PrP overdrive

Abstract

Knockout of the cellular prion protein (PrP^C) in mice is tolerated, as is complete elimination of the protein’s N-terminal domain. However, deletion of select short segments between the N- and C-terminal domains is lethal. How can one reconcile this apparent paradox? Research over the last few years demonstrates that PrP^C undergoes α-cleavage in the vicinity of residue 109 (mouse sequence) to release the bioactive N1 and C1 fragments. In biophysical studies, we recently characterized the action of relevant members of the ADAM (A Disintegrin And Metalloproteinase) enzyme family (ADAM8, 10, and 17) and found that they all produce α-cleavage, but at 3 distinct cleavage sites, with proteolytic efficiency modulated by the physiologic metals copper and zinc. Remarkably, the shortest lethal deletion segment in PrP^C fully encompasses the three α-cleavage sites. Analysis of all reported PrP^C deletion mutants suggests that elimination of α-cleavage, coupled with retention of the protein’s N-terminal residues, segments 23–31 and longer, confers the lethal phenotype. Interestingly, these N-terminal residues are implicated in the activation of several membrane proteins, including synaptic glutamate receptors. We propose that α-cleavage is a general mechanism essential for downregulating PrP^C’s intrinsic activity, and that blockage of proteolysis leads to constitutively active PrP^C and consequent dyshomeostasis.

Keywords: :

• ADAM enzyme
• AMPA receptor
• Alzheimer disease
• copper
• prion protein
• zinc
• α-cleavage

Introduction

To date, there is still no definitive function assigned to the prion protein (PrP). PrP has been implicated in transmembrane signaling,¹ metal ion redox regulation and trafficking,² cell adhesion,³ neuron myelination,⁴ and apoptosis⁵ (see ref. 6 for a general review of PrP function). Several recent discoveries, however, provide new perspectives that may not only assign a more definitive function to PrP, but may also give new insight into the processes that lead to prion disease. Among these discoveries are the findings that PrP is enzymatically cleaved to produce bioactive fragments,^7,8 and that full-length PrP directly interacts with a subfamily of glutamate receptors to regulate zinc transport.⁹ This brief review will describe these new perspectives, the connection to PrP structural features, and put forth a hypothesis in which cleavage serves as an essential regulatory mechanism of PrP action.

PrP^C Cleavage—the Standard Model

The full-length cellular prion protein, PrP^C, is approximately 210 amino acids long and possesses a helical C-terminal domain and a partially structured N-terminal domain (Fig. 1). A notable feature of the N-terminal domain is the octarepeat segment (residues 59–90, mouse sequence) capable of binding physiologic zinc and copper.^10,11 Between these 2 domains is a highly conserved hydrophobic segment often implicated in aggregation and the formation of PrP scrapie (PrP^Sc). Figure 1 presents the N- and C-terminal domains as spatially separated, however, recent structural work suggests an important tertiary contact promoted by zinc (see below). In addition to full-length PrP^C, the protein is also found in several distinct truncated forms in vivo, with proteolytic products possessing unique bioactive properties.^7,12 In healthy tissues, enzymatic cleavage of PrP^C at approximately K109↓H110 (mouse sequence), termed α-cleavage, produces the N-terminal and C-terminal fragments, N1 and C1, respectively. The preponderance of recent evidence suggests that α-cleavage, which separates most of the PrP N-terminus from the folded C-terminus, is due to action from one or more members of the ADAM (A Disintegrin And Metalloproteinase) family of enzymes, specifically ADAM8, ADAM10, and ADAM17.^13,14 α-Cleavage was originally assigned to ADAM10, but recent studies find that neither knocking out ADAM10 nor treatment with ADAM10 specific protease inhibitors are capable of blocking α-cleavage, and instead block proteolysis of PrP^C near the C-terminal GPI anchor at G227↓R228. More recently, ADAM8 was identified as the protease responsible for α-cleavage in skeletal muscle tissue.¹³

Figure 1. Ribbon diagram of the cellular prion protein (PrP^C) showing the N-terminal octarepeat domain (gold), the folded C-terminal domain (blue), and the central hydrophobic region (red). Note that residues 23–55 are unstructured and omitted for clarity. The 4 His residues (magenta) in the octarepeat domain coordinate zinc and copper ions, as shown. The additional His residues at 95 and 110 are also capable of coordinating Cu²⁺. Previously proposed sites for α-cleavage β-cleavage are indicated at the beginning of the hydrophobic segment and the end of the octarepeat domain, respectively.

