role, he added.

“Temporality means that infection precedes disease,” he said. “The studies showing infectious and inflammatory markers in the Alzheimer’s brain don’t tell us which came first. Alzheimer’s disease could be a state which predisposes to infection.”

The editorialists’ financial disclosures are available online. Dr Lipton has disclosed no relevant financial relationships.

Microbes and Alzheimer’s Disease

KEY POINTS

  • Herpes simplex virus 1 (HSV-1) encephalitis predominantly involves the orbital surface of the frontal lobes and medial surface of the temporal lobes, resulting in areas of increased T2 signal on MRI
  • Herpes simplex virus 2 (HSV-2) is the primary cause of recurrent meningitis
  • After varicella, the varicella zoster virus (VZV) becomes latent in ganglia along the entire neuraxis; its reactivation can lead to herpes zoster, vasculopathy, myelitis, necrotizing retinitis or zoster sine herpete
  • The neurological complications of Epstein–Barr virus are diverse, and include meningitis, encephalitis, myelitis, radiculoneuropathy, and even autonomic neuropathy
  • The most common neurological complication of cytomegalovirus (CMV) is poly-radiculoneuropathy in immunocompromised individuals
  • Virological confirmation of neurological disease relies on the detection of herpesvirus-specific DNA in bodily fluids or tissues, herpesvirus-specific IgM in blood, or herpesvirus-specific IgM or IgG antibody in cerebrospinal fluid
  • HSV-1, HSV-2, VZV and CMV are the most treatable herpesviruses

Most HHVs can cause serious neurological disease of the PNS and CNS through primary infection or following virus reactivation from latently infected human ganglia or lymphoid tissue. The neurological complications include meningitis, encephalitis, myelitis, vasculopathy, acute and chronic radiculoneuritis, and various inflammatory diseases of the eye. Disease can be monophasic, recurrent or chronic.

 

The researchers also add that a gene mutation – APOEe4 – which appears to makes some of the population more susceptible to Alzheimer’s disease, could also increase these people’s susceptibility to infectious diseases.

 As a counter view, Professor John Hardy, Teacher of Neuroscience, UCL, told the website Journal Focus he was doubtful about the claims: “This is a minority sight in Alzheimer research study. There had actually been no convincing evidence of infections triggering Alzheimer disease. We require constantly to maintain an open mind however this editorial does not show exactly what many scientists think of Alzheimer disease.”

However, another of the researchers, Resia Pretorius of the University of Pretoria, told Bioscience Technology: “The microbial presence in blood may also play a fundamental role as causative agent of systemic inflammation, which is a characteristic of Alzheimer’s disease. Furthermore, there is ample evidence that this can cause neuroinflammation and amyloid-β plaque formation.”

The possibility of transfer has been reported to the journal Nature. The paper is titled “Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy.”

The report explains that during the period from 1958 to 1985, 30,000 people worldwide — mainly children — were administered injections of human growth hormone. This was designed to treat short stature. The hormone was extracted from thousands of human pituitary glands, with the source material being recently deceased people.

It now appears, The Economist summarizes, that some of these hormonal extracts contained prions. Around one in 16 of the children developed the brain disorder Creutzfeldt-Jakob disease (CJD). The concern with CJD centered on prions.

Read more: http://www.digitaljournal.com/science/alzheimer-s-and-parkinson-s-diseases-may-be-transmissible/article/444338#ixzz43Y

Chain reaction

Evidence emerges that Alzheimer’s disease, and other neurodegenerative disorders such as Parkinson’s, may be transmissible

 

KAREN WEINTRAUB

Reporting from the frontiers of health and medicine

A rare disease killed her mother. Can this scientist save herself?

http://www.statnews.com/2016/01/20/prion-disease-genes/

CAMBRIDGE, Mass. — Five years ago, after watching her 51-year-old mother descend quickly into dementia, disability, and then death, Sonia Vallabh learned she was destined for the same fate. They both shared an extremely rare genetic mutation that leads a protein in the brain to turn toxic.

Vallabh, then a recent Harvard Law School graduate working as a consultant, decided to quit her job to spend time learning more about the mutation and nascent efforts to understand and treat it.

Now, she and her husband, Eric Minikel, a former transportation planner, are first authors on a paper about so-called prion diseases. Published Wednesday in Science Translational Medicine, the paper found that not all prion gene mutations are an early death sentence — though Vallabh’s variation is.

The husband-and-wife team, now both PhD students working in the same lab at the Broad Institute, also found that people can survive with only one copy of the prion gene, suggesting that a treatment to block the mutated version can be delivered safely.

Prion diseases were made famous by “mad cow disease,” outbreaks of which have led to mass killings of cattle. Eating sick cows can cause the fatal neurodegenerative illness known as Creutzfeldt-Jakob disease. But there are genetic versions of prion diseases that account for about 15 percent of cases. They come from mutations to the prion protein gene PRNP, which causes a protein in the brain to fold the wrong way, forming toxic clumps. Once these proteins get a foothold in the brain, they can cause extremely rapid damage.

Vallabh’s mother, who seemed completely normal at Christmastime in 2009, showed the first symptoms of disease in January 2010 and was demented and unable to speak clearly by March. She last recognized her daughter in May, Vallabh said, and died two days before Christmas that year, shortly after doctors finally identified the cause of her bizarre symptoms.

Vallabh, Minikel, and their coauthors compared a data set — painstakingly collected over decades — of gene sequences from 16,000 prion disease patients from all over the world, with two data sets of sequences from healthy people: more than 60,000 collected by the Broad-led Exome Aggregation Consortium and 530,000 from 23andMe, a consumer genetics company that invites clients to volunteer their gene sequences for research.

The size of the data sets allowed the researchers to draw conclusions even with a condition as rare as prion disease. Doctors had previously only known about 63 possible mutations in people with disease, so they had thought that all the mutations necessarily caused problems. But the researchers found 141 healthy people in the 23andMe dataset who had mutations to the PRNP gene — a rate far higher than the incidence of prion disease. That means some of the mutations must be harmless or at least not always cause disease, said J. Fah Sathirapongsasuti, a computational biologist at 23andMe and a study coauthor.

Out of 16 mutations for which there was evidence in the larger populations, they concluded that three were likely benign, three caused somewhat increased risk of disease, and four others, including Vallabh’s mutation, definitely do cause the fatal illness, they found.

They also discovered three older, healthy people who carried only one functional copy of the PRNP gene. That means that knocking out the mutated version of PRNP with gene therapy, or tamping down its activity with drugs, should be an effective way to eliminate the risk of disease without causing life-threatening problems.

Their paper has already helped at least one person, according to Dr. Robert Green, a medical geneticist at Brigham and Women’s Hospital, who cowrote an opinion piece published alongside the new study.

One of Green’s patients, whose mother died of prion disease, had been told her mom’s mutation — which she didn’t inherit, but her sister did — was always fatal. After seeing the new study, Green was able to inform the sister that her mutation was most likely harmless.

 

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Protein misfolding and prions

Larry H/ Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 4.9

Revised 9/30/2015

Susan L. Lindquist, Stanley B. Prusiner

Whitehead Member Susan Lindquist is a pioneer in the study of protein folding. She has shown that changes in protein folding can have profound and unexpected influences in fields as wide-ranging as human disease, evolution and nanotechnology.

Protein misfolding has been implicated as a major mechanism in many severe neurological disorders including Parkinson’s and Huntington’s diseases. Lindquist and colleagues have developed yeast strains that serve as living test tubes in which to study these disorders, unraveling how protein folding contributes to them.

Prions are proteins that can change into a self-perpetuating form. They have only been discovered recently, but one of them is already well known as the cause of mad cow disease. The Lindquist lab investigates both how prions form and the diseases they cause. In addition, Lindquist is convinced that other prion proteins play many important and positive roles in biological processes. The first evidence for this was shown in her work with Nobel Laureate Eric Kandel, which demonstrated that prions may be integral to memory storage in the brain.

Heat shock proteins are a group of molecular chaperone proteins that, as their name might suggest, guide other proteins to fold and mature correctly. Lindquist has established that heat shock protein 90 (Hsp90) can reveal hidden genetic variation in fruit flies and in cress plants (Arabidopsis) under certain environmental conditions.

Lindquist is a Member and former Director (2001-2004) of Whitehead Institute, a Professor of Biology at MIT, and a Howard Hughes Medical Institute investigator. Previously she was the Albert D. Lasker Professor of Medical Sciences from 1999-2001, and a Professor in the Department of Molecular Biology, University of Chicago, since 1978. She received a PhD in Biology from Harvard University in 1976, and was elected to the American Academy of Arts and Sciences in 1997, the National Academy of Sciences in 1997 and the Institute of Medicine in 2006.

QnAs with Susan L. Lindquist

http://www.pnas.org/content/108/50/19861.full  Dec 13, 2011  Susan L. Lindquist. PNAS: How did you suspect that prions might have a positive role in cells?
Lindquist: We were working on a heat shock …

Dr. Susan Lindquist – “Alzheimer’s Disease: An Entirely New Point of 

http://www.youtube.com/watch%3Fv%3DZ3tK50LQH_c  Nov 15, 2011 
Whitehead Institute Member Susan Lindquist’s keynote from the 2011 Whitehead Colloquium, November 5, 2011.

Susan Lindquist Lab uploaded a video 1 year ago

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Sue Lindquist Plenary Lecture at AAAS Annual Meeting 2014

by Susan Lindquist Lab

From Yeast Cells to Patient Neurons: A Powerful Discovery Platform for Parkinson’s and Alzheimer’s Disease

Susan Lindquist Lab uploaded a video 8 months ago

 45:12

Susan Lindquist ISSCR 6 21 14

by Susan Lindquist Lab

Susan Lindquist Keynote Lecture at ISSCR in Vancouver, June 2014

Stanley B. Prusiner, MD

Director, Institute for Neurodegenerative Diseases
Professor, Department of Neurology

Prusiner discovered an unprecedented class of pathogens that he named prions. Prions are proteins that acquire an alternative shape that becomes self-propagating. As prions accumulate, they cause neurodegenerative diseases in animals and humans. Prusiner’s discovery lead him to develop a novel disease paradigm: prions cause disorders such as Creutzfeldt-Jakob disease (CJD) in humans that manifest as (1) sporadic, (2) inherited and (3) infectious illnesses.  Based on his seminal discovery that prions can assemble into amyloid fibrils, Prusiner proposed that the more common neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases may be caused by prions.

