MINIREVIEW Molecular basis of cerebral neurodegeneration in prion diseases Jo ¨ rg Tatzelt 1 and Hermann M. Scha ¨ tzl 2 1 Department of Biochemistry, Neurobiochemistry, Ludwig-Maximilians-University Munich, Germany 2 Institute of Virology, Technical University of Munich, Germany Prion diseases in their ‘classical’ and naturally occur- ring forms are characterized by both neurodegenera- tion, with clinical symptoms, and propagation of infectious prions, the latter giving rise to the typical transmissibility within and between species [1–5]. The formation of the disease-associated isoform of prion protein (PrP Sc ) [i.e. the misfolded and partially protein- ase K (PK)-resistant isoform of the cellular prion pro- tein (PrP C )] is closely linked to the propagation of infectious prions, but apparently is not sufficient to induce neurodegeneration. Here, an important role in mediating the neurodegeneration process is increasing for PrP C . Evidence for this was found in neurografting approaches [6], in conditional prion protein (PrP) knockout studies [7] and in in vivo cross-linking experi- ments of PrP C [8]. Some genetic forms of human prion disease appear less transmissible, or even nontransmissible. With one exception this is also true for the transgenic animal mod- els established to mimic genetic prion diseases [1–5]. This nontransmissible character is reminiscent of ‘pro- teinopathies’, sometimes linked to PrP overexpression, rather than classical prion diseases, and is in line with the concept of ‘nontransmissible prionopathies’ Keywords amyloid; neurodegeneration; prion protein; prion; trafficking; transmissibility Correspondence J. Tatzelt, Department of Biochemistry, Ludwig-Maximilians-University Munich, Schillerstrasse 44, 80336 Munich, Germany Fax: +49 89 2180 75415 Tel: +49 89 2180 75442 E-mail: joerg.tatzelt@med.uni-muenchen.de H. M. Scha ¨ tzl, Institute of Virology, Technical University Munich (TUM), Trogerstraße 30, 81675 Munich, Germany Fax: +49 89 4140 6823 Tel: +49 89 4140 6820 E-mail: schaetzl@lrz.tum.de (Received 2 August 2006, revised 30 November 2006, accepted 4 December 2006) doi:10.1111/j.1742-4658.2007.05633.x The biochemical nature and the replication of infectious prions have been intensively studied in recent years. Much less is known about the cellular events underlying neuronal dysfunction and cell death. As the cellular func- tion of the normal cellular isoform of prion protein is not exactly known, the impact of gain of toxic function or loss of function, or a combination of both, in prion pathology is still controversial. There is increasing evi- dence that the normal cellular isoform of the prion protein is a key medi- ator in prion pathology. Transgenic models were instrumental in dissecting propagation of prions, disease-associated isoforms of prion protein and amyloid production, and induction of neurodegeneration. Four experimen- tal avenues will be discussed here which address scenarios of inappropriate trafficking, folding, or targeting of the prion protein. Abbreviations Ctm PrP, transmembrane form of PrP with the COOH-terminus in the endoplasmic reticulum lumen; cytoPrP, cytosolic PrP; Dpl, doppel protein; ER, endoplasmic reticulum; HD, hydrophobic domain; Ntm PrP, transmembrane form of PrP with the NH 2 -terminus in the endoplasmic reticulum lumen; PK, proteinase K; PrP, prion protein; PrP C , normal cellular isoform of PrP; PrP Sc , disease-associated isoform of PrP; secPrP, secretory PrP. 606 FEBS Journal 274 (2007) 606–611 ª 2007 The Authors Journal compilation ª 2007 FEBS introduced by C. Weissmann [3]. Two common phases in prion diseases can be described. In a first phase, with apparently obligate requirement for PrP C expression, profound conformational changes give rise to PrP aggregation and the formation of PrP Sc , resulting in more or less pronounced amyloid formation. This is paralleled by replication of infectious prions and trans- missibility. In a second phase this is transduced into physiological dysfunction in the central nervous system, and neuronal damage. On the other hand, transgenic mouse models have been helpful in revealing that these features can occur independently. There are models available which des- cribe scenarios for neurodegeneration alone, prion pro- pagation alone, or a combination of both (Fig. 1). In the following, such in vivo models are described which address certain aspects of membrane topology, folding, intracellular targeting and trafficking of PrP. The term ‘toxicity’ is used by us here for induction of neuro- degeneration, and PrP Sc is used synonymously for the pathological