Prion Protein at the Crossroads of Physiology

PRION PROTEIN AT THE CROSSROADS OF PHYSIOLOGY AND DISEASE Abstract The presence of the cellular prion protein (PrPC) on the cell surface is critical for the neurotoxicity of prions. Although a number of biological activities have been attributed to PrPC, a definitive demonstration of its physiological function remains elusive. In this review, we will discuss some of the proposed functions of PrPC, focusing on recently suggested roles in cell adhesion, regulation of ionic currents at the cell membrane, and neuroprotection. We will also discuss recent evidence supporting the idea that PrPC may function as a receptor for soluble oligomers of the amyloid β peptide and possibly other toxic protein aggregates. These data suggest surprising new connections between the physiological function of PrPC and its role in neurodegenerative diseases beyond those caused by prions. Introduction Prion diseases are a group of rare and invariably fatal neurodegenerative disorders of humans and animals, characterized by dementia, motor dysfunction, and cerebral amyloidosis. These disorders include Creutzfeldt-Jakob disease and kuru in humans, as well as bovine spongiform encephalopathy and scrapie in animals. Prion diseases can arise sporadically, as a result of mutations in the gene encoding the prion protein, or by infection from exogenous sources. A great deal of evidence indicates that the key event underlying all forms of prion diseases is conformational conversion of a normal cell surface glycoprotein called PrPC (cellular prion protein) into an aggregated, β-sheet-rich isoform called PrPSc (scrapie prion protein) (Box 1) [1]. The infectious spread of prions occurs via PrPSctemplated conversion of an endogenous pool of PrPC molecules, a process that has analogues in self-propagating proteins described in other species [2]. BOX 1 One protein, two forms: PrPC and PrPSc PrPC is the cellular form of the prion protein (Figure Ia). It is an endogenous glycoprotein that is expressed at highest levels in the CNS, and present in a wide range of species from fish to mammals [100–102]. The three-dimensional structure of PrPC includes a disordered N-terminal domain (residues 23–124, numbering for mouse PrP) and a C- Page 2 terminal globular region (residues 125–228) composed of three α-helices and two short β-strands (Figure Ib) [99, 103]. The N-terminal half encompasses a polybasic region (residues 23–27) and a series of histidine-containing octapeptide repeats (residues 51–90) that can bind metal ions like Cu2+ [104]. The central region encompasses a charged region (residues 105–111) followed by a highly conserved hydrophobic domain (residues 112–130) which serves as a transmembrane anchor in certain situations [69]. During its biosynthesis in the ER, the N-terminal signal peptide (residues 1–22) is removed and a GPI anchor is attached at residue 230 [105]. Two N-linked oligosaccharide chains are also added (at Asn-180 and Asn-196) [106]. Most PrP is found on the cell-surface where it is localized to lipid rafts, although a fraction is endocytosed via clathrin-coated pits [62, 107]. Some of the protein is proteolytically cleaved by cellular proteases near residue 111 to generate N- and C-terminal fragments called N1 and C1, respectively [108, 109]. PrPSc is the infectious isoform of the prion protein. It has the same amino acid sequence as PrPC, but has a higher content of β-sheet structure (Figure Ic), and is relatively resistant to protease digestion [110]. PrPSc acts as a molecular template by physically interacting with PrPC and converting the latter to more molecules of PrPSc. It is this process which accounts for the self-propagating nature of infectious prions [1]. A prionlike propagation mechanism has been described for several proteins in yeast and fungi (reviewed in [111, 112]). Furthemore, such a propagation mechanism may play a role in the neuroanatomical spread of protein aggregates in other neurodegenerative diseases like Alzheimer’s, Huntington’s, and Parkinson’s diseases (see [113] for a recent review). Despite compelling evidence for this mechanism of prion propagation, our current understanding of the primary causes of neurodegeneration in prion diseases is still limited. Several kinds of experiments suggest that PrPSc, or other pathological PrP conformers, require physiologically active PrPC on the cell membrane in order to exert their toxicity [3]. Interestingly, recent