Prion protein scrapie and the normal cellular prion protein

ABSTRACT

Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrP^C) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed.

INTRODUCTION

Some prion proteins have become well known for their causative role in a range of neurodegenerative diseases in humans and other mammals. Human prion diseases include Creutzfeldt-Jakob Disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), Familial Fatal Insomnia (FFI), kuru and variant CJD (vCJD).^1,2 Animal prion diseases include Scrapie in sheep, goats and moufflons (subspecies of wild sheep),³ Transmissible Mink Encephalopathy, Chronic Wasting Disease in deer (cervids), Bovine Spongiform Encephalopathy (BSE) in cattle, Exotic Ungulate Encephalopathy in nyala and kudu (sub-species of antelope). Feline Spongiform Encephalopathy in cats, Non-human Primate Transmissible Encephalopathy in lemurs.⁴ Over the past decade, significant research has been conducted to establish not only the types of prion diseases and their infection mechanisms, but to find an effective therapeutic strategy for treatment.

PRION DISEASE

Prion diseases have a number of common histopathological characteristics and neurological symptoms. These include spongiform degeneration of the central nervous system (CNS), formation of amyloid plaques, reactive gliosis (enlarged glial cells appearing after CNS injury) and neuronal loss.⁵ Other atypical properties characteristic of prion diseases are long incubation periods (which can extend from several months to several years), lack of inflammation and lack of disease-specific immune response.²

While at first these neuronal degeneration diseases were thought to be caused by “slow” viruses due to their long incubation periods,⁶ neither viral particles nor nucleic acids could be detected to support the hypothesis that these are viral diseases.² The viral hypothesis also failed to account for the finding that 95% of human neuronal degeneration disease cases are not linked to infection and lacked the typical histopathological features of viral encephalitis. Hence, the viral disease hypothesis failed to adequately account for the findings that emerged from studies of these diseases.^2,7 It was also discovered that between 10 and 15% of these neuronal degeneration diseases are dominantly inherited, including all cases of GSS and FFI and 10% of cases of CJD. The latter are referred to as familial CJD (fCJD). In summary, the data showed that these neuronal degenerative diseases can be both infectious and inherited.⁷

During the attempts to uncover the molecular basis of prion diseases over the past few decades, it was uncovered that the causative agent of scrapie, is a 27-30kDa protease-resistant protein designated as PrP 27-30.⁸ PrP 27-30 was found to be encoded by a single mammalian gene located on human chromosome 20.⁹ This gene was designated PRNP. Other animals have a homolog of the human PRNP gene and this gene was named Prnp. Furthermore, it was discovered that PrP27-30 was derived from a 30-35kDa protein in scrapie-infected animals, designated prion protein scrapie (PrP^Sc). PrP^Sc was discovered to be a derivative of the normal protease-sensitive form of PrP that was designated cell-surface glycoprotein (PrP^C).¹⁰ Upon further investigation, it was found that all dominantly inherited forms of prion diseases are linked to mutations of or insertions in the PRNP gene.¹⁰

Development of Prion Disease

Neurodegenerative prion diseases such as CJD, Kuru and BSE, result from the post-translational conversion of normal, glycosylphosphatidylinositol (GPI)-anchored PrP^C to a misfolded aggregated and pathogenic form, PrP^Sc.¹¹ This conversion followed by an accumulation of PrP^Sc within the central nervous system resulting in disease¹² (Fig. 1).

FIGURE 1.

PrPse propagation in the CNS. Schematic representation of the propagation of PrPsc in neuronal cells of the CNS. PrPSc acquired by infection, mutation or spontaneous conversion of cellular PrP (PrPC) combines with PrPc thereby converting it to PrPC is recruited and converted ti PrPSc. As PrPSc levels increase PrPC recruitment and conversion become more efficient, leading to an accumulation of PrPSc in neuronal cells. PrPSc accumulation causes cell dysfunction followed by death.

