Keywords

2.1 Introduction

Research over the last decade continues to find remarkable functional roles for the normal cellular form of the prion protein (PrPC). PrPC supports myelin development (Bremer et al. 2010), influences sleep–wake cycles (Tobler et al. 1996), is upregulated at sites of ischemic injury (McLennan et al. 2004), promotes neuron development (Kanaani et al. 2005) and protects nerve cells against chemical and oxidative assaults (Rachidi et al. 2003; Klamt et al. 2001). Although one cannot yet assign a sole function to PrPC as, say, a signaling molecule, enzyme or transporter, it is clear that the protein is required for normal neurological function. Most functional investigations link PrPC to metal ion binding, specifically to copper and zinc. This link was recently emphasized in an elegant X-ray fluorescence study that examined the spatial location and relative levels of iron, copper, and zinc in mouse brain (Pushie et al. 2011). Comparison of wild-type, PrP knockouts (KO) and 20X overexpressers revealed remarkable differences in specific brain regions, with each metal ion exhibiting a unique PrP-dependent profile. For example, PrP appears to drive copper levels near the ventricles and thalamus, whereas zinc is upregulated in cortical regions. And while there is scant evidence suggesting that PrP directly binds iron, levels are nevertheless influenced by PrP expression, perhaps suggesting a relationship between distinct metal transporters, as established in yeast (Bleackley and Macgillivray 2011).

This chapter will begin with a brief historical review of the PrP metal ion literature, with emphasis on works that frame current thinking. Next, I will describe the biophysical features of the copper and zinc sites in PrPC. Unlike most other metal binding proteins that present a unique high affinity site, PrP responds dynamically with a rich variation of coordination modes that depend on metal concentration and the presence of competing species. Recognition of these distinct coordination modes provides new insight into inherited disease resulting from octarepeat inserts. Finally, I will describe new electrochemical work that not only provides a detailed characterization of PrP–copper redox properties, but also suggests a mechanism for PrP-mediated signaling.

2.2 Brief History

PrPC is able to bind both copper and zinc, but most studies emphasize the specific interaction with Cu2+. (Copper possesses two common, biologically relevant oxidation states: Cu+ and Cu2+.) Hornshaw et al. recognized that the histidine-rich octarepeat domain, containing four tandem PHGGGWGQ segments, would likely bind Cu2+, and demonstrated this directly with mass spectrometry (Hornshaw et al. 1995a). Moreover, they showed a persistent 1:1 complex, although it was also noted that the OR region could take up additional equivalents. Next, using circular dichroism (CD), which detects conformational changes, and fluorescence quenching, they estimated a Cu2+ dissociation constant in the low micromolar range (Hornshaw et al. 1995b).

In 1997, Brown et al. published a remarkable study that clearly identified a physiological connection between PrP and copper (Brown et al. 1997). First, using a peptide corresponding to the PrP N-terminal domain, PrP(23–98), they showed that the protein takes up multiple Cu2+ equivalents with positive cooperativity, described by an unusually high Hill coefficient. Estimated affinity was higher than initially found by CD, as reflected in a low, submicromolar dissociation constant. Brown and colleagues further compared brain copper levels between wild-type and KO mice, and reported a severe reduction in brain copper in the transgenics. Many aspects of this work have been revisited in the last 15 years, but there is little doubt that this initial publication firmly established PrPC as a copper metalloprotein.

The lowered copper content in the mouse KO suggested that perhaps PrPC functions as a transporter. PrPC is attached to membrane surfaces through a GPI anchor and is cycled from the extracellular space to early endosomes through endocytosis, with approximately 90% of the protein returned to the surface by exocytosis. As monitored in N2a mouse neuroblastoma cells, Pauly and Harris showed that addition of 200 μM copper stimulated rapid PrPC internalization, while removal of the metal ion allowed the protein to redistribute back to the membrane surface (Pauly and Harris 1998). Elimination of the octarepeats, or the His residues within the repeats, fully disrupts these copper-dependent processes (Perera and Hooper 2001). Similarly, certain mutations in the octarepeat domain that give rise to familial prion disease also interfere with copper-stimulated endocytosis (Perera and Hooper 2001). Collectively, these findings suggest that PrPC may play a key role in copper trafficking. However, early examinations of tissue copper, and copper protein activity, in brain fractions derived from wild-type and transgenic mice possessing different levels of PrPC, failed to find a correlation between PrPC expression and copper content (Waggoner et al. 2000). Consequently, this promising line of research did not progress. However, the X-ray fluorescence imaging work described in the Introduction, certainly motivates a renewed look at the role of PrPC in neuronal copper distribution.