evidence indicates that PrPC may also mediate the toxicity of amyloid beta (Aβ) oligomers that are associated with Alzheimer’s disease (AD) [4], and possibly other β-rich protein aggregates [5]. Therefore, defining the physiological activity of PrPC is not only crucial for understanding the pathogenesis of prion diseases, but may also provide fundamental insights into the pathogenesis of other neurodegenerative disorders caused by misfolded protein assemblies. Here, we review recent findings that have provided fresh insights into potential physiological activities of PrPC, and how these might be subverted to produce pathological effects. A dual role for PrPC in prion diseases It is widely agreed that PrPC plays a crucial role in the pathogenesis of prion diseases by virtue of its ability to serve as substrate for generation of PrPSc. However, a number of reports have underscored the distinction between prion infectivity and prion toxicity, and provided evidence that alterations in the normal function of PrPC may lie at the root of prion-induced neurodegenerative processes. In particular, recent experiments suggest that accumulation of infectivity and neurodegeneration proceed in distinct chronological and mechanistic phases. Infectivity accumulates relatively rapidly, and requires only a minimum expression level of PrPC, while neurodegeneration takes much longer and is directly dependent on the amount of PrPC expressed in the brain [6]. A dramatic demonstration of the dissociation of infectivity and pathology is the observation that genetically depleting neuronal PrPC in mice with established prion infection reverses neuronal loss and progression of clinical signs, despite the continuous production of infectious PrPSc by surrounding astrocytes [7]. Similarly, the absence of endogenous PrPC renders host brain tissue resistant to the toxic effects of PrPSc emanating from implanted graft tissue [8]. Finally, the absence of the glycolipid membrane anchor on PrPC has been shown to cause Page 3 dramatic changes in the characteristics of scrapie-induced illness [9, 10]. Taken together, these lines of evidence indicate that the toxicity of PrPSc requires the presence of membraneanchored PrPC at the cell surface, and suggest that the normal, physiological activity of the protein could be subverted to create toxicity. Previous attempts to understand the function of PrPC Although PrPC was first identified as a normal cellular protein almost 20 years ago [11], its physiological function has remained uncertain. The creation of mice carrying targeted disruption of the gene encoding PrP represents a possible approach to this problem. Since 1992, several lines of PrP knockout mice have been engineered [12, 13]. These animals display resistance to prion infection, as predicted by the prion hypothesis, since they lack PrPC molecules that serve as substrates for the generation of PrPSc [14]. With only one notable exception, however (due to artifactual upregulation of an adjacent gene [15]), PrP knock-out mice display no major developmental or anatomical abnormalities, and lead a normal lifespan [12]. However, some of these animals have been reported to exhibit subtle phenotypic abnormalities at the behavioral and cellular levels (reviewed in [16]), for example alterations in olfactory function [17] or myelination [18]. As yet, the molecular basis for these defects is unclear. PrPC and cell adhesion: new insights from fish Several of the proposed functions of PrPC are related to the localization of the protein on the cell surface. Previous work has suggested that PrPC could act as a cellular adhesion molecule, participating in several interrelated processes including neurite outgrowth, neuronal survival, and neuronal differentiation (reviewed in [19]). Consistent with this idea, PrPC has been found to associate with several surface proteins including laminin [20], the 37 kDa laminin receptor precursor (LRP)/67 kDa laminin receptor [21, 22], and the neural cell adhesion molecule (NCAM) [23] (Figure 1a, b). The latter was recruited by PrPC into lipid rafts, producing activation of fyn kinase and promoting cell adhesion and neurite outgrowth [24]. PrPC was also found to bind to the contactin-associated protein (Caspr), a neurite outgrowth-inhibitory factor [25]. This interaction prevented Reelin-mediated shedding of Caspr from the cell surface, which resulted in increased surface levels of Caspr and potent