As shown in Figure 1, the newly formed PrP^Sc acts as a template to facilitate conversion of PrP^C to PrP^Sc, causing the accumulation of PrP^Sc.¹³ This process takes place regardless of the origin of the PrP^Sc, i.e. whether it is from an external source or a produced internally due to mutations in the PRNP gene (as in the case in fCJD and FFI)⁷ or due to a spontaneous conversion of wild-type PrP^C to PrP^Sc, which is reffered to as ‘de novo generation/synthesis’.¹⁴

PrP^Sc is characterized by its resistance to protease digestion, insolubility and its high content of β-sheet structure^15,16 (43% β-sheets compared to 30% α-helical). In contrast, PrP^C is an soluble protein high in α-helices (42%), low in β-sheet (3%) and highly susceptible to protease digestion.¹³ PrP^C contains 2 different domains that play different roles in the conversion of PrP^C to PrP^Sc. The first is a stable and ordered ‘core’ domain which contains a GPI lipid anchor that tethers PrP^C to the plasma membrane, 3 α-helices (helices A, B and C) (Fig. 2), 2 asparagines-linked oligosaccharides and a protein binding site capable of lowering the energy barrier for conversion of PrP^C to PrP^Sc when PrP^C binds to protein X (a species specific cofactor necessary for conversion of PrP^C to PrP^Sc).^17,18 The second domain is a ‘variable’ or disordered domain which interacts with PrP^Sc and changes PrP^C conformation from the unstructured form to the β-sheets of PrP^Sc (Fig. 2).¹⁶ During conversion of PrP^C to PrP^Sc, helix A of the core domain of PrP^C (Fig. 2) also gets converted into β-sheets.⁷

FIGURE 2.

Comparison of protein structure of PrPC and PRPSc. Shown is a schematic representation of the secondary structure of PrPC in comparison to PrPSc as inferred from a recombinant fragment of Syrian hamster PrP comprising amino cid residues 90-231.A,B and C indicate α-helices.

Types of Prion Disease

Prion diseases in humans and animals have thus far been classified into 3 broad categories, based on the properties of the corresponding pathogenic PrP proteins that accumulates in the brain and on their neuroanatomical features of the prion-infected brain⁷ as summarized in Table 1.

TABLE 1.

Summary of the 3 broad categories of prion diseases and the defining characteristics of each

Category	Prion Disease	Defining characteristics	References
1	Scrapie, sporadic, familial and iatrogenic CJD (sCJD, fCJD, iCJD), BSE, kuru and sporadic and familial fatal insomnia (sFI and fFI)	Vacuolar (spongiform) degeneration of gray matter, accumulation of protease resistant PrPSc in gray matter, little or no PrP amyloid plaque formation.	7
2	Dominantly inherited syndromes (GSS)	Deposition of numerous PrP immunopositive amyloid (abnormal protein) plaques in multiple cortical and subcortical brain regions. PRNP mutation.	7,19
3	Variant CJD (vCJD)	PrPSc amyloid deposition, vacuolation of gray matter, accumulation of protease resistant PrPSc in neuropil (space between neuronal and glial cell bodies comprised of dendrites, axons, synapses microvasculature and glial cell processes).	7,19-22

The oral route of infection is the major mode of transmission for most cases of acquired prion diseases in humans. The spread of the diseases via this route, both naturally and experimentally, has been described in many species. Kuru, for example, was shown to be spread among the Fore people who populate the eastern highlands of Papua New Guinea through ingestion of infected brain tissue during cannibalistic rituals which resulted in a particularly high incidence of the disease ^23-25

The sCJD accounts for more than 90% of all cases of sporadic prion disease. However, not all CJD is sporadic. Beginning in the 1980s when a considerable number of cattle were killed by BSE concerns about the spread of BSE to humans grew dramatically in the United Kingdom.²⁶ The concern was that BSE could spread to humans through ingestion of infected beef,²⁷ a fear which was realized in 1995.²⁸ A new form of CJD was first reported by the UK National CJD Research and Surveillance Unit in 1996, and is now known as variant CJD (vCJD).²⁸ It is believed that vCJD possesses distinct clinical and neuropathological characteristics from sporadic CJD (sCJD) and other forms of prion disease in human.²⁹ The main distinguishing neuropathological feature of vCJD is an extensive deposition of PrP^Sc amyloid in the brain in the form of large ‘florid’ plaques.³⁰ Other phenotypes include a younger average age of onset, and gliosis of the thalamis. Epidemiological studies supported the possibility that the outbreak of BSE in cattle in the UK in the same period may have been responsible for the emergence of vCJD. Subsequently, experimental transmission studies of vCJD and BSE in mice proved that the vCJD agent, unlike the agent responsible for sCJD, had biological properties closely similar to those of the BSE agent.^20,30 Therefore, vCJD has been confirmed as a novel prion disease and the only human prion disease acquired from another species. Subsequent studies suggested that the pathogen could be present in blood during the incubation period for vCJD,^31,32 and that exposure to such blood could result in the infection of humans or other animals. Therefore, the UK National CJD Surveillance Unit reported that vCJD is caused by exposure to BSE and that the primary source of exposure is the consumption of infected meat products.²⁹