The pro-domains released by α-cleavage exhibit potent activities. The N1 fragment is antiapoptotic, possibly acting through the inhibition of caspase-3.⁵ Conversely, the C1 fragment promotes apoptosis through p53 dependent caspase-3 activity, although it appears as though the protective effects of N1 significantly outweigh the pro-apoptotic effects of C1.¹⁵ Perhaps more importantly, substoichiometric levels of C1 protect against PrP^Sc propagation.¹⁶

PrP^C also undergoes β-cleavage, which takes place at multiple sites within and just following the octarepeat domain, producing N2 and C2 fragments.⁷ Experiments in CHO and SH-SY5Y cell lines expressing PrP^C find that levels of C2 are greatly enhanced upon the addition of H₂O₂, suggesting proteolysis by reactive oxygen species (ROS) generated by intrinsic copper, a metal ion that binds with high affinity to the N-terminal octarepeats.¹⁷ A separate pathway to β-cleavage of PrP^Sc is enzymatic, produced by calpains¹⁸ and cathepsin¹⁹ proteases (see review ref. 20). In general, β-cleavage is observed in normal brain tissue, but C2 is enriched in prion infection. Unlike the N1 and C1 fragments, N2 and C2 do not show any bioactivity or neuroprotection, although β-cleavage’s production of N2 and C2 may indirectly assert a biological effect by prohibiting the formation of N1.

Zinc Promoted Tertiary Contacts in PrP^C

The prion protein’s 2 domains are often thought to be noninteracting—a structured C terminus with an N-terminal tail. Cellular and biophysical studies, however, suggest that the 2 domains influence each other in a way that is relevant to both PrP^C function and misfolding in disease.²¹ It is now well established that physiologic concentrations of copper (Cu²⁺) and zinc (Zn²⁺) promote coordination of these metal ions to the octarepeat domain and, in the case of Cu²⁺, to 2 nearby sites, as well.^10,11,22 With the goal of trying to develop concepts for PrP^C function, our lab applied electron paramagnetic resonance (EPR) to evaluate the Cu²⁺ coordination features. At low copper concentration, the Cu²⁺ ion binds with high affinity (~0.10 nM dissociation constant) to the 4 histidine imidazoles in the octarepeat domain.^2,23 Using peptide mapping, we found a similar coordination sphere for Zn²⁺, although with a weaker dissociation constant of ~200 µM.²⁴

Given the potential for N-terminal—C-terminal interactions, and the fact that previous structural studies did not include metal ions, we investigated whether Zn²⁺ might promote higher order structure in PrP^C. NMR shows that the Zn²⁺ occupied octarepeat domain folds back on helices 2 and 3 of the C-terminal domain forming a previously unseen tertiary contact.²⁵ This newly identified contact is further supported by direct interdomain distance measurements using double electron-electron resonance (DEER) EPR. Our work parallels NMR studies with Cu²⁺ that also find a metal ion promoted interaction.²⁶ An energy-minimized model developed with constraints from our NMR and EPR experiments is shown in Figure 2. Interestingly, helices 2 and 3 possess the majority of the point mutations that cause familial prion disease, and NMR data suggest that these mutations weaken the interdomain interaction.²⁵

Figure 2. Global PrP^C fold promoted by Zn²⁺, as demonstrated by Spevacek et al. using NMR and double label electron paramagnetic resonance.²⁵ The Zn²⁺ occupied octarepeat domain makes a tertiary contact with the surface formed by C-terminal helices 2 and 3. Note that helices 2 and 3 present a cluster of negatively charged residues that facilitate the tertiary contact, and that familial mutations weaken the fold.

