Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Prion (PRNP)

  • Rafael Linden
  • Vilma R. Martins
  • Marco A. M. Prado
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_390

Synonyms

 CD230;  Prion protein;  Prn-i;  Prnp;  Prn-p;  PrP;  PrpC;  PrPC;  PrPSc;  Sinc

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. 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-rich structure known as PrPSc. The latter is believed to coerce PrPC molecules into conformational conversion, thus behaving as a proteinaceous infectious particle, or prion, which gave TSEs the epithet prion diseases (Colby and Prusiner 2011).

The much needed development of effective treatment for these still incurable diseases depends on the understanding of functional properties of the prion protein. 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 are believed 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 system, 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. Recent reviews of these functional properties suggest that the prion protein has a biological 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 nonmammalian species (Fig. 1). The globular domain of human PrPC contains three 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).
Prion (PRNP), Fig. 1

The structure of the prion protein is drawn at a roughly approximate scale, in a montage of elements taken from various sources: The globular C-terminal domain is a Pymol representation of mouse PrP121–231; the flexible N-terminal domain was freely drawn to scale from segments of unstructured sequences of aminoacids in the same Pymol representation, maintaining the number of aminoacids ascribed to each individually defined domain (see below). Abbreviations: CC charged cluster, Gly N-linked sugars, GPI GPI anchor, H1, H2, H3α-helix domains, HC hydrophobic core, OR octapeptide repeat domain

Full-length PrPC is found in non-, mono-, or di-glycosylated forms, corresponding to the variable occupancy of two asparagine residues. 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 thePrnp gene codes for PrPC, and the 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.

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, indicates 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).
Prion (PRNP), Table 1

Expression and distribution of prion protein

Species

Organ/tissue

Cell type/subcellular distribution

Technique

Detection level and regulation

Hamster

Brain

Neuron cell bodies

IHC, WB

 

Mouse, Hamster

Brain

Neurons, intracellular

IHC

 

Hamster, macaque, human

Brain

Presynaptic, not in cell bodies

WB, IHC (mAb 3F4), EM

 

Hamster

Developing brain

Mainly along axon tracts

IHC (mAb 3F4)

Developmentally regulated, remains high in adult olfactory bulb and hippocampus

Mouse

Embryonic brain, spinal cord, PNS

Neurons, nonneuronal cells

ISH

Developmentally regulated

Mouse

Brain

Neurons, neuronal processes, not in glia

IHC (pAbs GAx), ISH

Varied both among and within brain regions, depending on cell type and neurochemical phenotype. Protein often not correlated with mRNA

Hamster, human

Hippocampus

Presynaptic

IHC, EM

 

Hamster

Cerebellum

Pre- and postsynaptic

IHC, EM

 

Human

Cerebellum

Neurons

IHC (mAb 3F4)

Low level in cerebellar granule cells of normal brain

Rat

Cerebellum

Neuron and glial cell bodies and processes

IHC (mAb 8H4), EM

 

Rat

Neonatal retina

Retinal precursors and differentiating neurons

WB, IHC (mAb 6H4, pAb N10, pAbMo)

 

Hamster

Brain (hippocampus, septum, caudate nucleus, thalamus), DRG, blood, heart, skeletal muscle, lung, gut, spleen, testis, ovary, and others

Both in neuronal cell bodies and neuropil

WB, IHC (mAb 3F4 + two distinct pAbs), EM

Highest in hippocampus

Mouse

Olfactory bulb, PNS, bone marrow, lymphoreticular system, gut, lung, kidney, testis, skin, not liver

Peripheral axons, neuron cell bodies, not glia; hemopoietic progenitors, megakaryocytes, monocytes, not granulocytes in bone marrow; dendritic cells, pericytes, intraepithelial lymphocytes in various tissues

IHC (pAbs GAx, mAb SAF61), ISH

Varied both among and within distinct organs and tissues. Scattered cells with high expression. Protein correlated with mRNA

