help button home button Am J Pathol R & D Systems
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kovács, G. G.
Right arrow Articles by Budka, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kovács, G. G.
Right arrow Articles by Budka, H.
(American Journal of Pathology. 2005;166:287-294.)
© 2005 American Society for Investigative Pathology

Subcellular Localization of Disease-Associated Prion Protein in the Human Brain

Gábor G. Kovács*{dagger}, Matthias Preusser*, Michaela Strohschneider* and Herbert Budka*

From the Institute of Neurology, Medical University of Vienna, and Austrian Reference Centre for Human Prion Diseases,* Vienna, Austria; and the National Institute of Psychiatry and Neurology and Hungarian Reference Centre for Human Prion Diseases,{dagger} Budapest, Hungary


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Disease-associated prion protein (PrPTSE) deposits in distinct immunostaining patterns in the brain in Creutzfeldt-Jakob disease, including synaptic, extracellular, and cell-associated localizations. After having developed an appropriate pretreatment protocol to enhance immunostaining for PrPTSE without damaging epitopes of other antigens, we systematically evaluated co-localization patterns of distinct PrPTSE immunodeposits by confocal laser microscopy, including optical serial sectioning. As shown by quantification, the most prominent co-localization of PrPTSE is with synaptophysin, but PrPTSE may also co-deposit with connexin-32, a gap junction-related protein. Furthermore, neuronal cell bodies, dendrites, axons, astrocytes, and microglia harbor granular PrPTSE deposits. Highly aggregated deposits are focally ubiquitinated. We conclude that PrPTSE is not exclusively associated with chemical but also with electric synapses, axonal transport may be a relevant route of PrPTSE spread in the brain, and activated microglia and astrocytes may play a role in PrPTSE processing, degradation, or removal.

Post-translational conversion of the {alpha}-helix-dominant cellular prion protein (PrPC) into the ß-sheet-dominant PrPSc is a common characteristic of animal and human prion diseases including Creutzfeldt-Jakob disease (CJD).1 Both protease-resistant and protease-sensitive forms of PrPSc are known.2 Due to the lack of unequivocally conformation-specific antibodies, immunohistochemical differentiation of PrPC from PrPSc has remained a matter of debate and creates a barrier for the evaluation of disease process. However, detailed immunohistochemical descriptions define patterns which are specific for disease,3 thus the term disease-associated PrP (labeled PrPTSE in the present study) is suitable to label conformationally uncharacterized deposits of PrP observed only in prion-diseased brains by immunohistochemistry.

In the diseased human brain, main deposition patterns of PrPTSE are so-called diffuse/synaptic, patchy/perivacuolar, perineuronal, and plaque-like patterns.3 The first is characterized by a diffuse fine-granular distribution of anti-PrPTSE immunoreactivity resembling the distribution of the presynaptic protein synaptophysin (hence the term "synaptic pattern"). Presynaptic domains appear to be the privileged site of PrPC, as has been confirmed by double labeling with synaptophysin.4 Also a post-synaptic localization of PrPC is sometimes observed in synapses of the central nervous system (CNS) and muscle-nerve synapses.4 Others suggest that PrPC is almost excluded from synaptic vesicles and also describe its presence in the cytosol of certain neuronal subpopulations.5 Cell culture studies indicated a destiny of PrPC for the plasma membrane and caveolae-like membranous domains.6 Immunogold electron microscopy has demonstrated presence of PrP epitopes in a presynaptic localization in human variant CJD and bovine spongiform encephalopathy-infected monkey brain.4,7 Additionally, subcellular fractionation has shown that the highest concentration of PrPTSE in CJD brain is found in the synaptosomal fraction, however, without distinguishing between pre- and post-synaptic localization.8

