the misfolded and partially protein-ase K PK-resistant isoform of the cellular prion pro-tein PrPC] is closely linked to the propagation of infectious prions, but apparently is not suffic
Trang 1Molecular basis of cerebral neurodegeneration in prion
diseases
Jo¨rg Tatzelt1and Hermann M Scha¨tzl2
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 (PrPSc) [i.e the misfolded and partially
protein-ase K (PK)-resistant isoform of the cellular prion
pro-tein (PrPC)] 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 PrPC 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 PrPC[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 NH2-terminus in the
endoplasmic reticulum lumen; PK, proteinase K; PrP, prion protein; PrPC, normal cellular isoform of PrP; PrPSc, disease-associated isoform of PrP; secPrP, secretory PrP.
Trang 2introduced by C Weissmann [3] Two common phases
in prion diseases can be described In a first phase, with
apparently obligate requirement for PrPC expression,
profound conformational changes give rise to PrP
aggregation and the formation of PrPSc, 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 PrPSc is used synonymously for the
pathological form of PrP
Toxicity of transmembrane isoforms
of PrP
The evidence that PrPSc 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 (CtmPrP), has its COOH-terminus in the endoplasmic reticulum (ER) lumen The other form, termed N-trans transmembrane (NtmPrP), has its NH2-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 CtmPrP and the remainder asNtmPrP Naturally occurring and arti-ficial mutations in the membrane-spanning segment can lead to significantly increased generation of
CtmPrP In addition, several pieces of evidence have linked CtmPrP to neurodegeneration in transgenic mice
P r
P S c
prion propagation prion propagation
P r
P 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.
O C O C
S -I P G S
-R E
l p D
O C H
S -I P G
β1 α1 β2 α2 α3
β1 α1 β2 α2 α3
α1 β2 α2 α3
S
-R
E
P r
P c
O C H
S -I P G S
-R E
P r
P 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.
Trang 3and to some heritable prion diseases (mutation A117V
in the Gerstmann–Stra¨ussler–Scheinker syndrome)
The CtmPrP 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 PrPSc and of infectious prions On the other
hand, it was later shown that a prion infection
appar-ently can trigger the generation of ‘toxic’ CtmPrP [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 CtmPrP, 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 (NtmPrP and CtmPrP) and the
increased synthesis ofCtmPrP 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 NtmPrP and
CtmPrP 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 PrPCinto 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 PRNP0⁄ 0 mice [29] led to the discovery of doppel (Dpl), a protein structurally related to PrPC [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 PrPC, 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 PrPC Moreover, Dpl, containing either the whole N-terminal domain of PrPC or the HD only, was sorted basolaterally, indicating that this domain acts as a dominant sorting signal Vice versa, Dpl or PrPC lacking the HD were found mainly at the apical surface of MDCK cells [36] An interesting activity of PrPCemerged from co-expression experiments in trans-genic animals: full-length PrPC can antagonize both
Trang 4Dpl- and PrPDF-induced neurodegeneration [32,35].
This effect is difficult to understand in the light of the
differential sorting of PrPC and Dpl However, in
polarized cells expressing Dpl and PrPC, 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 PrPC They revealed that PrPChas
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 PrPC 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
PrPCtertiary or quaternary structure
No central nervous system toxicity of
PrP missing the GPI-anchor
A leading role of neuronally expressed PrPc 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 PrPC with
monoclonal antibodies in vivo demonstrated the
neuro-toxic signaling potential of PrPC [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 PrPSc[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 PrPScat 500 days post infection in the
scrapie-infec-ted PrPDGPI mice was higher than in scrapie-infecscrapie-infec-ted
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 PrPSc 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 PrPScis linked to its propagation at the plasma mem-brane or along the endocytic pathway There might be
an ‘undesired and deadly’ interaction between PrPScand PrPC, resulting in a ‘false’ or prolonged stimulation of PrPC, thereby transducing a neurotoxic signal via PrPC Alternatively, PrPSc 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, PrPSc, 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 PrPDGPI or PrPC 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 PrPC 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
Acknowledgements The work of the authors is supported by grants from the
‘Deutsche Forschungsgemeinschaft’, the ‘Bayerisches Staatsministerium fu¨r Wissenschaft, Forschung und
Trang 5Kunst’, the ‘Bayerisches Staatsministerium fu¨r
Verbr-aucherschutz’, the ‘Bundesministerium fu¨r Bildung und
Forschung’, and the European Union
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