Unlike vCJD, Iatrogenic CJD is an entirely person to person transmitted disease, identified to be transmitted through a number of mechanisms including contaminated surgical instruments,³³ and dura mater grafts,³⁴ blood transfusions³⁵ and injection of products from cadaveric pituitary glands.^36,37 The first case of iCJD was described in 1974 where a corneal graft recipient died after acquiring a dementia-like likness.³⁸ With improved screening and sterilisation techniques, rates of iCJD continue to decrease with new cases that occur often the result of longer incubation periods following infection acquired in the 1980s.^39,40

Whereas the causes of prion diseases that are genetically inherited or acquired by infection have been extensively studied and now quite well known, the cause of sporadic prion diseases is still a topic of speculation.⁴¹ Among the many mechanisms that have been proposed to provoke sporadic prion disease, the mechanism that is supported by the most compelling evidence is a mechanism in which malfunction of the so called quality control complex in cells is responsible for initiation of the disease.⁴² The cellular quality control complex acts like a set of enzymes which assist newly synthesized polypeptides to “grow” to their proper conformation and also play a role in disposal of those polypeptides that fail to adopt the native structure. Therefore, a lowered efficiency of the quality control complex, perhaps due to aging or if the quality control complex becomes overwhelmed by excessive protein production, may result in errors in protein folding occurring and the production of misfolded proteins.

One of the inherited prion diseases is Gerstmann–Sträussler–Scheinker disease (GSS). GSS is caused by a pathological mutation in the prion protein gene (PRNP) located on chromosome 20.It is a very rare disease with autosomal dominant inheritance. The age of onset of GSS is relatively early but the disease progresses slowly with an average illness duration of 49–57 months (until death).^43,44 The typical GSS syndrome includes prominent ataxia, gait disturbances, cognitive decline and spasticity in the lower extremities.^45,46 Rarely the syndrome may also include painful dysesthesias and visual disturbances, dystonia and myoclonus and dementia.^45,47,48 A GSS case attributable to an A133V mutation in PRNP resulted in an uncommon phenotype similar to that of progressive supranuclear palsy.⁴⁹ Several other pathological variations that cause GSS have been described. The most common mutation in PRNP is P102L, which is found in more than 80% of cases.⁵⁰ The P102L mutation was found in the original GSS pedigree.⁵¹ Other mutations in PRNP known to cause GSS include P105L, P105S, A117V, G131V, Y145*, H187R, D202N, Q212P, Q217R, M232T, and base-pairs insertions at positions 96, 192, or 216.⁵²

Recently, several GSS cases with novel PRNP mutations were reported. A 61-year-old British-born woman with no history of neurodegenerative disorder among her first-degree relatives in Australia presented with a rapidly progressive dementia. Sequencing of the PRNP gene demonstrated a V176G mutation. Subsequent Western blot analysis resulted in the detection of an 8 kDa atypical protease-resistant PrP band.⁵³ Sequencing of the PRNP gene of another GSS patient (this one in North America) revealed a 24-nucleotide insertion that when translated would result in a protein product with an 8-amino acid insertion.⁵⁴ Independent neuropathological studies of 2 GSS pedigrees with the P102L mutation obtained divergent findings.^55,56 The authors note that the variable clinical presentation of GSS patients (even those with the same PRNP mutation) makes diagnosis of GSS challenging and in some families the presence of GSS may be missed. Routine clinical and laboratory investigations, sequence analysis of the PRNP gene and post-mortem examination are recommended in all cases with a family history of any type of neuropsychiatric syndrome.

Treatment of Prion Disease

There are currently no treatments that have proven effective for curing prion diseases in humans or animals. However, monoclonal antibodies that recognize PrP^Sc and PrP^C have been shown to inhibit prion replication and delay prion disease development in animals models.⁵⁷ These antibodies block PrP^Sc replication by accelerating the degradation of PrP^C (i.e., through reduction of the half life of the PrP^C protein).⁵⁸

Another strategy has been the use of low molecular weight compounds. Compounds that have been undergoing clinical investigation as a possible therapy for prion disease include heterocyclic compounds, e.g. various tricyclic derivatives of acridine and phenothiazine, particularly quinacrine and chlorpromazine.^59,60 Treatments such as this have been shown to be effective in inhibiting the formation of nascent PrP^Sc from PrP^C in ScN2a (human neuroblastoma) cells.^59,61 These chemicals have been in clinical use for many years, quinacrine as an antimalarial drug and chlorpromazine as an antipsychotic drug, and both are capable of crossing the blood-brain barrier. Treatments with these chemicals are therefore considered to be attractive options for use essentially “as is” in treatment of prion infections.^59,62