Experimental data suggest that PrPTSE may also be found in other parts of neurons than their synaptic terminals, furthermore, there is also evidence for accumulation of PrPTSE in astrocytes and microglia.4 Due to methodological difficulties, confirmatory evaluation of PrPTSE deposition in the diseased human brain is lacking. After having developed an appropriate pretreatment protocol to visualize immunostaining for PrPTSE without damaging epitopes of other antigens, we systematically studied PrPTSE deposition by double immunofluorescent labeling and evaluated co-localization using laser confocal microscopy.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Four autopsy cases of sporadic Creutzfeld-Jakob disease (sCJD), with full-length analysis of the prion protein gene (PRNP), were selected from the tissue bank of the Institute of Neurology, Medical University Vienna, Austria. We selected CJD cases with relatively preserved cortical structure along with high amounts of immunohistochemically detectable PrPTSE. Cases 1 (72-year-old female, 5-month disease duration) and 2 (65-year-old female, 1-month disease duration) were methionine/methionine (MM) homozygotes at the polymorphic codon 129 of PRNP; case 3 (74-year-old male, 6-week disease duration) was a VV homozygote; and case 4 (67-year-old female, 12-month disease duration) was a methionine/valine (MV) heterozygote. Two control cases without prion disease were included as well (control case 1: 78-year-old male, cause of death, myocardiac insufficiency; control case 2: 78-year-old female, cause of death, myocardiac infarction). In all cases, brain tissue samples containing cerebral cortex and white matter were routinely fixed in 4% buffered formalin. This was performed within 12 hours (CJD cases 2 and 3), 24 hours (CJD case 4 and control case 1), 48 hours (CJD case 1 and control case 2) after death. Tissues were stored in formalin for 5 days (CJD case 1), 5 weeks (CJD case 2), 2 months (CJD case 3), 6 days (CJD case 4), 11 days (control case 1), and 4 days (control case 2) before paraffin-embedding. Paraffin-embedded tissue blocks were stored in darkness and at room temperature for 36 months (CJD case 1), 1 month (CJD case 2), 18 months (CJD case 3), 1 month (CJD case 4), 2 months (control case 1), and 2 weeks (control case 2) before being processed for this study as specified below.

Immunohistochemistry and Laser Confocal Microscopy

Five-µm thick sections were obtained from each tissue block for conventional immunohistochemistry and double immunofluorescent labeling. Twenty-µm thick tissue sections were used for optical serial sectioning ("z-stacking"). After a pilot fashion testing of different pretreatment protocols suitable to detect both PrPTSE and selected antigens, we defined a protocol that included 20 minutes of autoclaving (100°C) in citrate buffer (pH 6.0) and incubation for 2 minutes in formic acid (96%).

Commercially available primary antibodies with well-defined specificity used for this study are listed in Table 1 . We used the monoclonal anti-PrP antibody 12F10 that was previously shown to be the most reliable in detecting disease-associated PrP.3 Sections were incubated overnight at room temperature with each antibody.


View this table:
[in this window]
[in a new window]
  		Table 1. Summary of Antibodies Used in This Study