Prusiner’s contributions to scientific research have been internationally recognized: He is a member of the National Academy of Sciences, the Institute of Medicine, the American Academy of Arts and Sciences and the American Philosophical Society, and a foreign member of the Royal Society, London. He is the recipient of numerous prizes, including the Potamkin Prize for Alzheimer’s Disease Research from the American Academy of Neurology (1991); the Richard Lounsbery Award for Extraordinary Scientific Research in Biology and Medicine from the National Academy of Sciences (1993); the Gairdner Foundation International Award (1993); the Albert Lasker Award for Basic Medical Research (1994); the Wolf Prize in Medicine from the State of Israel (1996); the Nobel Prize in Physiology or Medicine (1997); and the United States Presidential National Medal of Science (2009).

Stanley Prusiner – National Medal of Science – YouTube

http://www.youtube.com/watch%3Fv%3DkghMfXrtvAY
Nov 29, 2010  2009 Medal of Science Laureate for his discovery of prions — a new class of infectious agents comprised only of proteins. Produced by Evolving ..

2011 Bay Area Council Outlook Conference – Dr. Stanley Prusiner 

http://www.youtube.com/watch%3Fv%3DcSZA8VUXxZ8
Apr 27, 2011  2011 Bay Area Council Outlook Conference – Dr. Stanley Prusiner …. President Obama Awards National Medal of Scienceand Medal of …

By Jennifer O’Brien on October 15, 2010

UCSF Nobel laureate Stanley B. Prusiner, MD, UCSF professor of neurology and director of the Institute for Neurodegenerative Diseases, today (Oct. 15, 2010) was named to receive the National Medal of Science, the nation’s highest honor for science and technology.

Prusiner received the medal for his discovery of and ongoing research on a novel infectious agent, which he named the prion (PREE-on). The prion, composed solely of protein, causes bovine spongiform encephalopathy, or “mad cow” disease, and other related fatal neurodegenerative diseases in animals and humans.

Prions

Stanley B. Prusiner

PNAS Nov 10, 1998; 95(23):13363–13383,    http://dx.doi.org:/10.1073/pnas.95.23.13363

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high β-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc.

The torturous path of the scientific investigation that led to an understanding of familial Creutzfeldt–Jakob disease (CJD) chronicles a remarkable scientific odyssey. By 1930, the high incidence of familial (f) CJD in some families was known (12). Almost 60 years were to pass before the significance of this finding could be appreciated (35). CJD remained a curious, rare neurodegenerative disease of unknown etiology throughout this period of three score years (6)(7).

Once CJD was shown to be an infectious disease, relatively little attention was paid to the familial form of the disease since most cases were not found in families(812). Libyan Jews living in Israel developed CJD about 30 times more frequently than other Israelis (13). This finding prompted some investigators to propose that the Libyan Jews had contracted CJD by eating lightly cooked brain from scrapie-infected sheep when they lived in Tripoli prior to emigration. Subsequently, the Libyan Jewish patients were all found to carry a mutation at codon 200 in their prion protein (PrP) gene (1416).

Slow Viruses.

The term “slow virus” had been coined by Bjorn Sigurdsson in 1954 while he was working in Iceland on scrapie and visna of sheep (17). Five years later, William Hadlow had suggested that kuru, a disease of New Guinea highlanders, was similar to scrapie and thus, it, too, was caused by a slow virus (18). Seven more years were to pass before the transmissibility of kuru was established by passaging the disease to chimpanzees inoculated intracerebrally (19). Just as Hadlow had made the intellectual leap between scrapie and kuru, Igor Klatzo made a similar connection between kuru and CJD (20). Neuropathologists were struck by the similarities in light microscopic pathology of the central nervous system (CNS) that kuru exhibited with scrapie or CJD. In 1968, the transmission of CJD to chimpanzees after intracerebral inoculation was reported (7).

In scrapie, kuru, CJD, and all of the other disorders now referred to as prion diseases (Table 1), spongiform degeneration and astrocytic gliosis is found upon microscopic examination of the CNS (Fig. 1) (2122).

Table 1

The prion disease

Figure 1

http://www.pnas.org/content/95/23/13363/F1.medium.gif

Neuropathologic changes in Swiss mice after inoculation with RML scrapie prions. (a) Hematoxylin and eosin stain of a serial section of the hippocampus shows spongiform degeneration of the neuropil, with vacuoles 10–30 μm in diameter.

Prions: A Brief Overview.

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases mediated by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP), a constituent of normal mammalian cells (23). CJD generally presents as progressive dementia, whereas scrapie of sheep and bovine spongiform encephalopathy (BSE) are generally manifest as ataxic illnesses (Table 1) (24).

Prions are devoid of nucleic acid and seem to be composed exclusively of a modified isoform of PrP designated PrPSc. The normal, cellular PrP, denoted PrPC, is converted into PrPSc through a process whereby a portion of its α-helical and coil structure is refolded into β-sheet (25). This structural transition is accompanied by profound changes in the physicochemical properties of the PrP. The amino acid sequence of PrPSc corresponds to that encoded by the PrP gene of the mammalian host in which it last replicated. In contrast to pathogens with a nucleic acid genome that encode strain-specific properties in genes, prions encipher these properties in the tertiary structure of PrPSc (2628). Transgenetic studies argue that PrPScacts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein.

More than 20 mutations of the PrP gene are now known to cause the inherited human prion diseases, and significant genetic linkage has been established for five of these mutations (4162931). The prion concept readily explains how a disease can be manifest as a heritable as well as an infectious illness.

Resistance of Scrapie Agent to Radiation.

My fascination with CJD quickly shifted to scrapie once I learned of the remarkable radiobiological data that Tikvah Alper and her colleagues had collected on the scrapie agent (3234). The scrapie agent had been found to be extremely resistant to inactivation by UV and ionizing radiation, as was later shown for the CJD agent (35). It seemed to me that the most intriguing question was the chemical nature of the scrapie agent. Suggestions as to the nature of the scrapie agent  (3642).

Scrapie of sheep and goats possesses a history no less fascinating than that of CJD. The resistance of the scrapie agent to inactivation by formalin and heat treatments (43), suggested that the scrapie agent might be different from viruses. British scientists had argued for many years about whether natural scrapie was a genetic or an infectious disease (4446). Scrapie, like kuru and CJD, produced death of the host without any sign of an immune response to a “foreign infectious agent.”

From studies on the sedimentation properties of scrapie infectivity in mouse spleens and brains  I concluded that hydrophobic interactions were responsible for the nonideal physical behavior of the scrapie particle (4748). The scrapie agent demanded new approaches and better assays (49).

Bioassays.

In 1972, when I became fascinated by the enigmatic nature of the scrapie agent, I thought that the most direct path to determining the molecular structure of the scrapie agent was purification. Few systematic investigations had been performed on the fundamental characteristics of the infectious scrapie particle (42). Knowledge of the sedimentation properties of the scrapie agent under defined conditions seemed mandatory.

Incubation time assays in hamsters.

In view of these daunting logistical problems, the identification of an inoculum that produced scrapie in the golden Syrian hamster (SHa) in ≈70 days after intracerebral inoculation proved to be an important advance (5354) once an incubation time assay was developed (55,56). In earlier studies, SHa had been inoculated with prions, but serial passage with short incubation times was not reported (57). Development of the incubation time bioassay reduced the time required to measure prions in samples with high titers by a factor of 5: only 70 days were required instead of the 360 days previously needed. Equally important, 4 animals could be used in place of the 60 that were required for endpoint titrations, making possible a large number of parallel experiments. With this bioassay, research on the nature of the scrapie agent was accelerated nearly 100-fold and the hamster with high prion titers in its brain became the experimental animal of choice for biochemical studies.

The incubation time assay enabled development of effective purification schemes for enriching fractions for scrapie infectivity. It provided a means to assess quantitatively those fractions that were enriched for infectivity and those that were not.  With a ≈100-fold purification of infectivity relative to protein, >98% of the proteins and polynucleotides were eliminated.

The Prion Concept.

As reproducible data began to accumulate indicating that scrapie infectivity could be reduced by procedures that hydrolyze or modify proteins but was resistant to procedures that alter nucleic acids, a family of hypotheses about the molecular architecture of the scrapie agent began to emerge (58). These data established, for the first time, that a particular macromolecule was required for infectivity and that this macromolecule was a protein. The experimental findings extended earlier observations on resistance of scrapie infectivity to UV irradiation at 250 nm (33) in that the four different procedures used to probe for a nucleic acid are based on physical principles that are independent of UV radiation damage.

Once the requirement of protein for infectivity was established, I thought that it was appropriate to give the infectious pathogen of scrapie a provisional name that would distinguish it from both viruses and viroids. After some contemplation, I suggested the term “prion,” derived from proteinaceous and infectious (58). At that time, I defined prions as proteinaceous infectious particles that resist inactivation by procedures that modify nucleic acids. I never imagined the irate reaction of some scientists to the word “prion”—it was truly remarkable!

Current definitions.

Perhaps the best current working definition of a prion is a proteinaceous infectious particle that lacks nucleic acid (28). Because a wealth of data supports the contention that scrapie prions are composed entirely of a protein that adopts an abnormal conformation, it is not unreasonable to define prions as infectious proteins (25275960). But I hasten to add that we still cannot eliminate a small ligand bound to PrPSc as an essential component of the infectious prion particle.  (61). From a broader perspective, prions are elements that impart and propagate conformational variability.

Although PrPSc is the only known component of the infectious prion particles, these unique pathogens share several phenotypic traits with other infectious entities such as viruses. (6267).

Families of hypotheses.

Once the requirement for a protein was established, it was possible to revisit the long list of hypothetical structures that had been proposed for the scrapie agent and to eliminate carbohydrates, lipids, and nucleic acids as the infective elements within a scrapie agent devoid of protein (58) (5868).

The family of hypotheses that remained after identifying a protein component was still large and required a continued consideration of all possibilities in which a protein was a critical element (49). The prion concept evolved from a family of hypotheses in which an infectious protein was only one of several possibilities. With the accumulation of experimental data on the molecular properties of the prion, it became possible to discard an increasing number of hypothetical structures. (69).

Genes and DNA.