inhibition of neurite outgrowth in cultured cerebellar neurons. Compelling evidence for an involvement of PrPC in cell adhesion has emerged recently from a niche of the animal kingdom that was not previously a focus of prion research: the zebrafish Danio rerio [26]. The zebrafish genome contains two PrP genes, called PrP-1 and PrP-2, both encoding glycosylphosphatidylinositol (GPI)-anchored proteins structurally similar to mammalian PrP. The two proteins differ in their spatiotemporal expression patterns, with PrP-1 being expressed primarily at cell contacts during early embryogenesis, while PrP-2 is up-regulated later in the developing nervous system where it is more widely distributed on the cell membrane [26]. Interestingly, experimental depletion of PrP-1 caused gastrulation arrest, a phenotype that was partially rescued by re-introduction of PrP-1, PrP-2 or mouse PrP [26], suggesting that a basic biological activity of PrPC is highly conserved from fish to mammals (Figure 1c, d). Further analysis revealed a role for PrP-1 in modulating cell adhesion by influencing delivery of E-cadherin to the plasma membrane, and by directly serving as a homophilic adhesion molecule (Figures 1e, f). These experiments demonstrate that PrPC function can be studied in zebrafish, a model vertebrate organism that allows direct visualization of developmental events, as well as ease of performing genetic and chemical screens. Importantly, and in contrast to mice, ablation of PrP-1 or PrP-2 produces robust loss-of-function phenotypes that can be dissected in greater detail at the molecular level. Page 4 A connection between PrPC, ion channels, and neuronal excitability Several lines of evidence support the idea that PrPC may play a role in the regulation of ion channels and neuronal excitability. PrPC is localized at synaptic sites where many kinds of ion channels are concentrated, and there is evidence that PrPC is important for normal synaptic development and function [27, 28]. In addition, a variety of electrophysiological abnormalities have been described in cerebellar or hippocampal neurons derived from PrP knock-out mice [29–38]. Perhaps the most compelling evidence for a functional and physical interaction between PrPC and receptors concerns receptors for the excitatory amino acid glutamate. Aberrant activation of the NMDA subtype of ionotropic glutamate receptors is thought to cause nerve cell damage and death in a number of acute and chronic neurological conditions by allowing abnormal entry of Ca2+ ions into neurons, a mechanism referred to as excitotoxicity [39]. In one recent study, hippocampal neurons from Prn-p0/0 mice were found to display enhanced NMDA-induced currents, an effect that was reversed by overexpression of PrPC [40]. Coimmunoprecipitation of PrPC and the NR2D subunit of the NMDA receptor suggested a direct modulation of NMDA receptors by PrPC. Additional evidence connecting PrPC to glutamate-mediated excitotoxicity has emerged from another recent study of transgenic (Tg) mice expressing a PrP form deleted for the central region (residues 105–125, referred to as ΔCR PrP) [41]. A neuropathological hallmark of these mice is massive degeneration of cerebellar granule neurons, which display a unique ultrastructural morphology, typical of non-apoptotic neuronal death occurring after excitotoxic stress [42]. There is also evidence that PrPC modulates the activity of kainate receptors [43] and metabotropic glutamate receptors [44], suggesting that PrPC may serve to suppress neuronal excitability mediated by multiple kinds of glutamate receptors. Other recent findings suggest the surprising possibility that the PrP molecule itself might form ion channels in the cell membrane, and that the currents associated with these channels might be responsible for certain kinds of PrP-related neurodegeneration. In particular, PrP molecules carrying neurotoxic deletions in the central region (such ΔCR PrP) have been found to induce spontaneous ionic currents in a variety of transfected cell lines [45, 46] (Figure 2a, b). Similar currents were induced by several different point mutations in the central region that are linked to familial prion diseases in humans [45, 46]. These spontaneous ionic currents were silenced by co-expression of wild type (WT) PrP (Figure 2c), parallel to the ability of WT PrP to suppress the neurodegenerative phenotypes of transgenic mice expressing deleted forms of PrP (see below). The