Early biophysical investigations into the thermodynamic basis for inherited prion disease hypothesized that mutations would thermodynamically destabilize the C-terminal structure of PrP^C rendering the protein susceptible to misfolding. However, structure and denaturation studies reveal only minimal changes in three-dimensional fold, and no systematic variation in stability.^27,28 Our investigations find that pathogenic mutants, such as E199K (mouse sequence; E200K in human), systematically alter the N-terminal–C-terminal tertiary interaction, likely by reducing the negative surface charge potential on helices 2 and 3 that attracts the Zn²⁺-bound N-terminus.²⁵ Thus, this class of charge altering mutations may reduce stability by affecting the metal ion promoted tertiary fold in PrP^C. Conversely, in wild type PrP perhaps the metal promoted interaction explains why both copper and zinc inhibit in vitro PrPres amplification.²⁹

PrP^C Function

Global perspectives of PrP function have been thoroughly reviewed elsewhere (see Aguzzi et al. for example, ref. 6). With regard to metal ions, it is now well established that both copper and zinc stimulate PrP^C internalization through endocytosis³⁰ and that copper activates transcription factors leading to an increase of PrP^C expression.^31,32 In general, PrP^C appears to be important in copper/zinc homeostasis and serves to protect cellular components when elevated metal ion levels would otherwise lead to cellular stress. Based on an analysis of coordination features, we’ve proposed that PrP^C acts to suppress copper electrochemistry that, if left unregulated, could lead to the production of free radicals and reactive oxygen species.² We also found that expansion of the octarepeat domain leads to altered copper uptake in a way that suggests a strong relationship to inherited prion disease.³³ Recent electrochemical studies lend support to this hypothesis and show further that the PrP^C-copper complex produces low levels of hydrogen peroxide, which may participate in cellular signaling.^34,35

Watt et al. identified a fundamentally new function for PrP^C in which the protein participates in neuronal zinc uptake. Using selective dyes, they showed that PrP^C enhances zinc uptake through AMPA receptors, a member of the multi-subunit glutamate receptor family.³⁶ PrP^C and AMPA co-precipitate, demonstrating a physical interaction between the 2 membrane proteins. Both the PrP octarepeat domain and the polybasic N-terminus, corresponding to residues 23–26 (Lys-Lys-Arg-Pro; mouse), were required for zinc transport, but only the polybasic region was required for co-precipitation. Watt et al. suggest that PrP^C provides neuroprotection by facilitating zinc reuptake following glutamate release, thereby regulating synaptic zinc levels. In this scenario, PrP^C docks to AMPA through its N-terminus and the octarepeat domain loads Zn²⁺ in preparation for transport through the AMPA channel.⁹ Interestingly, 2 familial mutants—P101L and D177N—shown by Spevacek et al. to weaken the Zn²⁺ promoted tertiary structure also lower zinc transport, likely by disrupting the PrP^C–AMPA physical interaction.

PrP^C Cleavage Revisited

As outlined above, the prevailing paradigm of PrP^C cleavage posits that α-cleavage is enzymatically driven and constitutes normal processing, while β-cleavage results from aberrant copper redox activity and is associated with the development of prion disease. But the identification of several ADAM family enzymes producing α-cleavage, along with the structural features promoted by copper and zinc, motivated a reassessment of PrP^C proteolysis. We used liquid chromatography/mass spectrometry (LC/MS) to analyze the rates and fragmentation patterns of PrP^C resulting from the redox activity of copper, and proteolysis from ADAM8, ADAM10, and ADAM17.⁸ Enzymes were assayed in the absence and presence of Cu²⁺ and Zn²⁺.

