Historical Background
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by neuron loss, glial reactions, and tissue spongiosis, which course with motor and/or cognitive symptoms (Knight and Will 2004). The TSEs are associated with conformational conversion of the prion protein (PrPC, the product of the Prnp gene), wherein the predominantly α-helical secondary structure of PrPC changes into an aggregation-prone, β-sheet richer structure known as PrPSc. The latter is believed to coerce PrPC molecules into conformational conversion, thus behaving as a proteinaceous infectious particle, or prion (Prusiner 1998), which gave TSEs the epithet prion diseases.
The much needed development of effective treatment for these still incurable diseases depends on the understanding of functional properties of the prion protein (Soto and Satani 2010). Most studies of physiological functions of PrPC have been directed at its major cell-surface GPI-anchored form, whereas minor transmembrane and cytosolic forms have usually been studied in the context of pathogenesis of prion diseases. The current account, therefore, focuses upon the physiological functions of the GPI-anchored PrPC.
Several dozen distinct molecules have been shown to bind PrPC, albeit on the basis of somewhat variable evidence. The expression and the engagement of PrPC with a variety of ligands activate numerous signal transduction pathways, thus leading to modulation of proliferation, differentiation, and cell death in the nervous and immune systems, as well as in many other organs and cell types. In addition, PrPC-mediated signaling is affected by trafficking of the protein both laterally between distinct plasma membrane domains and along endocytic pathways, as well as by its continuous and rapid recycling. A recent review of these functional properties suggests that the prion protein has a general function analogous to intracellular scaffolding proteins, as a dynamic cell surface platform for the assembly of signaling modules (Linden et al. 2008).
Structure, Expression, and Regulation of the Prion Protein
PrPC is an N-glycosylated, glycosyl-phosphatidylinositol (GPI)-anchored protein of 208–209 amino acids, containing an amino (N)-terminal flexible, random coil sequence and a carboxy (C)-terminal globular domain, the major structural features of which are preserved among both mammalian and non-mammalian species (Fig. 1). The globular domain of human PrPC contains 3 α-helices interspersed with an antiparallel β-pleated sheet formed by β-strands at two short stretches, and contains a single disulfide bond. The N-terminal flexible tail spans approximately half of the mature protein, and a short flexible C-terminal domain attaches to the GPI anchor (Wuthrich and Riek 2001).
Full length PrPC is found in non-, mono-, or di-glycosylated forms, corresponding to the variable occupancy of two asparagine residues (Rudd et al. 2001). A rather large variety of N-glycans were found attached to both full-length and truncated PrPC, which may be differentially distributed in various areas of the central nervous system (CNS).
A single exon within the Prnp gene codes for PrPC. Control of Prnp gene expression has been attributed to sequences within the 5′-flanking region, within the first intron, and to 3′-untranslated sequences, as well as to interactions between promoter and intronic regions. Differing from the Prnp open reading frame, the degree of homology of potential promoter sequences among various mammalian species is quite variable (Mastrangelo and Westaway 2001).
Prnp is often labeled as a housekeeping gene, but evidence that transcription of Prnp is modulated by chromatin structure, as well as the identification of potential binding sites for many transcription factors, indicate that expression of Prnp likely depends on a variety of cellular factors. Notwithstanding some variation between species, the following elements were reported both in the 5′-flanking region and within the first intron: Sp1, AP1, AP2, MZF-1, MEF2, MyT1, Oct-1, NFAT, POZ (BCL6); RP58 (ZNF238); NEUROG1; EGR4, Oct-1/Oct-2, NF-IL6, MyoD, p53, HSE, MRE, MLS (Linden et al. 2008 for review).
Expression of both Prnp messenger RNA (mRNA) and prion protein are developmentally regulated, and subject to modulation by growth factors such as NGF, PDGF, and various cytokines. Expression can also be modulated by stressful conditions, inclusive of heat shock, hypoglycemia, oxidative stress and inflammation, as well as copper overload.