biophysical features of the induced currents suggested that they are produced by a non-selective, cation-permeable channel or pore in the cell membrane. Notably, the currents were observed in both neuronal and non-neuronal cell lines derived from a variety of species, ranging from insects to mammals. Therefore, either mutant PrP regulates the activity of an endogenous channel that is widely expressed, or the PrP molecule itself forms a channel or pore in the membrane, independent of other cellular proteins (Figure 2b). Neuroprotective effects of PrPC Despite the fact that propagation of PrPSc is associated with neurodegeneration, several lines of evidence suggest that PrPC may have neuroprotective and pro-survival functions. Overexpression of PrPC has been shown to protect cell lines and primary neurons from several kinds of apoptotic stimuli [47–51]. Moreover, there is evidence that PrPC plays a role in regulating intracellular signaling cascades, including those mediating cellular survival [52]. A direct role for the N-terminal region of PrPC in protecting cells from reactive oxygen species (ROS) induced by serum deprivation has also been recently Page 5 proposed [53]. Finally, a cytoplasmic co-chaperone molecule called stress-inducible protein 1 (STI1) has been reported to be secreted from cells, where it binds to the central region of PrPC (residues 113–128) and promotes neuronal survival and differentiation [54]. The PrPCSTI1 complex was also recently implicated in self-renewal of neural progenitor/stem cells [55], a role that is consistent with a previously identified function of PrPC in regulating neural precursor proliferation during mammalian neurogenesis [56]. Collectively, these data suggest a role for PrPC, in particular its N-terminal and central regions, in protection from cellular stress, as well as neuronal survival, differentiation and proliferation. Neuroprotection and neurotoxicity: two sides of the same coin? In vitro and in vivo studies suggest that, although PrPC possesses neuroprotective activity, deletions and point mutations in the protein can endow it with cytotoxic properties in cultured cells and neurotoxic properties in mice. Interestingly, as will be discussed below, the same domains of PrPC may be responsible for both activities of the molecule, suggesting the intriguing possibility that prions may deliver their toxicity through corruption of the physiological function of PrPC [57]. Previous attempts to determine which parts of PrPC are necessary for prion replication led to the surprising observation that specific deletions encompassing the conserved central region endow the molecule with powerful toxic properties. Expression in transgenic mice on the Prn-p0/0 background of PrP molecules harboring deletion of residues 32–121 or 32–134 led to progressive neurodegeneration, marked by loss of cerebellar granular neurons and white matter vacuolation [58]. Transgenic mice expressing PrP molecules deleted for residues 105–125 (ΔCR) [41] or 94–134 [59] displayed a neonatal lethal phenotype, suggesting that the critical neurotoxicity-determining region encompasses a cluster of positively charged amino acids (residues 105–111), as well as part of the adjacent, central hydrophobic domain (residues 112–130) (Figure 3a; also see Box I). PrP molecules carrying these deletions are also cytotoxic in cell culture assays [60, 61]. A unique feature of transgenic mice expressing toxic PrP deletion mutants, originally described over 10 years ago [58], provides a clue that the neurotoxic and neuroprotective activities of PrPC may be related. In each of these transgenic lines, co-expression of WT PrPC abrogates clinical symptoms and neuropathology, with more toxic mutations requiring higher doses of WT PrPC to rescue the phenotype [41, 58, 59] (Figure 3b). This striking phenomenon suggests that there is a relationship between the physiological activity of PrPC and the toxic effects of the deletion mutants. Recent transgenic mouse models have implicated another small region in the neurotoxicity of PrP mutants: the N-terminal polybasic domain (residues 23–31) (Figure 3a). This region has been implicated in several aspects of PrPC biology, including regulation of endocytosis [62, 63], and binding to glycosaminoglycans (GAGs) [64]. When residues 23–31 are removed from PrP carrying the neurotoxic deletion Δ32–134, yielding Δ23–134 PrP, the protein is no longer toxic [65]. Moreover, deleting residues 