Prions and Prion Disease of the Central Nervous System (Transmissible Neurodegenerative Diseases)

Reviewed for currency February, 2020

Prions are the transmissible agents of a class of neurodegenerative diseases of humans and other mammals. Prions differ from other infectious agents in that they contain no information-bearing nucleic acid. Rather, they are composed mainly, perhaps only, of abnormal conformations of a normally produced cell surface glycoprotein called the prion protein (PrP). These abnormal conformations of PrP are able to trigger the abnormal folding of previously normal conformers of PrP. It is the abnormal conformation of PrP, rather than the polypeptide itself, that propagates in prion diseases. The human forms of prion disease are most commonly referred to as Creutzfeldt-Jakob disease (CJD), although some specific clinical forms carry other names, such as kuru, Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The most important prion diseases of animals are scrapie of sheep, bovine spongiform encephalopathy (BSE) of cattle, and chronic wasting disease (CWD) of deer and related species.

Brief History of Prion Disease Research

Scrapie, a disease of sheep and goats, is the prototypical prion disease. References to it are found at least as early as 1750 in Germany. Studies on scrapie in the late 19th through mid-20th century established that the disease was transmissible by a small filterable agent after incubation periods of up to 22 months. It was thus thought to be a virus or, because of the long latency between infection and the emergence of symptoms, a “slow virus.” However, studies in the mid-20th century revealed the scrapie agent to be resistant to ionizing radiation and other physical and chemical treatments that ordinarily inactivate viruses. This led Alper and coworkers to suggest that the agent might replicate despite lacking nucleic acid. The link between the agent of scrapie and human disease was established by Gajdusek and Zigas. Gajdusek and coworkers then identified kuru, a neurodegenerative condition endemic in certain cannibalistic tribes in the highlands of New Guinea, as a human prion disease when they transmitted the condition to primates. They were aided in this discovery by the insights of the veterinary neuropathologist Hadlow, who remarked on the histopathologic similarity of kuru to scrapie, triggering the search for the potential transmissibility of kuru. The similarities between kuru and scrapie that first drew Hadlow's attention included a “soap bubble”–like vacuolation of the neuropil, profound neuronal loss, and intense reactive astrogliosis in the absence of an associated inflammatory response. Modern neuropathologists would add only the immunohistochemical detection of abnormal forms of PrP to this list of key pathologic features of prion diseases. Prusiner and coworkers first purified the infectious agent. They found that a previously unidentified protein was the chief component of a highly infectious fraction isolated from brains of hamsters with scrapie. In 1982, Prusiner proposed the name prion (pronounced PREE-on) for the agent responsible for scrapie and related neurodegenerative diseases. The term prion initially was chosen to emphasize the hypothesis that the causative agents in these diseases were pr oteinaceous i nfectious particles that could be distinguished from viruses and viroids by their apparent lack of nucleic acid. The protein that comprised the dominant component of the infection fraction was thus named the “prion protein.”

Molecular Biology and Pathophysiology of Prion Diseases

The Prion Protein

The process of prion propagation involves the conversion of a normally produced form of the prion protein ( Fig. 179.1 ), usually designated PrP ^C (“C” for cellular), to an abnormal disease-causing form, often designated PrP ^Sc (“Sc” for scrapie). PrP ^C is a cell surface glycoprotein, transcribed off a chromosomal gene, PRNP, located at 20p13 in humans. The protein is modified during biosynthesis by the addition of a glycosylphosphatidylinositol moiety that anchors the carboxyl terminus of the mature protein to the external surface of the cell membrane. It may be further modified by oligosaccharides (glycans) linked to asparagine at positions 181 and 197 in the polypeptide. PrP ^C is constitutively expressed at high levels in the normal brain. The protein is also expressed at high levels in certain cells of the reticuloendothelial system, and this distribution contributes to the pathogenesis of epidemic prion disease (see “ Transmission by Oral Exposure ” later). The normal function of PrP ^C remains obscure. Mice in which production of PrP is abolished by disruption of the PRNP gene survive to adulthood, and show no gross anatomic or behavioral abnormalities. The PrP sequence is highly conserved in evolution, so it presumably confers an as-yet unrecognized fitness benefit on the host. Candidate functions for PrP ^C include cell adhesion, synaptic function, signal transduction, metabolism of amyloid β (the protein involved in Alzheimer disease pathogenesis), and a role in copper metabolism. High-resolution structures for PrP ^C have been obtained by nuclear magnetic resonance spectroscopy. This shows PrP ^C to be a globular protein composed of three α-helical segments and two short β strands that form a small antiparallel β sheet.

