Review article
Non-human primates in prion research

Introduction

Transmissible spongiform encephalopathies (TSEs) or prion diseases are transmissible, fatal neurodegenerative disorders that have been known for more than two centuries. In the last decades, the emergence of new prion diseases in animals (bovine spongiform encephalopathy, BSE) or humans (variant Creutzfeldt-Jakob disease, vCJD) brought this unique group of diseases into public focus and led to an unparalleled boost in scientific breakthroughs to unravel the basic mechanisms behind this fascinating class of diseases.

First to be considered of viral origin (slow virus disease), it became obvious that prion diseases are a prototype of cerebral proteinopathies [3,76]. This class of diseases is characterized by abnormal folding of an otherwise physiologically expressed protein into a misfolded conformer that is prone to aggregation and subsequently leads to cell dysfunction and death [76]. A key step in prion disease initiation is the conversion of the cellular prion protein (PrPC) into PrPSc, which is partially resistant to proteolytic digestion and represents an essential part of prion infectivity [1,51,78]. The fact that transmission of prions, from affected to healthy organisms, occurs by imposing its aberrant conformation to the host protein via templated misfolding and the fact that transmission of prion diseases occurs under natural con­ditions, make this disease unique [39]. Besides other factors, the efficiency of transmission seems to be dependent on the route of transmission, the amount of infectious prions and the degree of amino acid homology of the incoming PrPSc with the host encoded PrPC [82]. The latter may lead to a species barrier, where transmission cannot occur or the in­cu­bation time to clinical prion disease is considerably prolonged [49,80]. Nevertheless, interspecies transmission of prion diseases occurs not only between animals and humans but also between different animal species [9,85]. Although subtle alterations in amino acid homology lead to drastic changes in trans­mission of prions, major structural features of PrPC are remarkably preserved among both, mammalian and non-mammalian PrPC [16,64].

Human prion diseases

The first human prion disease was already des­cribed at the beginning of the twentieth century [22,53]. Creutzfeldt and Jakob published two papers describing a novel neurodegenerative disorder that was accordingly termed Creutzfeldt-Jakob disease (CJD). Now it is obvious that human prion diseases encompass a group of diseases with a wide phenotypical spectrum. Besides the most common form of CJD (sporadic, sCJD), there are genetic variants (genetic CJD) and acquired forms of the disease (variant and iatrogenic CJD) [7,32,42,52]. In genetic CJD, there are diseases with distinct clinicopathological profiles such as Gerstmann-Sträussler-Scheinker disease (GSS) or Fatal Familial Insomnia (FFI) [56]. Among the acquired forms of human prion diseases, two stand out: kuru, which was endemic in Papua New Guinea and where transmission occurred via ritualistic cannibalism [31, 62] and vCJD which is probably caused by transmission of BSE-prions to humans [5,50,90]. In both in­stanc­es, incubation times from putative exposure to­wards prions to onset of the disease are exceedingly long possibly surpass­ing decades [20]. For vCJD, iatro­genic transmission from subclinically-infected vCJD carriers to humans by blood transfusion has occurred raising a question as to whether transfusion-associated vCJD may lead to a second wave of vCJD in the future [63,91].

Thus, there are at least three ways to initiate prion disease:

1) Transmission of infectious prions from an exo-genous prion source. This is the case of acquired forms of human prion diseases [11,79].

2) Disease initiation associated with changes in the gene encoding PrPC. This is the case of genetic CJD, which is inherited as an autosomal dominant trait. The spectrum of disease-causing genetic changes includes point mutations with amino acid changes, mutations introducing a stop codon and leading to a truncated protein or deletions as well as insertions of octapeptide motives within the octarepeat region of the prion gene [56]. It is currently debated how these changes lead to prion disease. Possibly, mutant PrPC is less stable, thereby facilitating a cascade of events leading to misfolding of PrPC and disease [77].