form of PrP. Toxicity of transmembrane isoforms of PrP The evidence that PrP Sc is directly neurotoxic is con- troversial [6] and has fueled the search for other PrP conformers involved in pathophysiological scenarios. In the 1980s, it was shown, in cell-free translation- translocation systems, that PrP can be found in more than one topologic form [9]. The major form is the fully translocated isoform giving rise to the known, fully mature, PrP in the secretory pathway, located finally at the outer leaflet of the plasma membrane by its GPI-anchor (secPrP). In addition, the existence of two different transmembrane forms of PrP was verified [10]. One form, termed C-trans transmembrane ( Ctm PrP), has its COOH-terminus in the endoplasmic reticulum (ER) lumen. The other form, termed N-trans transmembrane ( Ntm PrP), has its NH 2 -terminus in the ER lumen. Both forms appear to span the membrane at the same hydrophobic stretch in PrP [in general, res- idues 110–135, previously termed TM1, now referred to as the hydrophobic domain (HD)] (Fig. 2). Interest- ingly, it was shown that during normal biogenesis of PrP, only about two-thirds is expressed as the secre- tory form (secPrP), less than 10% as the Ctm PrP and the remainder as Ntm PrP. Naturally occurring and arti- ficial mutations in the membrane-spanning segment can lead to significantly increased generation of Ctm PrP. In addition, several pieces of evidence have linked Ctm PrP to neurodegeneration in transgenic mice PrP cS prion propagation prion propagation PrP c neurodegeneration neurodegeneration stress sensitive? Fig. 1. Scheme illustrating putative scenarios in PrP pathology. In the middle, the ‘classical’ pathway, resulting in neurodegeneration and PrP propagation, is depicted. The other pathways are from transgenic mouse models characterized by either PrP-induced neu- rodegeneration or prion propagation. A loss of function of PrP C might result in sensitizing neurons to stress stimuli. OHCOHC SS-IPGSS-RE lpD OHCOHC SS-IPG β 1 α 1 β 2 α 2 α 3 β 1 α 1 β 2 α 2 α 3 α 1 β 2 α 2 α 3 RODHSS-RE PrP c O H C OHC SS-IPGSS - RE PrP F( 32-134) Fig. 2. Structure of PrP c , Dpl, and PrPDF(D32–134). Schematic presentation of the proteins mentioned in the text. a, alpha helix; b, beta strand; ER-SS, endoplasmic reticulum signal sequence; GPI-SS, GPI anchor signal sequence; HD, hydrophobic domain (putative transmem- brane domain of Ctm PrP); OR, octarepeat. J. Tatzelt & H. M. Scha ¨ tzl Cerebral neurodegeneration in prion diseases FEBS Journal 274 (2007) 606–611 ª 2007 The Authors Journal compilation ª 2007 FEBS 607 and to some heritable prion diseases (mutation A117V in the Gerstmann–Stra ¨ ussler–Scheinker syndrome). The Ctm PrP isoform has been hypothesized to repre- sent an important intermediate in the pathway of prion- induced neurodegeneration, by escaping ER resident quality control mechanisms [10,11]. Of note, this takes place in the absence of generation of ‘classical’ PK- resistant PrP Sc and of infectious prions. On the other hand, it was later shown that a prion infection appar- ently can trigger the generation of ‘toxic’ Ctm PrP [11]. This would link transmissible and genetic prion diseases and provide a common pathway of neurodegeneration in prion disease. Of note, another group has found, in an additional transgenic model for Ctm PrP, that the neurodegenerative phenotype is strongly dependent on the co-expression of endogenous wild-type PrP [12]. Toxicity of PrP located in the cytosol During the initial characterization of the biosynthesis of PrP, in vitro studies revealed that PrP could, at least in part, be localized in the cytosolic compartment. As mentioned above, two different transmembrane topo- logies were also found ( Ntm PrP and Ctm PrP) and the increased synthesis of Ctm PrP has been shown to coin- cide with progressive neurodegeneration [10]. In these isoforms, the internal HD (amino acids 112–135) of PrP serves as a transmembrane domain [13]. In a yeast model the HD interfered with the post-translational import of PrP into the ER, and as a consequence yeast growth was impaired and misfolded PrP accumulated in the cytosol [14]. Interestingly, both Ntm PrP and Ctm PrP are partly cytosolic proteins. Nearly half of the PrP molecule is exposed to the cytoplasm in the trans- membrane configuration and could thereby facilitate ‘toxic’ signaling events residing in the cytoplasm. Strong evidence that cytosolic PrP (cytoPrP) is neu- rotoxic emerged from a transgenic mouse model. Mice expressing a PrP mutant with a deleted N-terminal ER targeting signal acquired severe ataxia owing to cere- bellar degeneration