In parallel to cellular assays were several notable structural and biophysical investigations (Stöckel et al. 1998; Garnett and Viles 2003; Viles et al. 1999; Valensin et al. 2004; Aronoff-Spencer et al. 2000; Burns et al. 2002, 2003; Chattopadhyay et al. 2005; Van Doorslaer et al. 2001). Early work focused primarily on the octarepeat domain, although newer research finds copper sites outside this region. Viles et al. performed a wide array of spectroscopic experiments including CD, nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR) (Viles et al. 1999). This work demonstrated a 1:1 stoichiometry between each histidine (His) containing repeat segment and Cu2+, and suggested a micromolar dissociation constant. Moreover, they identified a strong pH dependence, with tight copper binding only at pH 6.0 and above. These findings have endured many follow-up studies. To account for cooperative uptake, they proposed a ring-like structure of alternating His imidazole side chains and Cu2+ ions. While there is precedence for this type of structure in the inorganic chemistry literature, it is now considered unlikely to be a significant biological conformation.

Most copper binding proteins exhibit a very high affinity, reflected by a low dissociation constant (K d). For example, the K d for copper at the active site of superoxide dismutase is approximately 10–14 M. Early work with PrP N-terminal peptides pointed to a much weaker affinity, suggesting that perhaps PrP might not take up copper in vivo. This was addressed with detailed MS and fluorescence assays to carefully assess copper binding thermodynamics in full-length PrP (Kramer et al. 2001). Analysis of the observed fluorescence quenching revealed both affinity and detailed stoichiometry, with five Cu2+ per protein. Copper uptake showed positive cooperativity with the last equivalent exhibiting a K d of  ∼  2 μM, well below the level of Cu2+ in blood estimated at 18 μM. It is not clear, though, how relevant the comparison to blood copper levels is, given that high levels of PrP are localized to extracellular presynaptic surfaces in the CNS (Herms et al. 1999). As will be discussed, more recent analyses find specific binding modes that display very high affinity, below 1.0 nM, and thus eliminate doubt that PrP takes up Cu2+ in vivo.

2.3 Features of Cu2+ and Zn2+ Coordination in PrP

Copper binds within PrP’s N-terminal region, with the relevant segment from the human sequence shown below:

PrP(51–111) PQGGGGWGQP H GGGWGQP H GGGWGQP H GGGWGQP H GGGWGQGGGTHSQWNKPSKPKTNMKH

There are five tandem eight-residue repeats, each with the canonical sequence PXGGGWGQ, but in the first repeat, a Gln fills the X position. Since histidine, with its imidazole side chain, is required for copper uptake, the first repeat does not participate. Thus, from a sequence or genetics perspective, there are five N-terminal octarepeats, but from a metal ion coordination perspective, there are four (underlined in the sequence). Beyond the octarepeat domain, copper also interacts with high affinity at the His residues at positions 96 and 111 (Walter et al. 2009; Jones et al. 2005). The current consensus is that all copper coordination is within the segment PrP(61–111) (human) bounded by the histidines (His, bold H) in the sequence shown above.

A number of early investigations used peptide design, NMR, mass spectrometry, circular dichroism, Raman spectroscopy, molecular modeling, and related biophysical approaches to develop insight into the structure of the Cu2+–octarepeat complex. Ultimately, though, EPR provided the essential insights leading to the current models. EPR is sensitive to the chemical environment at paramagnetic Cu2+ centers and, through hyperfine couplings to copper’s unpaired electron, can directly reveal nearby nuclei and atomic features of the coordination environment. Details of the relevant EPR techniques have been reviewed elsewhere (Millhauser 2004, 2007); a summary of the coordination features is given in Fig. 2.1. The copper coordination environment depends critically on the ratio of copper to protein. At low copper concentration, the four octarepeat His imidazole side chains bind simultaneously to a single Cu2+, as shown in the figure and inset (Chattopadhyay et al. 2005). This is often referred to as the low occupancy binding mode or “component 3,” based on component analysis of the EPR spectra. The affinity for this mode is very high, with a dissociation constant of approximately 0.10 nM (Walter et al. 2006).

