The elusive role of the prion protein and the mechanism of toxicity in prion disease.

PrPSc, a misfolded, aggregation-prone isoform of the cellular prion protein (PrPC), is the infectious prion agent responsible for incurable brain diseases such as scrapie of sheep, bovine spongiform encephalopathy, and its human counterpart, variant Creutzfeldt-Jakob disease. In these disorders, collectively known as prion diseases, exogenous PrPSc propagates in the infected host by imprinting its aberrant conformation onto endogenous PrPC, eventually triggering a rapidly progressing neurodegenerative process that invariably leads to death. But what is the function of PrPC besides serving as a substrate for the generation of PrPSc? And how does PrPC misfolding cause neurological disease?

The Cellular Prion Protein

PrPC is a cell surface glycoprotein expressed in neurons and many other body cells. It is synthesized in the endoplasmic reticulum (ER), where it undergoes oxidative folding, N-linked glycosylation, and addition of a glycosyl-phosphatidyl-inositol (GPI) anchor that attaches the protein’s C terminus to the lipid bilayer. After transit in the Golgi, PrPC is delivered to the plasma membrane, where it resides in lipid rafts, which are membrane microdomains rich in cholesterol and sphingolipids. Some PrPC molecules are constitutively endocytosed and either recycled to the plasma membrane or delivered to lysosomes for degradation. PrPC has a flexible N terminus (residues 23–127, mouse PrP numbering) that can interact with copper and zinc ions, and a C-terminal globular domain (residues 128–231) comprising three α-helices and two short anti-parallel β-strands.

Inactivation of the PrPC gene in living organisms produced variable phenotypes. Knockdown of the PrPC-related genes PrP-1 and PrP-2 in the zebrafish Danio rerio caused, respectively, gastrulation arrest and malformed brains and eyes, indicative of essential roles in the fish’s development [1,2]. In contrast, PrPC knockout mice and cows had no major developmental or neuroanatomical defects (reviewed in [3]), indicating non-essential or redundant functions in higher vertebrates.

Based on the analysis of mild phenotypic traits that develop in PrPC knockout mice and on cell culture studies, mammalian PrPC has been assigned roles in many biological processes, including neurotransmission, olfaction, proliferation and differentiation of neural precursor cells, myelin maintenance, copper and zinc ion transport, and calcium homeostasis, as well as neuroprotective activities against several toxic insults, such as oxidative and excitotoxic damage [2-4]. How can PrPC serve so many different functions? Perhaps the answer lies in its ability to interact with a number of membrane proteins, potentially influencing their cellular localization and activity [4]. These include glutamate receptors of the N-methyl-D-aspartate (NMDA) subclass [5] and voltage-gated calcium channels (VGCCs) [6]. Interaction with these channels may account for some of the functional activities of PrPC, but may also activate toxic responses when PrPC misfolds (see below).

PrPC Mediates PrPSc Neurotoxicity

The fact that inactivation of the PrPC gene in mice or cows does not cause neurodegeneration indicates that prion pathogenesis is not due to loss of PrPC function, but to a gain of toxicity upon its conversion to PrPSc. Interestingly, extracellular PrPSc kills only neurons that express PrPC. This was first shown by a neurografting experiment in which neural tissue from PrPC-expressing mice was transplanted into the brains of PrP knockout mice, which do not replicate prions since they lack the PrPC substrate for PrPSc production [7]. After intracerebral prion infection, the transplanted mice developed neuropathology in the PrPSc-replicating graft but not in the surrounding PrP knockout tissue, even though this tissue accumulated substantial amounts of graft-derived PrPSc [8]. Consistently with this, switching off neuronal PrPC expression in mice with established prion infection rescued clinical disease and prevented neuronal loss, despite continuous production of PrPSc by surrounding astrocytes [9]. Moreover, prion-infected mice expressing a form of PrPC that lacks the GPI anchor and is secreted into the extracellular space did not develop the typical prion pathology despite large amounts of extracellular PrPSc [10,11]. Thus, PrPSc is not directly toxic to neurons; it is the endogenous PrPC conversion that causes neuronal dysfunction and death.