Mouse

Intestine

Submucosa, muscularis mucosa

ISH

 

Hamster

Stomach, intestine, lung, kidney

Secretory globules

IHC (pAbs Br-1, R073, P38, mAb 3F4), EM

 

Human

Stomach, kidney, spleen

Secretory globules

IHC (pAbs Br-1, R073, P38, mAb 3F4), EM

 

Bovine

Ovary

Ovarian follicles

Microarray, real-time PCR, WB (mAb HumP)

Upregulated in theca cells of dominant, as compared with subordinate follicles

Sheep

Spleen, lymph node, lung, heart, kidney, skeletal muscle, uterus, adrenal gland, parotid gland, intestine, mammary gland, not liver, not pancreas

 

WB (home-made pAbs and mAbs), NB

 

Mouse, hamster, human

Muscle

Subsynaptic sarcoplasm, not postsynaptic plasma membrane

IHC (pAb R254, R073), EM

 

Mouse

Muscle

Myoblast cell lines

WB (pAb Ra5)

Upregulated with differentiation of myotubes from myoblasts

Human

Blood

Lymphocytes and lymphoid cell lines, monocytes, not erythrocytes, not mature granulocytes

FC (mAb 3F4), NB

Downregulated with differentiation in granulocytes

Human

Blood

Monocytes, T cells, NK cells, B cells

FC (mAbs 3F4, 3F5)

Upregulated in activated T cells and monocytes, not uniform among NK cells, low in B cells

Human

Blood

Monocytes, T cells, B cells, DC

FC (four distinct mAbs)

Upregulated in activated T cells

Human

Blood

CD34+ cells, megakaryocytes, platelets

WB, FC, IHC(mAb 6H4), EM (pAb P3), RT-PCR

Surface PrPC increases upon platelet activation

Mouse

Bone marrow, thymus, fetal liver, not spleen, not peritoneum

Hemopoietic stem cells, immature thymocytes, not peripheral blood leukocytes, not gut intraepithelial lymphocytes

FC (mAb 6H4)

 

Mouse

Skin, thymus, spleen, lymph nodes

DC

FC (mAb SAF83)

Heterogeneous distribution among DC subtypes; absent in B220+ DCs; upregulated with DC maturation

Bovine

Spleen, lymph nodes, blood

Follicular DC, B cells, lymphocytes, monocytes, PMN

IHC, FC (mAb 6H4)

Monocytes and PMN immunoreactivity low, B cells high

Sheep

Blood

B cells, T cells, monocytes, not granulocytes, not erythrocytes

FC (mAbs 8H4, 5B2, 7A12), RT-PCR

Platelet fraction contained PrP mRNA, not surface PrPC

Sheep

Blood

PBMC, platelets, not granulocytes

WB, FC (mAbs FH11, 4F2. 8G8, 6H4)

PrPCin platelets intracellular only; level of PrPC expression in B cells higher in scrapie-susceptible than in scrapie-resistant genotype

Modified from Linden et al. (2008)

Abbreviations: DC dendritic cell, EM electron microscopy, FC flow cytometry, GAx glutaraldehyde cross-linked epitopes, IHC immunohistochemistry, ISH in situ hybridization, mAb monoclonal antibody, NBNorthern blot, pAb polyclonal antiserum, pAbMo polyclonal antiserum raised in PrP-null mouse, PMN polmorphonuclear, WB Western blot, 3F4, 3F5, 4F2, 5B2, 6H4, 7A12, 8G8, 8H4, FH11, HumP, SAF61, SAF83 designations of monoclonal antibodies, Br-1, N10, p3, P38, Ra5, R254, R073 designations of polyclonal antisera

Many reports are available on putative ligands of the prion protein, and candidate physiological ligands are listed in Table 2, and binding domains for a number of ligands were identified 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).
Prion (PRNP), Table 2