Quinacrine has been shown the ability to inhibit the formation of PrP^Sc in ScN2a cells with an IC₅₀ of 0.3–0.4 µM.^59,61

A study by Barrett et al.⁶² re-evaluated the potential of quinacrine and chlorpromazine as treatments for prion diseases. The efficacies of these drugs were assessed in vitro using 2 cell line models (ScN2a and ScGT1) and in vivo using a mouse model. While the results obtained from the previous studies using the ScN2a cell line were replicated in this study for the ScN2a cell line, this study obtained different results from previous studies for the ScGT1 cell line. In contrast to the findings from the previous studies using ScGT1 cells, this study found that only higher doses of chlorpromazine and quinacrine (10 times the concentration previously described, 4 µM) decreased PrP^Sc accumulation in ScGT1 cells in a single treatment. Lower doses of quinacrine (0.4 µM) cured ScGT1 cells only over longer treatment times (i.e. daily treatment for 3 weeks) and the effect was not permanent as PrP^Sc infection was re-stablished after approximately 3 months. Furthermore, quinacrine and chlorpromazine failed to inhibit PrP^Sc accumulation in vivo (either individually or even in combination). It was also noted that quinacrine treatment did not affect the proteinase K-resistance or accumulation of PrP^Sc in the spleens of mice inoculated with scrapie. Thus overall the studies show that quinacrine and chlorpromazine, individually or in combination, do not have therapeutic anti-prion effects in animal models and highlights the need for new and more effective therapeutics.

Doxycycline has been used as a compassionate treatment for CJD patients and was observed to increase mean survival times by 4-7 months in comparison to historical controls. Furthermore, a patient with variably protease-sensitive prionopathy was treated from an early stage and for 4 years in total with doxycycline and not only lived one year longer than the longest surviving patient with that subgroup of prion disease, but also had less severe and widespread lesions.⁶³ However, while indeed promising these beneficial effects were not confirmed in randomized, double-blind trials.⁶⁴ In experimental rodent models of prion disease, treatment with doxycycline at early stages (i.e., pre-clinical onset) showed good efficacy, while there was little or no apparent effect once clinical signs had emerged.⁶⁵ This suggests that treatment with doxycycline may be most useful as a preventive measure, for example for those patients who are carriers of the PRNP mutation that causes Familial Fatal Insomnia,⁶⁶ and this trial is currently underway.⁶⁷

A recent study has shown that treatment of prion disease in humans with non-human prion proteins may be a viable treatment option. This approach is based on the knowledge that conversion of PrP^C to PrP^Sc has a strong dependence on protein sequence homology between the prion inoculum and host PrP^C.^68-70 Skinner et al.⁷¹ showed that animals treated with a heterologous prion protein (bacterially expressed and purified recombinant hamster prion protein), demonstrated reduced prion-disease-associated pathology, decreased accumulation of protease-resistant disease associated prion protein and delayed onset of clinical symptoms (including motor deficits), as well as significantly increased mean survival times in comparison to mock-treated control animals.

THE NORMAL CELLULAR PRION PROTEIN

PrP^C, as mentioned previously, is the normal cellular form of the causative agent of PrP^Sc and is required for the development of all the above-mentioned prion diseases. Expression of PrP^C begins in embryogenesis, and expression reaches its highest level in adulthood. In adults, PrP^C is highly expressed in the neurons of the nervous system,⁷² and lower, or no expression is observed in other peripheral organs.⁷³ While the 3-dimensional structure and a number of putative roles of PrP^C have been reported the exact biochemical function of PrP^C is yet to be elucidated.

Processing, 3D Structure and Putative Physiological Role of PrP^C

3D Structure of PrP^C

PrP^C is generally located on the cell membrane and associates with cholesterol-rich microdomains (rafts) in cultured non-neuronal and neuronal cells.⁷⁴ The immature PrP^C protein is approximately 253amino acid residues long and 32-35kDa in mass and comprises of an unstructured N-terminal region and a structured C-terminal domain. The C-terminal domains consists of 3 α-helices, a β-sheet comprising 2 antiparallel β-stands⁷⁵ and a signal sequence for attachment of the GPI anchor.⁷⁶ The unstructured N-terminal domain contains an octarepeat and a hydrophobic region.

