Prion Protein at the Crossroads of Physiology Box 2

BOX 2 Outstanding questions • What is the physiological function of PrPC? A number of functions have been proposed, based largely on subtle abnormalities in PrP knock-out mice or in cells derived from them, including roles in cell adhesion, neurite outgrowth, neuronal excitability, and neuroprotection. However, the underlying molecular mechanisms remain uncertain. A recent addition to the list of experimental models for studying the function of PrPC is the zebrafish, which, unlike the mouse, displays dramatic phenotypes when PrP gene expression is knockeddown. How the different effects of PrP deletion can be reconciled is unclear. PrPC may operate in several, distinct cellular processes, or it may have a more general modulatory role (e.g., protection from cellular stress). • What molecules interact with PrPC? A key to deciphering the function of PrPC is the identification of interacting proteins. A number of candidate interactors have been identified based on co-immunoprecipitation, yeast-two hybrid analysis, and molecular cross-linking [19, 114], but the physiological significance of many of these remains unclear. The interactors include cell surface or secreted molecules involved in adhesion [24, 115, 116]), receptor signaling and trafficking [117], and ion channel/transporter activity [40, 118]. Other proposed PrPC interactors are localized primarily to the cytoplasmic compartment. These might associate with the polypeptide chain of cell-surface PrPC via transmembrane linker proteins, for example as part of a signal transduction complex [119–124]. Intracellular proteins might also bind directly to cytosolic forms of PrP, or the intracellular domains of transmembrane variants (CtmPrP and NtmPrP). The functional activity of PrPC may also depend on interaction with non-protein components, such as lipids of GAGs [64]. • How do alterations of the function of PrPC contribute to the neurotoxicity of prions? In addition to its well-known role as a precursor to PrPSc, PrPC is required for transducing prion-related neurotoxic signals. Thus, the normal, physiological function of PrPC may be subverted as part of the pathological process. How this occurs is still poorly understood. PrP molecules missing certain critical regions produce dramatic neurodegenerative phenotypes when expressed in transgenic mice, which may reflect alterations in the normal function of PrPC. It remains to be determined whether the same pathways are affected by infectious PrPSc. If so, then compounds that affect the functional activities of PrPC may represent potent therapeutic agents in prion diseases. • Does PrPC mediate the neurotoxicity of Aβ oligomers in AD, and possibly of β-rich protein assemblies in other neurodegenerative disorders? It is likely that PrPC binds Aβ oligomers, but whether this interaction represents a major pathway for synaptotoxicity is unresolved. Relevant to this issue, it would be useful to know how oligomer binding affects the physiological and cell biological properties of PrPC, for example its localization, processing, and assayable functional activities. Regardless of whether cell-surface PrPC mediates Aβ-induced synaptotoxicity, soluble forms of PrP could have therapeutic benefit by sequestering oligomers in the extracellular space. This effect may extend to β-rich protein oligomers in other neurodegenerative disorders, some of which have also been shown to bind PrPC [5]. FIGURE 1. A role for PrPC in cell adhesion The figure provides three examples of how PrPC could participate in cellular adhesion. (a) Schematic illustration of PrPC interaction with neural cell adhesion molecule (NCAM), which results in activation of Fyn kinase and enhancement of neurite outgrowth in cultured hippocampal neurons (not shown) [24]. (b) PrPC partially co-localizes with NCAM along neurites and in growth cones (arrows) of cultured hippocampal neurons [22]. Bar, 10 μm. (c) Schematic model, based on experiments performed in zebrafish [26], illustrating how PrPC could modulate cell adhesion by influencing delivery of E-cadherin to the plasma membrane. Homodimers of E-cadherin can be anchored to F-actin via α and β catenins [127]; thus, PrPC may indirectly participate in rearrangement of the actin cytoskeleton [26]. The precise mechanism by which PrPC on the plasma membrane regulates the delivery of intracellular vesicles is currently unknown, but may involve activation of a signal transduction cascade involving