Putative physiological ligands of the prion protein

Ligand

Description

Method

αB-crystalline

Stress-induced, small heat shock protein

Two-hybrid screen, N-PAGE, optical biosensor

β-dystroglycan

Transmembrane protein

coIP, detergent sensitivity

14-3-3

Intracellular scaffolding protein

Overlay, MS

50, 56, 64, 72, 110 kDa proteins

Unidentified

Overlay

5-HT5a

Serotonin receptor

Overlay, colocalization

Aldolase C/zebrin II

Glycolytic pathway enzyme

Overlay, coIP, MS

APLP1

Amyloid precursor-like protein

Lambda-gt11 mouse brain cDNA library

Bcl-2

Antiapoptotic protein

Two-hybrid screen

BiP/Grp78

Endoplasmic reticulum chaperone

coIP

CHIP/Stub1

Cochaperone

Two-hybrid screen, pull-down assay, coIP

CK2

Protein kinase

Overlay, SPR

Cu+2 ions

Metal

 

DNA

Nucleic acid

Biophysical measurements

D1R

Dopamine receptor

Overlay, colocalization

Fbx6/Fbxo2

Substrate recognition unit of ubiquitin ligase complex (Fig. 2c)

Lambda-gt11 mouse brain cDNA library

Fyn, ZAP-70

Protein tyrosine kinases, soluble

coIP

GASP

G protein-coupled receptor-associated sorting protein (Fig. 2b)

Lambda-gt11 mouse brain cDNA library

GFAP

Intermediate filament protein

Overlay

Glycosaminoglycans

Glycosaminoglycans

 

Grb

Adaptor protein for tyrosine kinase receptors

Two-hybrid screen

Heparin/heparan sulfate

Glycosaminoglycans

SPR, ELISA

hnRNP A2/B1

RNA-binding protein

Overlay, coIP, MS

Hop/STI1

Cochaperone

Complementary hydropathy, binding assays, SAXS, NMR

Hsp60

Chaperone

Two-hybrid screen

Laminin

Extracellular matrix component

Binding assay

LRP/LR

Laminin receptor precursor/laminin receptor

Two-hybrid, cell binding

LRP1

Scavenger receptor

Cross-linking, coIP, binding assay

mGluR1/R5

Glutamate receptor

Phage display; coIP

nAChRα7

Acetylcholine receptor

Phage display; coIP

N-CAM

Cell adhesion molecule

Cross-linking, coIP

NMDAR

Glutamate receptor

coIP

Nrf2

Transcription factor

Lambda-gt11 mouse brain cDNA library

P2X4R

Purinergic receptor

Overlay, CoIP, colocalization

Pint1

Exonuclease motif (Fig. 2a)

Two-hybrid screen

PSD-95

Postsynaptic density scaffolding protein

Lambda-gt11 mouse brain cDNA library

PTPD1

Protein tyrosine phosphatase, soluble

Lambda-gt11 mouse brain cDNA library

RNA

Nucleic acid

EMSA

Synapsin Ib

Synaptic vesicle release regulator

Two-hybrid screen

Vitronectin

Extracellular matrix component

Binding assay

Updated from Linden et al. (2008)

Abbreviations: ELISA enzyme-linked immunosorbent assay, EMSA electrophoretic mobility shift assay, coI P coimmuno precipitation, MS mass spectrometry, NMR nuclear magnetic resonance, SAXS small-angle X ray scattering, SPR surface plasmon ressonance, N-PAGE nondenaturing polyacrylamide gel electrophoresis, n.d. not determined

Prion (PRNP), Fig. 2

The translated sequence of the mouse prion protein is depicted as a rod-like shape, with major domains shown in color such as in Fig. 1. Each binding partner is indicated together with the stretch of aa. residues that contain their binding domain in mouse PrPC. Pink stars indicate the position of the glycosylation residues. The double arrow straddling the whole mature molecule represents undetermined binding site for the partners listed at the bottom of the figure. Abbreviations as in Fig. 1, and Table 2, plus: SP signal peptide, GPIp GPI anchor-signaling peptide (modified from Fig. 3 in Linden et al. (2008))