FIG. 179.1, Structural elements of prion protein (PrP) and mutations associated with genetic human prion disease.

Infectious Prions

Prion diseases are marked by the appearance of a pathologic form of PrP, designated PrP ^Sc . PrP ^Sc has biochemical properties different from those of PrP ^C ( Table 179.1 ). In particular, the C-terminal portion of PrP ^Sc is resistant to digestion by proteases, so that after treatment with the serine endopeptidase proteinase K, only the N-terminal portion of PrP ^Sc is digested, whereas PrP ^C is completely hydrolyzed. The generation of protease-resistant C-terminal fragments of characteristic size is a widely used assay for PrP ^Sc . Spectroscopic studies indicate the PrP ^Sc polypeptide has acquired extensive β sheet structure that is not present in PrP ^C . These and other data indicate that PrP ^Sc is composed of aggregates of PrP tightly bound by interpeptide β sheets. In contrast, PrP ^C exists as monomers or perhaps low-order multimers. The tertiary structure of PrP ^Sc (i.e., the conformation of the polypeptide chain in aggregates) has proved difficult to determine, in part because available methods to determine high-resolution protein structure require that the protein be soluble, but the aggregates of which PrP ^Sc is composed are insoluble. The conformation of PrP ^Sc appears to be central to the process of prion propagation, so efforts to decipher this structure using advanced techniques are underway.

TABLE 179.1

The Properties of the Normal and Scrapie-Associated PrP Isoforms

Modified from Reisner D. Biochemistry and structure of PrP(C) and PrP(Sc). Br Med Bull. 2003;66:21–33.

FEATURE	PrP ^C (NORMAL ISOFORM)	PrP ^Sc (SCRAPIE ISOFORM)
Present in normal brain	Yes	No
Present in scrapie-infected brain	Yes	Yes
Covalent modifications	GPI anchor, Asn-linked polysaccharides, single intramolecular disulfide bond	Probably identical to PrP ^C
Soluble in mild detergent	Yes	No
Effect of protease	Hydrolyzed to small peptides	Protease resistant C-terminal portion of variable length
Conformation
Secondary structure: Local conformation of the polypeptide chain	About 40% α-helical, little β sheet	30% β sheet, 20% α-helix
Tertiary structure: Overall fold of the polypeptide	3 α-helical regions, unstructured N-terminus	Not determined
Quaternary structure: Association with other polypeptides	Monomeric or few-subunit oligomer	Aggregated

Asn, Asparagine; GPI, glycosylphosphatidylinositol.

Propagation of Prions

The precise mechanism of prion propagation is not completely understood. A popular model views prion propagation as similar to the “seeded polymerization” mechanism of amyloid formation. “Amyloid” is defined as a fibrillary protein aggregate, in which the protein monomers are bound together in β sheets oriented perpendicularly to the long axis of the fibril. Such aggregates may form the large “plaques” that are histologically identifiable by apple green birefringence under polarized light when stained with the dye Congo red (the classical definition of amyloid), but need not do so. The kinetics of amyloid fibril formation in vitro suggests a mechanism for prion propagation. Typically, a pure solution of monomers of an amyloidogenic peptide will exhibit a lag phase, often days in duration, before the rapid conversion of monomers into amyloid fibrils. If a small “seed” fibril is introduced into a solution of monomeric peptides, the lag phase is eliminated and the monomers rapidly polymerize. By analogy, an inoculum of PrP ^Sc acts as seed to trigger aggregation of PrP ^C . PrP amyloid of the classical type is seen in some forms of human and animal prion disease. Uncertainty persists over whether amyloid fibrils or a protofibrillary state may be the actual infectious moiety of prion diseases. A conceptual model of prion propagation is presented in Fig. 179.2 .

