HC switches PrP from good to evil – and beyond

The proposed transmission of a protective and the a PrP signal by one or two receptors leads to a common concept for PrP toxicity and function, based on the pivotal role of the flexible region around HC, which serves as a “hinge” or “switch” for the different physiological or pathological conformational states of the prion protein (Figure 48):

1.) In the physiological and protective conformation, termed PrP^P, the flexible structure of the “hinge” actively supports cell survival via its interaction with a signaling molecule and the subsequent transmission of a protective signal, simultaneously counteracting the toxic property of the C-terminal globular domain of PrP.

2.) The transformation of PrP into toxic PrP^L by the conformational change of the flexible hinge region into a rigid -sheet structure or by its deletion in the toxic mutants, leads to the loss of its interaction with the protective receptor site.

On the other hand, the altered structure of the “hinge” no longer prevents the toxic influence of the C-terminal globular domain, leading to a pathological instead of a protective receptor signaling.

3.) The subsequent conversion of PrP^L into a non-functional but infectious conformation, PrP^Sc, leads to additional changes in the C-terminal globular domain of PrP. These structural alterations eliminate the binding of the C-terminus to the receptor, leading to the dissociation of the “mature” PrP^Sc molecule form the receptor, which in turn is free to convert further PrP^P molecules.

132 4.) An additional “neutral” PrP^N conformation is manifested by the deletion of HC in PrP 114-121, which leads to a reduced structural flexibility of the “hinge” or “switch”

region, rendering the mutant slightly more stable (Thaa et al., 2007).

The slight increase in stability not only prevents the major structural change of PrP into the toxic PrP^L or the subsequent infectious PrP^Sc conversion; it also leads to an improper interaction with both, the protective receptor site and the toxic receptor activity, which impairs or even completely abolishes the activation of a protective, as well as a pathological signal.

Therefore, the structural change of the N-terminal flexible region by the deletion of HC arrests the conformational “switch” of PrPin an almost “neutral” position, in-between the fully flexible physiological and the solid -sheet rich pathological conformations.

The non-functional conformation of PrP^N therefore resembles a Prnp knockout situation in many, but not in all aspects (Chapter 5.5.2).

Discussion

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Figure 48: The hydrophobic core is part of the conformational “switch” of PrP^C

(a) The N-terminal flexible region of PrP^C, including the stop transfer effector (STE) and the adjacent hydrophobic core (HC), forms a conformational “hinge” or “switch”, which allows PrP^C to adopt distinct functional conformations.

(b) The physiological, flexible state of the “hinge” results in a protective PrP^P conformation. The change in -sheet structure leads the protein to a toxic, lethal PrP^L state. This structural change enables further C-terminal conformational alterations, finally resulting in an infectious PrP^Sc state. The deletion of HC arrests the “hinge” in a non-functional, “neutral” conformation.

134 5.5.2 Complex interactions of different conformations lead to PrP disease The concept of different conformational PrP states, interacting with one (Figure 50) or two (Figure 49) protective and toxic signaling molecule(s) provides also the basis for the mechanisms leading to distinct pathological phenotypes in the examined mouse genotypes:

 PrP-wt mice only express the physiological PrP^P conformation, which exerts a protective influence on the cell, especially on neurons. This protection might not be essential in young and middle age, probably by the compensation of a second protective mechanism, such as the expression of a redundant protein (Shmerling et al., 1998). In old age however, this additional protection might decrease, for example by the reduced expression of anti-apoptotic Bcl-2 (see Chapter 5.2.3) and/ or by an increase of toxic stressors, such as ROS, requiring the additional protective function of PrP^P for the maintenance of a balanced health status.

The redundant protective function of PrP could also become vital in young mice by an unexpected, sudden toxic insult, such as ischemic stroke, which can be counteracted by the protective impact of PrP^P (see Chapter 5.2.3, Figure 46) (Shyu et al., 2005; Spudich et al., 2005; Weise et al., 2004).

- A prion inoculation of PrP-wt mice leads in the first conversional step to the formation of the toxic PrP^L by the structural change of the “hinge” region.