Conformational conversion of PrPC starts on the neuronal surface, where PrPC interacts with exogenous PrPSc, and proceeds within endocytic compartments. Thus, neurotoxicity may be triggered by PrPC misfolding at the cell surface or inside the cell.

Toxicity Induced by PrPC Misfolding at the Neuronal Surface

Two kinds of evidence suggest that alterations in the structure of cell surface PrPC can lead to neuronal death. PrPC molecules with certain internal deletions, including Δ94–134 and Δ105–125, induce dramatic neurodegeneration when expressed in transgenic mice [12,13]. These mutant molecules are efficiently trafficked to lipid raft regions of the plasma membrane, suggesting that their toxicity stems from abnormal activity at the neuronal surface rather than from mislocalization or intracellular retention.

PrPC attenuates the activity of NMDA receptors (NMDARs), protecting neurons from glutamate-induced excitotoxicity [5]. Supporting the idea that the internal deletions may corrupt this function, PrPΔ105–125 sensitized neurons to glutamate-induced, calcium-mediated cell death [14]. It was also found that PrPΔ105–125 induced non-selective ionic currents that depended on the integrity of the N-terminal 23–31 region [15]. A possible interpretation is that the toxic deletions promote a conformational change of the PrPC N terminus, altering its interaction with NMDARs and enabling the 23–31 segment to interact abnormally with the lipid bilayer, generating pores in the plasma membrane. Thus, a structural change in cell surface PrPC would simultaneously corrupt NMDAR function and plasma membrane permeability, leading to dysregulation of ion homeostasis and neuronal death.

Another set of experiments showed that monoclonal antibodies against specific epitopes in the C-terminal globular domain of PrPC induce rapid neurodegeneration when injected into the mouse brain or applied to cultured cerebellar slices [16]. Neurodegeneration was prevented by deleting the PrPC N terminus or by antibodies against this region. The latter also attenuated the toxicity of PrPΔ94–134 [16], suggesting that the globular domain antibodies and the internal deletions activate a similar pathogenic cascade, involving a structural rearrangement of the N terminus.

Supporting the idea that PrPSc docking onto cell surface PrPC may elicit a similar structural change and downstream toxic effects (Fig 1), it was found that when PrPSc was exogenously presented to cultured neurons, the resulting neurotoxicity was blocked by NMDAR antagonists or by deletion of the N-terminal domain of neuronal PrPC [17,18].

Fig 1

Theoretical model for how cell surface PrPC misfolding could result in neurotoxicity.

(A) PrP consists of a flexible N terminus (mauve) and a globular C-terminal domain (green) attached to the plasma membrane (PM) by a GPI anchor (black line). PrP associates with NMDARs, attenuating their activity [5]. (B–C) Interaction with extracellular PrP causes the N terminus of PrP to undergo a structural rearrangement. This leads to aberrant interaction of PrP with NMDARs and their hyperactivation (B) and/or abnormal insertion of the PrP N terminus into the lipid bilayer with generation of a toxic pore (C). In addition to NMDARs, PrP misfolding at the cell surface may corrupt the activity of other PrP-interacting ion channels or signaling complexes.

Neurotoxicity Induced by Intracellular PrPC Misfolding

A study in prion-infected mice gave information about a neurotoxic mechanism potentially triggered by intracellular accumulation of misfolded PrPC. Prions inoculated into the mouse hippocampus activates the translational repression pathway of the unfolded protein response (UPR). The UPR is a signal transduction cascade set in motion when misfolded proteins accumulate in the ER. A crucial step is auto-phosphorylation of the ER-associated kinase PERK, which phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α). This inhibits protein translation, reducing the overload of misfolded proteins. In the case of protracted UPR, however, sustained translational attenuation can have detrimental effects. In prion-infected mice, prolonged activation of the PERK/eIF2α pathway caused drops in the levels of pre- and post-synaptic proteins in the hippocampus, deficits in hippocampal synaptic transmission, and behavioral decline [19].

But what activates the UPR? PrPSc is unlikely to be the instigating factor, since it accumulates in the extracellular space or in endocytic compartments, rather than in the ER. The level of PrPC mRNA rises during prion infection, and the PrPC mRNA molecules escape eIF2α-P-induced translational inhibition [19]. Thus, ER overload with misfolded PrPC due to increased biosynthesis may be the actual cause of UPR activation. Alternatively, the UPR could be triggered by ER accumulation of CtmPrP, a transmembrane form of PrPC whose biogenesis at the ER membrane increases in prion-infected mice [20].