3) Spontaneous generation of PrPSc. Interestingly, the events underlying the most common form of human prion disease (sCJD) are not yet fully understood [19,42]. It was proposed that a rare stochastic event of misfolding of endogenous PrPC to PrPSc lies at the basis of this disease [3,39].

Pathology of prion diseases is limited to the central nervous system (CNS) and characterized by neuronal loss, the typical name-giving spongiform lesions, as­trogliosis and deposition of PrPSc [15]. The latter can differ in the glycotype ratio and core fragment size between different prion diseases [71]. Moreover, these different types of PrPSc show specific patterns of deposition and neuronal cell tropism, which are re­flected by disease-specific lesion profiles that can be utilized for diagnostic purposes and to determine CJD subtypes [15,81].

In human prion diseases in general, but especially in vCJD, deposition of PrPSc in peripheral sites such as lymphoid and muscular compartments have been described [37,74].

Prion diseases in animals and transmission to humans

Non-human prion diseases include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, transmissible mink encephalopathy (TME) in minks and chronic wasting disease (CWD) in elk and deer.

Scrapie in sheep is the oldest known prion disease. It was already described as early as in 1732 and affects sheep, goats and mouflons. The transmissibility of scrapie was shown in 1939 [23]. Neuro­pathological findings include spongiform vacuolation, astrogliosis and the deposition of PrPSc in the CNS, but also in peripheral tissues such as the lymphoreticular system, the neuromuscular system and the digestive tract [46,72]. Moreover, secretions and excretions have also been found to contain prion infectivity and may account for horizontal transmission within flocks [66]. Similarities between the neuropathological and clinical presentation of scrapie and the human prion disease, kuru, were already noted in 1959 [44].

Transmissible mink encephalopathy was first described in 1947 and is restricted to farmed animals [10,84]. Neuropathological findings include typical spongiform vacuolation, astrogliosis and detection of PrPSc. As in scrapie, widespread colonization of non-CNS compartments occurs [84].

Bovine spongiform encephalopathy or “mad cow disease” was first reported in the early 1980s in the UK and soon afterwards developed epidemic proportions [24,89]. A ban on feeding meat and bone meal to cattle resulted in a progressive decline in the number of diagnosed cases of BSE. Disease-associated PrPSc is mainly found in the CNS with a limited involvement of peripheral organs [6]. Beside the classical form of BSE, other atypical BSE-types, termed bovine amyloidotic spongiform encephalopathy (BASE)/L-BSE and H-BSE have been described [17].

Chronic wasting disease is a prion disease affecting captive and free-ranging cervids such as deer and elk, mainly in the US. The origin of CWD is still unknown [83]. CWD prions can be found in the CNS and other non-CNS compartments including bodily fluids or excretions such as saliva, feces, urine and blood [45]. This is of relevance since horizontal transmission seems to occur in CWD.

The first wave of prion research using non-human primates

The first experiments with prions involving primates were started after the description of kuru, which challenged the view on neurodegenerative diseases [31].

Kuru and sCJD can be transmitted to non-human primates

The transmissibility of human prion diseases to non-human primates was demonstrated in 1966 when Gajdusek et al. transmitted kuru to chimpanzees [27]. This was the first demonstration of the transmissibility of a neurodegenerative disorder affecting humans. One year later, it could be demonstrated that the incubation time of kuru shortened upon the second passage into chimpanzees hinting to adaptation [29]. In 1968, Gibbs et al. proved that intracerebral transmission to chimpanzees occurred in another human prion disease: CJD [36]. Incubation time to onset of disease was 13 months with the disease duration of only two months. The neuropathological analysis showed typical signs of prion disease with severe spongiosis. The two other chimpanzees which were caged together with the former ape were inoculated with pooled urine from kuru patients and brain homogenate from a patient with amyotrophic lateral sclerosis and failed to develop any neurological disease. When these primate studies were performed, the infectious entity was considered to be a slow virus as infectivity passed through small pore filters and transmitted easily with long incubation times. Efficient transmission of kuru and CJD to other primate species like marmoset monkeys, stump-tail macaques, gibbons, spider monkeys, sooty mangabey, pigtail macaques and others followed [25,26,28,30,68,75].