FIG. 179.2, A conceptual model of prion replication.

In vitro Synthesis of Prions

The seeded aggregation approach has been used to synthesize infectious prions in vitro. In the simplest application of this in vitro method, termed prion misfolding cyclic amplification, a small inoculum of infectious prions is added to a larger volume of brain homogenate. This mixture is incubated at a warm temperature and subject to cycles of sonication. In this process, PrP ^C in the brain homogenate is converted to PrP ^Sc . A small amount of this treated mixture is then inoculated into another volume of brain homogenate. The process is repeated through many cycles. Infectious prions and PrP ^Sc are present in the final mixture, yet the original inoculum has been diluted to nothingness. This method has been further refined to produce infectious prions using only defined components, confirming the prion hypothesis. Further modifications of this method have been exploited to develop sensitive and specific minimally invasive (typically lumbar puncture) tests for prion disease (see “ Laboratory Diagnosis of Prion Disease ” later).

Other Macromolecules Contributing to Prion Propagation

Whether proteins or macromolecules other than PrP play a role in prion propagation is uncertain. In the yeast prion state [PSI+] (see “ Yeast Prions ” later), protein folding chaperones such as heat shock proteins can influence the rate of prion propagation. The evidence of similar effects for mammalian prion disease is less clear. To date, no protein other than PrP has been unequivocally demonstrated to participate in mammalian prion propagation. Genome-wide surveys of human populations have suggested that genes other than PRNP may contribute to a risk for prion disease, but conclusive evidence is lacking. In vitro studies of prion replication indicate that phosphatidylethanolamine and RNA (of any sequence) may be important cofactors to produce infectious prions.

Species Barrier to Transmission of Prion Diseases

Prion diseases are enzootic in several species of ruminants to which humans are exposed through food consumption and other routes, raising concerns of transmission of these conditions to humans (see “ Variant Creutzfeldt-Jakob Disease ” later). The amino-acid sequence of PrP can have a dramatic influence on prion propagation. The transmission of prion diseases across species is generally less efficient than transmission within the species. This phenomenon is known as the “species barrier.” A crucial determinant of the species barrier is differences in the amino-acid sequence of PrP between species. Studies in transgenic mice demonstrate that the species barrier can be abrogated by the introduction of a gene directing expression of PrP of the prion donor species into the host. Thus mice, which are usually not susceptible to human sporadic CJD (sCJD), are readily infected if the mouse Prnp gene has been replaced by a transgene coding for the human PrP sequence. This phenomenon has been exploited to generate transgenic mice that can be used to model prion diseases of several species. The efficiency of cross-species transmission of prions is also determined by the strain of prion involved, as discussed in the next section.

Prion Strains

One of the most enigmatic features of prion diseases is the existence of what are termed prion strains ( Table 179.2 ). In experimental animals, prion strains can be distinguished by clinical features such as disease incubation time in animals or the pattern of clinical signs at disease onset. Pathologically, strains can be characterized by the region of the brain in which PrP ^Sc accumulates, by the degree of brain vacuolization, and by the morphology and distribution of PrP amyloid accumulation. Of particular importance to human health in the presence of animal prion disease epidemics, the species barrier to prion disease transmission may vary considerably between prion strains. Thus the resistance of humans to prions of other species cannot be predicted simply from a comparison of PRNP sequences.