Trafficking, Endocytosis, and Recycling of the Prion Protein

An N-terminus peptide (aa. 1–22) drives 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 independent of clathrin. Although caveolae- and flotilin-derived vesicles may participate in the internalization of PrPC in nonneural cells and in astrocytes, mounting evidence suggests that, in neurons, clathrin-mediated endocytosis plays an important role in 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).
Prion (PRNP), Fig. 3

Subcellular trafficking of PrPC. The plasma membrane is represented by thick lines, intracellular membranes by thinner lines, and lipid rafts by a gray-shaded wider bilayer. The cytoplasm is colored light blue. PrPC is depicted similar to Fig. 1, including a loose representation of the N-terminal flexible domain. The major vesicular systems involved in both the synthesis (bottom right), the endocytic cycle of PrPC, and release of PrPC-containing exosomes (bottom left) are indicated with black lettering, and the major pathways of trafficking are indicated in colored letters. Molecules involved in the clathrin-dependent pathway are indicated in dark blue lettering. Arrows indicate trafficking, the most likely pathways based on current experimental evidence are shown with dark blue arrows. Clathrin is represented by red circles, caveolin is depicted in purple. Note that PrPC is associated with rafts early in the secretory pathway and that cell surface PrPC leaves the rafts and associated with LRP1 on its way to internalization via clathrin. MVB multivesicular bodies, ER endoplasmic reticulum, LRP1 low-density lipoprotein receptor-like protein-1 (modified from Linden et al. (2008))

The hypothesis that GPI-anchored PrPC may “piggy-back” on an integral membrane protein had long been raised, and several studies indicated that LRP1 may participate in clathrin-mediated endocytosis of PrPC, because knockdown of LRP1, but not LRP1b, reduced internalization of PrPC. LRP1 has also been implicated in the entry PrP fibrils into cells.

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 nonraft 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 nonraft 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 nonraft protein(Taylor et al. 2005). It is not clear yet whether 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. Remarkably, amyloid β oligomers can function as a ligand for PrPC leading to toxic signaling via metabotropic glutamate receptors (mGluR5) and the soluble tyrosine kinase Fyn (Um et al. 2013). A binding site was identified at aa. 90–110 and, more recently, the endocytic motif 23–27 was also shown to mediate binding to peptide, albeit this last binding site is not as established in the literature. This suggests that amyloid β may be able to regulate PrPC trafficking, and indeed oligomers of amyloid β led to clustering of PrPC at the cell surface in both cell lines and hippocampal neurons in culture (Caetano et al. 2011). Given that laminin gamma 1 chain, another ligand of PrPC can trigger survival signaling via mGluR5, it remains to be established if regulation of PrPC trafficking may underlie the switch between toxic and nontoxic signaling in neurons. Moreover, an additional PrPC ligand, the cochaperone hop/STI1, signals for neuronal survival by coupling PrPC to the alpha7 nicotinic receptor, triggers the internalization of PrPC, which regulates the extent of PrPC modulated neuronal signaling (Linden et al. 2008 for review).

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; Martins et al. 2010) for reviews). Although no overt phenotypic changes were described in the first generated PrP-null mice, later studies of such animals 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 are 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 synaptic level, impairment of long-term potentiation (LTP) was found in the CA1 area of the hippocampus of PrPC-null mice when experiments were done at physiological temperature, albeit not at room temperature. LTP in hippocampal CA1 is at the root of formation of memory in the context 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 after hyperpolarization potential (AHP) were also detected both in constitutive PrP-null mice and in conditional knockouts in which the expression of PrPC is abolished at 12 week of age. The latter indicates that this phenotype is caused by neural dysfunction, rather than by a developmental deficit.