 
For double immunolabeling, the fluorescent-labeled secondary antibody for the anti-PrP antibody 12F10 was Alexa Fluor 488 goat anti-mouse IgG (1:200, Molecular Probes, USA), for anti-SMI-31, anti-SMI-32, anti-MAP-2, anti-connexin-32, anti-synaptophysin, and anti-HLA-DR Alexa Fluor 546 goat anti-mouse IgG (1:200, Molecular Probes), and for synapsin, GFAP, and ubiquitin goat anti-rabbit IgG (1:200, Molecular Probes). Primary antibodies were visualized with fluorophore-labeled, anti-mouse Fc, Fab fragments, using the Zenon Alexa Fluor 488 Mouse IgG2a Labeling Kit (Molecular Probes, Leiden, The Netherlands) for 12F10, and the Zenon Alexa Fluor 546 Mouse IgG1 Labeling Kit (Molecular Probes, Leiden, The Netherlands) for other antibodies. We used argon 488-nm and helium/neon 633-nm and 543-nm lasers to elicit immunofluorescent staining. Immunofluorescence labeling was evaluated using a Zeiss LSM 510 laser scanning confocal microscope. The optical pinhole was adjusted for each scanning experiment to obtain optical slices with a 0.2-µm thickness. Optical serial sections with distances of 0.5 µm between individual sections were obtained to confirm intracellular localization of PrPTSE deposits. To compare the proportion of co-localized pixels of PrPTSE/synaptophysin and PrPTSE/connexin-32 we extracted 10 randomly selected 1000 µm2 areas for quantification from the scanned images of double immunolabeling. Accepting bright yellow color as a marker of co-labeling of green and red particles, we used Adobe Photoshop 6.0 to select one bright yellow-colored pixelbox from each of the saved images. Pixelboxes containing bright yellow components were highlighted and counted manually on the screen. A similar procedure was performed for green color. We then calculated the percentage of pixelboxes containing a yellow component from the total number (yellow+green) of counted pixelboxes.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
By testing pretreatment protocols, we have defined a protocol suitable for our study. This enabled us to detect either diffuse/synaptic, patchy/perivacuolar, perineuronal, or plaque-type PrPTSE deposits sufficiently after this pretreatment (Figure 1, a to d) . In comparison with our standard immunohistochemical pretreatment protocol,3 the signal was slightly less intense but labeled all structures similarly (Figure 1, m and n) . In addition, all other antibodies used in our study convincingly detected their respective antigens with this protocol (Figure 1, e to l) in contrast to the loss of immunoreactivity after using the three-tiered pretreatment protocol for PrPTSE (Figure 1, o to t) . In normal control brains we found no immunoreactivity for PrPTSE, and we did not observe immunolabeling of any cells in the CNS (Figure 4) .



View larger version (85K):
[in this window]
[in a new window]
  		Figure 1. a–l: The modified pretreatment protocol allows satisfactory immunohistochemical detection of diffuse/synaptic (a–c), patchy/perivacuolar (b), perineuronal (c) PrPTSE deposits in sporadic, codon 129 MM (a–b), and VV (c) homozygote CJD cases. Plaque-type PrPTSE deposits are observed in a codon 129 MV (d) CJD case (magnification, x10, a–d, left; magnification, x40, a—d, right). In addition, synaptophysin (e), connexin-32 (f), phosphorylated neurofilaments (g), unphosphorylated neurofilaments (h), MAP-2 (i), HLA-DR (j), GFAP (k), and ubiquitin (l) are satisfactorily immunolabeled using the same pretreatment protocol (all case 3 except h which is case 4; magnification, x40). m–t: The standard three-tiered pretreatment protocol for PrPTSE allows immunohistochemical detection of synaptic (m) and perivacuolar/patchy (n) PrPTSE deposits using antibody 12F10 but strongly reduces or abolishes immunoreactivity against synaptophysin (o), connexin-32 (p), phosphorylated neurofilaments (q), unphosphorylated neurofilaments (r), MAP-2 (s), and HLA-DR (t) (m and o–t show case 3, n shows case 2; magnification, x40).

 


View larger version (74K):
[in this window]
[in a new window]
  		Figure 4. Confocal laser scanning microscopy of double immunofluorescent labeling with 12F10 (a–d) and anti-GFAP (a), SMI 31 (b) anti-synaptophysin (c), and anti-connexin-32 using the modified pretreatment protocol in a non-CJD brain (control case 1; blue signals indicate autofluorescence, eg, lipopigment). No green PrP deposits are detectable, while astrocytes (a), axons (b), presynaptic structures (c), and gap junction-related components (d) are immunolabeled (red).

 
PrPTSE and Synapses: Chemical and Electric

In all cases, fine granular, so-called diffuse/synaptic type, of PrPTSE deposits co-localized with synaptophysin (Figure 2a) . A similar co-labeling pattern was seen with synapsin I (data not shown). Quantification analysis revealed co-localization of PrPTSE with synaptophysin in 72.91 + 5.9% of pixels. Coarse PrPTSE deposits like patchy/perivacuolar or plaque type did not show co-localization with synaptophysin. Co-labeling of fine granular PrPTSE and connexin-32 was shown in 10.78 + 3.8% of pixels (Figure 2b) .