Figure 3 shows the identified proteolysis sites. all three enzymes produce α-cleavage, but at distinct locations, which we refer to as α₁ (following residue 109; ADAM8 primary site), α₂ (following residue 116; ADAM8 minor site), and α₃ (following residue 119; ADAM10 and ADAM17). ADAM10 also functions as a sheddase, cleaving PrP^C near the C-terminus to release the protein from the membrane surface.³⁷ Under reducing conditions, produced by the addition of ascorbic acid, copper alone produces extensive β-cleavage, consistent with prevailing hypotheses. Unexpectedly, ADAM8 also produces extensive β-cleavage with multiple proteolysis sites throughout the octarepeat domain. Consequently, β-cleavage may result from both copper redox activity and ADAM8 proteolysis.

Figure 3. Summary of the findings of McDonald et al. on the α-cleavage and β-cleavage by ADAM family enzymes.⁸ N-terminal (white) and C-terminal (green) domains are shown, with octarepeats (gold), as is the C-terminal GPI moiety that anchors the protein to the extracellular membrane leaflet. (A) ADAM8, ADAM10, and ADAM17 produce α-cleavage at distinct sites, noted as α₁, α₂, and α₃. ADAM10 also acts as a sheddase by cleaving near the C-terminus to release the protein from the membrane. Also shown are the sites in the octarepeat domain where ADAM8 produces β-cleavage. (B) Relative cleavage activities are indicated by arrow thickness. ADAM8 alone (no metal ions) produces extensive α₁- and β-cleavage, with α₂-cleavage as a minor product. (C) In the presence of Cu²⁺, α₁- and β-cleavage are suppressed, while α₂-cleavage is enhanced. (D) Zn²⁺ also inhibits β-cleavage but, in contrast to Cu²⁺, promotes α₁- over α₂-cleavage. In contrast to the previous paradigm, these findings show that metal ions suppress β-cleavage and template a structure that promotes α-cleavage.

Zinc is inert with respect to redox activity, and so is copper in the absence of the ascorbic acid reductant, but both metal ions strongly influence the proteolytic susceptibility to ADAM8, the primary enzyme in muscle responsible for α-cleavage. As shown in Figure 3, Cu²⁺ inhibits β-cleavage and promotes α₂-cleavage while Zn²⁺ similarly inhibits β-cleavage but promotes α₁-cleavage. Interestingly, elimination of the octarepeat domain in parallel experiments with Cu²⁺ fails to find an enhancement of α₂-cleavage suggesting that the metal ion promoted tertiary fold in PrP^C facilitates specific cleavage patterns. Together, our results find that α-cleavage takes place at several nearby sites between residues 109 and 120, and that metal ions actually suppress β-cleavage, while modulating the α₁/α₂ product ratios.

Emphasizing the importance of α-cleavage to release the N1 and C1 PrP^C fragments, Oliveira-Martins et al. examined PrP^C proteolysis in a wide range of cell lines.³⁸ Production of the N1/C1 fragments was universal, thereby underscoring the physiological importance of α-cleavage. It was thought at the time that cleavage was localized exclusively to the 109↓110 position, yet replacing the residues flanking this site only lowered proteolytic yields, but did not fully eliminate it. In general, mutations producing changes in local charge or hydrophobicity exerted only a modest reduction in α-cleavage efficiency. In contrast, polypeptide deletions were effective and removal of progressively larger stretches within the segment 106–119 produced a linear reduction in cleavage, with complete deletion of this segment required to fully suppress proteolysis. The authors proposed that perhaps several PrPase enzymes might be active in this region. Following our characterization of PrP^C proteolysis, one would need to eliminate the α₁, α₂, and α₃ sites, in the segment 109–120, to fully eliminate α-cleavage. Perhaps the physiologic requirement for α-cleavage is sufficiently important to be preserved through the evolution of redundant mechanisms.