Although changes in the central nervous system are subtle, and do not compromise the general health of the PrP-null mice, expression in knockout mice of certain deletion mutants such as PrPΔ32–121, PrPΔ32–134, or PrPΔ94–134 causes neurodegeneration. In particular, the deletion of amino acids 105–125 causes cerebellar atrophy, loss of cerebellar granule cells, gliosis, and astrocyte hypertrophy, with decreased body size, immobility, myoclonus, and death within 1 month. 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 neuronal survival 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. Thus, PrP-null mice undergo 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 and mice. Independent published work suggests that this effect of PrPC may depend on activation of signaling by STI1 (Beraldo et al. 2013).

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).

On the other hand, the binding of oligomers of Aβ peptide to PrPC was shown to be involved on the transduction of neurotoxic signals (Um and Strittmatter 2013). Such a property widens the neuropathological roles of the prion protein to at least both TSEs and Alzheimer’s disease, in an extended context of neurodegeneration. Furthermore, a review of current evidence suggests that PrPC may play an important role in mechanisms that regulate clinical depression associated with neurodegeneration (Beckman and Linden 2016).

The absence of PrPC is associated with altered sensitivity to injury not only in the central nervous system but also in peripheral nerves (Bremer et al. 2010) and 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 (Nitta et al. 2009; Isaacs et al. 2006 for reviews).

A critical caveat was, nonetheless, raised with respect to the identification of PrPC functions based solely on the phenotype of PrPC-null mice. Concerns are based on the so-called flanking gene problem, which is the persistence in congenic knockout mice of allelotypes of bystander genes closely linked to the targeted gene. Such an effect has been shown to confound our previous proposal of a role of PrPC upon phagocytosis, which has subsequently been traced to the Prnp-flanking gene Sirpa that encodes signal regulatory protein alpha (SIRPα). Thus, unambiguous attribution of true systemic functions of PrPC depend on further evidence in addition to loss of function in widely used strains of PrPC-null mice (Nuvolone et al. 2016).

Cellular Functions of the Prion Protein

Conflicting results have been reported with regard to both the expression of PrPC during development, as well as to its cellular distribution in neuronal cell body, dendrites, and axons, thus 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).
Prion (PRNP), Table 3

Cellular functions of PrPC

Cell type/organism

Function

Type of interaction/ligand

Neuronal precursors; subventricular zone; dentate gyrus from PrP-null and wt mice

↑Proliferation

Cis-acting PrP

Splenic lymphocytes from PrP-null and wt mice

↑ Mitogen-induced proliferation

Cis-acting PrP

Splenocytes from PrP-null and wt mice

↑ or ↓ mitogen-induced proliferation

Cis-acting PrP

Human glioblastoma cell line

↑Proliferation

Cis-acting/PrP-hop/STI1 binding

Cultured human enterocytes

Balance between proliferation and polarization–differentiation

Cis-acting/interacts with cytoskeleton and desmosome-associated proteins

Multipotent neuronal precursors from PrP-null, -wt, and PrP-overexpressor mice

↑Differentiation

Cis-acting PrP

Neurons from hamster developing brain

↑Expression and localization suggest involvement in axon growth, guidance, and synaptogenesis

Cis-acting PrP

Hippocampal neurons from PrP-null and wt mice

↑ Neurite outgrowth

Cis-acting PrP

Cerebellum granule cells and hippocampal mouse neurons from PrP-null, wt mice, and PrP-overexpressor mice

↑Neurite outgrowth

Trans-acting:PrP-Fc or PrP expressed by monolayers of CHO cells

Embryonic rat hippocampal neurons

↑ Neurite outgrowth, dendrite like and single axon like process, enhanced synaptogenesis