Processing of PrP^C

 In order to form a mature protein PrP^C undergoes a number of posttranslational modifications and these are initiated by the removal of the N-terminal and C-terminal signal peptides which is coincident with import of the nascent chain into the endoplasmic reticulum and attachment of the GPI anchor. Two N-linked glycans are also attached and this is followed by a disulphide bond between Cys178 and Cys213.^77,78 This disulphide bond is important as it connects the C-terminal α-helices, and serves to stabilize the fold of the PrP^C protein.⁷⁹ PrP^C (which is 210 amino acid residues in length⁷⁷) is then targeted to the outer leaflet of the plasma membrane by the GPI-anchor.^77,78 PrP^c can also undergo 2 endoproteolytic cleavage events.⁸⁰ The normal constitutive cleavage, known as α-cleavage⁸¹ (Fig. 3B), occurs in the brain and in cultured cells between residues 110 and 111. This cleavage is stimulated by agonists of the protein kinase C pathway⁸² and results in the formation of a 9kDa soluble N-terminal fragment and a 17kDa C-terminal fragment that remains attached to the cell membrane via the GPI anchor.^83-85 The second cleavage, known as β-cleavage⁸¹ (Fig. 3C), is mediated by reactive oxygen species (ROS)^81,86 and leads to the formation of a 19kDa GPI-anchored C-terminal fragment and a 7kDa N-terminal fragment.^84,85,87

FIGURE 3.

Schematic representation of the protein structure and the proteolytic processing of the PrPC in human cells. (A) Whole PrPC moleule. Post-translational modification is initiated by the removal of N-terminal and C-terminal signal p eptides. (B) Normal constitutive cleavage occurring in brain and culture cells. (C) β-cleavage mediated by reactive oxygen species (ROS). (D) Ecotodomain Shecking of the PrPC where nearly whole PrPc molecule is released from the cellular membrane.

PrP^C can then undergo a third cleavage (known as ectodomain shedding) in which PrP^C is cleaved at a site close to the GPI anchor thus releasing the nearly full-length PrP^C protein from the plasma membrane into the extracellular medium. This proteolytic cleavage has been shown to be performed by the sheddase ADAM10.^88-90 Release of PrP^C from the cell surface has not only been demonstrated in cell culture, but also in neuronal and lymphoid cells in vivo.^88,91-93

Copper Regulation

PrP^C is a metal ion-binding protein. It binds copper and zinc with high affinity and manganese and nickel cations with a lower affinity.^94-97 Copper binding involves the histidine residues located within the octarepeat region of the N-terminal domain^98,99 although recent studies reveal additional copper-binding sites.¹⁰⁰ Since the N-terminal domain is also involved in the binding of PrP^C to a number of protein ligands, it has been hypothesized that copper binding may play a structural role and influence the binding of PrP^C to these other proteins.¹⁰¹

In support of a possible physiological role for PrPC in copper homeostasis, it has been shown that PrP^C-deficient (PRNP null) mice exhibit a 50% lower copper concentration in synaptosomal fractions in comparison to wild type mice. This suggests that PrP^C may be involved in the regulation of copper concentrations in the synaptic region of the neuron, e.g., by playing a role in the uptake of copper into presynaptic cells.¹⁰¹ Furthermore, PrP^C endocytosis has been shown to be stimulated when copper is added to cultured neuroblastoma cells,¹⁰² suggesting that PrP^C internalisation may be involved in the transport of copper from extracellular to intracellular compartments. It may also indicate that PrP^C functions as a copper buffer, binding the copper and transferring it to another membrane transporter.¹⁰³

Qin and colleagues¹⁰⁴ reported that in murine neuro-2a and human HeLa cells, endogenous PrP^C rapidly reacts with Cu²⁺. Cu²⁺ elevates PrP^C expression through transcriptional up-regulation mediated by the ataxia-telangiectasia mutated (ATM) transcription factor. Elevation of PrP^C expression protects the cell against copper-induced oxidative stress (and therefore prevents cell death) by playing a role in the modulation of intracellular copper concentrations.

Recently, PrP^C has further been shown to function as a modulator of heavy metal concentrations, protecting cells against heavy metal build-up and thus oxidative stress. It was shown in cells with full-length PrP^C were more resistant to chronic overload of heavy metals (copper, sinc, nickel and manganese) than their PrP-knock out counterparts.¹⁰⁵

Signal Transduction

PrP^C has been hypothesized to modulate various signaling pathway components involved in proliferation, cell adhesion, transmembrane signaling, differentiation, and trafficking. For example, PrP^C has been shown to have a functional link to phosphatidylinositol 3 kinase (PI-3), a protein kinase involved in cell survival, with in vitro (cell line) and in vivo (mouse) studies showing cells that express PrP^C have higher PI-3 levels than those without PrP^C.¹⁰⁶ PrP^C has further been shown to transduce neuroprotective signals through the cyclic AMP-dependent protein kinase/protein kinase A (PKA) pathway¹⁰⁷ as well as Fyn¹⁰⁸ and many others.