In some respects, the early development of the prion concept mirrors the story of DNA (7075). Those who attacked the hypothesis that the prion is composed only of protein had more than 30 years of cumulative evidence showing that genetic information in all organisms on our planet is encoded in DNA and that biological diversity resides in DNA. Studies of viruses and eventually viroids extended this concept to these small infectious pathogens (76) and showed that genes could also be composed of RNA (7778).

Discovery of the Prion Protein.

The discovery of the prion protein transformed research on scrapie and related diseases (7980). It provided a molecular marker that was subsequently shown to be specific for these illnesses as well as the major, and very likely the only, constituent of the infectious prion.

PrP 27–30 was discovered by enriching fractions from SHa brain for scrapie infectivity (7980). This protein is the protease-resistant core of PrPSc and has an apparent molecular mass of 27–30 kDa (8182). Although resistance to limited proteolysis proved to be a convenient tool for many but not all studies, use of proteases to enrich fractions for scrapie infectivity created a problem when the NH2-terminal sequence of PrP 27–30 was determined (81). The ragged NH2 terminus of PrP 27–30 yielded three sets of signals in almost every cycle of the Edman degradation. Only after these signals were properly interpreted and placed in correct register could a unique sequence be assigned for the NH2 terminus of PrP 27–30. The determination of the amino acid sequence of the NH2 terminus of PrP 27–30 made subsequent molecular cloning studies of the PrP gene possible (8384).

The finding that PrP mRNA levels were similar in normal uninfected and scrapie-infected tissues caused some investigators to argue that PrP 27–30 was not related to the infectious prion particle (83). An alternate interpretation prompted a search for a prion protein in uninfected animals that was found to be protease sensitive and soluble in nondenaturing detergents, unlike PrP 27–30. This isoform was designated PrPC(Fig. 2) (8485). Deduced amino acid sequences from PrP cDNA as well as immunoblotting studies revealed that PrP 27–30 was NH2-terminally truncated and was derived from a larger molecule, designated PrPSc, that was unique to infected animals (81828486).

Figure 2

http://www.pnas.org/content/95/23/13363/F2.medium.gif

With the discovery of PrP 27–30 and the production of antiserum (87), brains from humans and animals with putative prion diseases were examined for the presence of this protein. In each case, PrP 27–30 was found, and it was absent in other neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (8891). The extreme specificity of PrPSc for prion disease is an important feature of the protein and is consistent with the postulated role of PrPSc in both the transmission and pathogenesis of these illnesses (Table 2) (92).

Table 2

Arguments for prions being composed largely, if not entirely, of PrPSc molecules and devoid of nucleic acid

The accumulation of PrPSc contrasts markedly with that of glial fibrillary acidic protein (GFAP) in prion disease. In scrapie, GFAP mRNA and protein levels rise as the disease progresses (93), but the accumulation of GFAP is neither specific nor necessary for either the transmission or the pathogenesis of disease. Mice deficient for GFAP show no alteration in their incubation times (9495).

Except for PrPSc, no macromolecule has been found in tissues of patients dying of the prion diseases that is specific for these encephalopathies. In searches for a scrapie-specific nucleic acid, cDNAs have been identified that are complementary to mRNAs encoding other proteins with increased expression in prion disease (9698). Yet none of the proteins has been found to be specific for prion disease.

Attempts to Falsify the Prion Hypothesis.

Numerous attempts to disprove the prion hypothesis over the past 15 years have failed (99), and no replication of prions in PrP-deficient (Prnp0/0) mice was found (100104).

Copurification of PrP 27–30 and scrapie infectivity demands that the physicochemical properties as well as antigenicity of these two entities be similar (105) (Table 2). The results of a wide array of inactivation experiments demonstrated the similarities in the properties of PrP 27–30 and scrapie infectivity (61,106109). To explain these findings in terms of the virus hypothesis, the PrP-like coat protein or the PrPSc/virus complex must display properties indistinguishable from PrPSc alone. With each species that the putative virus invades, it must incorporate a new PrP sequence during replication.

Search for a scrapie-specific nucleic acid.

The inability to inactivate preparations highly enriched for scrapie infectivity by procedures that modify nucleic acids militates against the existence of a scrapie-specific nucleic acid (58110111). Both UV and ionizing radiation inactivation studies as well as physical studies have eliminated the possibility of a large nucleic acid hiding within purified preparations of prions (11011263115).

PrP amyloid.

In preparations highly enriched for scrapie infectivity and containing only PrP 27–30 by silver staining of gels after SDS/PAGE, numerous rod-shaped particles were seen by electron microscopy after negative staining (Fig. 3) (107),(116). These irregular rods, composed largely, if not entirely, of PrP 27–30, were indistinguishable morphologically from many other purified amyloids (117). Studies of the prion rods with Congo red dye demonstrated that the rods also fulfilled the tinctorial criteria for amyloid (107), and immunostaining later showed that PrP is a major component of amyloid plaques in some animals and humans with prion disease (118120). Subsequently, it was recognized that the prion rods were not required for scrapie infectivity (121); (Fig. 3) (122). 

Figure 3

http://www.pnas.org/content/95/23/13363/F3.medium.gif

The idea that scrapie prions were composed of an amyloidogenic protein was truly heretical when it was introduced (107). Since the prevailing view at the time was that scrapie is caused by an atypical virus, many argued that amyloid proteins are mammalian polypeptides and not viral proteins!

Some investigators have argued that the prion rods are synonymous with scrapie-associated fibrils (123125) even though morphologic and tinctorial features of these fibrils clearly differentiated them from amyloid and as such from the prion rods (126127). The scrapie-associated fibrils were identified by their unique ultrastructure in which two or four subfilaments were helically wound around each other (126) and were proposed to represent the first example of a filamentous animal virus (128).

Search for the ubiquitous “scrapie virus.”

When PrP gene mutations were discovered to cause familial prion diseases (4), it was postulated that PrPC is a receptor for the ubiquitous scrapie virus that binds more tightly to mutant than to wt PrPC (131). A similar hypothesis was proposed to explain why the length of the scrapie incubation time was found to be inversely proportional to the level of PrP expression in transgenic (Tg) mice and why Prnp0/0 mice are resistant to scrapie (132). The higher the level of PrP expression, the faster the spread of the putative virus, which results in shorter incubation times; conversely, mice deficient for PrP lack the receptor required for spread of the virus (63). The inability to find virus-like particles in purified preparations of PrPSc was attributed to these particles being hidden (115) even though tobacco mosaic viruses could be detected when one virion was added per ID50 unit of scrapie prions (121).

Recent studies on the transmission of mutant prions from FFI and fCJD(E200K) to Tg(MHu2M) mice, which results in the formation of two different PrPSc molecules (27), has forced a corollary to the ubiquitous virus postulate. To accommodate this result, at least two different viruses must reside worldwide, each of which binds to a different mutant HuPrPC and each of which induces a different MHu2M PrPSc conformer when transferred to Tg mice. Even more difficult to imagine is how one ubiquitous virus might acquire different mutant PrPSc molecules corresponding to FFI or fCJD(E200K) and then induce different MHu2M PrPScconformers upon transmission to Tg mice.

Artificial prions.

To explain the production of artificial prions from chimeric or mutant PrP transgenes in terms of a virus (133135), mutated PrPSc molecules must be incorporated into the virus. In the case of mice expressing chimeric PrP transgenes, artificial prions are produced with host ranges not previously found in nature. Similarly, deleting specific regions of PrP resulted in the formation of “miniprions” with a unique host range and neuropathology as described below. The production of artificial prions that were generated by modifying the PrP gene sequence and exhibit unique biological properties is another compelling argument against the proposition that scrapie and CJD are caused by viruses.

Prions Defy Rules of Protein Structure.

Once cDNA probes for PrP became available, the PrP gene was found to be constitutively expressed in adult, uninfected brain (8384). This finding eliminated the possibility that PrPSc stimulated production of more of itself by initiating transcription of the PrP gene as proposed nearly two decades earlier (37). Determination of the structure of the PrP gene eliminated a second possible mechanism that might explain the appearance of PrPSc in brains already synthesizing PrPC. Since the entire protein coding region was contained within a single exon, there was no possibility for the two PrP isoforms to be the products of alternatively spliced mRNAs (82). Next, a posttranslational chemical modification that distinguishes PrPScfrom PrPC was considered, but none was found in an exhaustive study (59) and we considered it likely that PrPC and PrPSc differed only in their conformation, a hypothesis also proposed earlier (37). However, this idea was no less heretical than that of an infectious protein.

For more than 25 years, it had been widely accepted that the amino acid sequence specifies one biologically active conformation of a protein (136). Yet in scrapie we were faced with the possibility that one primary structure for PrP might adopt at least two different conformations to explain the existence of both PrPC and PrPSc. When the secondary structures of the PrP isoforms were compared by optical spectroscopy, they were found to be markedly different (25). Fourier-transform infrared (FTIR) and circular dichroism (CD) studies showed that PrPC contains about 40% α-helix and little β-sheet, whereas PrPSc is composed of about 30% α-helix and 45% β-sheet (25137). Nevertheless, these two proteins have the same amino acid sequence!

Prior to comparative studies on the structures of PrPC and PrPSc, we found by metabolic labeling studies that the acquisition of PrPSc protease resistance is a posttranslational process (138). In our quest for a chemical difference that would distinguish PrPSc from PrPC, we found ethanolamine in hydrolysates of PrP 27–30, which signaled the possibility that PrP might contain a GPI anchor (139). Both PrP isoforms were found to carry GPI anchors, and PrPC was found on the surface of cells where it could be released by cleavage of the anchor. Subsequent studies showed that PrPSc formation occurs after PrPC reaches the cell surface (140141) and is localized to caveolae-like domains (142145).

Modeling PrP structures.

Molecular modeling studies predicted that PrPC is a four-helix bundle protein containing four regions of secondary structure denoted H1, H2, H3, and H4 (Fig. 4) (146147). Subsequent NMR studies of a synthetic PrP peptide containing residues 90–145 provided good evidence for H1 (148). This peptide contains the residues 113–128, which are most highly conserved among all species studied (Fig. 4A) (147149150) and correspond to a transmembrane region of PrP that was delineated in cell-free translation studies (151152). Recent studies show that a transmembrane form of PrP accumulates in GSS caused by the A117V mutation and in Tg mice overexpressing either mutant or wild-type (wt)PrP (153). The paradoxical lack of evidence for an α-helix in this region from NMR studies of recombinant PrP in aqueous buffers (154156) could be explained if the recombinant PrPs correspond to the secreted form of PrP that was also identified in cell-free translation studies. This contention is supported by studies with recombinant antibody fragments (Fabs) showing the GPI-anchored PrPC on the surface of cells exhibits an immunoreactivity similar to that of recombinant PrP that was prepared with an α-helical conformation (157,158). GPI-anchored PrPC is synthesized within the secretory pathway and transported to the surface of the cell (139159).