Since more and more protective PrP molecules are exponentially converted into PrP^L the amount of PrP^P conformations and their counterbalancing protective effect decreases, finally resulting in prion disease.

The subsequent second conversional change in the C-terminal globular domain of PrP leads to the loss of receptor binding, transforming the intermediate PrP^L state into the non-toxic but infectious PrP^Sc form.

Discussion

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 PrP-ko mice have lost the physiological PrP^P function, becoming susceptible to toxic stressors by an insufficient protection in old age or by the emerging of abnormal toxic insults, such as ischemic stroke or the expression of toxic PrP deletion mutants.

- However, an inoculation with the infectious but non-toxic PrP^Sc conformation has no impact on these mice, since no PrP molecule can be converted into PrP^L.

 The expression of the non-functional conformation PrP 114-121 has no decisive influence when expressed on the knockout background. The obstructive influence of PrP^N on both, the protective as well as the toxic receptor activity, leads to an almost non-functional state of PrP, consequently resulting in the same pathological phenotype as in aged Prnp-ko mice.

- Its increased structural stability by the deletion of a decisive part of the “hinge” prevents in addition the conversion into a toxic PrP^L and the subsequent infectious PrP^Sc state upon prion inoculation.

 A co-expression of PrP-wt and PrP -121 results in a slight reduction of the protective effect of PrP^P by the mutant, since both molecules compete with each other for the same receptor binding sites. The impaired but not completely abolished protective function of PrP^P does not result in a severe pathology of aged mice, such as hind-limb paresis or paralysis; it can however elicit milder pathological signs, explaining the observed itching of aged mice, when co-expressing both conformations.

- A prion inoculation of these mice on the other hand, leads also to a dominant negative inhibition of the conversion of PrP-wt into the pathological states due to the “trapping” of PrP^Sc molecules by the inconvertible PrP^N conformation of PrP 114-121 (see Chapter 5.3.4), resulting in a delayed disease progress.

136 Interestingly, the co-expression of mutant PrP 114-121 with the toxic deletion mutants PrP 32-134 or PrP 94-134, modified their toxicity in two different ways: Its co-expression with PrP 32-134 on the Prnp-ko background slowed down the disease progress. In contrast, the pathology in animals expressing PrP 94-134 on the Prnp-wt background was enhanced by the additional expression of PrP 114-121 (Baumann et al., 2007). These contradictory observations further demonstrate the non-functional, “neutral” effect of the HC deletion:

When co-expressed, the “neutral” PrP 114-121 reduces the signaling of the toxic mutants, for example by interfering for the same receptor binding site. However, a co-expression of PrP-wt exerts a stronger inhibition of toxic mutants due to its additional protective, counteracting signaling. The additional expression of the less protective “neutral” PrP 114-121 on the Prnp-wt background, which competes with PrP-wt for the inhibition of the toxic mutants, diminishes the more potent protective effect of full-length PrP-wt expression, leading to a lower protection than the solely expression of PrP-wt.

Discussion

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Figure 49: Two-receptor model for the functional effect of PrP conformations in the examined mice In a Prnp-ko situation, neither the protective (green), nor the toxic (red) receptor can be activated. No or only a slight putative constitutive activity of the protective receptor (Baumann et al., 2007) is not sufficient to protect ko mice from late pathology (a). An infection with prions has no effect on Prnp-ko mice, since no PrP molecules can be converted into a toxic PrP^L conformation (b).

The expression of PrP-wt mediates a protective signal via the binding of its N-terminal region to the protective receptor, which is sufficient to counteract toxic stressors in old age. The protective PrP^P conformation might also prevent the correct binding of the C-terminal globular domain to the toxic receptor, resulting in receptor blocking (a). Upon prion inoculation, the protective PrP^P conformation is converted by PrP^Sc into an intermediate PrP^L state. The conformational change of the “hinge” region abrogates the interaction of PrP with the N-terminal protective receptor site but no longer prevents the binding of the C-terminal domain to the toxic receptor, resulting in toxic signaling and subsequently in severe, lethal prion pathology (b).