PrP^C Deletions and Toxicity

Complete elimination of the prion protein in transgenic mice is well tolerated, yet paradoxically, deletion of certain N-terminal PrP segments can be lethal. Schmerling et al. explored a series of N-terminal deletions starting at residue 32, and extending progressively toward the C-terminal domain, and found that those ending at residue 121 or beyond produce ataxia, cerebellar degeneration, and death after several months.³⁹ Interestingly, as shown by Baumann et al., retaining more of the PrP^C N-terminus (Δ94–134) leads to an even more toxic phenotype, with myelin degeneration and death within 20–30 d.⁴ Consistent with this trend, Li et al. found that mice expressing PrP^C(Δ105–125)—a deletion of only 21 residues—develop severe neurodegenerative symptoms leading to lethality approximately a week after birth.⁴⁰ In all cases, PrP^C deletion toxicity was rescued by coexpression with wild type PrP^C, with the more toxic mutants requiring higher relative levels of wild type protein.

We collected the specific sequences from studies of mouse PrP^C deletion mutants, focusing on those with the N-terminal signal peptide (residues 1–22) and the C-terminal GPI signal sequence (to ensure export and attachment to the extracellular membrane), and show them aligned in Figure 4. Sequences are ordered with benign deletion mutants at the top, moving to progressively more toxic phenotypes at the bottom. Sites for the recently characterized α₁, α₂, and α₃-cleavage are shown. There are 2 complementary patterns. First, deletions that fully remove all three α-cleavage sites are toxic, but only if N-terminal residues 23–31 are retained. Second, with deletion of the α-cleavage sites, the longer the retained N-terminal segment, the greater the toxicity, as measured by the copy number of wild type PrP^C required for rescue. Highlighting this pattern, PrP^C(Δ23–121) is not toxic, whereas PrP^C(Δ105–125) gives the most lethal phenotype. It’s interesting to compare 2 tolerated deletion proteins PrP^C(Δ104–114) and PrP^C(Δ114–121). While each possesses most of the N-terminus, but with partial elimination of the α-cleavage sites, they retain the α₂ and α₁ sites, respectively, and thus remain susceptible to ADAM8 proteolysis.

Figure 4. Summary of PrP^C deletion mutants and resulting phenotypes (sequence details summarized in Aguzzi et al. ref. 6). The N-terminal domain after removal of the signal peptide starts at residue 23, and is shown in blue, while the structured C-terminal domain, starting at approximately residue 125 is shown in green. Deleted segments, as noted by the numbering (left), are gray. Locations for α₁, α₂, and α₃-cleavage are shown. The green checks, right, indicate tolerated deletion mutants, whereas the red X’s are the lethal mutants. The number of Xs qualitatively represents the lethal potency of each mutant, as indicated by the relative amount of wildtype PrP^C required for rescue. This alignment shows that deletion of the full α-cleavage segment, 109–120, but with retention of N-terminal residues starting at position 23 confers toxicity.

PrP^C Overdrive—Toxic Deletions Eliminate α-Cleavage

Deletion studies have prompted several different hypotheses, with suggestions of a PrP^C binding partner where interaction is facilitated by a segment in the vicinity of 105–125. Either the segment alone is essential for binding⁴⁰ or, alternatively, that PrP^C binds in a bivalent fashion through its N- and C-terminal domains with a region encompassing 105–125 serving as a linker.^4,39 Another line of inquiry suggests that PrP^C(Δ105–125) facilitates membrane leakiness by forming spontaneous ion channels, an effect that is blocked by interaction with wildtype PrP^C.⁴¹

The recent appreciation of the physiologic importance of proteolysis leading to N1 and C1, along with redundant cleavage sites and enzymes targeted to 109–120, suggest a different scenario. We propose that full-length PrP^C modulates the activity of one or more nearby companion membrane proteins, such as the AMPA receptor, and that this activity is downregulated by α-cleavage. Any deletion that fully interrupts α-cleavage leads to constitutive activity of PrP^C and its binding partner, which promotes the toxic response and subsequent neurodegeneration. For example, in the case of the AMPA receptor, neurons may hyper accumulate zinc. Similarly, any molecular species that blocks protease access to the α-cleavage sites will also produce a toxic response.