Figure 4

http://www.pnas.org/content/95/23/13363/F4.medium.gif

Species variations and mutations of the prion protein gene. (A) Species variations. The x-axis represents the human PrP sequence, with the five octarepeats and H1–H4 regions of putative secondary structure shown as well as the three α-helices A, B, and C and the two β-strands S1 and S2 as determined by NMR. The precise residues corresponding to each region of secondary structure are given in Fig. 5. Vertical bars above the axis indicate the number of species that differ from the human sequence at each position. Below the axis, the length of the bars indicates the number of alternative amino acids at each position in the alignment. (B) Mutations causing inherited human prion disease and polymorphisms in human, mouse, and sheep. Above the line of the human sequence are mutations that cause prion disease. Below the lines are polymorphisms, some but not all of which are known to influence the onset as well as the phenotype of disease. Data were compiled by Paul Bamborough and Fred E. Cohen.

Optical spectroscopic measurements of PrPC provided the necessary background for more detailed structural studies (25). Unable to produce crystals of PrP, we and others utilized NMR to determine the structure of an α-helical form of a recombinant PrP. The NMR structure of a COOH-terminal fragment of MoPrP consisting of 111 residues showed three helices, two of which corresponded to H3 and H4 in the PrPC model, and two small β-strands each consisting of three residues (154). How the structure of this MoPrP(121–231) fragment differs from PrPC is of interest because this fragment is lethal when expressed in Tg mice (160). Subsequently, structural studies were performed on a longer fragment of PrP containing residues 90–231 and corresponding to SHaPrP 27–30 (155161162). Expression of PrP(90–231) in Tg mice did not produce spontaneous disease (163164). More recently, NMR structures of recombinant full-length PrP have been reported (156165).

Models of PrPSc suggested that formation of the disease-causing isoform involves refolding of residues within the region between residues 90 and 140 into β-sheets (166); the single disulfide bond joining COOH-terminal helices would remain intact because the disulfide is required for PrPSc formation (Fig. 5E) (167,168). The high β-sheet content of PrPSc was predicted from the ability of PrP 27–30 to polymerize into amyloid fibrils (107). Subsequent optical spectroscopy confirmed the presence of β-sheet in both PrPSc and PrP 27–30 (25169171). Deletion of each of the regions of putative secondary structure in PrP, except for the NH2-terminal 66 amino acids (residues 23–88) (163172) and a 36-amino acid region (mouse residues 141–176) prevented formation of PrPSc as measured in scrapie–infected cultured neuroblastoma cells (168). With anti-PrP Fabs selected from phage display libraries (157) and two monoclonal antibodies (mAbs) derived from hybridomas (173175), the major conformational change that occurs during conversion of PrPC into PrPSc has been localized largely, but not entirely, to a region bounded by residues 90 and 112 (158). Similar conclusions were drawn from studies with an anti-PrP IgM mAb (176). While these results indicate that PrPSc formation involves primarily a conformational change in the domain composed of residues 90–112, mutations causing inherited prion diseases have been found throughout the protein (Fig. 4B). Interestingly, all of the known point mutations in PrP with biological significance occur either within or adjacent to regions of putative secondary structure in PrP and as such, appear to destabilize the structure of PrP (147148154). 

Figure 5

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Structures of prion proteins. (A) NMR structure of SHa recombinant (r) PrP(90–231). Presumably, the structure of the α-helical form of rPrP(90–231) resembles that of PrPC. rPrP(90–231) is viewed from the interface where PrPSc is thought to bind to PrPC. The color scheme is as follows: α-helices A (residues 144–157), B (172193), and C (200227) in pink; disulfide between Cys-179 and Cys-214 in yellow; conserved hydrophobic region composed of residues 113–126 in red; loops in gray; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassing strand S2; the arrows span residues 129–131 and 161–163, as these show a closer resemblance to β-sheet (155). (B) NMR structure of rPrP(90–231) is viewed from the interface where protein X is thought to bind to PrPC. Protein X appears to bind to the side chains of residues that form a discontinuous epitope: some amino acids are in the loop composed of residues 165–171 and at the end of helix B (Gln-168 and Gln-172 with a low-density van der Waals rendering), whereas others are on the surface of helix C (Thr-215 and Gln-219 with a high-density van der Waals rendering) (178). (C) PrP residues governing the transmission of prions (180). NMR structure of recombinant SHaPrP region 121–231 (155) shown with the putative epitope formed by residues 184, 186, 203, and 205 highlighted in red. Residue numbers correspond to SHaPrP. Additional residues (138, 139, 143, 145, 148, and 155) that might participate in controlling the transmission of prions across species are depicted in green. Residues 168, 172, 215, and 219 that form the epitope for the binding of protein X are shown in blue. The three helices (A, B, and C) are highlighted in pink. (D) Schematic diagram showing the flexibility of the polypeptide chain for PrP(29–231) (156). The structure of the portion of the protein representing residues 90–231 was taken from the coordinates of PrP(90–231) (155). The remainder of the sequence was hand-built for illustration purposes only. The color scale corresponds to the heteronuclear {1H}-15N nuclear Overhauser enhancement data: red for the lowest (most negative) values, where the polypeptide is most flexible, to blue for the highest (most positive) values in the most structured and rigid regions of the protein. (E) Plausible model for the tertiary structure of HuPrPSc(166). Color scheme is as follows: S1 β-strands are 108–113 and 116–122 in red; S2 β-strands are 128–135 and 138–144 in green; α-helices H3 (residues 178–191) and H4 (residues 202–218) in gray; loop (residues 142–177) in yellow. Four residues implicated in the species barrier are shown in ball-and-stick form (Asn-108, Met-112, Met-129, Ala-133).

NMR structure of recombinant PrP.

The NMR structure of recombinant (r) SHaPrP(90–231) derived from Escherichia coli was determined after the protein was purified and refolded (Fig. 5A). Residues 90–112 are not shown because marked conformational heterogeneity was found in this region, while residues 113–126 constitute the conserved hydrophobic region, which also displays some structural plasticity (162). Although some features of the structure of rPrP(90–231) are similar to those reported earlier for the smaller recombinant MoPrP(121–231) fragment (154177), substantial differences were found. For example, the loop at the NH2 terminus of helix B is well defined in rPrP(90–231) but is disordered in MoPrP(121–231); in addition, helix C is composed of residues 200–227 in rPrP(90–231) but extends only from 200–217 in MoPrP(121–231). The loop and the COOH-terminal portion of helix C are particularly important because they form the site to which protein X binds as described below (Fig. 5B) (178). Whether the differences between the two recombinant PrP fragments are because of (i) their different lengths, (ii) species-specific differences in sequences, or (iii) the conditions used for solving the structures remains to be determined.

Studies of chimeric SHa/Mo and Hu/Mo PrP transgenes identified a domain composed of residues 95–170, where PrPC binds to PrPSc (133179). When chimeric bovine (Bo)/Mo PrP transgenes failed to render mice sensitive to BSE prions, we examined the differences among the sequences in the chimeric and parent PrP genes (180). The findings identified a second domain in PrP composed of residues 180–205 that seems to modulate the interaction between PrPC and PrPSc (Fig. 5C).

Recent NMR studies of full-length MoPrP(23–231) and SHaPrP(29–231) have shown that the NH2 termini are highly flexible and lack identifiable secondary structure under the experimental conditions employed (Fig. 5D) (156165). Studies of SHaPrP(29–231) indicate transient interactions between the COOH terminus of helix B and the highly flexible, NH2-terminal random-coil containing the octareapeats (residues 29–125) (156); such interactions were not reported for MoPrP(23–231) (165).

PrP appears to bind copper.

The highly flexible NH2 terminus of recombinant PrP may be more structured in the presence of copper. Each SHaPrP(29–231) molecule was found to bind two atoms of Cu2+; other divalent cations did not bind to PrP (181). Earlier studies with synthetic peptides corresponding to the octarepeat sequence demonstrated the binding of Cu2+ ions (182183), and optical spectroscopy showed that Cu2+ induced an α-helix formation in these peptides (184). More recently, PrP-deficient (Prnp0/0) mice were found to have lower levels of Zn/Cu superoxide dismutase (SOD) activity than do controls (185); SOD activity has been shown to mirror the state of copper metabolism (186). Measurements of membrane extracts from brains of Prnp0/0 mice showed low levels of Cu, whereas Fe and Zn were unchanged suggesting PrPC might function as a Cu2+-binding protein (187).

Disturbances in Cu2+ homeostasis leading to dysfunction of the CNS are well documented in humans and animals but are not known to be due to abnormalities in PrP metabolism: Menkes disease is manifest at birth and is due to a mutation of the MNK gene on the X chromosome, whereas Wilson’s disease appears in childhood and is due to a mutation of the WD gene on chromosome 13 (188191). Both the MNK and WDgenes encode copper-transporting ATPases. While both Menkes and Wilson’s diseases are recessive disorders, only Wilson’s disease can be treated with copper-chelating reagents. Interestingly, cuprizone, a Cu2+-chelating reagent, has been used in mice to induce neuropathological changes similar to those found in the prion diseases (192193).

PrP Gene Structure and Expression.

The entire open reading frame (ORF) of all known mammalian and avian PrP genes resides within a single exon (482194195). The mouse, sheep, cattle, and rat PrP genes contain three exons with the ORFs in exon 3 (196200) which is analogous to exon 2 of the SHa gene (82). The two exons of the SHaPrP gene are separated by a 10-kb intron: exon 1 includes a portion of the 5′ untranslated leader sequence, while exon 2 includes the ORF and 3′ untranslated region (82). Recently, a low abundance SHaPrP mRNA containing an additional small exon in the 5′ untranslated region was discovered that is encoded by the SHaPrP gene (201). Comparative sequencing of sheep and Hu cosmid clones containing PrP genes revealed an additional putative, small untranslated 5′ exon in the HuPrP gene (202). The promoters of both the SHa- and MoPrP genes contain multiple copies of G+C-rich repeats and are devoid of TATA boxes. These G+C nonamers represent a motif that may function as a canonical binding site for the transcription factor Sp1 (203). Mapping of PrP genes to the short arm of Hu chromosome 20 and to the homologous region of Mo chromosome 2 argues for the existence of PrP genes prior to the speciation of mammals (204,205).