and gliosis [15]. Cytotoxic effects of cytoPrP were also observed in some cell culture models [15–19], whereas in other studies the expression of cytoPrP seemed not to interfere with cellular viabil- ity [20,21]. Of interest, a small fraction of wild-type PrP can also be found in the cytosol of cultured cells [22,23] and neurons [24]. Moreover, some pathogenic muta- tions linked to Gerstmann–Stra ¨ ussler–Scheinker syn- drome in humans, such as Q160Stop and W145Stop, significantly increase the fraction of cytosolically locali- zed PrP [25,26]. These mutations do not change the N-terminal ER signal sequence but delete parts of the highly ordered C-terminal domain, revealing that this region is necessary for the import of PrP C into the ER [25]. What is the mechanism of cytoPrP-induced toxicity? The first studies addressing this important issue were recently described. By employing cytoPrP transgenic mice [15], it was shown that toxicity correlates with membrane localization of cytoPrP [19]. In a different study, apoptotic effects were linked to the association of cytoPrP with Bcl-2, an anti-apoptotic protein localized at the cytosolic site of ER and mitochondria membranes [17]. It also appeared that proteasomal activity and cytosolic chaperones, such as Hsp70 and Hsp40, can modulate the toxic potential of cytoPrP [17]. Of note, a variety of previous reports indicated that PrP can inter- act with chaperones, and that chaperones can modulate the formation of misfolded PrP conformers [27]. Another important question involves the possible link between the demise of scrapie-infected neurons and the formation of cytosolically localized PrP. The first clues from cell culture work show that aggresome formation in scrapie-infected mouse neuroblastoma (ScN2a) cells induces caspase-3 activation and apoptosis [28]. Toxicity of PrP located at the plasma membrane Spontaneous cerebellar neurodegeneration in certain strains of PRNP 0 ⁄ 0 mice [29] led to the discovery of doppel (Dpl), a protein structurally related to PrP C [30]. Under physiological conditions, Dpl seems not to be expressed in the brain; however, ectopic neuronal expression of Dpl induces Purkinje cell degeneration [31,32]. Dpl is complex glycosylated, harbors a GPI-anchor and shows structural homology with the C-terminal globular domain of PrP C , but lacks the N-terminal octarepeats and the internal HD [33] (Fig. 2). Interestingly, the expression of PrPDF, a mutant devoid of the octarepeats and the HD (D32– 134), induces cerebellar degeneration similarly to Dpl [32,34,35]. The neurotoxic potential of PrP variants was found to correlate with the disruption of the HD, indicating that the deletion of this domain, rather then the absence of the octarepeat region, is linked to the neurotoxic properties of PrPDF. The internal HD was identified as an important domain for basolateral sort- ing of PrP C . Moreover, Dpl, containing either the whole N-terminal domain of PrP C or the HD only, was sorted basolaterally, indicating that this domain acts as a dominant sorting signal. Vice versa, Dpl or PrP C lacking the HD were found mainly at the apical surface of MDCK cells [36]. An interesting activity of PrP C emerged from co-expression experiments in trans- genic animals: full-length PrP C can antagonize both Cerebral neurodegeneration in prion diseases J. Tatzelt & H. M. Scha ¨ tzl 608 FEBS Journal 274 (2007) 606–611 ª 2007 The Authors Journal compilation ª 2007 FEBS Dpl- and PrPDF-induced neurodegeneration [32,35]. This effect is difficult to understand in the light of the differential sorting of PrP C and Dpl. However, in polarized cells expressing Dpl and PrP C , both proteins are found at the same cellular locale, which could be a prerequisite for a functional interaction [36]. Several studies have indicated that Dpl or PrPDF can induce apoptotic cell death [37–39]. However, the major question remains how these molecules, possibly located at the plasma membrane, can activate pro- apoptotic signaling pathways. In this context it might be interesting to recall studies addressing the physiolo- gical role of PrP C . They revealed that PrP C has neuro- protective activity after an ischemic insult [40–43], supports self-renewal of hematopoietic stem cells and positively regulates neural precursor proliferation [44,45]. This indicates that the deletion of the internal HD could change a neuroprotective activity of wild- type PrP to the pro-apoptotic activity of mutants, such as PrPDF. The HD might directly mediate an interac- tion of PrP C with accessory proteins, such as trans- membrane proteins involved in PrP-induced