Immune System

PrP^C has recently been reported to play a role in the development, activation and proliferation of T lymphocytes.¹⁰⁹ While PrP^C is widely expressed in the immune system including in human T and B lymphocytes, natural killer cells, platelets, monocytes, and dendritic cells, it has been found to be up-regulated during T-lymphocyte activation and at even more upregulated during natural killer cell differentiation.¹¹⁰ Follicular dendritic cells show high expression of PrP^C, however, mice with follicular dendritic cell specific PrP^C knock down, showed no alteration in on maturation or function of follicular dendridic cells,¹¹¹ indicating the high expression or PrP^C is non-essential. In contrast, PrP^C expression has been shown to be important for macrophage function, modulating phagocytosis in vitro and in vivo.¹¹²

PrP^C has been found to physically interact with a signal transduction protein with an important role in T lymphocyte activation and proliferation: zeta-chain-associated protein (ZAP)-70.¹⁰⁹ In addition, the expression of interleukin-2 is increased when PrP^C is expressed.¹¹³ These observations suggest that PrP^C is involved in the development, activation and proliferation of T-lymphocytes. Furthermore, a recent study showed a soluble recombinant form of PrP^C activates human natural killer cells via the ERK and JNK signaling pathways, facilitating IL-15 induced proliferation of natural killer cells, as well as inducing phosphoryation of ERK1/2 and JNK.¹¹⁴

Protection from Programmed Cell Death

When PRNP was knocked out in mice, there was no alteration of life span or observed change in the phenotype of mice,¹¹⁵ indicating that PrP^C has a non-critical function or its function is taken over by another protein in its absence. However, further research into the function of PrP^C in the CNS demonstrated that its absence in hippocampal neurons resulted in apoptotic (programmed) cell death.¹¹⁶

PrP^C also has a structural similarity to the BH2 domain of B-cell lymphoma (Bcl)-2 family members, resulting in the suggestion that PrP^C may also function as a member of this family of proteins.¹¹⁷ It was demonstrated that in vitro, PrP^C protects human neurons against Bcl^-2-associated X protein (Bax)-mediated cell death,¹¹⁸ a pro-apoptotic protein that accelerates cell death by initiating the release of apoptogenic factors by mitochondria.¹¹⁹ When Bcl^-2 and Bax are co-expressed, hyperactivation of Bax-induced apoptosis is prevented. Similarly, co-expression of PrP^c with Bcl^-2 also prevented Bax-induced cell death, implying that PrP^C may play a role in protection of neurons against Bax-induced cell death.^117,120

Role of PrP^C in Central Nervous System Functions

Electrophysiological in vivo studies on PrP-null mice have found that PrP^C influences a number of processes within the CNS. These studies demonstrated a number of functional abnormalities in the hippocampus. First there was reduced gamma-aminobutyric acid type A/GABAa receptor-mediated synaptic transmission (GABAa is a ligand-gated ion channel and voltage-dependent calcium channel that play a role in synaptic transmission and regulation of neuronal excitability). Another defect observed in PrP^C neurons was attenuation of long-term potentiation. Long-term potentiation is the long-lasting signal transmission between neurons involved in memory. Furthermore, the PrP^C neurons exhibited slow after hyperpolarization (i.e. prolonged phase of an action potential in which the membrane potential of a neuron falls below resting potential). Other defects in PrP^C-null neurons included: disruption of calcium currents, activation of potassium currents, reduction in the amplitude of inhibitory postsynaptic potentials^121-125 and abnormal reorganization of the mossy fiber circuitry in the hippocampus.¹²²

Impairment in hippocampus-dependent spatial learning and altered excitatory and inhibitory neurotransmission in PrP-null mice further support the proposed role of PrP^C in synaptic function.¹²⁶ Studies have also found changes in motility, anxiety and equilibrium in adult mice that were attributable to reduced levels of PrP^C.¹²⁷