Although PrP mRNA is constitutively expressed in the brains of adult animals (8384), it is highly regulated during development. In the septum, levels of PrP mRNA and choline acetyltransferase were found to increase in parallel during development (206). In other brain regions, PrP gene expression occurred at an earlier age. In situ hybridization studies show that the highest levels of PrP mRNA are found in neurons (207).

PrPC expression in brain was defined by standard immunohistochemistry (208) and by histoblotting in the brains of uninfected controls (209). Immunostaining of PrPC in the SHa brain was most intense in the stratum radiatum and stratum oriens of the CA1 region of the hippocampus and was virtually absent from the granule cell layer of the dentate gyrus and the pyramidal cell layer throughout Ammon’s horn. PrPScstaining was minimal in those regions that were intensely stained for PrPC. A similar relationship between PrPC and PrPSc was found in the amygdala. In contrast, PrPSc accumulated in the medial habenular nucleus, the medial septal nuclei, and the diagonal band of Broca; in contrast, these areas were virtually devoid of PrPC. In the white matter, bundles of myelinated axons contained PrPSc but were devoid of PrPC. These findings suggest that prions are transported along axons and are in agreement with earlier findings in which scrapie infectivity was found to migrate in a pattern consistent with retrograde transport (210212).

Molecular Genetics of Prion Diseases.

Independent of enriching brain fractions for scrapie infectivity that led to the discovery of PrPSc, the PrP gene was shown to be genetically linked to a locus controlling scrapie incubation times (213). Subsequently, mutation of the PrP gene was shown to be genetically linked to the development of familial prion disease (4). At the same time, expression of a SHaPrP transgene in mice was shown to render the animals highly susceptible to SHa prions, demonstrating that expression of a foreign PrP gene could abrogate the species barrier (214). Later, PrP-deficient (Prnp0/0) mice were found to be resistant to prion infection and failed to replicate prions, as expected (100101). The results of these studies indicated PrP must play a central role in the transmission and pathogenesis of prion disease, but equally important, they argued that the abnormal isoform is an essential component of the prion particle (23).

PrP gene dosage controls length of incubation time.

Scrapie incubation times in mice were used to distinguish prion strains and to identify a gene controlling its length (135215).  With a PrP cDNA probe, we demonstrated genetic linkage between the PrP gene and a locus controlling the incubation time in crosses between NZW/LacJ and I/Ln mice (213). We provisionally designated these genes as components of the Prn complex but eventually found that the incubation time gene, Prn-i, is either congruent with or closely linked to the PrP gene, Prnp (195).

Although the amino acid substitutions in PrP that distinguish NZW (Prnpa ) from I/Ln (Prnpb) mice argued for congruency of Prnp and Prn-i, experiments with Prnpa mice expressing Prnpb transgenes demonstrated a “paradoxical” shortening of incubation times (196). We had predicted that these Tg mice would exhibit a prolongation of the incubation time after inoculation with RML prions on the basis of (Prnpa × Prnpb)F1mice, which do exhibit long incubation times. We described those findings as “paradoxical shortening” because we and others had believed for many years that long incubation times are dominant traits (213,215). From studies of congenic and transgenic mice expressing different numbers of the a and b alleles ofPrnp, we learned that these findings were not paradoxical; indeed, they resulted from increased PrP gene dosage (218). We concluded that both Sincand Prn-i are congruent with PrP (218), and recent gene targeting studies have confirmed this view (219).

Overexpression of wtPrP transgenes.

Mice were constructed expressing different levels of the wt SHaPrP transgene (214). Inoculation of these Tg(SHaPrP) mice with SHa prions demonstrated abrogation of the species barrier, resulting in abbreviated incubation times (220). The length of the incubation time after inoculation with SHa prions was inversely proportional to the level of SHaPrPC in the brains of Tg(SHaPrP) mice (220). Bioassays of brain extracts from clinically ill Tg(SHaPrP) mice inoculated with Mo prions revealed that only Mo prions but no SHa prions were produced. Conversely, inoculation of Tg(SHaPrP) mice with SHa prions led only to the synthesis of SHa prions. Although the rate of PrPScsynthesis appears to be a function of the level of PrPC expression in Tg mice, the level to which PrPSc finally accumulates seems to be independent of PrPC concentration (220).

During transgenetic studies, we discovered that uninoculated older mice harboring numerous copies of wtPrP transgenes derived from Syrian hamsters, sheep, and Prnpb mice spontaneously developed truncal ataxia, hind-limb paralysis, and tremors (198). These Tg mice exhibited a profound necrotizing myopathy involving skeletal muscle, a demyelinating polyneuropathy, and focal vacuolation of the CNS. Development of disease was dependent on transgene dosage. For example, Tg(SHaPrP+/+)7 mice homozygous for the SHaPrP transgene array regularly developed disease between 400 and 600 days of age, whereas hemizygous Tg(SHaPrP+/0)7 mice also developed disease, but only after >650 days.

PrP-deficient mice.

The development and lifespan of two lines of PrP-deficient (Prnp0/0) mice were indistinguishable from those of controls (221222), whereas two other lines exhibited ataxia and Purkinje cell degeneration at ≈70 weeks of age (223) (R. Moore and D. Melton, personal communication). In the former two lines with normal development, altered sleep–wake cycles have been reported (224), and altered synaptic behavior in brain slices was reported (225226) but could not be confirmed by others (227,228).

Prnp0/0 mice are resistant to prions (100101). Prnp0/0 mice were sacrificed 5, 60, 120, and 315 days after inoculation with RML prions, and brain extracts were bioassayed in CD-1 Swiss mice. Except for residual infectivity from the inoculum detected at 5 days after inoculation, no infectivity was detected in the brains ofPrnp0/0 mice (101). One group of investigators found that Prnp0/0 mice inoculated with RML prions and sacrificed 20 weeks later had 103.6 ID50 units/ml of homogenate by bioassay (100). Others have used this report to argue that prion infectivity replicates in the absence of PrP (67132). Neither we nor the authors of the initial report could confirm the finding of prion replication in Prnp0/0 mice (101103).

Prion Species Barrier and Protein X.

The passage of prions between species is often a stochastic process, almost always characterized by prolonged incubation times during the first passage in the new host (36). This prolongation is often referred to as the “species barrier” (36229). Prions synthesized de novo reflect the sequence of the host PrP gene and not that of the PrPSc molecules in the inoculum derived from the donor (90). On subsequent passage in a homologous host, the incubation time shortens to that recorded for all subsequent passages. From studies with Tg mice, three factors have been identified that contribute to the species barrier: (i) the difference in PrP sequences between the prion donor and recipient, (ii) the strain of prion, and (iii) the species specificity of protein X, a factor defined by molecular genetic studies that binds to PrPC and facilitates PrPSc formation. This factor is likely to be a protein, hence the provisional designation protein X (134178). The prion donor is the last mammal in which the prion was passaged and its PrP sequence represents the “species” of the prion. The strain of prion, which seems to be enciphered in the conformation of PrPSc, conspires with the PrP sequence, which is specified by the recipient, to determine the tertiary structure of nascent PrPSc. These principles are demonstrated by studies on the transmission of SHa prions to mice showing that expression of a SHaPrP transgene in mice abrogated the species barrier (Table 3) (214). Besides the PrP sequence, the strain of prion modified transmission of SHa prions to mice (Table 3) (135230231).

Table 3

Influence of prion species and strains on transmission across a species barrier

Transmission of Hu prions.

Protein X was postulated to explain the results on the transmission of Hu prions to Tg mice (Table 4) (134179). Mice expressing both Mo and HuPrP were resistant to Hu prions, whereas those expressing only HuPrP were susceptible. These results argue that MoPrPC inhibited transmission of Hu prions—i.e., the formation of nascent HuPrPSc. In contrast to the foregoing studies, mice expressing both MoPrP and chimeric MHu2MPrP were susceptible to Hu prions and mice expressing MHu2MPrP alone were only slightly more susceptible. These findings contend that MoPrPC has only a minimal effect on the formation of chimeric MHu2MPrPSc.

Table 4

Evidence for protein X from transmission studies of human prions

Genetic evidence for protein X.

When the data on Hu prion transmission to Tg mice were considered together, they suggested that MoPrPC prevented the conversion of HuPrPC into PrPSc but had little effect on the conversion of MHu2M into PrPSc by binding to another Mo protein. We interpreted these results in terms of MoPrPC binding to this Mo protein with a higher affinity than does HuPrPC. We postulated that MoPrPC had little effect on the formation of PrPSc from MHu2M (Table 4) because MoPrP and MHu2M share the same amino acid sequence at the COOH terminus. We hypothesized that MoPrPC only weakly inhibited transmission of SHa prions to Tg(SHaPrP) mice (Table 3) because SHaPrP is more closely related to MoPrP than is HuPrP.

Using scrapie-infected Mo neuroblastoma cells transfected with chimeric Hu/Mo PrP genes, we extended our studies of protein X. Substitution of a Hu residue at position 214 or 218 prevented PrPSc formation (Fig.5B) (178). The side chains of these residues protrude from the same surface of the COOH-terminal α-helix, forming a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at position 167, 171, or 218 prevented PrPSc formation; these mutant PrPs appear to act as “dominant negatives” by binding protein X and rendering it unavailable for prion propagation. Our findings within the context of protein X explain the protective effects of basic polymorphic residues in PrP of humans and sheep (199232233).

Is protein X a molecular chaperone?

Since PrP undergoes a profound structural transition during prion propagation, it seems likely that other proteins such as chaperones participate in this process. Whether protein X functions as a molecular chaperone is unknown. Interestingly, scrapie-infected cells in culture display marked differences in the induction of heat-shock proteins (234235), and Hsp70 mRNA has been reported to increase in scrapie of mice (236). While attempts to isolate specific proteins that bind to PrP have been disappointing (237), PrP has been shown to interact with Bcl-2 and Hsp60 by two-hybrid analysis in yeast (238239). Although these studies are suggestive, no molecular chaperone involved in prion formation in mammalian cells has been identified.