The prion protein is highly expressed within the nervous system, although its content varies among distinct brain regions, among differing cell types and among neurochemically distinct neurons. In addition, substantial amounts of PrPC are expressed in various cellular components of the immune system, the bone marrow, blood, and peripheral tissues. Other organs and tissues also express PrPC (Table 1).
Many reports are available on putative ligands of the prion protein, and candidate physiological ligands are listed in Table 2. It can be appreciated that binding domains identified for a number of ligands extend along the entire PrPC molecule (Fig. 2). It should, however, be noted that the techniques used for those studies were quite variable, and many interactions detected by screening methods have yet to be confirmed by biochemical and cell biological approaches. In particular, some putative ligands appear not to be accessible from the usual topology of PrPC, which constitutes a critical question to be addressed by future studies (Rutishauser et al. 2009).
Trafficking, Endocytosis, and Recycling of the Prion Protein
An N-terminus peptide (aa. 1–22) is used to drive nascent PrPC into the endoplasmic reticulum, where its GPI-anchor is added at the C-terminus. Distinct topologies of PrPC have been described, some of which are of pathological interest. PrPC follows the classical pathway for its insertion at the plasma membrane, passing through the Golgi and following a Brefeldin A-sensitive pathway to reach the cell surface (Prado et al. 2004 for review).
At the cell surface, PrPC molecules are found predominantly anchored to low density, detergent insoluble membrane domains, rich in cholesterol and sphingolipids (lipid rafts). GPI-anchored proteins located in rafts are thought to recycle between the plasma membrane and intracellular organelles, in particularly to the Golgi. In neurons, endogenous PrPC appears to internalize as fast as classical membrane receptors, such as the transferrin receptor, with a T1/2 of approximately 3–5 min.
Initial data pointed to the possibility that PrPC, similar to other GPI-anchored proteins, is internalized by a raft-mediated mechanism that is independent from clathrin. Although caveolae anf flotilin-derived vesicles may participate in the internalization of PrPC in non-neuronal cells and in astrocytes, mounting evidence suggests that, in neurons, clathrin-mediated endocytosis plays an important role on PrPC internalization. Cell surface biotinylation, live cell microscopy, GFP-tagged PrPs, and electron microscopy support the view that clathrin-coated vesicles and classical endosomal organelles are involved in endocytosis of PrPC (reviewed by Linden et al. 2008). Dominant negative approaches indicated a role for the activities of dynamin and clathrin in the internalization of PrPC in distinct cell lines. It was proposed that an N-terminal, positively charged domain of PrPC (KKRPKP) is responsible for the constitutive endocytosis of PrPC by clathrin-coated vesicles. Remarkably, a number of reports indicated a role for the N-terminal region of PrPC upon endocytosis and cellular trafficking, and this basic region of the protein has been previously implicated in the binding of negatively charged proteoglycans, which are thought to modulate PrPC sequestration (Prado et al. 2004) (Fig. 3).
The hypothesis that GPI-anchored PrPC may “piggy-back” on an integral membrane protein had long been raised, and recent studies indicated that the low-density lipoprotein receptor-related protein 1 (LRP1) may participate in clathrin-mediated endocytosis of PrPC, because knock-down of LRP1, but not LRP1b, reduced internalization of PrPC. LRP1 has also been implicated in PrP fibril entry in cells (Parkyn et al. 2008; Taylor and Hooper 2007).
High extracellular levels of Cu2+ induce the endocytosis of PrPC to intracellular organelles and the Golgi. It was reported that Cu2+-induced endocytosis of PrPC expressed in neuroblastoma cells caused its movement from raft to non-raft membrane regions. Although the KKRP motif was shown to be important for endocytosis, this motif is not essential for the lateral displacement of PrPC to non-raft membrane, indicating that this movement occurs prior to PrPC endocytosis. It was also suggested that Cu2+ may destabilize a putative PrPC interaction within rafts, rather than inducing PrPC to interact with a non-raft protein. It is not clear yet if the KKRP domain is required for binding to LRP1 or to other membrane proteins that may be accessory in this process. Interestingly, amyloid β peptide 1–42, a major culprit in Alzheimer’s disease was recently shown to bind PrPC. A binding site was identified at aa. 90–110 and, more recently, the endocytic motif 23–27 was also shown to mediate binding to amyloid β peptide. This suggests that amyloid β may be able to regulate PrPC trafficking. Moreover, one of the ligands of PrPC, hop/STI1, which signals in cells by coupling PrPC to the alpha7 nicotinic acetylcholine receptor, triggers the internalization of PrPC, which regulates the extent of PrPC modulated neuronal signaling.