23–31 within ΔCR PrP or a disease-associated PrP point mutant (G130V) completely abrogates the channel-inducing activity and cytotoxicity of the latter mutations [46] (Figure 2d). Interestingly, removal of the 23–31 region greatly diminishes the ability of WT PrP to suppress neurodegeneration in mice expressing a PrP deletion mutant (Δ32-134) [66]. In summary, two different regions of PrP (the central region and the N-terminal polybasic domain) are essential for the neurotoxic effects produced by deleted forms of PrP, as well as for the ability of WT PrPC to suppress these effects. What is the explanation for this correlation? One possibility is that toxic mutations in PrP disrupt a normal physiological function of the protein (e.g., neuroprotection), for example by altering interactions with Page 6 downstream signaling components, resulting in neurotoxic effects. In this scenario, WT and mutant PrP might compete for binding to cell surface receptors that control cell death or survival via the N-terminal and central domains. Another hypothesis is that PrPC functional activity depends on multimerization via these two domains [57], and incorporation of mutant PrP molecules into the complex somehow results in a neurotoxic signal. Future studies will be necessary to test these and other models, and to determine whether these same two regions of PrP also play a role in the neurotoxicity of infectious prions. Membrane interactions and PrP toxicity Most molecules of PrPC are attached to the plasma membrane exclusively via a GPI anchor at the C-terminus, and can be released by phospholipase-mediated cleavage of the anchor [67, 68]. However, there is also evidence that PrPC can associate with the membrane independently of the GPI-anchor, and that the polypeptide chain can permeate the lipid bilayer under some conditions. Such interactions may represent an aspect of the normal cell biology of PrPC, but they may also be the source of cytotoxic effects. There is extensive evidence that the polypeptide chain of PrPC can span the lipid bilayer under certain circumstances [68]. Two topological variants of PrPC have been described, designated CtmPrP and NtmPrP, with their N- or C-termini, respectively, on the extracellular side of the membrane [69, 70]. Both forms span the membrane via the central, hydrophobic domain (amino acids 112–130). The proportion of CtmPrP synthesized in the endoplasmic reticulum (ER) can be enhanced by mutations in the central, hydrophobic domain as well as the N-terminal signal peptide [69, 70], and expression of these mutants in transgenic mice induces neurodegenerative phenotypes [70, 71]. Interestingly, the phenotype associated with one of these mutants is dependent on the co-expression of WT PrPC, suggesting that CtmPrP subverts the normal function of PrPC function to generate toxicity [72]. Forms of PrP that have been engineered to remain in the cytoplasm by removal of their N- and C-terminal signal peptides also cause neurodegeneration when expressed in transgenic mice [73], but whether such forms exist naturally is unclear. The toxic effects of cytosolic as well as transmembrane forms of PrP may be related to inappropriate interactions between the PrP polypeptide chain and proteins in the cytoplasm [74, 75]. Another potential interaction between the PrP polypeptide chain and the lipid bilayer involves the ability of two different segments of PrP to function as protein transduction domains (PTDs). PTDs are polybasic peptide segments, exemplified by the nine amino acid transactivator of transcription (TAT) peptide derived from the TAT protein of human immunodeficiency virus (HIV-1). These domains are able to penetrate membranes and promote cellular internalization of proteins to which they are fused or covalently attached [76]. Two polybasic domains of PrPC (residues 23–28 and 100–109) resemble prototypic PTDs, and indeed the 23–28 region has been shown to permeabilize membranes of bacteria [77] and to mediate transduction of attached reporter proteins into mammalian cells [78]. It is possible that the PTD activity of the N-terminal domain is related to the ability of PrP molecules with deletions in the central region (like ΔCR) to induce channels or pores in the cell membrane, an effect that is abolished when residues 23–31 are deleted [79]. It will be important to investigate whether the PTD-like domains of PrPC mediate protein transduction events at the cell membrane under