Miniprions.

By using the four-helix bundle model of PrPC (Fig. 4A) (147), each region of proposed secondary structure was systematically deleted and the mutant constructs were expressed in scrapie-infected neuroblastoma (ScN2a) cells and Tg mice (164168). Deletion of any of the four putative helical regions prevented PrPScformation, whereas deletion of the NH2-terminal region containing residues 23–89 did not affect the yield of PrPSc. In addition to the 67 residues at the NH2 terminus, 36 residues from positions 141–176 could be deleted without altering PrPSc formation (Figs. 6 and 7). The resulting PrP molecule of 106 amino acids was designated PrP106. In this mutant PrP, helix A as well as the S2 β-strand were removed. When PrP106 was expressed in ScN2a cells, PrPSc106 was soluble in 1% Sarkosyl. Whether the structure of PrPSc106 can be more readily determined than that of full-length PrPSc remains uncertain. 

Figure 6

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Miniprions produced by deleting PrP residues 23–89 and 141–176. The deletion of residues 141–176 (green) containing helix A and the S2 β-strand is shown. Side chains of residues 168, 172, 215, and 219, which are thought to bind protein X, are shown in cyan. 

Figure 7

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Transgene-specified susceptibility.

Tg(PrP106)Prnp0/0 mice that expressed PrP106 developed neurological dysfunction ≈300 days after inoculation with RML prions previously passaged in CD-1 Swiss mice (S. Supattapone, T. Muramoto, D. Peretz, S. J. DeArmond, A. Wallace, F. E. Cohen, S.B.P., and M. R. Scott, unpublished results). The resulting prions containing PrPSc106 produced CNS disease in ≈66 days upon subsequent passage in Tg(PrP106)Prnp0/0 mice (Table 5). Besides widespread spongiform degeneration and PrP deposits, the pyramidal cells of the hippocampus constituting the CA-1, CA-2, and CA-3 fields disappeared in Tg(PrP106)Prnp0/0 mice inoculated with prions containing PrPSc106 (Fig. 7). In no previous study of Tg mice have we seen similar neuropathological lesions. The Tg(MoPrP-A) mice overexpressing MoPrP are resistant to RML106 miniprions but are highly susceptible to RML prions. These mice require more than 250 days to produce illness after inoculation with miniprions but develop disease in ≈50 days when inoculated with RML prions containing full-length MoPrPSc.

Table 5

Susceptibility and resistance of Tg mice to artificial miniprions

Smaller prions and mythical viruses.

The unique incubation times and neuropathology in Tg mice caused by miniprions are difficult to reconcile with the notion that scrapie is caused by an as-yet-unidentified virus. When the mutant or wt PrPC of the host matched PrPSc in the inoculum, the mice were highly susceptible (Table 5). However, when there was a mismatch between PrPC and PrPSc, the mice were resistant to the prions. This principle of homologous PrP interactions, which underlies the species barrier (Table 3), is recapitulated in studies of PrP106 where the amino acid sequence has been drastically changed by deleting nearly 50% of the residues. Indeed, the unique properties of the miniprions provide another persuasive argument supporting the contention that prions are infectious proteins.

Human Prion Diseases.

Most humans afflicted with prion disease present with rapidly progressive dementia, but some manifest cerebellar ataxia. Although the brains of patients appear grossly normal upon postmortem examination, they usually show spongiform degeneration and astrocytic gliosis under the microscope. The human prion diseases can present as sporadic, genetic, or infectious disorders (5) (Table 1).

Sporadic CJD.

Sporadic forms of prion disease constitute most cases of CJD and possibly a few cases of Gerstmann–Sträussler–Scheinker disease (GSS) (Table 1) (4240241). In these patients, mutations of the PrP gene are not found. How prions causing disease arise in patients with sporadic forms is unknown; hypotheses include horizontal transmission of prions from humans or animals (242), somatic mutation of the PrP gene, and spontaneous conversion of PrPC into PrPSc (515). Because numerous attempts to establish an infectious link between sporadic CJD and a preexisting prion disease in animals or humans have been unrewarding, it seems unlikely that transmission features in the pathogenesis of sporadic prion disease (912243).

Inherited prion diseases.

To date, 20 different mutations in the human PrP gene resulting in nonconservative substitutions have been found that segregate with the inherited prion diseases (Fig. 4B). Familial CJD cases suggested that genetic factors might influence pathogenesis (12244), but this was difficult to reconcile with the transmissibility of fCJD and GSS (3). The discovery of genetic linkage between the PrP gene and scrapie incubation times in mice (213) raised the possibility that mutation might feature in the hereditary human prion diseases. The P102L mutation was the first PrP mutation to be genetically linked to CNS dysfunction in GSS (Fig. 4B) (4) and has since been found in many GSS families throughout the world (245247). Indeed, a mutation in the protein coding region of the PrP gene has been found in all reported kindred with familial human prion disease; besides the P102L mutation, genetic linkage has been established for four other mutations (162931).

Tg mouse studies confirmed that mutations of the PrP gene can cause neurodegeneration. The P102L mutation of GSS was introduced into the MoPrP transgene, and five lines of Tg(MoPrP-P101L) mice expressing high levels of mutant PrP developed spontaneous CNS degeneration consisting of widespread vacuolation of the neuropil, astrocytic gliosis, and numerous PrP amyloid plaques similar to those seen in the brains of humans who die from GSS(P102L) (248250). Brain extracts prepared from spontaneously ill Tg(MoPrP-P101L) mice transmitted CNS degeneration to Tg196 mice but contained no protease-resistant PrP (249250). The Tg196 mice do not develop spontaneous disease but express low levels of the mutant transgene MoPrP-P101L and are deficient for mouse PrP (Prnp0/0) (221). These studies, combined with the transmission of prions from patients who died of GSS to apes and monkeys (3) or to Tg(MHu2M-P101L) mice (134), demonstrate that prions are generated de novo by mutations in PrP. Additionally, brain extracts from patients with some other inherited prion diseases, fCJD(E200K) or FFI, transmit disease to Tg(MHu2M) mice (27), (153251).

Infectious prion diseases.

The infectious prion diseases include kuru of the Fore people in New Guinea, where prions were transmitted by ritualistic cannibalism (242252253). With the cessation of cannibalism at the urging of missionaries, kuru began to decline long before it was known to be transmissible (Fig. 8). Sources of prions causing infectious CJD on several different continents include improperly sterilized depth electrodes, transplanted corneas, human growth hormone (HGH) and gonadotropin derived from cadaveric pituitaries, and dura mater grafts (254). Over 90 young adults have developed CJD after treatment with cadaveric HGH; the incubation periods range from 3 to more than 20 years (255256). Dura mater grafts implanted during neurosurgical procedures seem to have caused more than 60 cases of CJD; these incubation periods range from 1 to more than 14 years (257259). 

Figure 8

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Disappearance of the kuru and BSE epidemics. (A) Annual number of cases of BSE in cattle in Great Britain. (B) Biannual number of cases of kuru in Papua New Guinea. Data were compiled for BSE by John Wilesmith and for kuru by Michael Alpers.

Prion Diversity.

The existence of prion strains raises the question of how heritable biological information can be enciphered in a molecule other than nucleic acid (131215260265, 231266267). The typing of prion strains in C57BL, VM, and (C57BL × VM)F1 inbred mice began with isolates from sheep with scrapie. The prototypic strains called Me7 and 22A gave incubation times of ≈150 and ≈400 days in C57BL mice, respectively (215268269). The PrP genes of C57BL and I/Ln (and later VM) mice encode proteins differing at two residues and control scrapie incubation times (195213217219270).

Until recently, support for the hypothesis that the tertiary structure of PrPSc enciphers strain-specific information (23) was minimal except for the DY strain isolated from mink with transmissible encephalopathy by passage in Syrian hamsters (26271272).  Also notable was the generation of new strains during passage of prions through animals with different PrP genes (135230).

PrPSc conformation enciphers diversity.

Persuasive evidence that strain-specific information is enciphered in the tertiary structure of PrPSc comes from transmission of two different inherited human prion diseases to mice expressing a chimeric MHu2M PrP transgene (27). In FFI, the protease-resistant fragment of PrPSc after deglycosylation has a mass of 19 kDa, whereas in fCJD(E200K) and most sporadic prion diseases it is 21 kDa (Table 6) (273274). This difference in molecular size was shown to be due to different sites of proteolytic cleavage at the NH2 termini of the two human PrPSc molecules reflecting different tertiary structures (273).

Table 6

Distinct prion strains generated in humans with inherited prion diseases and transmitted to Tg mice

Extracts from the brains of FFI patients transmitted disease to mice expressing a chimeric MHu2M PrP gene about 200 days after inoculation and induced formation of the 19-kDa PrPSc, whereas fCJD(E200K) and sCJD produced the 21-kDa PrPSc in mice expressing the same transgene (27). On second passage, Tg(MHu2M) mice inoculated with FFI prions showed an incubation time of ≈130 days and a 19-kDa PrPSc, whereas those inoculated with fCJD(E200K) prions exhibited an incubation time of ≈170 days and a 21-kDa PrPSc (28). The experimental data demonstrate that MHu2MPrPSc can exist in two different conformations based on the sizes of the protease-resistant fragments, yet the amino acid sequence of MHu2MPrPSc is invariant.

The results of our studies argue that PrPSc acts as a template for the conversion of PrPC into nascent PrPSc. Imparting the size of the protease-resistant fragment of PrPSc through conformational templating provides a mechanism for both the generation and propagation of prion strains.

Interestingly, the protease-resistant fragment of PrPSc after deglycosylation with a mass of 19 kDa has been found in a patient who developed a sporadic case of prion disease similar to FFI but with no family history. Because both PrP alleles encoded the wt sequence and a Met at position 129, we labeled this case fatal sporadic insomnia (FSI). At autopsy, the spongiform degeneration, reactive astrogliosis, and PrPScdeposition were confined to the thalamus (275). These findings argue that the clinicopathologic phenotype is determined by the conformation of PrPSc in accord with the results of the transmission of human prions from patients with FFI to Tg mice (27).

Selective neuronal targeting.