Systemic Functions of the Prion Protein
The systemic functions of the prion protein have been addressed in PrP-null mice, in transgenic mice overexpressing PrPC, or in transgenic mice expressing deletion mutants of PrPC (Linden et al. 2008; Weissmann et al. 1998; Wadsworth and Asante 2010; Martins et al. 2010 for reviews). Although no overt phenotypic changes were described in the first generated PrP-null animals, later studies showed altered patterns of sleep, enhanced locomotor activity, and increased anxiety, possibly because of changes in the glutamatergic system. Impairment of both short- and long-term memory is found in old PrP-null mice. On the other hand, spatial memory is impaired in young PrP-null mice, which can be rescued upon PrPC expression in neurons. Impairment of memory formation and retention in rats also followed the blockade of PrPC interaction with its ligands, by direct hippocampal infusion of anti-PrPC antibodies or competitor peptides.
At the synaptic level, impairment in long-term potentiation (LTP) was found in the CA1 area of the hippocampus of PrPC-null mice when experiments were done at physiological temperature. These results were, however, not reproduced when LTP was examined at room temperature. LTP in hippocampal CA1 is at the root of memory formation of one-trial inhibitory (passive) avoidance in the rat. Thus, changes in LTP in PrP-null mice may explain at least in part their memory impairment, as may be the case following PrPC blockade by specific antibodies. Changes in the afterhyperpolarization potential (AHP) were also detected both in constitutive PrPC-null mice, and in conditional knockouts in which the expression of PrPC is abolished at 12 weeks of age. This indicates that this phenotype was caused by neural dysfunction, rather than by a developmental deficit.
Although the changes in the central nervous system are subtle, and do not compromise the life of the PrP-null mice, the expression of some deletion mutants such as PrPΔ32-121, PrPΔ32-134, PrPΔ94-134 in knockout mice causes neurodegeneration. In particular, the deletion of amino acids 105–125 causes cerebellar atrophy, loss of cerebellar granule cells, gliosis, and astrocytic hypertrophy, with decreased body size, immobility, myoclonus, and death within one month after birth. This suggests that compensatory mechanisms effective in PrP-null mice do not function upon expression of specific deletion mutants. Remarkably, these deleted domains contain sites for binding of ligands that promote specific responses at the cellular level (Linden et al. 2008; Martins et al. 2010 for reviews).
PrPC has also been implicated in protection against brain insults. In PrP-null mice the threshold for seizures is lower than in wild-type. In addition, PrP-null mice suffered more extensive damage in the brain than wild-type following either hypoxic-ischemic insult or administration of ethanol. Remarkably, overexpression of PrPC reduced infarct volume and improved neurobehavioral signals after cerebral ischemia in rats.
In transgenic mouse models of amyotrophic lateral sclerosis, the expression of human mutated superoxide dismutase 1 (SOD1) in the absence of PrPC causes significantly reduced life span, an earlier onset, and accelerated progression of disease when compared with control transgenic mice expressing PrPC. In this case, PrPC has a pivotal function in the control of neuronal and/or glial factors associated with antioxidant defenses (Steinacker et al. 2010). Recently, it was also shown that the expression of the prion protein in axons is required for maintenance of peripheral myelin (Bremer et al. 2010).