Genetic CJD can be transmitted to non-human primates

In 1980, the oral transmission to primates via consumption of PrPSc containing tissue was demonstrated for kuru, CJD and scrapie [33]. Interestingly, scrapie has never been reported to be transmitted to humans, although incidence of scrapie in certain regions is high and consumption of meat from diseased animals has certainly occurred.

In 1981, the transmission of GSS to non-human primates was successful, subsequently GSS was classified as a transmissible spongiform encephalo­pathy [68].

Altogether 1,914 primates were infected with a total of 440 cases of human prion diseases be­tween 1963 and 1993 at the NIH [12]. The main findings of this and other transmission studies are summarized in Table I. Inoculations were performed intracerebrally in most cases with fresh-frozen brain homogenate, although other preparations (e.g. formalin-fixed brain) as well as other tissue were investigated, too. These include tissues from sCJD, gCJD, iCJD, FFI, GSS and kuru patients. From these cases, in 291 individuals, transmission occurred without doubt. Primates utilized in this study in­clude chimpanzees, the new world monkeys squirrel, spider and capuchin monkey and the old world monkeys African green, rhesus and cynomolgus monkey. Susceptibility was highest in chimpanzees, squirrel and spider monkeys. Experimental transmission rates were 100% for iCJD, 95% for kuru, 90% for sCJD and about 68% for the genetic forms (GSS, gCJD, FFI). Data on transmission of FFI were inconclusive. Transmission efficiencies have been shown to be influenced by a number of factors including the amount of disease-associated PrPSc contained in the tissue used for inoculation [38]. Many brain regions remain unaffected in FFI and diseased brain tissue contains relatively low amounts of PrPSc, which might explain failure to transmit FFI to primates [13]. However, the transmissibility of FFI and therefore its correct classification as a prion disease could be proven by the transmission of FFI to mice [86].

Within the framework of the NIH study, a series of 1,113 human cases of neurological disorders and syndromes (besides human prion diseases, other disorders such as Alzheimer’s disease, multiple sclerosis, schizophrenia, amyotrophic lateral sclerosis, epilepsy, and others) were inoculated into 3,418 primates to investigate which of these disorders have transmission potential [12]. Only prion diseases have shown to be transmissible. After inoculation of tissue samples from more than 600 patients with non-spongiform disorders, 9 cases of non-prion neurological diseases also produced spongiform dege­neration upon inoculation in 9 out of 1,504 animals. None of these cases could be confirmed by subsequent multiple reinoculations of the human tissue in additional animals [12,14,43]. This needs to be taken into account when concepts of transmissibility of other neurodegenerative diseases are currently being discussed [54].

Determination of prion infectivity in peripheral human tissues

Within the NIH study, the distribution of prion-infectivity in prion-diseased humans was also as­sessed by bioassay into primates. Infectivity was, as expected, found in the brain, spinal cord and eye.

In all other tissues tested, infectivity was only irre­gularly detectable in the lung, liver, kidney, spleen and lymph nodes after inoculation into mainly squirrel monkeys [12]. In contrast, no infectivity could be detected in peripheral nerves, intestine, bone marrow, whole blood, leukocytes, serum, thyroid gland, adrenal gland, heart, skeletal muscle, adipose tissue, gingiva, prostate, testis and placenta/amnion. Diverse human body fluids and secretions were also tested by bioassay in non-human primates. These included tears, nasal mucus, saliva, sputum, urine, feces, semen, vaginal secretion and milk. None of these samples contained detectable levels of infectivity in non-human primate bioassay.