Besides incubation times, profiles of spongiform change (Fig. 1) have been used to characterize prion strains (276), but recent studies argue that such profiles are not an intrinsic feature of strains (277278). The mechanism by which prion strains modify the pattern of spongiform degeneration was perplexing, since earlier investigations had shown that PrPSc deposition precedes neuronal vacuolation and reactive gliosis (212231). When FFI prions were inoculated into Tg(MHu2M) mice, PrPSc was confined largely to the thalamus (Fig. 9A) as is the case for FFI in humans (27279). In contrast, fCJD(E200K) prions inoculated into Tg(MHu2M) mice produced widespread deposition of PrPScthroughout the cortical mantle and many of the deep structures of the CNS (Fig. 9B) as is seen in fCJD(E200K) of humans. To examine whether the diverse patterns of PrPSc deposition are influenced by Asn-linked glycosylation of PrPC, we constructed Tg mice expressing PrPs mutated at one or both of the Asn-linked glycosylation consensus sites (278). These mutations resulted in aberrant neuroanatomic topologies of PrPC within the CNS, whereas pathologic point mutations adjacent to the consensus sites did not alter the distribution of PrPC. Tg mice with mutation of the second PrP glycosylation site exhibited prion incubation times of >500 days and unusual patterns of PrPSc deposition. These findings raise two possible scenarios. First, glycosylation can modify the conformation of PrPC and affect its affinity for a particular conformer of PrPSc, which results in specific patterns of PrPSc deposition; such interactions between PrPScand PrPC are likely to determine the rate of nascent PrPSc formation. Second, glycosylation modifies the stability of PrPSc and, hence, the rate of PrPSc clearance. This latter explanation is consistent with the proposal that the binding of PrPC to protein X is the rate-limiting step in PrPSc formation under most circumstances (280345).

Figure 9

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BSE.

Prion strains and the species barrier are of paramount importance in understanding the BSE epidemic in Great Britain, in which it is estimated that almost one million cattle were infected with prions (281282). The mean incubation time for BSE is about 5 years. Most cattle therefore did not manifest disease because they were slaughtered between 2 and 3 years of age (283). Nevertheless, more than 160,000 cattle, primarily dairy cows, have died of BSE over the past decade (Fig. 8A) (281). BSE is a massive common-source epidemic caused by meat and bone meal (MBM) fed primarily to dairy cows (282284). The MBM was prepared from the offal of sheep, cattle, pigs, and chickens as a high-protein nutritional supplement. In the late 1970s, the hydrocarbon-solvent extraction method used in the rendering of offal began to be abandoned, resulting in MBM with a much higher fat content (284). It is now thought that this change in the rendering process allowed scrapie prions from sheep to survive rendering and to be passed into cattle. Alternatively, bovine prions were present at low levels prior to modification of the rendering process and with the processing change survived in sufficient numbers to initiate the BSE epidemic when inoculated back into cattle orally through MBM. Against the latter hypothesis is the widespread geographical distribution throughout England of the initial 17 cases of BSE, which occurred almost simultaneously (282285286). Furthermore, there is no evidence of a preexisting prion disease of cattle, either in Great Britain or elsewhere.

Origin of BSE prions?

Brain extracts from BSE cattle cause disease in cattle, sheep, mice, pigs, and mink after intracerebral inoculation (291295), but prions in brain extracts from sheep with scrapie fed to cattle produced illness substantially different from BSE (296). However, no exhaustive effort has been made to test different strains of sheep prions or to examine the disease after bovine-to-bovine passage. In July 1988, the practice of feeding MBM to sheep and cattle was banned. Recent statistics argue that the epidemic is now disappearing as a result of this ruminant feed ban (Fig. 8A) (281), reminiscent of the disappearance of kuru in the Fore people of New Guinea (242253) (Fig. 8B).

Monitoring cattle for BSE prions.

Although many plans have been offered for the culling of older cattle to minimize the spread of BSE (281), it seems more important to monitor the frequency of prion disease in cattle as they are slaughtered for human consumption. No reliable, specific test for prion disease in live animals is available, but immunoassays for PrPSc in the brainstems of cattle might provide a reasonable approach to establishing the incidence of subclinical BSE in cattle entering the human food chain (176209,289297299345). Determining how early in the incubation period PrPSc can be detected by immunological methods is now possible, since a reliable bioassay has been created by expressing the BoPrP gene in Tg mice (180). Prior to development of Tg(BoPrP)Prnp0/0 mice, non-Tg mice inoculated intracerebrally with BSE brain extracts required more than 300 days to develop disease (67295300301). Depending on the titer of the inoculum, the structures of PrPC and PrPSc, and the structure of protein X, the number of inoculated animals developing disease can vary over a wide range (Table 3).

Have bovine prions been transmitted to humans?

In 1994, the first cases of CJD in teenagers and young adults that were eventually labeled new variant (v) CJD occurred in Britain (304). Besides these patients being young (305306), their brains showed numerous PrP amyloid plaques surrounded by a halo of intense spongiform degeneration (Fig. 10) (307 – 309).

Figure 10

http://www.pnas.org/content/95/23/13363/F10.medium.gif

The restricted geographical occurrence and chronology of vCJD have raised the possibility that BSE prions have been transmitted to humans. That only ≈25 vCJD cases have been recorded and the incidence has remained relatively constant make establishing the origin of vCJD difficult. It is noteworthy that epidemiological studies over the past three decades have failed to find evidence for transmission of sheep prions to humans (912). Attempts to predict the future number of cases of vCJD, assuming exposure to bovine prions prior to the offal ban, have been uninformative because so few cases of vCJD have occurred (312314). Are we at the beginning of a human prion disease epidemic in Britain like those seen for BSE and kuru (Fig. 8), or will the number of vCJD cases remain small as seen with iCJD caused by cadaveric HGH (255256)?

Strain of BSE prions.

Was a particular conformation of bovine PrPSc selected for heat resistance during the rendering process and then reselected multiple times as cattle infected by ingesting prion-contaminated MBM were slaughtered and their offal rendered into more MBM? Recent studies of PrPSc from brains of patients who died of vCJD show a pattern of PrP glycoforms different from those found for sCJD or iCJD (315316). But the utility of measuring PrP glycoforms is questionable in trying to relate BSE to vCJD (317,318) because PrPSc is formed after the protein is glycosylated (138140) and enzymatic deglycosylation of PrPSc requires denaturation (319320). Alternatively, it may be possible to establish a relationship between the conformations of PrPSc from cattle with BSE and those from humans with vCJD by using Tg mice as was done for strains generated in the brains of patients with FFI or fCJD (27180).

Yeast and Other Prions.

Although prions were originally defined in the context of an infectious pathogen (58), it is now becoming widely accepted that prions are elements that impart and propagate variability through multiple conformers of a normal cellular protein. Such a mechanism must surely not be restricted to a single class of transmissible pathogens. Indeed, it is likely that the original definition will need to be extended to encompass other situations in which a similar mechanism of information transfer occurs.

Two notable prion-like determinants, [URE3] and [PSI], have already been described in yeast and one in another fungus denoted [Het-s*] (321326). Studies of candidate prion proteins in yeast may prove particularly helpful in the dissection of some of the events that feature in PrPSc formation. Interestingly, different strains of yeast prions have been identified (327). Conversion to the prion-like [PSI] state in yeast requires the molecular chaperone Hsp104; however, no homolog of Hsp104 has been found in mammals (322328). The NH2-terminal prion domains of Ure2p and Sup35 that are responsible for the [URE3] and [PSI] phenotypes in yeast have been identified. In contrast to PrP, which is a GPI-anchored membrane protein, both Ure2p and Sup35 are cytosolic proteins (329). When the prion domains of these yeast proteins were expressed in E. coli, the proteins were found to polymerize into fibrils with properties similar to those of PrP and other amyloids (323325).

Prevention of and Therapeutics for Prion Diseases.

As our understanding of prion propagation increases, it should be possible to design effective therapeutics. Because people at risk for inherited prion diseases can now be identified decades before neurologic dysfunction is evident, the development of an effective therapy for these fully penetrant disorders is imperative (334335). Although we have no way of predicting the number of individuals who may develop neurologic dysfunction from bovine prions in the future (313), it would be prudent to seek an effective therapy now (28336). Interfering with the conversion of PrPC into PrPSc seems to be the most attractive therapeutic target (60). Either stabilizing the structure of PrPC by binding a drug or modifying the action of protein X, which might function as a molecular chaperone (Fig. 5B), is a reasonable strategy. Whether it is more efficacious to design a drug that binds to PrPC at the protein X binding site or one that mimics the structure of PrPC with basic polymorphic residues that seem to prevent scrapie and CJD remains to be determined (178233).

The production of domestic animals that do not replicate prions may also be important with respect to preventing prion disease. Sheep encoding the R/R polymorphism at position 171 seem to be resistant to scrapie (199232338344); presumably, this was the genetic basis of Parry’s scrapie eradication program in Great Britain 30 years ago (4446). A more effective approach using dominant negatives for producing prion-resistant domestic animals, including sheep and cattle, is probably the expression of PrP transgenes encoding R171 as well as additional basic residues at the putative protein X binding site (Fig. 5B) (178).

Whether gene therapy for the human prion diseases by using the dominant-negative approach described above for prion-resistant animals will prove feasible depends on the availability of efficient vectors for delivery of the transgene to the CNS.

Concluding Remarks—Looking to the Future.

While learning the details of the structures of PrPs and deciphering the mechanism of PrPC transformation into PrPSc will be important, the fundamental principles of prion biology have become reasonably clear. Though some investigators prefer to view the composition of the infectious prion particle as unresolved (336346). But the discovery of prion-like phenomena mediated by proteins unrelated to PrP in yeast and fungi serves not only to strengthen the prion concept but also to widen it (329).

Hallmark of prion diseases.

The hallmark of all prion diseases—whether sporadic, dominantly inherited, or acquired by infection—is that they involve the aberrant metabolism and resulting accumulation of the prion protein (Table 1) (23). The conversion of PrPC into PrPSc involves a conformation change whereby the α-helical content diminishes and the amount of β-sheet increases (25).

Understanding how PrPC unfolds and refolds into PrPSc will be of paramount importance in transferring advances in the prion diseases to studies of other degenerative illnesses. The mechanism by which PrPSc is formed must involve a templating process whereby existing PrPSc directs the refolding of PrPC into a nascent PrPSc with the same conformation. Indeed, the expanding list of prion diseases and their novel modes of transmission and pathogenesis (Table1), as well as the unprecedented mechanisms of prion propagation and information transfer (Tables 5 and6), indicate that much more attention to these fatal disorders of protein conformation is urgently needed.