Trans-acting recombinant PrP

Embryonic hippocampal mouse neurons from PrP-null and wt mice

Adhesion and neuritogenesis

PrP-laminin

Dorsal root ganglia neurons from PrP-null and wt mice

Axonal growth

PrP-vitronectin

PC-12

↑Neurite outgrowth

PrP-laminin

PC-12

↑Neurite outgrowth

PrP-NCAM

Bone marrow-derived granulocytes and HL60 leukemia cells

↓ Expression

↑ Differentiation

Cis-acting PrP

Bone marrow-derived macrophage cell line

↑Phagocytosis

Cis-acting PrP

Murine erythroleukemia, neuro-ectodermal, and myeloid cell lines

↑ Expression

↑ Differentiation

Cis-acting PrP

myc and ras transformed fibroblasts from wt and PrP-nullmice

↑ Aggregation, embolization, and metastasis

Cis-acting

Tissue samples from gastric adenocarcinoma

↑ Expression

↑ Histopathological differentiation and tumor progression

Cis-acting PrP

Embryonic hippocampal neurons from PrP-null and wt mice

↑ Differentiation and survival

Cis-acting binding to hop/STI1

Embryonic astrocytes from PrP-null and wt mice

↑ Differentiation and survival

Cis-acting binding to hop/STI1 or laminin

Embryonic astrocytes from PrP-null and wt mice

Regulation of glutamate-dependent lactate transport

Cis-acting PrP, complex of GluR2, α2/β2-ATPase, basigin, and MCT1

Immortalized hippocampal neurons from PrP-null and wt mice

Higher sensitivity to apoptosis upon serum deprivation

Cis-acting PrP

Fetal human neurons

Protection against bax-induced apoptosis

Cis- and Trans-acting: GPI-anchored, cytosolic, and anchorless PrP

Retinal explants from PrP-null and wt mice

↑ Survival of undifferentiated postmitotic cells subject to inhibition of protein synthesis

Cis-acting

Human neurons, mouse neuronal cell lines

↑ Survival, delays conformational change of Bax

Cis-acting

Cerebellar granule neurons from PrP-null and wt mice

↑ Survival

Trans-acting soluble PrP

Undifferentiated postmitotic retinal neurons from PrP-null and wt mice

↑ Survival

Cis-acting binding to Hop/STI1

Retinal ganglion cells

↑ Survival in hypoxia-induced apoptosis

Trans-acting, soluble, alpha-secretase-derived N-terminal PrP

Embryonic primary hippocampal neurons or immortalized neuronal cell lines from PrP-null and wt mice

↑ Survival

Cis-acting PrP:hop/STI1 binding

Cell line derived from neocortical neuronal precursors expressing SV40 large-T antigen v-Src and v-Myc or HEK293

Proapoptotic, ↑ sensitivity to staurosporine-induced cell death

Ectopic overexpression of PrP

1C11 neuroectodermal cell line

Survival, proliferation, and neuronal plasticity

Cis-acting

HEK293 and rabbit kidney epithelial cell line

Proapoptotic, ↑ sensitivity to staurosporine-induced cell death

Ectopic overexpression of PrP

Gastric cancer cells

↑ Protection against apoptosis

Overexpression of PrP

Human breast cancer cell lines

↑Resistance to TNF-α induce cell death

Overexpression of PrP in resistant cells

Human breast cancer cell line

No effect upon staurosporin-induced cell death

Overexpression of PrP

Pancreatic tumors and melanona

↑ Expression of unprocessed signal peptide PrPC (Pro-PrPC) associated with tumorigenesis

Pro-PrP binding to filamin A

Yeast

Survival against Bax-induced cell death

Ectopic expression of mammalian PrP

Abbreviations: wt wild-type, Fc Fc chain of IgG, N-CAM neural cell adhesion molecule, GluR2 glutamate receptor 2, MCT1 monocarboxylate transporter 1

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. Evidence that PrPC can be secreted by exosomes from various cell types also extends the possibilities of trans effects at both the tissue and system levels.