In contrast to the body fluids, it could be shown in 1994 that stereotactic electrodes used for recordings of electrical activity in the cerebral cortex of a patient with progressive dementia, easily transmitted a prion disease when implanted in the cortex of a chimpanzee [34]. Remarkably, this transmission occurred two years after the last use in humans and after repeated cleanings and sterilization procedures. This finding highlights the extreme resistance of prion-infectivity especially on metal surfaces and underscores the possibility of accidentally transmitting human prion diseases by contaminated metal instruments [61].

The first wave of prion research using non-human primates: conclusions

The main findings of this first wave of prion research using non-human primates can be summarized as follows:

1) Human prion diseases can be transmitted experimentally to a variety of non-human primates.

2) Efficiency of transmission depends on the primate species (host) as well as the origin of inoculated material (donor) with iCJD being most and familial cases being least efficient.

3) Efficiency also depends on the inoculation route with highest transmission rates after intracerebral inoculation.

4) The same material injected into multiple primates leads to a range of incubation times.

5) Characteristics of the original disease are preserved upon transmission (e.g. lesion profile).

6) Incubation time to clinical disease is shortened upon secondary transmission (= adaptation).

The second wave of prion research using non-human primates

The development of BSE into an epidemic and the putative transmission of BSE to humans giving rise to a novel human prion disease (vCJD) initiated a second round of experiments with prions involving primates [24,89].

SE and non-human primates

The main focus of the initial experiments using primates was to address the zoonotic potential and risk of transmissibility of BSE and vCJD. Since the use of chimpanzees for these types of studies was practically banned and the use of other non-human primates drastically reduced due to ethical considerations, animal numbers were usually much smaller in these studies [88]. Instead of chimpanzees, cy­no­molgus and rhesus macaques were used in most of the studies. Cynomolgus and rhesus macaques are ideally suited since these species are evolutionary very close to humans, have a high degree of amino acid homology in PrPC and are also polymorphic on codon 129 of the gene encoding PrP, an important genetic determinant of susceptibility towards prion diseases in humans [41].

Thus, cynomolgus macaques were used to investigate the transmissibility of BSE. In 1996, it became obvious that BSE may be transmitted by intracerebral inoculation [59]. Moreover, the clinical presentation and the pathological signature in primates with typical so called “florid” plaques in brain tissue was identical to that of humans with vCJD and differed from that seen after transmission of sCJD. In addition, the PrPSc-type indicative of the predominant species of infectious prions present also supported the link between BSE and vCJD, since PrPSc-types between BSE-diseased cattle, vCJD and BSE-inoculated primates were identical and differed significantly from sCJD in humans and primates (see Fig. 1). Five years later, it could be shown that secondary transmission of macaque adapted BSE leads to a shortening in incubation time, as seen for other transmission studies [60]. In addition, the successful transmission of vCJD with a comparable short incubation time in primates suggested that BSE easily adapted to primates with enhanced virulence. In this study, it could also be shown that primate adapted BSE transmit via intravenous inoculation with incubation times only slightly higher than in i.c. inoculations [60]. These experiments showed that BSE can be easily transmitted by the intravenous route from primate to primate and suggested that secondary i.v. transmission of vCJD in humans may occur. In 2004 and 2005, the capability of BSE to be orally transmitted to primates via consumption of prioncontaining tissue was proven [48, 58]. However, in­cu­bation time to clinical disease was considerably longer than with i.c. or i.v. inoculation.

The discovery of L-BSE/BASE and H-BSE gave rise to the question of transmissibility to humans. This was first evaluated in mice transgenic for human prion protein [8,55]. These mice were susceptible towards BASE much more efficiently than classical BSE. However, BSE-H failed to transmit in these experiments [8]. The transmission of BASE via intracerebral injection to cynomolgus macaques was demonstrated in 2008 and 2011 [21,73]. Moreover, the oral transmission of BASE was demonstrated only recently in another primate model (mouse lemur) [70].