Figure 5 provides a schematic using zinc transport through the AMPA receptor as an example. Considering the previous sections, we note that (1) the PrP^C N-terminus makes direct contact with the receptor, (2) the octarepeat domain is required for enhanced zinc transport through the AMPA receptor, and (3) that copper and zinc promote α-cleavage. As shown stepwise in Figure 5, and in accord with a model previously advanced by Watt et al.,⁹ Zn²⁺ binds to the PrP^C octarepeat domain, which in turn, drives the compact fold identified by Spevacek et al.²⁵ PrP^C then associates with the AMPA receptor, thorough its N-terminal segment, leading to enhanced Zn²⁺ transport. Zn²⁺ structures the segment 109–120 so that it is more susceptible to ADAM cleavage, which turns off PrP^C enhanced AMPA receptor activity. PrP^C fragments (N1 and C1) then dissociate from the AMPA receptor. The net effect of this mechanism is that PrP^C facilitates the tightly controlled trafficking of Zn²⁺ into the cell. Interestingly, an evolutionary cousin of PrP^C, the zinc transporter ZIP10 also employs a Zn²⁺ mediated interdomain interaction, as well as Zn²⁺ dependent cleavage of the N-terminus to modulate the ability of ZIP10 to uptake zinc.⁴² We note that with PrP^C, the specific enzyme responsible for α-cleavage is certain to be tissue dependent, and there is yet no evidence of significant ADAM8 activity in the brain.

Figure 5. A proposal for PrP^C regulation through α-cleavage. (A) The AMPA receptor (AMPAR), a cation channel, is one of several membrane proteins thought to be modulated by PrP^C binding. The folded PrP^C C-terminal domain (square) is GPI linked to the extracellular membrane. The N-terminal domain is unstructured in the absence of Zn²⁺. (B) Zn²⁺ binds to the octarepeats, structuring the N-terminal domain, which then associates with a negatively charged patch on the C-terminal domain. Residues 23 – 31 bind to the AMPAR facilitating Zn²⁺ transport. The PrP^C fold promoted by Zn²⁺ exposes the α-cleavage site, which becomes susceptible to the ADAM proteases (8, 10, and 17). (C) α-Cleavage releases N1, thus downregulating PrP^C facilitated Zn²⁺ transport. C-Terminal PrP^C (the C1 fragment) is degraded and replaced by full-length PrP^C. (D) Occlusion of α-cleavage (skull and crossbones) by the peptide PrP(106–126) or Aβo, for example, or by misfolding to PrP^Sc, leaves PrP in a constitutively active state, thus driving dyshomeostasis.

A consequence of the model in Figure 5 is that interfering with access to the α-cleavage would lead to persistent and elevated Zn²⁺ transport, perhaps disrupting cellular homeostasis. A partial test of toxicity arising from proteolytically resistant PrP^C would be select point mutations, as opposed to deletions, designed to suppress α-cleavage with minimal alteration of the native protein sequence or structure. We recently developed the mutants PrP^C(H110Y) and PrP^C(V120D), which block α₁- and α₃-cleavage, respectively, thereby offering strategies for disentangling the toxic responses from different cleavage patterns.⁸

The scheme presented in Figure 5 offers mechanisms for how coexpression of wild type PrP^C rescues the syndrome resulting from deletion of the proteolytic segment. Release of the wild type PrP^C N1 peptide, which alone is unlikely to facilitate zinc transport, could compete with mutant PrP^C for the AMPA receptor binding site. Similarly, full-length or C1 wild type PrP^C could associate with the AMPA receptor driving off the mutant protein.