Prion infections are extremely rare in humans, in whom approximately 99% of all cases occur sporadically or are inherited because of mutations in the gene encoding PrPC. In these illnesses, PrPC misfolds spontaneously without the need for contact with exogenous PrPSc. When expressed in transgenic mice, PrPC molecules with certain genetic prion disease-associated mutations cause neurological syndromes that recapitulate key features of the corresponding human disorders [21-23]. These mutant PrPs misfold spontaneously in the ER lumen and are partly retained in the secretory pathway; surprisingly, however, they do not trigger the UPR [24,25]. How do they cause neurological disease? PrPC interacts physically with the α2δ-1 subunit of VGCCs [6]. This is a GPI-anchored protein which promotes the anterograde trafficking and correct synaptic localization and function of the channel complex. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of VGCCs to synapses. This leads to inefficient depolarization-induced calcium influx, abnormal cerebellar neurotransmission, and motor disease [6]. Since PrPC interacts with a number of other proteins that transit the secretory pathway, such as glutamate receptors and signaling complexes, its intracellular retention may have broader effects on neuronal function (Fig 2) [25].

Fig 2

A role for intracellular PrPC retention in neuronal dysfunction.

(A) PrP on the plasma membrane (PM) influences the activity of neurotransmitter receptors, ion channels, and signaling complexes with which it interacts. (B) Owing to retention in transport organelles (ER/Golgi), misfolded/aggregated PrP sequesters the interacting protein in intracellular compartments, leading to loss of normal function on the cell membrane [6]. Intracellular retention might also cause the complex to function abnormally and generate a toxic signal.

Thus, in both acquired and genetic prion diseases, intracellular PrPC misfolding would ultimately alter synaptic proteostasis, either through an indirect, UPR-mediated mechanism, or by directly interfering with secretory trafficking of PrPC-interacting cargoes.

A Complex Interplay of Extracellular and Intracellular Toxicities

The experimental studies outlined above indicate different neurotoxic mechanisms that may be activated by misfolded PrPC in distinct cellular compartments, including corruption of PrPC interactions on the cell surface, disruption of plasma membrane permeability, impairment of secretory protein transport, and dysregulation of generic proteostatic pathways, such as the UPR.

These mechanisms are likely to co-exist, but may contribute differently to pathogenesis in different prion diseases. UPR-induced transcriptional attenuation may account for the synaptic dysfunction and degeneration that precedes neuronal death in the early stages of prion infection. As the disease progresses and PrPSc accumulates in the extracellular space, additional mechanisms may be engaged. PrPSc-induced misfolding of cell surface PrPC may be a key mediator of cell death [10] and cause rapid neuron demise by corrupting ion channel or signaling activities, and/or by generating toxic pores (Fig 1). In sporadic and genetic prion diseases, in which PrPSc formation is not obligatory for pathogenesis [26-28], spontaneous accumulation of misfolded PrPC molecules in transport organelles may be more important. Misfolded/aggregated PrPC may sequester ion channels or signaling complexes in intracellular compartments, leading to loss of their normal functions on the cell membrane and/or gain of toxic intracellular activities (Fig 2) [6].

This neurotoxic modality may contribute to the clinical variability of prion diseases. Different misfolded PrPC variants may be produced in different prion disorders, which may have different effects on neuronal function—hence, on the clinical presentation of disease—depending on their propensity to accumulate in intracellular organelles and interfere with the transport of the molecules with which they interact [25].

In view of their complex pathogenesis, what would be the best therapeutic option for prion diseases? Several compounds inhibit PrPSc propagation in cultured cells, but show little or no efficacy in vivo, and no therapeutically useful drug is currently available. The “mutability” of prions [29] means that molecules that target PrPSc can lead to the selection of drug-resistant variants that propagate more efficiently in the presence of the drug [30]. Given the emerging role of PrPC misfolding in neurotoxicity, drugs that stabilize its native conformation or down-regulate its expression may prove more effective, and applicable to the sporadic, genetic, and acquired forms.