View larger version (89K):
[in this window]
[in a new window]
  		Figure 2. Confocal laser scanning microscopy of double immunofluorescent labeling for PrPTSE (a–g: green) and synaptophysin (a), connexin-32 (b), phosphorylated neurofilaments (c–e), unphosphorylated neurofilaments (f), and MAP-2 (g) using an appropriate pretreatment protocol. Presynaptic structures (a), gap junction-related components (b), axons (c–e), neurons and neuronal processes (f–g) are labeled with red. Autofluorescence (eg, lipofuscin) is labeled with blue. Co-localization is shown by yellow color. Optical sections are 0.2-µm thick. c: Split image showing PrPTSE granules following the outline of an axon, thus indicating intra-axonal localization of PrPTSE granules. Intra-axonal localization of PrPTSE granules is confirmed by optical serial sections (individual sections are 0.2-µm thick and 0.5 µm apart) showing that individual PrPTSE granules are surrounded by axoplasm on all sides (d–e). The area highlighted in (d) by a white box is shown in (e). Further, we observed clustering of granular PrPTSE immunoreactivity within the perikaryon in close association with the surface of neurons (f) and within dendrites (g). Additionally there is perineuronal and periaxonal deposition of PrpTSE (f). On the right of (a), (b), (f), and (g), pixelboxes of three selected areas showing co-localization are enlarged. Fine granular (diffuse/synaptic) PrP immunodeposits are seen in all pictures.

 
PrPTSE and Neurons: Processes and Cell Body

Using the SMI-31 antibody to immunolabel axons, we noted co-localization with PrPTSE within the axon (Figure 2, c to e) . This was also evident after creating split images where the incorporated PrPTSE granular deposits follow the outline of an axon (Figure 2c) . Intra-axonal localization of PrPTSE granules was confirmed by optical serial sections showing that individual PrPTSE granules are surrounded by axoplasm on all sides (Figure 2, d and e) . Infrequently, we observed clustering of granular PrPTSE immunoreactivity within the perikaryon in close association with the surface of neurons (Figure 2f) and dendrites (Figure 2g) of neurons. Additionally there is perineuronal and periaxonal deposition of PrPTSE (Figure 2f) . None of the neuronal markers showed co-labeling with PrPTSE in aggregated patchy/perivacuolar deposits.

PrPTSE and Glia: Astro- and Microgliosis

Rarely, we observed PrPTSE deposits within micro- (Figure 3, a and b) and astroglial (Figure 3, c to e) cells. Similarly to the intra-axonal distribution, intracytoplasmic astro- and microglial localization of PrPTSE deposits was confirmed by optical serial sectioning, showing that PrPTSE deposits are surrounded by cytoplasm on all sides. Occasionally PrPTSE deposits were clustered in close vicinity of astroglial cells that also showed intracytoplasmic accumulation of PrPTSE (Figure 3e) .



View larger version (97K):
[in this window]
[in a new window]
  		Figure 3. Confocal laser scanning microscopy of double immunofluorescent labeling with 12F10 (a–f, green) and HLA-DR (a–b), GFAP (c–e), and ubiquitin (f) using an appropriate pretreatment protocol. Microglia (a–b), astrocytes (c–e), and ubiquitin (f) are labeled with red. Autofluorescence is demonstrated by blue color in the right lower corner inset (e). Optical sections are 0.2-µm thick. We observed granular PrPTSE deposits in the cytoplasms of microglial cells (a) and astrocytes (c–e). Periastrocytic accentuation of PrPTSE deposits is noted (c). On the right of (a–f), pixelboxes of three selected areas are enlarged. Fine granular (diffuse/synaptic) PrP immunodeposits are seen in (a and c) (case 1), (e) (case 3), while coarse patchy/perivacuolar immunodeposits are seen in (b, d, and f) (case 2). Magnification, x100 (a–f).