The absence of PrPC is associated with altered sensitivity to injury not only in the central nervous system, but also in other tissues. PrP-null mice showed impairment of locomotor activity under extreme exercise conditions. PrPC is also a relevant regeneration factor in acutely damaged muscle (Stella et al. 2010). Moreover, a uniform pattern of increased PrPC expression was described in a series of muscular disorders, as well as in an experimental model of chloroquine-induced myopathy, which suggests a role for PrPC in muscle physiology (Linden et al. 2008 for review).
The expression of PrPC is variable both across species and among subsets and states of maturation of immune cells. Although PrP-null mice do not present gross defects in the immune system, PrPC modulates the ability of long-term hematopoietic stem cells (HSC) to sustain self-renewal under stress. PrP-null mice also showed altered inflammatory responses, and it has been shown that PrPC in dendritic cells is a positive regulator of the immunological synapse (Linden et al. 2008; Nitta et al. 2009; Isaacs et al. 2006 for reviews).
Cellular Functions of the Prion Protein
Studies of the expression of PrPC during development, and also its cellular distribution in neuronal cell body, dendrites, and axons provided conflicting results, highlighting the difficulties in attributing function to PrPC on the basis of its distribution. Nonetheless, a large number of studies have identified roles of PrPC in cell proliferation, differentiation, and cell death in both neural precursors and central neurons. In addition, functions were also attributed to PrPC in peripheral neurons, lymphoid cells, and some tumor cells (Linden et al. 2008; Martins et al. 2010; Mehrpour and Codogno 2010) (Table 3).
PrPC in a cell can modulate cellular functions of either the same (in cis) or of a distinct cell (in trans). The trans effects were demonstrated in experiments with recombinant, Fc-bound soluble PrPC or its fragments, GPI-anchorless recombinant forms, or using a feeder layer of PrPC expressing cells. The evidence that PrPC can be secreted by exosomes from various cell types extends the possibilities of trans effects at both the tissue and system levels (Porto-Carreiro et al. 2005).
The rate of proliferation of neuronal precursors correlated with PrPC content in both the subventricular zone and dentate gyrus of the hippocampus of adult mouse brain, but PrPC expression in proliferating zones was restricted to postmitotic neurons. Thus, the effect of PrPC upon proliferation of neuronal precursors are probably indirect, a possibility that must be considered when dealing with complex tissues. One hypothesis, not yet explored, is the potential effect of exosomes containing PrPC from postmitotic neurons. PrPC can also affect the proliferation of splenic lymphocytes, splenocytes, and enterocytes.
The role of PrPC in neurite and axon outgrowth has been demonstrated both in human and mouse neurons from hippocampus, cerebellum, cortex, dorsal root ganglia, as well as in neuronal derived cell lines. These effects are mediated by PrPC acting in cis upon binding to specific ligands, such as hop/STI1, laminin, and NCAM, and in trans together with either NCAM or other unidentified ligands at the cell surface. PrPC also modulates astrocyte differentiation, secretion of neurotrophic factors, and metabolism. These results are consistent with PrPC functions at system levels (see Sect. Systemic functions of the prion protein).
One of the clearest roles of PrPC is upon cell survival. This is particularly relevant, since PrPC is associated with TSEs, and, more recently was suggested to play a role in Alzheimer’s disease. Although the importance of PrPC loss-of function in neurodegeneration is still debatable, most studies using primary neuronal brain and retina cultures, immortalized neuronal cells, tumor cell lines, and even yeast support a cytoprotective function for PrPC. Nonetheless, the expression of PrPC may also be associated with increased cell death in certain circumstances. Remarkably, in recent years PrPC has been implicated in tumor proliferation, progression, invasiveness, and resistance to drug treatment (Mehrpour and Codogno 2010 for review).
Together these results suggest that PrPC may be a therapeutic target to increase neuronal survival in either acute or chronic neurodegenerative diseases. In addition, this ubiquitously expressed molecule may also represent both a new biomarker and a therapeutic target in cancer.
To explain all these possible functions of PrPC we have raised the possibility that this molecule acts in a much more fundamental level coordinating signaling pathways at the cell surface. Proteins with unstructured domains can bind multiple partners, and are critical as intracellular scaffolds to regulate intracellular signaling. The unstructured N-terminal region of PrPC may play a similar role and position the protein to organize signaling with multiple partners.