The rate of proliferation of neuronal precursors correlated with the content of PrPC in both the subventricular zone and dentate gyrus of the hippocampus of adult mouse brain, but expression of PrPC in proliferating zones was restricted to postmitotic neurons. Thus, the effect of PrPC upon proliferation of neuronal precursors is probably indirect, a possibility that must always be considered when dealing with complex tissues. One hypothesis, not yet explored, is the potential effect of exosomes containing PrPC from postmitotic neurons. In addition, 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, cerebral cortex, dorsal root ganglia, as well as in neuronal-derived cell lines. These effects are mediated by cis-acting PrPC upon its binding to specific ligands, such as hop/STI1, laminin, and NCAM, as well as in trans together with either NCAM or other unidentified ligands at the cell surface. In the case of extracellular ligands, such as laminin or hop/STI1, cell signaling requires intermediation by mGluR1/5 or nAChR7, respectively. PrPC also modulates the differentiation of astrocytes, secretion of neurotrophic factors, and metabolism. These results are consistent with proposed PrPC functions at system levels (see section “Systemy Functions of the Prion Protein”).

One of the most widely accepted roles of PrPC is upon cell survival. This is particularly relevant, due to the association of PrPC with neurodegenerative diseases. Although the importance of PrPC loss-of-function in neurodegeneration is still debatable, most studies using primary neuronal cultures from both brain and retina, 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, PrPC has been also implicated in tumor proliferation, progression, invasiveness, and resistance to drug treatment (Mehrpour and Codogno 2010 for review). Together, these results suggest that PrPC may constitute 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 the pleiotropic effects of the prion protein, we have raised the hypothesis that PrPC acts at a much more fundamental level, coordinating signaling pathways at the cell surface. Such a biological function is consistent with the evidence for multiple PrPC-binding partners (Table 2), which, however, are variably present in distinct cell types and may be present in differing amounts depending on context. Granted that the evidence in favor of several proposed partners is debatable, a multiplicity of PrPC-based molecular complexes is likely even if considering only a handful of accepted, strictly validated binding partners among those currently under study.

Mechanisms of PrPC-Mediated Signal Transduction

Roles of the prion protein in signal transduction have been unraveled by various approaches. The main procedures include 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 these reagents 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 are now believed to be a target of modulation by the prion protein (Table 4).
Prion (PRNP), Table 4

Examples of signaling mediated by the prion protein

Signaling pathway

Cell type

Method

Intracellular messenger

Transmembrane signal transfer

Tyrosine phosphorylation

Embryonic carcinoma 1C11 transformed cell line

Antibody cross-linking

Fyn tyrosine kinase

Caveolin, N-CAM?

Tyrosine phosphorylation

Central nervous system neurons

Overexpression, antibody cross-linking, ligand binding assays, co-IP

Fyn

N-CAM, receptor type protein phosphatase α

Protein kinase A

Central nervous system neurons

Peptide or hop/STI1 binding to PrPC, PKA antagonist

cAMP

Unknown

Erk MAP kinase

Central nervous system neurons

Peptide or hop/STI1 binding to PrPC, MEK inhibitor

Unknown

Unknown

Erk MAP kinase

Embryonic carcinoma 1C11, neurohypothalamic GTI-7, lymphoid BW5147, and Jurkat T cell lines

Antibody cross-linking

Fyn? ROS?

NADPH oxidase, EGFR?

Erk MAP kinase

Macrophage-like P388D-1 cell line

Soluble PrPC-Fc fusion protein, Src and PI3-K antagonists

Src family soluble tyrosine kinases Syk and Pyk2

PI3-kinase

Calcium influx

Central nervous system neurons

Comparison PrP-null vs. wild type; recombinant soluble PrPC

 

Voltage-gated calcium channels

Calcium influx

CEM-T and Jurkat cell lines

Antibody cross-linking

 

Unknown

Calcium influx

Hippocampal neurons; transfected HEK293 cells

Ligand binding

 

Alpha-7 nicotinic cholinergic receptor

Calcium homeostasis

Hippocampal neurons; transfected HEK293 cells

Ligand binding

 

Group I metabotropic glutamate receptors

Calcium homeostasis

Chinese hamster ovary cells; neuron-derived cell lines

Overexpression

ER release and mitochondrial uptake

Store-operated calcium channels?