 
Ubiquitination of PrPTSE

Parts of extensive aggregated deposits of PrPTSE co-localized with ubiquitin frequently (Figure 2f) while fine granular deposits do not appear to be ubiquitinated.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Immunohistochemistry for PrP is a powerful tool for diagnosing prion disease.9 To avoid misinterpretation caused by the inability of available antibodies to distinguish between normal and pathological conformers of PrP, tissues must undergo special pretreatment before immunostaining to abolish PrPC and to enhance PrPTSE immunoreactivities. Commonly used pretreatment protocols use the combination of hydrated or hydrolytic autoclaving at 121° C of formic acid, guanidine thiocyanate, and proteinase K.3 However, these pretreatment protocols also damage other antigens present in the tissue, making their immunodetection impossible. This is one reason for the paucity of co-labeling studies defining the exact localization of PrPTSE in the CNS.

Regional distribution of PrPTSE was shown to be different in sporadic, iatrogenic, and genetic prion disease cases.10 Later molecular classification based on Western blot characteristics of the protease-resistant PrP and the PRNP polymorphism at codon 129 has revealed that the PrP immunostaining patterns are distinguishable in anatomical regions and molecular phenotypes of sporadic CJD cases.11,12 In our present study we collected samples harboring all major morphological subtypes of PrPTSE immunodeposits related to sporadic CJD brains.

Co-localization of PrPTSE deposits with synaptophysin and synapsin I confirms previous ultrastructural studies that PrPTSE is present in presynaptic components. Quantification analysis, however, suggests the existence of other sites of PrPTSE deposition. Surprisingly, we observed co-labeling of fine granular PrPTSE and connexin-32. Connexins are molecular constituents of gap junctions (electric synapses) that are distributed in astrocytes, oligodendrocytes, and neurons.13,14 Connexin-32 is predominantly related to neurons, less to oligo-, and not to astrocytes.14 Interestingly, mainly GABAergic interneurons are interconnected through electric synapses, a subset of which is particularly vulnerable in human and experimental prion diseases.15,16 Accumulation of PrPTSE in gap junctions may be a substrate for neuronal damage as the role of these structures involves intra- and extracellular homeostasis, trafficking and supply of energy-producing substrates to neurons, or neuroprotection.14

We observed unequivocal localization of PrPTSE granules within axons. Previous ultrastructural studies have suggested association of PrPTSE with the plasmalemma of neurites,17,18 however, PrPTSE has never been detected inside neurites. Presence of PrPTSE in the cytosol of axons is not unexpected, since a recent study demonstrated the existence of cytosolic PrPC probably following a retro-translocation from the endoplasmic reticulum.5 As PrPTSE has been shown to traffic also in endosome-like organelles,19 it is also conceivable that our demonstration of intra-axonal PrPTSE has such subcellular basis. However, our observation merits (immuno)-ultrastructural confirmation to clarify whether PrPTSE within the axons is free in the cytosol or related to vesicles (eg, endosomal). Recent observations in experimental models suggest that transport of PrPTSE along peripheral nerves to the CNS is following a domino-like manner along PrPC-expressing nerve membranes rather than being achieved via conventional axonal transport.20 In human prion disease PrPTSE deposits in the CNS may be observed in both fine and coarse perineuronal patterns supporting a domino-like manner of propagation. However, in human disease the perineuronal pattern is not predominant. It is seen mainly in VV or MV type 2 subtypes of sporadic CJD cases and variant CJD, suggesting that this might be a strain-dependent phenomenon.3,21,22 In contrast, fine granular, so-called diffuse/synaptic, deposits of PrPTSE represent the major morphological appearance. We thus suggest that PrPTSE in the CNS follows at least two pathways, one along and one within neurites. Although PrPC is subject to fast anterograde transport,23 other possibilities need to be elucidated. One important route is probably ad-axonal, however, in circumstances with overwhelming production of PrPTSE, which is more likely to happen in the CNS and not in the periphery, conventional axonal transport systems may participate in distribution of PrPTSE.