In addition, PrP^C has been shown to bind to the neural cell adhesion molecule (NCAM) which is a signaling receptor¹²⁸ in the nervous system that takes part in a number of developmental processes including cell migration, synaptic plasticity and neurite outgrowth.^129-131 In summary, these various studies suggests that PrP^C may play a role in CNS development^128,132 through effect on directed cell migration of neural progenitor cells and the spatial coordination of the outgrowth of neurites as well as playing a role in neuronal survival.¹³³

Potential Role or PrP^C in Cancer Development, Progression And Multi-Drug Resistance

Evidence supporting a role of prions and/or prion-like proteins in cancer is becoming increasingly significant. PrP^C has been shown to be highly expressed in a number of cancers including pancreatic, gastric, breast, prostate and colorectal and multi-drug resistant forms of breast and gastric. Consistant with this, over-expression of PrP^C has also been reported in a number of human gastric cancer cell lines including SCG7901 and AGS.^73,134,135 PrP^C was also found to be expressed in gastric carcinoma tissues using immunohistochemical staining.^73,134 Du et al.⁷³ discovered that PrP^C is expressed more strongly in gastric adenocarcinoma tissues than in adjacent non-tumorous tissues and is weakly, or not expressed, in normal gastric mucosa.

PRNP was found to be over-expressed in pancreatic ductal adenocarcinoma (PDAC) in a microarray study by Han et al.¹³⁶ Another study investigated 7 human PDAC cell lines to determine whether PrP^C is overexpressed. While PrP^C is over-expressed in the PDAC cell lines, it exists in the form of a pro-protein (Pro-PrP) and is neither glycosylated nor GP- anchored.¹³⁷

RT-PCR analysis conducted on surgically removed colorectal cancer specimens revealed that PRNP expression is up-regulated in colorectal carcinoma when compared to normal colorectal tissue. This suggests a role for PrP^C in the development of colorectal cancer.¹³⁸ Examination of PrP^C expression in colorectal cancer was conducted using formalin-fixed paraffin-embedded colonic neoplastic tissue sample from 110 patients. This study also found that PrP^C protein expression increased in cancerous colorectal tissues in comparison to normal colorectal tissues¹³⁹ and, moreover, that the differential expression in these tissues was even greater than that observed for PRNP mRNA levels in the previous study.¹³⁸

Study of differential gene expression profiles revealed that the PRNP gene is upregulated in the adriamycin-resistant gastric carcinoma cell line (SGC7901/ADR) when compared to its parental cell line SGC7901.¹⁴⁰ This indicates that PrP^C may have a role in the development of multi-drug resistance (MDR) phenotypes in gastric carcinomas and has led to a focus on PrP^C expression levels in gastric cancer and the mechanisms of PrP^C action within MDR gastric carcinoma relvant to its acquisition of MDR phenotypes.⁷³

PrP^C has been shown to promote the metastasis of colorectal cancer by mediating epithelial-mesenchymal transition (EMT). It was shown that PrP^C expression is associated with the invasive front of colorectal cancer where cells display the characteristics of EMT and that PrP^C expression facilitates tumor invasion. Furthermore, functional assays showed that ectopic PrP^C expression promotes metastatic potential while knock-down reduces motility of cells. Additionally, knock down of PrP^C in implanted colorectal cancer cells in orthotopic xenograft mouse model abolished the number of distant metastases. It was shown that PrP^C accelerates tumor metastasis by upregulation of SATB1 expression via the Fyn-SP1 pathway.¹⁴¹ SATB1, a matrix attachment region binding protein important for tissue-specific gene expression, has been shown to alter the gene expression profile of cancer cells to induce an aggressive phenotype and promote metastasis. Depletion of SATB1 reduces cancer progression and results in a metastatic cells undergoing transition to a normal phenotype.¹⁴² The molecular signaling events downstream of PrP^C were abolished when the Fyn tyrosine kinase was inhibited. This indicates a requirement for Fyn kinase signaling for PrP^C-mediated SATB1 expression. Furthermore, SP1 inhibition also reduced SATB1 expression and the metastatic potential of colorectal cancer cells. Overall, this study showed for the first time a link between PrP^C expression levels and colorectal cancer metastasis and furthermore showed that the signaling is through a PrP^C -Fyn-SP1-SATB1 axis. This signaling pathway may represent a potential target for the future development of anti-metastasis drugs.¹⁴¹