Protein kinase C

Splenocytes

Comparison PrP-null vs. wild type;

Calcium-dependent PKC forms

Unknown

PI3-kinase

Brain; neural cell lines; hippocampal neurons

Comparison PrP-null vs. wild type; transfection of cell lines; recombinant soluble PrPC

 

Unknown

PI3-kinase

P388D-1 macrophage-like cell line

Recombinant soluble PrPC; PI3-K inhibitor

Erk (reciprocal effect with Akt)

Unknown

PI3-kinase

Hippocampal neurons

Ligand binding

Akt; mTOR; p70S6 kinase; 4E-BP1

Unknown

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: (a) The level of expression and distribution of PrPC, as indicated in Table 1; (b) The availability of ligands amongst at least those indicated in Table 2, which may interact with PrPC either in cis or in trans; (c) Structural rearrangements caused by multiple ligands; (d) 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 (Linden et al. 2012).

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. An integrated approach to the pleiotropic effects of PrPC will likely explain systems-level phenotypes associated with either engagement or loss of function of the prion protein.

Summary and Future Directions

The currently available data suggest that the prion protein plays a significant role in signal transduction. PrPC appears to function as a cell surface scaffolding protein, with the ability to assemble multicomponent complexes at the cell surface, with 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, responses to oxidative stress, synaptic modulation, and immunomodulation, with widespread physiological and pathophysiological consequences.
Prion (PRNP), Fig. 4

Schematic representation of cell surface signaling modules scaffolded by the prion protein. a–d represent cis-acting PrPC. (a) NCAM-Fyn interaction induced by cross-linking of the prion protein; (b) Laminin receptors, including integrin, the laminin receptor, and PrPC itself may interact in various combinations, and binding of the laminin γ-chain to PrPC induces calcium release from intracellular stores, transduced by PrPC-interacting, group 1 metabotropic glutamate receptors, via phospholipase C; (c) Interaction of hop/STI1 with PrPC leads to calcium influx mediated by the α7 nicotinic acetylcholine receptor; (d) Certain signals induced by the interaction of hop/STI1 with PrPC require endocytosis and are probably mediated by a hitherto unidentified transmembrane protein; (e) Trans-acting PrPC may be located in either neighboring cells or exosomes and may scaffold various signaling modules. Molecular structures are used only for illustration purposes and are not to scale. The N-terminal flexible domain of PrPCwas omitted for clarity. Abbreviations as in Table 2, plus: exo exosome, endo signaling endosome, ER endoplasmic reticulum. Lipid rafts are represented by a slightly thicker membrane depiction containing blue cholesterol molecules, only where there is evidence for the location therein of assembled complexes at the cell surface (modified from Linden et al. (2008))

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 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 scaffolding protein, may contribute to a better understanding of its physiological functions, as well as to the establishment of effective therapeutic options for still incurable neurodegenerative conditions.

Notes

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), Canadian Institutes of Health Research (CIHR), the Weston Brain Institute, Brain Canada, The Alzheimer’s Association (USA), The Alzheimer’s Society (Canada), and PrioNet-Canada. V.R.M. is an International Scholar of the Howard Hughes Medical Institute.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Rafael Linden
    • 1
  • Vilma R. Martins
    • 2
  • Marco A. M. Prado
    • 3
  1. 1.Laboratorio de NeurogeneseInstituto de Biofisica da UFRJRio de JaneiroBrazil
  2. 2.International Research CenterA.C. Camargo Cancer CenterSão PauloBrazil
  3. 3.Department of Physiology and Pharmacology and Anatomy and Cell Biology, Schulich School of MedicineUniversity of Western OntarioLondonCanada