normal circumstances. Intriguing connections between PrPC and Aβ Recent observations have raised the unexpected possibility that PrPC also plays an important role in the biology of AD. AD is the most prevalent form of dementia in the aging population, and is characterized pathologically by the accumulation of the Aβ peptide, a cleavage product of the amyloid precursor protein (APP) [80, 81]. Considerable evidence Page 7 indicates that oligomeric assemblies of Aβ, rather than large amyloid fibrils, are the key toxic species in AD, and that these assemblies cause neurodegenerative changes by delivering a toxic signal at synapses [82]. However, the identity of the receptors on neurons that bind Aβ to transduce neurotoxic signals has remained enigmatic. Recent data have suggested that PrPC is a high affinity receptor for Aβ oligomers, and may also mediate the synaptotoxic effects of these assemblies [4] (Figure 4a, b). In the initial study [4], PrPC emerged from an expression cloning screen as a receptor capable of binding Aβ oligomers with nanomolar affinity. Binding was not observed with Aβ monomers or fibrils, suggesting that PrPC was specifically a receptor for oligomers. Importantly, PrPC was also found to be a mediator of Aβ-induced synaptotoxicity. In support of this conclusion, hippocampal slices derived from Prn-p0/0 mice were found to be resistant to Aβ oligomerinduced suppression of long-term potentiation (LTP) (Figure 4c). In a second study from the same group [83], Prn-p0/0 mice were crossed with transgenic mice that model AD, in which mutant forms of APP and presenilin-1 are co-expressed. The results showed that PrPC was required for both the cognitive deficits and reduced survival observed in AD mice, although the presence of PrPC did not influence the rate of Aβ plaque formation in the brain. These initial findings motivated other groups to test the link between PrPC and Aβ oligomers. Several studies have confirmed that PrPC plays an essential role in mediating the alterations in synaptic plasticity induced by Aβ oligomers [84], and have provided evidence that immuno-targeting PrPC in vitro or in vivo can rescue Aβ-dependent toxic effects [85– 87]. In contrast, however, another study showed that synthetic Aβ oligomers injected intraventricularly into mice impaired consolidation of long-term recognition memory regardless of whether the animals expressed PrPC [88]. In two other recent studies, genetic ablation of PrPC had no effect on Aβ-induced inhibiton of hippocampal LTP in brain slices [89], or learning and memory deficits in a line of AD transgenic mice expressing mutant APP [90]. Using organotypic brain slices, another group reported that PrPC was not required for any of several synaptotoxic effects of Aβ oligomers, including depression of basal synaptic transmission, reduction in the number of dendritic spines, and blockade of LTP [91]. The evident discrepancy that emerges from these studies may, at least in part, be explained by the fact that preparation of synthetic Aβ oligomers is notoriously challenging, and the product obtained can differ from one laboratory to another [92, 93]. The ability of synthetic Aβ oligomers to bind PrPC has been observed in all of the published studies, although it is unclear why only some preparations of Aβ oligomers appear to display PrPC-dependent synaptotoxicity. It is possible that only a specific conformation or size of synthetic oligomers operate through a PrPC-dependent mechanism. The discrepancy between results obtained in vivo by different laboratories may also be due to the use of different transgenic models of AD. It is possible that multiple receptors mediate the toxic effect of Aβ oligomers, and eliminating a PrPC-dependent pathway may produce only a partial rescue of the AD phenotype, or none at all, depending on the mouse model being studied. Given these uncertainties, the role of PrPC in mediating the synaptic toxicity of Aβ clearly requires further clarification. Two, distinct Aβ oligomer binding sites have been identified on PrPC (residues 95–105 and 23–27), based on deletion analysis, antibody inhibition, and biophysical techniques [4, 94]. It is noteworthy that these two sites are coincident with or lie immediately adjacent to regions that are important determinants of PrPC activity and mutant PrP toxicity (as discussed in previous sections). Moreover, residues 23–31 have been implicated in internalization of PrP via clathrin-mediated