TABLE 179.2

Comparison of Two Hamster Prion Strains ^a

	STRAIN
PROPERTY	HYPER	DROWSY
Incubation time	65 days	165 days
Clinical signs	Hyperexcitability and ataxia	Lethargy
Size of protease-resistant fragment (nonglycosylated band)	21 kDa	20 kDa
Sensitivity of resistant carboxyl-terminal fragment to prolonged exposure to protease	Present after 24 h of digestion	Hydrolyzed after 12 h
Resistance to denaturation (concentration of guanidine HCl that denatures 50% of PrP ^Sc )	1.5 M	1.1 M
Distribution of PrP ^Sc in the brains of clinically affected hamsters	Most in medial geniculate nucleus and deep cerebellar nuclei	Most in regions of hippocampus, cerebellar granular layer, and occipital cortex
Distribution of PrP ^Sc outside the CNS	In spleen and other lymphoreticular organs	Not found in lymphoreticular system
Species barrier	Nonpathogenic in mink	Pathogenic in mink

CNS, Central nervous system; PrP ^Sc , scrapie prion protein.

a Perhaps the two most well-studied prion strains are “hyper” and “drowsy,” which are adapted to hamsters from transmissible mink encephalopathy, a prion disease of mink. The two strains are typically propagated in Syrian golden hamsters. Some characteristic properties are compared in the table. The number of potential strains that can exist on a single genetic background is not known, but evidence indicates that it is more than two.

Because the prion consists of host-derived PrP, the means by which the properties of individual strains are maintained is puzzling. The biochemical basis of prion strain variety appears to reflect differences in PrP ^Sc conformation between various strains ( Fig. 179.2 ). Evidence for this includes differences in size of the protease-resistant carboxyl-terminal fragment between strains, different stabilities of the PrP aggregate to denaturation with chaotropic salts, and differential binding of antibodies to PrP ^Sc of different strains. Taken together, these and other observations suggest that subtle conformational differences between the PrP ^Sc associated with the various strains somehow dictate the clinical and pathologic differences.

It appears that differences in the clinical manifestation of certain human prion diseases (e.g., sCJD, sporadic fatal insomnia, variant CJD [vCJD], and so-called variable protease-sensitive prionopathy) may relate to differences in the strain of the prion involved.

Transmission by Oral Exposure

Only a small proportion of cases of human prion disease are infectiously transmitted, but intraspecies transmission through the oral route is the major cause of epidemic prion disease in animals. After dietary exposure to prions, the reticuloendothelial system plays a major role in the initial propagation of prions and in carrying infection to the central nervous system (CNS). Prion titers rise first in gut-associated lymphoid tissue. Mice deficient in the number of functional Peyer patches show increased resistance to oral prion challenge. Similarly, a number of studies have demonstrated that follicular dendritic cells are necessary for mice inoculated intraperitoneally with mouse-adapted scrapie to propagate prions to the brain. Prion infection is carried to the brain from lymphoid tissue by axoplasmic transport in neurons of the sympathetic nervous system.

Neurodegeneration in Prion Disease

How propagation of PrP ^Sc leads to neurodegeneration remains unknown. Although the phenotypic expression of yeast prions, as discussed in the next section, is in several instances due to the loss of the protein's normal function, this does not appear to be the case with mammalian prions. Rather, the conversion of PrP ^C to PrP ^Sc causes a toxic “gain of function.” The nature of this toxic function remains poorly understood. Toxicity requires expression of PrP, and this PrP must be glycosylphosphatidylinositol anchored. Substantial evidence suggests that the toxic species of PrP associated with prion infection may be different from the infectious species. As with other neurodegenerative diseases associated with protein aggregates, dysfunction of the ubiquitin-proteasome system has been invoked as a cause of neurodegeneration in prion disease.

Yeast Prions

Wickner first proposed that certain epigenetic traits of yeast could be manifestations of a process analogous to that which occurs in mammalian prion diseases. To date, at least 10 yeast prions and 1 prion of the filamentous fungus Podospora anserina ([Het-s]) have been identified. These traits are transmitted through exchange of cytoplasm but are not linked to the mitochondrial genome. Each particular prion trait is linked to a different protein, encoded by the nuclear genome, that is found to be in an aggregated state when the prion trait is expressed. Unlike the mammalian PrP prion, the phenotype of at least some yeast prions is equivalent to an inactivating mutation in the cognate protein. The [Het-s] prion is particularly interesting in that it appears to convey a useful property, mating incompatibility, upon its host. Yeast prions are intensively studied as models of mammalian prions and the processes of protein folding and aggregation in general. Yeast prions do not play any role in the initiation or transmission of mammalian prion diseases.