Src-family tyrosine kinases. (d) Morpholino knockdown of zebrafish PrP-1 causes gastrulation arrest, which can be rescued by re-introduction of several kinds of PrP molecules [26]. (i) Control embryos at the 1–4 cell stage display normal progression of the blastodermal margin (red arrowheads), while morphant (MO) embryos knocked down for zebrafish PrP-1 (ii), but not PrP-2 (iii), are severely arrested. Gastrulation arrest caused by depletion of PrP-1 can be rescued by microinjection of RNA encoding zebrafish (zf) PrP-1 (iv), zebrafish PrP-2 (v) or mouse PrP (m PrP) (vi), suggesting that a biological function of the PrP molecule is conserved from fish to mammals. (e) Model for how PrPC molecules on adjacent cells engage in homophilic interactions that promote cell adhesion, based on experiments in cultured cells [26]. (f) Green fluorescent protein (GFP)tagged forms of mouse PrPC (i) or zebrafish PrP-1 (ii) expressed in non-adhesive Drosophila S2 cells accumulate at cell junctions (arrowheads). Panels (i) and (ii) show fluorescence images, and panels (iii) and (iv) show the corresponding phase-contrast images. Scale bars, 5 μm. Reproduced, with permission, from [24] (panel b) and [26] (panels d and f). FIGURE 2. Model illustrating how PrP molecules may form ion channels or pores The figure illustrates how mutant PrP molecules carrying a deletion (referred to as ΔCR) of residues 105–125 in the central region induce spontaneous ionic currents by the proposed formation of channels or pores in the cell membrane, and how this activity is dependent on residues 23–31 and is suppressed by WT PrP. For each panel, the upper graph shows wholecell patch-clamp recordings of human embryonic kidney 293 (HEK293) cells expressing the corresponding mouse PrP protein. The schematic below each graph represents one possible model for interpreting the data at the molecular level. In contrast to WT PrP (a), ΔCR PrP (b) induces spontaneous inward currents, which may reach amplitudes of up to several thousand pA [45]. This phenomenon could be explained by hypothesizing that the lack of the central region promotes the aberrant insertion of ΔCR PrP molecules into the plasma membrane, which results in the formation of a channel or pore. (c) The spontaneous ionic currents induced by ΔCR PrP are silenced by co-expression of WT PrP [45, 46]. WT PrP may inhibit formation of the pore by physically interacting with the ΔCR mutant. (d) Deleting residues 23–31 within ΔCR PrP abrogates the ability of this mutant to induce spontaneous currents [46]. One hypothesis to explain this observation is that interaction of ΔCR PrP molecules with the cell membrane requires the polybasic N-terminal region, which has been shown to act as a protein transduction domain (PTD). Whole-cell patch-clamp recordings are reproduced, with permission, from [45] (a–c), and [46] (d). In each cartoon, the blue ball indicates the C-terminal globular domain of PrP (residues 125–231). The Nterminal flexible domain is divided in three segments corresponding to residues 23–31 (green), 32–104 (black) and 105–125 (red). FIGURE 3. Deletions within the central region of PrP are associated with neurotoxicity in transgenic mice (a) Deletions up to residue 106 have no adverse effect [58], while deletions extending through residues 121 or 134 cause spontaneous neurodegenerative illness in the absence of endogenous PrPC [58]. Additional deletions (Δ94–134 [59] and Δ105–125 [41]) narrow the critical, neurotoxicity-determining region to a highly conserved segment of 21 amino acids in the central region (residues 105–125). Schematics show the structures of WT mouse PrP and each of the deletion mutants (with the deleted region indicated by dotted lines). The column labeled “Onset” gives the age (in weeks) at which spontaneous neurological illness occurs in mice expressing the corresponding deletion mutant. The column labeled “WT PrP Rescue” gives the fold-expression of WT PrPC needed to prevent the appearance of neurological illness. A minus symbol (“−“) indicates that no spontaneous disease is observed. (b) The graph, which is a representation of the data shown in the first two columns, shows the inverse correlation between age of onset in the different mouse lines and amount of WT PrP expression required for rescuing the neurological phenotype. FIGURE 4. PrPC may mediate the toxicity of both Aβ oligomers and PrPSc The diagram illustrates how cell-surface PrPC, as a consequence of its binding to either Aβ oligomers (green) or