Fig. 2.1
figure 00021

Structural features of PrPC at low and high Cu2+ concentrations. The C-terminal domain is helical, whereas the N-terminal domain is flexible and able to restructure to accommodate different copper coordination modes. At low [Cu2+], the metal ion coordinates to sites localized to His96 and His111. In addition, a single equivalent of Cu2+ binds within the octarepeat domain, coordinated by the four His imidazole side chains (“component 3,” details shown in the inset). The affinity in the octarepeat domain is high, as characterized by a low K d of approximately 100 pM. At high [Cu2+], the octarepeat domain restructures to take up four copper equivalents, each coordinated to single His side chain and backbone nitrogens (“component 1,” inset). The affinity for this coordination mode is lower than that of component 3

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At intermediate Cu2+ concentration, the octarepeats take up two copper equivalents, with each coordinated by two His side chains (not shown) (Chattopadhyay et al. 2005). At high copper concentrations, the octarepeat domain saturates at 4 equiv., with each His binding to a single Cu2+, as shown in Fig. 2.1 (Aronoff-Spencer et al. 2000; Burns et al. 2002, 2003; Chattopadhyay et al. 2005). This high occupancy binding mode is referred to as “component 1.” The copper affinity for this state is lower than that of component 3, with a dissociation constant of approximately 10 μM (Walter et al. 2006). The specific coordination features of this high occupancy site, shown in the inset, were determined by isotopic labeling, in combination with a range of EPR techniques (Aronoff-Spencer et al. 2000), and confirmed by X-ray crystallography of the Cu2+–HGGGW complex (Burns et al. 2002).

The specific features of the component 1 site are unusual compared to previously characterized protein copper sites. In most copper metalloproteins, the metal ion is coordinated to His or Cys side chains. For example, copper superoxide dismutase contains the metal ion with four tetrahedrally placed His imidazoles. As seen in the inset, the Cu2+ ion coordinates to the His side chain, the deprotonated amide nitrogens of the two Gly residues that immediately follow the His, and a Gly carbonyl. In addition, there is an axially coordinated water molecule that hydrogen bonds to the Trp indole hydrogen (not shown). A coordination sphere with deprotonated amides has been seen previously with the N-terminal copper binding segment of albumin (Harford and Sarkar 1997), and also in peptides, but not in the interior polypeptide segments of a protein. The involvement of amide nitrogens confers significant pH sensitivity since an increase in the H+ concentration (lower pH) protonates at the nitrogen and competes with copper complexation. Consequently, high occupancy copper binding is unstable below pH  ∼  6.0. It has been proposed that this might provide a chemical mechanism for release of Cu2+ in the endosomal compartments (Burns et al. 2002).

In addition to Cu2+ uptake in the octarepeats, there are two additional binding sites localized to His96 and His111 (human PrP numbering), and these also exhibit sub-nanomolar affinity. These two sites are often referred to as the “5th sites,” since early studies suggested only the involvement of His96, beyond that of the four sites in the octarepeat domain (Burns et al. 2003). We prefer to label these as “non-octarepeat” coordination sites, thus underscoring their distinct location and chemical properties (Walter et al. 2009). At both of these non-octarepeat sites, copper coordinates to the imidazole side chain, the His backbone nitrogen, and two additional backbone nitrogens from the residues on the N-terminal side of the His (Burns et al. 2003). Affinity at these sites is high, with a K d that is similar to that found for the multi-His component 3 mode in the octarepeat domain. Titration studies show that these non-octarepeat sites take up copper simultaneously with component 3 (Walter et al. 2009). Once PrPC is saturated with Cu2+, the octarepeat domain restructures to component 1 coordination thus enabling additional binding equivalents, as shown in Fig. 2.1.