PrP^Sc in peripheral tissue of non-human primates

Improvements in the detection of minute amounts of PrPSc by selective precipitation (NaPTA) or highly sensitive ELISA led to the demonstration of PrPSc in peripheral tissues [37]. Subsequently, these methods were used to assess the degree of peripheral deposition of PrPSc in non-human primates challenged with various human prion-isolates [47]. Interestingly, spreading of PrPSc to the periphery seems to be prion strain and host dependant, since vCJD could be detected in tongue and skeletal muscle in a primate model of human prion diseases [57] (see Fig. 1). In this model, very small amounts of aberrantly folded PrPSc could also be detected in heart tissue. In contrast, BSE-infected monkeys repeatedly failed to show any detectable PrPSc deposition in heart tissue and the ultrastructural changes in the brain were minimal (Fig. 3). This is in line with data from mice inoculated with mouse-adapted BSE, where no heart-associated PrPSc could be found in preclinical or clinical stages [18]. PrPSc found in skeletal muscle mainly associated to lymphatic tissues and only at later time points to neuromuscular junctions and small nerves in this model.

Preclinical distribution and deposition of PrP^Sc

Data about the preclinical phase of prion diseases are difficult to assess in humans, thus small laboratory rodents have been used to address these questions instead [2]. In these experiments, it could be shown that PrPSc plateaus during the development of clinical symptoms [40]. Moreover, it could be shown that peripheral deposition of PrPSc occurs before the onset of clinical symptoms [65]. Interestingly, the degree of peripheral deposition is highly dependant on the host and prion strain combination. In hamsters, the deposition of PrPSc in the peri­phery is higher than in mice even with the use of transgenic approaches [18,87]. Recently, we could demonstrate that for PrPSc amounts and plateaus in preclinical prion disease in primates infected with vCJD [57] (see Fig. 1A). In these experiments, we were also able to assess the preclinical deposition of PrPSc in muscle tissue. We could show that peripheral deposition is prion strain dependant also in non-human primates with most abundant prion-colonisation of the periphery after inoculation with vCJD. This study furthermore showed that muscular PrPSc occurs long before the onset of clinical symptoms and, as in sCJD, positively correlates with long disease duration [37].

The second wave of prion research using non-human primates: conclusions

The main focus of the second round of transmission studies to non-human primates was to assess the zoonotic potential of the novel and emerging prion diseases BSE and vCJD [48,58]. For a summary of the main transmission studies see Table I. Due to novel sensitive methods to detect PrPSc it was also possible to investigate the spread of PrPSc from or to peripheral tissues [57]. Moreover, the preclinical de­position of PrPSc in brain and peripheral tissue was assessed for the first time.

Conclusions

Prion diseases are intensively studied neurodegenerative disorders. A variety of different animal models and ex vivo models exists for this prototype of cerebral proteinopathy. Non-human primate models reproduce main characteristics of human prion diseases and have proven valuable to study their pathophysiology [48,57,58,60]. Although the idea of a simple protein as a transmissible entity that is

able to impose its conformation to an otherwise normally folded protein was quite revolutionary [1,78], the prion concept has spread to other diseases such as Alzheimer’s disease. On the other hand, it has to be said that these diseases lack the natural transmissibility of prion diseases although transmission can be achieved in experimental conditions [4].

Although ethical obstacles to perform primate studies are high, and usage of primates in biome-dical research should be considered very carefully, some aspects of primate models are superior to studies in small laboratory animals. In prion disease-research, primate studies have laid the ground for a wealth of studies using genetically modified mice and cell-based methods. Thus, primate studies have contributed to the commonly heard notion that prion research is now the driving force of biomedical research in the field of neurodegenerative diseases.

Acknowledgements

S.K. was supported by “Pro Exzellenzia” (City of Hamburg) and BMBF-DLR grant 01GZ0712.

P.P.L. and B.S. are supported by the Polish-German grant P-N/035/2006.

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