PrPSc (orange), may deliver a neurotoxic signal in Alzheimer’s disease (a) and prion diseases (b), respectively. The N-terminal polybasic domain of PrPC (depicted as a small blue ball carrying positive charges) has been shown to participate in binding to both PrPSc [128] and Aβ oligomers [94]. In addition to acting as a transducer of PrPScinduced neurotoxicty, PrPC also serves as a precursor for the generation of more molecules of PrPSc, which allows prion propagation [1] [(red arrow in part (b)]. (c) PrPC is required for inhibition of hippocampal LTP induced by Aβ oligomers. Field potentials were recorded from the CA1 region of hippocampal slices derived from wild-type mice (green) or Prn-p−/− mice (red), which had both been treated with Aβ oligomers [4]. The red trace is similar to the one recorded from untreated control (Prn-p+/+) slices (not shown). (d) Depletion of neuronal PrPC reverses spongiosis, but not accumulation of PrPSc, in prion-infected mice [7]. Neuronal PrPC expression in mice previously infected with scrapie prions was eliminated by means of Cre-Lox recombination at approximately 12 weeks of age. Hippocampal sections from PrP-expressing mice (i and iii) or PrP-ablated mice (ii and iv) (at 8 and 48 weeks post infection, respectively) were stained with hematoxylin and eosin (i and ii) or an anti-PrP antibody (iii and iv). The severe loss of CA1 to CA3 neurons caused by scrapie infection (arrows) can be rescued by depleting neuronal PrPC, despite continued PrPSc accumulation. Reproduced, with permission, from [4] (c) and [7] (d). FIGURE 5. Important functional domains of PrPC The figure summarizes the functions attributed to two regions of PrPC (highlighted in yellow shading): the N-terminal polybasic domain (residues 23–31) and the central region (residues 95–135). These two regions influence several cell biological and functional properties of the protein, including transmembrane anchoring (resulting in formation of CtmPrP and NtmPrP) [69, 70], interaction with glycosaminoglycans (GAGs) [64], endocytosis [129], cytotoxicity and cytoprotection [41, 58, 59], binding to Aβ oligomers [4, 88, 89, 119], and regulation of the enzymatic steps involved in the APP processing [97]. In addition, the N-terminal polybasic domain of PrPC (residues 23–28) resembles prototypic protein transduction domains (PTDs) [76–78], short polypeptide segments capable of penetrating cellular membranes. Residues are based on the mouse sequence. Box 1 FIGURE I. Structure of PrPC and PrPSc (a) Schematic illustration of PrPC. Residues correspond to the mouse sequence. A signal peptide (SP, residues 1–22), removed during PrP biosynthesis in the ER, precedes a polybasic region (residues 23–27, green) and five histidine-containing octapeptide repeats (residues 51–90, gray) that can bind Cu2+ and other bivalent metal ions. The central part of the molecule includes a positively charged region (residues 95–111, cyan) followed by a highly conserved hydrophobic domain (HD, residues 111–130). The C-terminal part includes two short β-strands (residues 127–129 yellow; and 166–168, purple) and three αhelices (residues 143–152, blue; 171–191, orange; 199–221, red). A C-terminal peptide (residues 231–254) is removed during biosynthesis, followed by attachment of a glycosylphosphatidylinositol (GPI) moiety, which anchors the protein to the outer leaflet of the plasma membrane. PrPC also contains two N-linked oligosaccharide chains (at Asn-180 and Asn-196, black lollipops) and a disulfide bond between residues 178 and 231). (b) Threedimensional structure of PrPC, based on nuclear magnetic resonance (NMR) analysis. The structure of mouse prion protein fragment 121–231 was taken from entry 1XYX of the Protein Data Bank (PDB, http://www.ebi.ac.uk/pdbe), and was modified using the RasMol 7.4 software (www.rasmol.org). Colors correspond to the structural motifs described in (a). (c) Models of aggregated PrPSc. Although a high-resolution structure for PrPSc has not been determined, several different models have been proposed. Two of these are illustrated here. (i) In the first model, derived from a molecular dynamics simulation, the core of the PrPSc aggregate is formed by parallel and antiparallel β-strands, organized in a spiral shape. Reproduced, with permission, from [125]. (ii) In the second model, which is based on electron crystallographic studies, the core is formed by left-handed βhelices. Reproduced, with permission, from [126]. Both pictures show trimers of PrPSc.