The deletion of HC, shifts PrP in a non-functional, “neutral” PrP^N state, which has no effect or even aborts the slight constitutive protective signaling, resulting in late pathology (a). Transgenic mice, expressing PrP 114-121 are resistant to the toxic effect of PrP^Sc (b) but also exhibit pathology in old age.

A co-expression of PrP-wt and PrP 114-121 leads to a slight inhibition of the protective function of PrP-wt by non-functional PrP 114-121 (grey arrows), resulting in the observed pathology of aged mice (a), whereas PrP 114-121 inhibits the conversion of PrP-wt into PrP^L upon prion inoculation (b) in a dominant negative manner (see Chapter 5.3.4, Figure 47).

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Figure 50: One-receptor model for the examined mice and two additional mutations

A postulated model of a single receptor (Li et al., 2007b), which is able to elicit two opposing signals, results in the same functional mechanism as the proposed two-receptor concept of Figure 49.

A PrP^P conformation of the wild-type protein is able to bind to an N-terminal site of the receptor, eliciting a protective signal (a). The correct binding of converted PrP^L by its C-terminal globular domain to an additional C-terminal receptor sit leads to a toxic signal (b).

PrP 114-121 cannot correctly interact with either the protective or the toxic site, leading to a non-functional receptor (a, b).

The effects of two additional mutations involving the N-terminal flexible region furthermore highlight the pivotal role of the “hinge” for PrP function and infectivity. The effects of these mutants are illustrated for one receptor, whereas the same mechanism would also apply for the two-receptor model:

A deletion within the “hinge” segment, which encompasses in addition to HC further adjacent regions, such as the stop transfer effector (STE) in PrP105-125 (Li et al., 2007b), leads to the complete loss of the structural flexibility and to its arrest in a toxic PrP^L state (a). Similar to PrP 114-121, the deletion of the flexible “hinge”

renders this protein resistant to prion conversion (b).

The sole expression of the segment PrP105-125 does not elicit prion pathology (a). However, the expression of PrP105-125 in an “aged” (Sakudo et al., 2007), -sheet conformation, which resembles the decisive part of PrP^Sc for the conversion process, allows PrP105-125 to convert PrP-wt molecules from the protective PrP^P in a toxic PrP^L conformation (b). Since PrP105-125 lacks the C-terminal region, it is not able to further convert PrP-wt in a “mature”, infectious PrP^Sc conformation.

Discussion

139 5.5.3 Distinct pathological phenotypes by different prion conformations The property of the prion protein to elicit a protective, as well as a toxic effect on neuronal survival also explains the two kinds of pathology observed in the different genotypes:

The toxic and the protective effects of PrP mainly influence the same pathological mechanisms, manifested for example by vacuolation and astrocytic gliosis in the cerebellum and the pons (Figure 41). Besides this common major pathway however, both properties of PrP exhibit also distinct functions, most clearly demonstrated by the different pattern of astrocytic gliosis in the cerebral cortex and the corpus callosum (CC) (Figure 42, Figure 43).

The loss of the physiological PrP function leads to the observed astrocytic gliosis of CC in aged mice on the knockout background.

The toxic effect of PrP^L conformations on the other hand, seems not to notably result in this pathological phenotype, but leads instead to severe astrocyte activation in the cerebral cortex.

Interestingly, inoculated transgenic mice on the Prnp-wt background exhibited a “mixed”

pathological pattern, characterized by astrocytic gliosis in both brain regions. As pointed out above (Chapter 5.5.2), the protective function of PrP in these mice is impaired by the interference of non-functional PrP 114-121, which results in the activation of astrocytes in CC.

Upon prion inoculation, these mice suffered in addition from the toxic impact of the PrP-wt conversion into PrP^L,leading to an additional astrocytic gliosis in the cerebral cortex.

The “mixed” pathology in these mice therefore reflects both sides of the coin, i.e., both pathological mechanisms of PrP: The loss of protective function due to the inhibition of PrP^P by “neutral” PrP 114-121 and the gain of PrP toxicity by its conversion into PrP^L.

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