Prion-Like Behavior of Other Neurodegenerative Diseases

Prions formed of PrP remain the only proven mammalian prions, but there is some evidence that prion-like behavior of other proteins may play a role in neurodegenerative diseases such as Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis. Limited evidence suggests that under unusual circumstances these conditions might be iatrogenically transmitted.

Human Prion Diseases

Classification and Epidemiology of Human Prion Disease

Traditionally, human prion diseases have been classified by a combination of epidemiology, clinical features, histopathology, and family history. This has led to a proliferation of named syndromes that fundamentally share a similar pathophysiology. A more useful and consistent classification considers the various manifestations of human prion disease in terms of origin: sporadic, genetic, or infectiously transmitted. Most human prion disease is sporadic, that is, there is no determined infectious or genetic cause. In general, the term Creutzfeldt-Jakob disease refers to human prion disease and includes sporadic, genetic, and infectiously acquired forms that have not been given another name. The substantial clinical and pathologic diversity of human prion disease may be the manifestations of different strains of prions. However, typing of human prion strains is an emerging technology, and the basis of prion strain differences is incompletely understood. It is not clear whether the diverse manifestations of human prion disease can be attributed to differences in the properties of the initiating prion strains or result from other as-yet unidentified factors.

Epidemiology of Creutzfeldt-Jakob Disease

The term Creutzfeldt-Jakob disease was first used by Spielmeyer in 1922 to refer to a puzzling, rapidly progressive neurodegenerative syndrome initially described separately by the German neurologists Creutzfeldt and Jakob. CJD is rare, with an annual prevalence and incidence typically said to be 1 case per 1 million population worldwide. In several countries, the rate of CJD has increased since the mid-1990s. This most likely is the result of improved surveillance in response to the outbreak of BSE and vCJD. Based on these studies, the actual incidence of human prion disease may approach 2 cases per million annually. In the United States, studies find a substantially lower rate of CJD among African Americans and other nonwhites than among whites. Whether this reflects reduced ascertainment in these groups or a truly reduced incidence is not certain.

Sporadic Creutzfeldt-Jakob Disease

Epidemiology

sCJD comprises approximately 85% to 94% of all cases of human prion disease. It shows no gender predilection. Mean age at onset is 57 to 66 years, although patients as young as 17 years and older than 80 years with sCJD have been reported. Several studies found a peak incidence, approaching 6 per 1 million population, in the eighth decade and then a distinct decline in incidence in those older than 80 years.

Clinical Features

The most distinctive clinical feature of sCJD is the pace of its progression, typically described as “rapid” or “subacute.” In the context of neurodegenerative conditions, these terms refer to perceptible declines in cognitive and motor function that are obvious over a period of a few weeks. In contrast, in more common neurodegenerative conditions, such as Alzheimer or Parkinson disease, decline is typically only apparent over periods ranging from months to years. Some observers have noted that the pace of decline in sCJD accelerates until the later stages of akinetic mutism, when neurologic dysfunction is so severe that it is difficult to appreciate further decline. A second distinctive feature of sCJD is the prominent involvement of multiple brain systems in which motor signs, such as ataxia, bradykinesia, or spasticity, are combined with memory and other cognitive deficits. There is a great deal of variability in the clinical manifestations of sCJD, and this has led to attempts to describe a variety of clinical subtypes, including those with predominance of visual, cerebellar, thalamic, and striatal features. The existence of these syndromes indicates that sCJD may affect particular brain regions disproportionately. In some cases, the particular regional predominance of pathology may reflect the strain of prion involved.