Like copper, zinc also binds to PrPC and stimulates endocytosis (Pauly and Harris 1998). Because this metal ion is found only as diamagnetic Zn2+, EPR is of limited use in directly evaluating its coordination features. To address this, we applied two different approaches (Walter et al. 2007). First, using an octarepeat peptide, as well as full-length PrPC, we competed Zn2+ against Cu2+ and monitored by copper EPR. Interestingly, we found that regardless of concentration, Zn2+ was not able to displace Cu2+, which shows that copper has a much higher affinity than zinc. However, Zn2+ was able to influence the Cu2+ coordination mode, shifting the distribution to favor component 1 binding. Next, we tested Zn2+ coordination to a range of octarepeat-derived peptides and monitored binding with the reagent diethylpyrocarbonate (DEPC). DEPC chemically modifies free imidazole groups, but only if they are not involved in metal ion coordination. Analysis by mass spectrometry showed protection against DEPC modification only with the full octarepeat domain. Collectively, these experiments demonstrate that Zn2+ coordinates to the four octarepeat His imidazoles, equivalent to that observed for Cu2+ in its low occupancy mode. With a K d of approximately 200 μM, the affinity is substantially lower than any of the coordination modes found for Cu2+. However, because Zn2+ competes with Cu2+, it is able to influence copper coordination in a concentration-dependent fashion. These results, summarized in the scheme in Fig. 2.2, show that when copper levels are low, PrP can simultaneously bind both copper and zinc. At higher copper levels, the protein accommodates the zinc by shifting to the high occupancy binding mode that minimizes the ratio of histidines to copper. However, when no rearrangement can accommodate both zinc and the available copper, it is the zinc that is displaced, not the copper. These results are consistent with previous screens that identified copper and zinc as the sole biologically relevant metal ions that coordinate to PrPC, and perhaps suggest mechanisms by which both metal ions may stimulate endocytosis. What is also clear is that copper exhibits a substantially higher coordination affinity, thus arguing against zinc as the dominant species in PrP metallobiochemistry.

Fig. 2.2
figure 00022

Models representing metal binding in the N-terminal domain of PrP. Top row (high zinc); zinc (red) is bound by the octarepeat region (left), while non-octarepeat sites (H96 and H111) are available for copper binding (blue, middle). Copper at high concentration will displace zinc from octarepeats to form up to 4 equiv. of component 1 (right). Bottom row (low zinc); copper (blue) is bound by the octarepeats in component 3 when copper is low (left), with increasing copper loads in the non-octarepeat sites (middle). High copper (right column) results in component 1 copper binding by the octarepeats. Approximate molar metal concentrations are shown in the arrows

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2.4 A Role for Altered Copper Coordination in Octarepeat Expansion Disease

Approximately 10–15% of human TSE cases are inherited and arise from mutations in the PRNP gene (Prusiner 2004). Of these, most are missense mutations in the folded C-terminal domain. For example, the E200K mutation causes midlife development of CJD, with most patients dying 6–24 months after onset (Colombo 2000). In addition to these point mutations are insertional mutations of one to nine PHGGGWGQ segments in the octarepeat domain (Goldfarb et al. 1991). This class of mutations is enigmatic insofar that they modify a region of the protein that is not essential for propagating prion disease. Treatment of PrPSc with proteinase K cleaves the protein at approximately residue 90, thereby removing the octarepeat domain, but the remaining protease resistant aggregate retains infectivity. Despite these results, early studies with transgenic mice showed that the PrP octarepeats modulate the disease process. Specifically, inoculated mice expressing a modified PrPC lacking residues 32–93 develop disease with longer incubation times than wild type, produce tissues with lower prion titers, and exhibit a reduced presentation of prion plaques (Flechsig et al. 2000).

Disease progression in individuals with octarepeat expansions depends on the number of inserts. Individuals with one to four extra octarepeats develop disease with an average onset age of 64 years, whereas five to nine extra octarepeats result in an average onset age of 38 years, a difference of almost three decades (Croes et al. 2004; Kong et al. 2004). A number of previous studies examined the biophysical properties of expanded octarepeat domains with emphasis on either the rate of amyloid production or its uncomplexed backbone conformation (Leliveld et al. 2006, 2008; Dong et al. 2007). However, none of these identified a quantitative link between octarepeat length and age of disease onset.

Given the profound influence of octarepeat domain length on expansion disease, we explored whether the domain’s response to copper is altered by insertion number (Stevens et al. 2009). We also reevaluated all known cases of human prion disease resulting from octapeptide insertions, and compared the findings to biophysical studies that examined the balance between component 1 and component 3 coordination, as a function of octarepeat domain length. Beginning with statistical data from two existing studies (Croes et al. 2004; Kong et al. 2004), we surveyed the recent clinical literature, pooled the data, and established a new data set covering approximately 30 families and 108 individuals. Onset age for individual cases is shown in Fig. 2.3a. The red line is drawn at 55.5 years. All cases up to four octarepeat inserts (eight repeats total) are above this line and 96% of the cases of five or more octarepeat inserts are below the line. Although there is significant scatter in reported onset age for each specific octarepeat length, the dramatic shift to early onset disease between four and five inserts is apparent. A detailed statistical analysis shows that the results are indeed consistent with the presence of two groups, one composed of individuals with one to four OR inserts and another of individuals with five to eight inserts.