Once it had been confirmed that PrP^C is overexpressed in gastric cancer tissues relative to adjacent non-tumor tissues and normal gastric mucosa,⁷³ studies were performed to assess whether PrP^C expression level influences the invasive and metastatic properties of gastric cancer. While PrP^C was found to be more highly expressed in metastatic cancer than non-metastatic cancer, there was no significant correlation between PrP^C expression levels at the primary site and at the metastatic site of the same metastatic gastric cancer. This suggests that an increase in PrP^C expression is an early determinant of metastasis and may be useful as a prognostic factor. PrP^C expression was also shown to promote adhesive, invasive and metastatic properties of gastric cancer cells in vivo. The N-terminal region of PrP^C exhibits an invasion-promoting effect through the activation of the mitogen-activated protein kinase (MAPK)/ extracellular-signal-regulated kinase (ERK) pathway or the MEK/ERK pathway [11]. The MEK/ERK pathway controls cellular processes such as proliferation, apoptosis, survival and differentiation.¹⁴³ In part, MEK/ERK signaling controls these processes by the transactivation of expression of MMP11 (matrix metalloproteinase 11) [11]. MMP11 in turn is promotes matrix degradation, inflammation and tissue remodeling.¹⁴⁴

Potential Role of PrP^C in Alzheimer's

Alzheimer's disease accounts for 60% to 70% of all cases of dementia.¹⁴⁵ There are 2 core pathological hallmarks of Alzheimer's disease: amyloid plaques and neurofibrillary tangles. The cause of the disease is complex, but the amyloid hypothesis proposes that extracellular amyloid plaques comprising the protein amyloid β (Aβ) is the fundamental cause of the disease. The deposition of Aβ in the brain could trigger neuronal dysfunction and cause neuronal cell death.¹⁴⁶ Several studies have shown a physical interaction between Aβ oligomers and PrP^C. These studies have provided evidence that the Aβ-PrP^C interaction may play an important role in the Aβ toxicity at the synapse by perforating membranes, increasing intracellular calcium ion concentrations and release of synaptic vesicles.¹⁴⁷ Recently, it has been shown that sodium dextran sulphate inhibits the binding of Aβ to PrP^C and furthermore that simultaneous treatment with sodium dextran sulphate protects long-term potentiation in mouse hippocampal slices from the toxic effects of Aβ oligomers.¹⁴⁸

Like Alzheimer's disease,^149,150 human prion diseases are associated with a number of psychiatric and behavioral symptoms such as depression and anxiety.¹⁵¹ It has been speculated that the monoaminergic system plays a role in the development of some of these symptoms. It has been shown that PrP^C-null mice display depressive-like behavior,¹⁵² and recently, PrP^C has been shown to regulate the functions of the monoaminergic systems. The authors suggest that the loss of PrP^C function in neurodegenerative diseases, attributable to the accumulation of PrP^Sc in prion diseases and binding of Aβ oligomer to PrP^C in Alzheimer's disease interferes with the monoaminergic signaling thus resulting in the neuropsychotic manifestations of these diseases.¹⁵³ In support of this hypothesis, the behavioral phenotypes of PrP^C-null mice have similarities to the behavioral effects observed following injection of wild type Aβ peptide oligomers.¹⁵⁴ Overall, recent studies indicate that PrP^C may be a potential target for the future development of drugs to treat depression and depression-related disorders.

CONCLUSION

Investigations into prion disease and suitable treatments continue to advance. However, a cure for these diseases still appears to be a distant possibility. Additionally, although decades of research have uncovered a vast range of information about PrP^C, there is still much that remains to uncover, particularly in relation to the exact molecular function of PrP^C. While PrP^C does not appear to be essential, with some studies showing PRNP knock out mice displaying no apparent deficiencies, and others showing minor behavioral changes, PrP^C does appear to have a range of important functions. Even more interestingly, the role of PrPC in the development and progression of cancer as well as other diseases such as Alzheimer's disease, is receiving increasing research attention. While the exact role of PrP^C in these other diseases remains undetermined, existing research has already shown PrP^C to be a potential target for the future development of drug treatments for these other diseases.

ABBREVIATIONS

BSE

Bovine Spongiform Encephalopathy

CJD

Creutzfeldt-Jakob Disease

Fcjd

familial CJD

FFI

Familial Fatal Insomnia

GSS

Gerstmann-Sträussler-Scheinker disease

iCJD

iatrogenic CJD

PrP

Prion Protein

PrP^C

Cellular Prion Protein

PrP^Sc

Prion protein scrapie

sFI

sporadic fatal insomnia

vCJD

variant CJD

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Authors confirm there is no conflict of interest.

Funding

This work was supported by grants from the Australian Research Council (DP110100389) to ALM. Molecular Basis of Disease Program of the Menzies Health Institute Strategic Project Grant to Trina Stewart and ALM.