In many patients with sCJD, there is a prodromal phase of psychiatric disturbance before the onset of neurologic signs. Of 126 mostly sporadic cases of CJD, 26% of patients had psychiatric signs in the prodromal or presenting phase. Most common were sleep disturbance, depression, and anxiety. In approximately one-third of patients, initially prominent visual or cerebellar symptoms may overshadow dementia. Mental deterioration typically is rapidly progressive, and the average duration of illness from onset of symptoms to death is 7 to 9 months. In addition to profound and rapidly progressive mental deterioration, another very common feature is involuntary twitches or jerks of muscles, known as myoclonus. However, myoclonus in demented patients is not pathognomonic of sCJD. It can occasionally occur in Alzheimer disease and is common in Lewy body dementia. Extrapyramidal and cerebellar signs, including bradykinesia, rigidity, ataxia, nystagmus, and tremor, ultimately develop in approximately two-thirds of patients. Approximately 40% to 80% of patients have signs of corticospinal tract dysfunction, including hyperreflexia, spasticity, and extensor plantar responses. Prominent visual disturbances, which can include visual field cuts, cortical blindness, and visual agnosia, occur in 50% of sCJD patients.

Some patients have vague sensory complaints, including pruritus and aching limbs. It is unclear if these sensations are of peripheral or central origin. Signs of a motor or sensory peripheral neuropathy are occasionally found in sCJD, although these signs are almost always overshadowed by the dramatic signs of CNS dysfunction. One study found clinical evidence of peripheral neuropathy in approximately 20% of cases examined and electrophysiologic abnormalities in 14 of 16 surveyed cases of sCJD. On occasion, signs of sensory or motor neuropathy may dominate the early disease course. Rarely, fasciculations and muscle wasting will be so prominent as to suggest amyotrophic lateral sclerosis. Some cases of sCJD also have been reported in which the clinical features indicated prominent autonomic nervous system involvement. These features included hypohidrosis, bowel dysfunction, abnormal pupillary responses to autonomic drugs, abnormal diurnal blood pressure variation, and electrocardiogram abnormalities. Such findings are cardinal features of FFI.

Certain neurologic disturbances occur only rarely as prominent features in sCJD, and their presence should prompt clinicians to consider other diagnostic possibilities. Although seizures occur in 10% to 20% of cases, they are rarely a dominant feature and typically are amenable to therapy. Cranial nerve involvement is never prominent, although isolated cases have been reported with involvement of the pupils, extraocular movements, and involvement of the auditory and vestibular systems.

Strains

At least two distinctive sporadic forms of sCJD bear the hallmarks of discrete prion strains. First, very rare sporadic cases of sporadic fatal insomnia, a clinical and pathologic syndrome indistinguishable from FFI (see “ Familial Prion Disease ” next), will be encountered in patients without PRNP mutations. This form of sporadic prion disease maintains a biochemical signature identical to that of FFI upon serial transmission in transgenic mice expressing human PrP. It thus behaves as a distinct human prion strain.

Second, variably protease-sensitive prionopathy, a recently described condition, is a form of sCJD with fewer and less prominent motor and sensory signs and a pattern of cognitive impairment said to resemble frontotemporal dementia. Progression is slower than typical sCJD, with a mean duration of 30 months. PrP ^Sc from these patients is more sensitive to protease digestion than in typical sCJD. The predominant protease-resistant fragment is 8 kDa and cleaved at both the amino and carboxyl termini, whereas in sCJD the predominant fragment is cleaved only at the amino terminus and is 19 or 21 kDa in size. This 8-kDa fragment is also observed in the genetic prion disease GSS (see “ Gerstmann-Sträussler-Scheinker Syndrome ” later) leading some to speculate that variably protease-sensitive prionopathy is a sporadic form of GSS. The disease is quite rare; 13 cases were identified by the main US prion disease pathology center over an 8-year period, and 5 cases were identified in Britain in a retrospective review of 20 years. Ignoring ascertainment problems, which may be significant, these translate into an annual incidence of approximately 0.5 per 100 million in either population. Attempts to transmit the condition to rodents, including transgenic mice expressing only human PrP, have been minimally successful, with some evidence of prion replication on first passage, but no propagation on later passages.

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