Fig. 2.3
figure 00023

The relationship between onset age for familial prion disease resulting from octarepeat inserts and copper coordination modes. (a) Onset age for individual cases as a function of extra octarepeat inserts. Note that wild type corresponds to four repeats, so three inserts correspond to seven total repeat segments. The horizontal red line is at 55.5 years and represents a statistically defined separation between late and early onset. (b) Average onset age, with standard deviation (blue circles, left axis), and component 1 coordination (orange diamonds and red squares, right axis, for 3.0 and 4.0 equiv. Cu2+, respectively) as a function of extra octarepeat inserts. At both copper concentrations, component 1 coordination drops suddenly at approximately the same OR length threshold as average onset age

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We then performed EPR analysis on a series of PrP-derived constructs from four to nine repeats, corresponding to zero to nine insertions. The experiments showed that domains with four to seven repeats (i.e., zero to three insertions) behave much like the wild type. However, constructs of eight or nine repeats exhibit persistent component 3 coordination. Moreover, these constructs take up approximately twice as much copper as wild type. Equivalent trends were observed with full-length recombinant protein, where we compared wild type with mutant PrPC containing five repeat inserts. To underscore these findings, we compared the average onset age and standard deviation, as a function of octarepeat length, to Cu2+ binding properties. The longest OR expansions favor component 3 coordination and resist component 1. Thus, component 1 coordination serves as a convenient measure of altered Cu2+ binding properties. Figure 2.3b shows the relative population of component 1 coordination for each OR construct superimposed on the average age of onset. For wild type and expansions involving up to seven repeats (three inserts beyond wild-type), component 1 coordination is dominant for both 3.0 and 4.0 equiv. Cu2+. However, at eight and nine ORs (four and five inserts, respectively), the population of component 1 coordination drops precipitously.

These data reveal a remarkable relationship, where decreased onset age and persistent component 3 coordination take place at a threshold of eight or more total repeats. In turn, our findings suggest an important protective role for component 1 coordination that may be lost in cases of octarepeat expansion disease with four or more inserts. Together, these findings motivate a careful examination of the distinct chemical properties and reactivity of component 1 vs. component 3 copper coordination.

2.5 Electrochemical Properties of the PrP Copper Sites

Copper’s ability to cycle between the Cu+ and Cu2+ oxidation sites is essential for life. For example, cellular respiration relies on cytochrome c oxidase, a copper-dependent enzyme that converts molecular oxygen to water, ultimately leading to the production of ATP. Since the earliest studies connecting PrPC to copper uptake, there has been interest in understanding reduction–oxidation (redox) cycling at the copper sites. One line of enquiry suggests that PrPC functions as a superoxide dismutase (SOD), which inactivates toxic O −2 , converting it to the more benign hydrogen peroxide (H2O2). This hypothesis has been controversial, and is reviewed elsewhere (Brown 2009; Daniels and Brown 2002). The connection between copper coordination mode and onset age for octarepeat expansion disease, discussed above, certainly motivates an evaluation whether component 1 and component 3 coordination sites give rise to distinct redox properties.

Initial electrochemical studies used cyclic voltammetry to evaluate short single repeat peptides as models of component 1 coordination (Bonomo et al. 2000). Reduction of Cu2+ to Cu+ was found to be energetically unfavorable, leading to the possibility that PrPC may stabilize copper in its oxidized form. From a neuroprotective perspective, this could be important since weakly complexed copper readily cycles between oxidation states, resulting in the production of reactive oxygen species that are often cytotoxic. By stabilizing copper in a single oxidation state, PrPC may quench this deleterious chemistry.