Mechanisms of Prpc-Mediated Signal Transduction
Roles for the prion protein in signal transduction have been unraveled by various approaches. The main procedures involve the use of antibodies, modulation of protein content via either the knockout or overexpression of the Prnp gene, the use of anchorless, soluble recombinant forms of PrPC, and engagement of PrPC with one of its ligands.
In certain cases, modulation of signaling was shown by direct measurements, whereas in others, the effect of either pharmacological or molecular inhibitors was used to infer a role of PrPC upon cellular responses. Both approaches may, in fact, unravel downstream responses networked to signaling pathways, instead of direct activation by the PrPC. Also, when antibodies were used, some caution is necessary to interpret the results, since antibodies may have either a blocking or agonist effect, as well as either cross-linking or non-cross-linking activity. Nonetheless, a variety of signaling pathways have been shown to be modulated by the prion protein (Table 4).
Thus, current data strongly support the hypothesis that the prion protein may be physiologically engaged by a variety of extracellular and cell surface ligands, and mediates signal transduction through interaction with various transmembrane partners. The PrPC-dependent signaling complexes are likely to vary among distinct cell types, depending on: the level of expression and distribution of PrPC, as indicated in Table 1; the availability of ligands amongst, at least, those indicated in Table 2; structural rearrangements caused by multiple ligands; and kinetics of endocytosis/recycling. Indeed, the resulting signals likely depend on a complex interplay of allosteric effects caused by the binding to PrPC of multiple partners with varying kinetics. Interactions are likely to be of significance both in cis and in trans. The latter are inferred mostly from results unraveled with the use of the soluble recombinant forms of PrPC, which may correspond to PrPC located at the surface of either neighboring cells, exosomes, or other exocytic particles (Linden et al. 2008).
Further analysis of signaling mediated by the prion protein is likely to unravel the mechanisms by which modulation of expression, engagement, or exposure to soluble PrPC trigger proliferative, differentiating, or death/survival responses, as well as other effects upon cell metabolism, such as modulation of responses to oxidative stress, synaptic modulation, and immunomodulation. These, in turn, will likely explain the systems-level functions of PrPC. An integrated systems approach may help defining signaling patterns generated by multiple pathways.
Summary and Future Directions
The currently available data suggest that the prion protein plays a significant role in signal transduction. PrPC likely works as a cell surface scaffold protein, with the ability to organize multi-component complexes at the cell surface, which include other proteins, glycosaminoglycans, and free ions (Fig. 4). This role of PrPC probably involves dynamic changes along its path of trafficking among distinct plasma membrane domains and endosomes. The resulting signals contribute to biological responses such as cell proliferation, differentiation, and modulation of cell death and responses to oxidative stress, synaptic modulation, and immunomodulation.
The relative lack of a spontaneous phenotype reported after deletion of the Prnp gene, which, for many years, has driven research interest off the physiological roles of the prion protein, may indeed invite one or more among several explanations, such as: a compensatory role of other members of the prion family; complex regulatory changes among networked signaling pathways; or a more subtle role of PrPC as a modulator of signal transduction, particularly in the processing of either systemic or cellular stress and danger signals. Further investigation of these hypotheses, as well as of the proposed role of PrPC as a cell surface scaffold protein, may contribute to a better understanding of its physiological functions, as well as to the establishment of effective therapeutic options for the still incurable transmissible spongiform encephalopathies.
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Acknowledgments
The authors’ research groups have been supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Programa Institutos Nacionais de Ciência e Tecnologia (CNPq/MCT), Ludwig Institute for Cancer Research, and PrioNet-Canada. V.R.M. is an International Scholar of the Howard Hughes Medical Institute.
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Linden, R., Martins, V.R., Prado, M.A.M. (2012). Prion Protein (PRNP). In: Choi, S. (eds) Encyclopedia of Signaling Molecules. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-0461-4_390
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