endocytosis [63, 95]. Interestingly, another Page 8 recent study showed that Aβ oligomers can affect the localization of PrPC by increasing the formation of clusters of PrPC on the cell surface [96]. Taken together, these results suggest that the same structural domains involved in the binding to Aβ oligomers may also govern PrPC function, cellular trafficking, and toxicity. Furthermore, a recent study provided evidence that PrPC could mediate the toxicity not only of Aβ oligomers, but also of other β-sheet-rich protein conformers [5]. Therefore, it is possible that oligomeric forms of several different neurotoxic proteins could exert their effects by blocking, enhancing or subverting the normal function of PrPC. Although considerable attention has been focused on the ability of PrPC to serve as a receptor for Aβ oligomers, there is also evidence that PrPC might play a role in the processing events that generate Aβ from its APP precursor. In two recent studies, PrPC was reported to physically interact with the β-site APP cleaving enzyme 1 (BACE1), an enzyme responsible for cleaving APP and initiating the amyloidogenic cascade [97, 98]. As a result of this interaction, BACE1 activity was inhibited, and the overall production of Aβ peptides decreased. In conclusion, despite the debate surrounding the recent studies, defining the potential connections between PrPC and Aβ, and determining whether these interactions play a role in pathological conditions is undoubtedly an exciting future line of investigation. Concluding remarks The possibility that alterations in the normal function of PrPC play a role in the pathogenesis of prion and other neurodegenerative diseases is sparking renewed efforts to elucidate the cellular and molecular pathways in which this protein is involved. Traditional approaches involving analysis of PrP knock-out mice and biochemical identification of PrPC interacting molecules have been supplemented by the development of new assays for PrP-related functions in cultured cells and transgenic mice, as well as by the use of zebrafish as a genetically tractable model system. Taken together, these studies have uncovered roles for PrPC in cell adhesion, neurite outgrowth, ion channel activity, neuronal excitability, cytotoxicity and cytoprotection. The disease relevance of these efforts has been heightened by recent reports that PrPC, aside from serving as a precursor of PrPSc and mediating prion-induced neurodegeneration, may also play an important role in the pathology of AD. There is evidence in both prion diseases and AD that the disease phenotype may depend on some alteration in the physiological activity of PrPC. Thus, binding to cell-surface PrPC of either oligomeric Aβ or PrPSc (or other pathogenic PrP aggregates) may initiate toxic signals that lead to neuronal loss and/or synaptic dysfunction, changes that are ameliorated by elimination of PrPC (Figure 4). Interestingly, the same domains of PrPC that are critical for its physiological and cellular functions have also been implicated in mediating neurodegenerative processes. Two regions, which lie almost entirely within the unstructured half of the protein [99], seem to be particularly important: the N-terminal polybasic domain (residues 23–31) and the central domain (residues 95–135) (Figure 5). These two regions influence several cell biological features of the protein, including transmembrane anchoring, protein transduction, interaction with GAGs, and endocytic trafficking. Manipulation of these domains creates or alters cytotoxic and cytoprotective activities of the molecule that can be detected in several different assays in vitro and in vivo. They are also responsible for binding to Aβ oligomers, possibly mediating the synaptotoxic effects of the latter, and may regulate the enzymatic processing steps responsible for production of the Aβ peptide. Page 9 Although several outstanding questions remain (Box 3), these new results highlight a close connection between the normal biology of PrPC and its role in several neurodegenerative diseases. This connection has important therapeutic implications. Compounds that bind to specific functional domains of PrPC and modulate its physiological activity may retard the neurotoxic effects of several kinds of β-rich protein aggregates. Pursuing this novel therapeutic approach will clearly require a deeper understanding of PrPC and its position at the crossroads of physiology and disease.