Component 3 coordination, with four His residues, appears somewhat similar to the active site in SOD and initially suggested that it might readily undergo redox cycling. Redox kinetics, as measured by bathocuproine absorbance, suggested that indeed component 3 was more easily reduced than component 1 (Miura et al. 2005). Building from these results, it was proposed that PrPC might function in concert with endocytosis as a copper reductase. In this scenario, extracellular Cu2+ binds to PrPC with component 1 coordination, and the complex is internalized by endocytosis. Next, the low pH drives rearrangement in the octarepeat domain to favor component 3 coordination, leading to reduction to Cu+. Finally, the copper is released and internalized through a copper transporter.

In a collaborative work with Zhou and coworkers, we recently revisited the detailed electrochemical features of the component 1 and component 3 coordination modes (Liu et al. 2011). The full octarepeat domain with 1 equiv. of Cu2+ served as a model for component 3 coordination. Cyclic voltammetry performed in the presence of ascorbate, with and without oxygen, and under nearly reversible conditions showed facile reduction to Cu+, along with a significant increase in affinity. Thus, as opposed to cycling copper, these data suggest that Cu+ is very stable in this low occupancy mode, and unlikely to be reoxidized back to Cu2+. Next, we used the same conditions to examine component 1 coordination and found reduction potentials consistent with a copper center that supports cycling between its oxidation states. However, when we compared the findings to free copper, or simple copper–peptide complexes like those found in blood or cerebral spinal fluid, we observed that the reaction was controlled and less likely to produce cytotoxic species such as hydroxyl radicals. Additional assays demonstrated that copper bound to PrP with component 1 coordination, under reducing conditions by ascorbate, gently converts dissolved oxygen to hydrogen peroxide. A summary of these findings is shown in Fig. 2.4.

Fig. 2.4
figure 00024

Schematic representation of the possible roles of PrPC–Cu2+ complexes in quenching the Cu2+ redox cycling or gradual production of H2O2 for signal transduction. PrP is tethered to cell membrane via the GPI anchor (green) with its α-helices in the C terminus shown in orange, N-linked carbohydrates in purple, and the N-terminal copper binding segment depicted in white. When [Cu2+] is at a low level (nM or lower), Cu2+ (blue sphere) remains bound in the component 3 mode (left), quenching the Cu2+ redox cycling. At higher [Cu2+] (μM), the binding mode transitions to component 1 (right), leading to a gradual and controlled production of H2O2”(Coordinates for the PrPC-terminal domain, along with carbohydrates, GPI anchor, and membrane, were kindly provided by Professor Valerie Daggett (U. Washington))

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The ability to bind copper and facilitate redox cycling is shared with the Aβ peptide and α-synuclein, which are causative in Alzheimer’s and Parkinson’s disease, respectively. Unlike PrPC, however, these species exhibit only a single binding mode and, therefore, a single profile for producing hydrogen peroxide. Comparing these two neurodegenerative species with PrPC, we find that component 3 is by far the least reactive, producing hydrogen peroxide at the lowest rate, whereas component 1 is the most reactive (Liu et al. 2011). Thus, PrPC exhibits vastly different electrochemical profiles, depending on copper occupancy. Both modes are neuroprotective with component 3 coordination completely inhibiting copper redox activity, and component 1 regulating activity with the controlled formation of hydrogen peroxide.

Together, these findings support a role for PrPC in suppressing copper’s inherent redox activity that would otherwise be very damaging to cellular components. However, the discovery that high copper occupancy PrPC produces hydrogen peroxide suggests additional biochemical control. Similar to nitric oxide, hydrogen peroxide is now considered a signaling species of particular importance in the immune system and also in protein localization (Veal et al. 2007). There are likely several possible mechanisms for H2O2 action. However, PrPC has been linked to transmembrane signaling (Mouillet-Richard et al. 2000) and it is noteworthy that hydrogen peroxide readily crosses membrane bilayers and inactivates phosphatase and kinase active sites by reaction with catalytic residues.

The cumulative findings reviewed here emphasize the complex connection between zinc and copper uptake, and the variability in copper binding as controlled by concentration. The relationship between copper coordination modes and onset age for prion disease, resulting from octarepeat expansion, suggests that metal ion regulation may also factor into the development of disease. New electrochemical findings provide a foundation for understanding how PrPC protects cells against oxidative assaults, and also reveal a possible mechanism for transmembrane signaling. Further refinement of these concepts is sure to lead to a precise function for PrPC and perhaps insight into how the loss of function contributes to neurodegenerative disease.