Multiple conformers.

The discovery that proteins may have multiple biologically active conformations may prove no less important than the implications of prions for diseases. How many different tertiary structures can PrPSc adopt? This query not only addresses the issue of the limits of prion diversity (345) but also applies to proteins as they normally function within the cell or act to affect homeostasis in multicellular organisms. The expanding list of chaperones that assist the folding and unfolding of proteins promises much new knowledge about this process. For example, it is now clear that proproteases can carry their own chaperone activity where the pro portion of the protein functions as a chaperone in cis to guide the folding of the proteolytically active portion before it is cleaved (347). Such a mechanism might well feature in the maturation of polypeptide hormones. Interestingly, mutation of the chaperone portion of prosubtilisin resulted in the folding of a subtilisin protease with different properties than the one folded by the wild-type chaperone. Such chaperones have also been shown to work in trans (347). Additionally, apoptosis during development and throughout adult life might also be regulated, at least in part, by alternative tertiary structures of proteins.

http://www.pnas.org/content/95/23/13363.full

Copper and its role on “progressive neurodegeneration” and death

Reported by: Dr. Venkat S. Karra, Ph.D.

Many of us are familiar with prion disease from its most startling and unusual incarnations—the outbreaks of “mad cow” disease (bovine spongiform encephalopathy) that created a crisis in the global beef industry. Or the strange story of Kuru, a fatal illness affecting a tribe in Papua New Guinea known for its cannibalism. Both are forms of prion disease, caused by the abnormal folding of a protein and resulting in progressive neurodegeneration and death.

While exactly how the protein malfunctions has been shrouded in mystery, scientists at The Scripps Research Institute now report in the journal Proceedings of the National Academy of Sciences (PNAS) that reducing copper in the body delays the onset of disease. Mice lacking a copper-transport gene lived significantly longer when infected with a prion disease than did normal mice.

“This conclusively shows that copper plays a role in the misfolding of the protein, but is not essential to that misfolding,” said Scripps Research Professor Michael Oldstone, who led the new study.

“We’ve known for many years that prion proteins bind copper,” said Scripps Research graduate student Owen Siggs, first author of the paper with former Oldstone lab member Justin Cruite. “But what scientists couldn’t agree on was whether this was a good thing or a bad thing during prion disease. By creating a mutation in mice that lowers the amount of circulating copper by 60%, we’ve shown that reducing copper can delay the onset of prion disease.”

Zombie Proteins
Unlike most infections, which are caused by bacteria, viruses, or parasites, prion disease stems from the dysfunction of a naturally occurring protein.

“We all contain a normal prion protein, and when that’s converted to an abnormal prion protein, you get a chronic nervous system disease,” said Oldstone. “That occurs genetically (spontaneously in some people) or is acquired by passage of infectious prions. Passage can occur by eating infected meat; in the past, by cannibalism in the Fore population in New Guinea through the ingestion or smearing of infectious brains; or by introduction of infectious prions on surgical instruments or with medical products made from infected individuals.”

When introduced into the body, the abnormal prion protein causes the misfolding of other, normal prion proteins, which then aggregate into plaques in the brain and nervous system, causing tremors, agitation, and failure of motor function, and leads invariably to death.

Like a prion, Alzheimer’s protein seeds itself in the brain

Misshapen amyloid-beta self-propagates in mice

BY LAURA SANDERS  JUNE 19, 2012

The Alzheimer’s-related protein amyloid-beta is an infectious instigator in the brain, gradually contorting its harmless brethren into dangerous versions, new evidence suggests. The study adds to the argument that A-beta is a prion, a misfolded protein that behaves like the contagious culprits behind Creutzfeldt-Jakob disease in people, scrapie in sheep and mad cow disease.

https://www.sciencenews.org/article/prion-alzheimers-protein-seeds-itself-brain

Memories Cemented by Flux of Prion-Like Proteins

Persistent memories are doubly paradoxical. They are stable because they are built on physical connections that are dynamically maintained. Also, long-term memories are maintained through the work of prion-like proteins, even though prions are notorious for their contribution to neurodegenerative diseases—Alzheimer’s, Parkinson’s, and Huntington’s—and the destruction of memory.

Prion-like proteins, assert researchers at Columbia University, can have a functional role within neurons instead of contributing to disease. These researchers first identified a functional prion within Aplysia, a giant sea slug. Then they found a similar prion within mice. And now, in a new study, the researchers have proposed a mechanism for how this prion maintains long-term memories.

The new study—“The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3”—appeared June 17 in the journal Neuron. It explains how CPEB3, which stands for cytoplasmic polyadenylation element-binding protein, can account for the persistence of memory even though memories are built on molecular substrates that undergo rapid turnover.

A similar protein exists in humans, suggesting that human memories, too, rely on functional prions. This protein, said Eric Kandel, M.D., the leader of the Columbia team, may have the same role in memory. “Until this has been examined,” he prudently added, “we won’t know.”

When disease-causing prions form within a neuron, they cause damage by grouping together in sticky aggregates that disrupt cellular processes. Prion aggregates are highly stable and accumulate in infected tissue, causing tissue damage and cell death. The dying cell releases the prion proteins, which are then taken up by other cells—and are thus considered infectious.

Sourced through Scoop.it from: www.genengnews.com

PLoS One. 2014; 9(2): e89286. doi:  10.1371/journal.pone.0089286

Increasing Prion Propensity by Hydrophobic Insertion

Aaron C. Gonzalez Nelson,# Kacy R. Paul,# Michelina PetriNoe FloresRyan A. RoggeSean M. Cascarina, and Eric D. Ross*

Prion formation involves the conversion of proteins from a soluble form into an infectious amyloid form. Most yeast prion proteins contain glutamine/asparagine-rich regions that are responsible for prion aggregation. Prion formation by these domains is driven primarily by amino acid composition, not primary sequence, yet there is a surprising disconnect between the amino acids thought to have the highest aggregation propensity and those that are actually found in yeast prion domains. Specifically, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobic residues are strongly under-represented. Here, we directly test the effects of hydrophobic and aromatic residues on prion formation. Remarkably, we found that insertion of as few as two hydrophobic residues resulted in a multiple orders-of-magnitude increase in prion formation, and significant acceleration of in vitro amyloid formation. Thus, insertion or deletion of hydrophobic residues provides a simple tool to control the prion activity of a protein.

Protein disorder, prion propensities, and self-organizing macromolecular collectives.

Malinovska L1, Kroschwald S, Alberti S.

Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition.

PAPA – a method for predicting prion forming propensity

Biologists Find Unexpected Role for Amyloid-Forming Protein
http://www.technologynetworks.com/Proteomics/news.aspx?ID=183415
Yeast protein could offer clues to how Alzheimer’s plaques form in the brain.
Fibrous protein clumps known as amyloids are most often associated with diseases such as Alzheimer’s disease, where they form characteristic plaques in the brain.

Scientists first described amyloids about 150 years ago; they have since been tagged as key players in Parkinson’s disease, Huntington’s disease, and rheumatoid arthritis, as well as Alzheimer’s. However, recent findings suggest that this class of proteins may also have critical biological functions in healthy cells.

MIT biologists have discovered that yeast cells need to build amyloid-like structures during the production of reproductive cells called spores. Learning more about how yeast build and then break down these protein structures could help scientists develop drugs that destroy disease-causing amyloids, the researchers say.

http://www.technologynetworks.com/images/videos/News%20Images/PT/MIT-Amyloid-Protein_0.jpg

“Amyloids in the brain persist for decades. We just can’t get rid of them, yet yeast cells seem to have a mechanism for getting rid of them in 15 minutes,” says Luke Berchowitz, a postdoc at MIT’s Koch Institute for Integrative Cancer Research and the paper’s lead author. “If we can harness that mechanism, and really understand it, that could lead to anti-amyloid therapeutic opportunities.”

Reproductive role

Berchowitz and colleagues came across the yeast amyloid-forming protein known as Rim4 while investigating how sexual reproduction works in yeast. Rim4 is a protein containing long regions of disorder and stretches rich in the amino acid asparagine, which is a hallmark of a type of amyloid-forming proteins known as prions.

Berchowitz and Amon had previously discovered that Rim4 latches onto messenger RNA (mRNA) molecules, which carry genetic information to the cell’s protein-building machinery. In the new Cell paper, the researchers found that Rim4 uses amyloid-like clusters to prevent these mRNA molecules from being transcribed into proteins.

This process regulates the formation of spores — reproductive cells that are analogous to eggs and sperm, the researchers found.

Yeast usually reproduce asexually, through a process called budding, but under certain high-stress conditions, they can also undergo sexual reproduction through creation of spores that fuse to form new cells. The MIT team found that as yeast cells near completion of sexual reproduction, Rim4 amyloid-like clusters are broken down, releasing mRNA required for the cells to complete meiosis — the specialized type of cell division that produces spores.

“None of us anticipated that the way Rim4 actually works is by formation of these aggregates,” says Scott Keeney, a member of the Memorial Sloan Kettering Cancer Center, who was not involved in the research. “We’re used to thinking of these as toxic aggregates, so to demonstrate that they actually have a useful function in cells is intriguing.”

The researchers also found preliminary data suggesting that an amyloid protein known as DAZL plays the same role in sperm formation in mice; they believe that similar proteins are probably found in every organism that reproduces sexually, including humans.

Berchowitz says it is still unclear why cells rely on amyloid-forming proteins for this type of regulation, but one advantage amyloids offer is their ability to withstand the harsh environments in which sexual reproduction cells are formed. “Amyloids are stable and they’re able to sequester things,” he says. “They’re very tough guardians.”

“A great opportunity”

Previously, scientists have found a few other examples of amyloid-forming proteins that have critical roles in normal cell functions: In fruit flies, persistence of memory can rely on formation of amyloid-like structures in the brain, and amyloids are also involved in the formation of the skin pigment melanin in humans.

Learning more about how cells break down those amyloids could help scientists develop new drugs for disease such as Alzheimer’s, Parkinson’s, Huntington’s, and rheumatoid arthritis. Berchowitz is now working on figuring out how yeast cells regulate the breakdown of Rim4 aggregates.

“It’s a great opportunity to study assembly, regulation, and function of amyloids in living cells,” he says. “It’s pretty exciting that we can form them rapidly, synchronously, and abundantly.”

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