Could this model, which posits the essential need for PrP^C inactivation by α-cleavage, help explain other experiments demonstrating neurotoxic effects and perhaps certain aspects of neurodegenerative disease? The short peptide PrP(106–126) is toxic when dosed into cultured neurons, but only if wild type PrP is present.⁴³ This particular stretch of PrP is hydrophobic and amyloidogenic and, consistent with fibril forming peptides, forms self-complementary ultra structures. Thus, it likely targets the PrP^C segment 106–126, which perfectly overlaps the α-cleavage region thereby reducing susceptibility to proteolysis. In prion disease, the prion protein misfolds and assembles into aggregates forming PrP^Sc, which is partially protease resistant. Experimental treatment of purified PrP^Sc with proteinase K cleaves at approximately residue 90,⁴⁴ suggesting that the N-terminal segment is largely exposed but that the α-cleavage region beyond residue 90 is occluded. As with PrP(106–126) this would inhibit normal proteolysis needed to inactivate PrP’s influence on the AMPA receptor. Recent work in Alzheimer disease finds that the most toxic species formed by Aβ aggregates are oligomers, often noted as Aβo.⁴⁵ In hippocampal slices Aβo inhibits long-term potentiation, but only in the presence of PrP^C; brain slices derived from PrP knockouts are resistant to Aβo toxicity.⁴⁶ Fluharty et al. have demonstrated direct binding between Aβo and PrP^C and mapped the interaction to 2 N-terminal regions, residues 23–27 and 95–105.⁴⁷ The segment 95–105 is just on the N-terminal side of the α₁-cleavage site and Aβo binding would likely block access for proteolysis. Additionally, Aβo sequesters N1, removing the ability of N1 to regulate PrP^C activation of membrane receptors. With regard to the specific activation of the AMPA receptor, emerging theories of Alzheimer disease point to the importance of maintaining zinc homeostasis, the breakdown of which promotes the formation of neurofibrillary tangles.⁴⁸

Much of our discussion above has centered on PrP^C activation of zinc transport through the AMPA receptor. However, PrP^C may have other membrane targets. For example, Um et al. find that PrP^C-Aβo couples to Fyn kinase activation through the metabotropic glutamate receptor 5 (mGluR5).⁴⁵ At this juncture, it is not clear whether PrP^C binds to mGluR5 in the absence of Aβo. PrP^C also modulates currents through the NMDA receptor in a copper-dependent fashion.⁴⁹ Specifically, copper occupied PrP^C reduces the receptor’s sensitivity to glycine, which promotes persistent cationic currents, an effect that is tempered by both monomeric Aβ and Aβo. PrP^C is trafficked by endocytosis through interaction with the LDL receptor-related protein 1 (LRP1), identifying yet another membrane partner. Taken together, PrP^C may have multiple interaction partners and the scenario we developed for inactivation may be a universal regulatory mechanism.

We have emphasized constitutive PrP^C activity through elimination of the α-cleavage sites as a primary mechanism for driving dyshomeostasis. This concept supports the notion of an executive function for the PrP^C N-terminal domain, where interference between the protein’s 2 intact domains enhances toxicity.⁵⁰ However, there is ample evidence that N1, in particular, is an important signaling species.⁵ Without α-cleavage, N1 production is eliminated, which could facilitate apoptosis. If this mechanism alone were responsible for the neurodegeneration resulting from toxic PrP^C deletion mutants, it would be difficult to understand the relatively mild phenotype resulting from PrP^C knockout.

Prion disease and Alzheimer disease are marked by a wide set of neurological derangements and it is not clear whether constitutive activity driven by hindered proteolysis of PrP^C is a major component. However, brain zinc levels are lowered in prion disease suggesting altered PrP^C function. In general, there are ongoing discussions as to whether neurodegenerative disease arises from a loss of normal protein function or a gain of function from toxic aggregates. We argue here for a third possibility where intrinsic PrP^C function is constitutively maintained thereby leading to neuronal stress. If correct, this scheme could lead to new avenues for treating neurodegenerative processes.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Eric G.B. Evans for suggestions and editorial comments on the manuscript. This work was supported by a grant from the National Institutes of Health (GM065790).

10.4161/pri.28796