signaling. Alternatively, deletion of the HD could indirectly affect intermolecular interactions by modulating the PrP C tertiary or quaternary structure. No central nervous system toxicity of PrP missing the GPI-anchor A leading role of neuronally expressed PrP c in medi- ating neurodegeneration first emerged from neurograft- ing studies [6] and later was reinforced by a conditional PrP knockout analysis [7]. In line with these findings, cross-linking studies of PrP C with monoclonal antibodies in vivo demonstrated the neuro- toxic signaling potential of PrP C [8]. An unexpected twist came very recently by re-addressing an old obser- vation. In prion-infected cultured mouse cells, it was found that the absence of the GPI moiety of PrP redu- ces the formation of PrP Sc [46,47]. Recently, two lines of transgenic mice were produced which expressed a PrP mutant devoid of the GPI-anchor. PrPDGPI [named GPI(–)PrPsen in the mouse study] was expressed in these mice and, similarly to the findings in cultured cells, was efficiently secreted [48,49]. After infection with three different prion strains, the trans- genic mice did not develop clinical symptoms. Quite unexpectedly, however, the brains of these mice con- tained high prion titers, about 1 : 10 compared with scrapie-infected wild-type mice. Moreover, the amount of PrP Sc at 500 days post infection in the scrapie-infec- ted PrPDGPI mice was higher than in scrapie-infected wild-type mice. This was reflected by a high load of amyloid plaques, which are less frequent in PrP wild- type mice. Interestingly, the pathological features were most pronounced along blood vessels [48]. In conclusion, although many more PrP plaques and more PK-resistant PrP Sc were present than usual, the mice harboured less prion infectivity in the brain and showed no clinical signs. How does this all fit together? First, the form of amyloid was different, reflected by a different biophysical behaviour of nonglycosylated PrP apparently highly prone to the formation of higher aggregates. It seems to be a common underlying idea in neurodegeneration that amyloid plaques are more an end-product and that smaller units on the road of aggre- gation (‘toxic folding intermediates’) are crucial players. Second, the findings could indicate that neurotoxicity of PrP Sc is linked to its propagation at the plasma mem- brane or along the endocytic pathway. There might be an ‘undesired and deadly’ interaction between PrP Sc and PrP C , resulting in a ‘false’ or prolonged stimulation of PrP C , thereby transducing a neurotoxic signal via PrP C . Alternatively, PrP Sc or precursors thereof directly interact with other cell-associated signaling molecules. Regardless, the exact mechanism of the study clearly emphasizes a critical role of the GPI anchor of PrP in the pathogenesis of prion diseases. Concluding remarks The puzzle of how infectious prions, PrP Sc , and neuro- degeneration are interconnected is still far from being solved. Obviously, prion-induced neurodegeneration may require membrane-anchored PrP in neurons, whereas expression of secreted PrP DGPI or PrP C in glia cells can promote the propagation of infectious prions without clinical symptoms, or at least with a significantly delayed onset. On the other hand, the des- cribed transgenic mice models revealed neurodegenera- tion induced by aberrant PrP conformers in the absence of prion propagation. It will now be important to show that neurotoxicity induced by alterations in folding or trafficking of PrP C is indeed relevant to neuronal cell death in a prion-diseased brain. How- ever, they are valuable models to systematically study pathways induced by neurotoxic protein conforma- tions, a challenging question also in other neurodegen- erative disorders, such as Alzheimer’s, polyglutamine and Parkinson’s disease. 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Scha ¨ tzl Cerebral neurodegeneration in prion diseases FEBS Journal 274 (2007) 606–611 ª 2007 The Authors Journal compilation ª 2007 FEBS 611 . cellular isoform of the prion protein is a key medi- ator in prion pathology. Transgenic models were instrumental in dissecting propagation of prions, disease-associated isoforms of prion protein and amyloid. isoform of prion protein is not exactly known, the impact of gain of toxic function or loss of function, or a combination of both, in prion pathology is still controversial. There is increasing. an interac- tion of PrP C with accessory proteins, such as trans- membrane proteins involved in PrP-induced signaling. Alternatively, deletion of the HD could indirectly affect intermolecular interactions