Clusters of granular PrPTSE immunoreactivity within the perikaryon in close association with the surface of neurons and dendrites of neurons must be distinguished from an occasional diffuse cytoplasmic immunolabeling of neurons which has also been described in non-diseased brains and is considered to represent cellular PrP.24,25 The anti-PrP antibody (12F10) applied in our present study does not show this type of diffuse neuronal labeling.3 The granular deposits in the cell body and dendrites suggest a post-synaptic localization of PrPTSE. This is compatible with previous descriptions of a post-synaptic localization of cellular PrP,4 which is considered to be a prerequisite for formation of PrPTSE. We confirm previous ultrastructural studies that have already noted PrPTSE in neuronal perikarya that was proposed to be mainly related to the lysosomal-endosomal system there.4 The PrPC-PrPSc conversion was suggested to use the endocytic pathway via the caveola-like domain membrane or coated pits to the endosomal-lysosomal system. The latter were suggested to be key organelles in the processing route toward production of PrPSc not only in cell culture, but also in animal TSE and human variant CJD brains.7,26 Having found no neuronal markers within aggregated patchy/perivacuolar or plaque-like deposits we excluded the possibility that neuronal debris is a component of these amorphous materials.

Our observations that micro- and astroglial cells harbor intracytoplasmic PrPTSE granules and that there is periastrocytic accumulation of PrPTSE deposits suggest that both microglial and astroglial cells may have a role in the processing, degradation, or removal of PrPTSE. Whether microglia are capable of phagocytosing aggregated PrPTSE material has rarely been addressed in research, although prominent microgliosis is a characteristic finding in CJD brains. Only a recent study demonstrated that spleen macrophages actively participate in the clearance of intraperitoneal scrapie inoculum.27 Activation of micro- and astroglial cells at sites of aggregated PrPTSE deposits and accumulation of PrPTSE in lysosomes have been documented previously.28 In an in vitro model using prion fragment 106–126 it has been shown that microglia is needed for neurotoxicity, while the same fragment stimulates proliferation of astrocytes.29,30 Microglia activation occurs early in disease but there is neither direct in vivo evidence that PrPSc is proinflammatory nor was it demonstrated to what microglial cells actually respond.29

Ubiquitination of PrPTSE was already suggested by demonstrating ubiquitin immunoreactivity in areas where PrPTSE deposits are observed (eg, as perivacuolar immunopositivity).31 Here we confirm that highly aggregated PrPTSE deposits indeed co-localize with ubiqutin frequently. That disease-associated PrP may be ubiquitinated is supported by a recent study, using immunoprecipitation and sandwich ELISA, showing ubiqituination of PrP after developing protease resistance in the brains of scrapie-infected mice.32

In sum, our study suggests alteration of both chemical, indicated by the presynaptic protein synaptophysin, and electric synapses, indicated by the gap junction-related protein connexin-32, by accumulated PrPTSE. This might be a central substrate of prion disease pathogenesis and clinical deterioration either by abolishing homeostatic communication between cells or by releasing inhibition. By demonstrating PrPTSE in the neuronal cell body and dendrites, a role of post-synaptic, in addition to previously assumed presynaptic, structures in CJD pathogenesis is proposed. The observation of both intra- and ad-axonal localization of PrPTSE suggests that both intra-axonal transport as well as domino-like propagation along the axonal surface may occur in the CNS. Moreover, our results suggest that PrPTSE may be processed or degraded in micro- or astroglial cells.


   Acknowledgements
 
We thank Gerda Ricken for excellent technical assistance.


   Footnotes
 
Address reprint requests to Professor Herbert Budka, Institute of Neurology, AKH 4J, Währinger Gürtel 18–20, POB 48, A-1097 Vienna, Austria. E-mail: herbert.budka{at}meduniwien.ac.at

Supported by Hungarian-Austrian intergovernmental S&T cooperation programme (A14/02) (to G.G.K., M.P., and H.B.), Bolyai fellowship, and ETT 69/03 (to G.G.K.). This work was supported by the EU FP 5 QoL projects TSELAB and SEEC-CJD, and has been performed within the framework of the EU FP6 Network of Excellence NeuroPrion.

G.K. and M.P. contributed equally to this study.

Accepted for publication September 16, 2004.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Prusiner SB: Prions. Proc Natl Acad Sci USA 1998, 95:13363-13383[Abstract/Free Full Text]
  2. Safar J, Wille H, Itri V, Groth D, Serban H, Torchia M, Cohen FE, Prusiner SB: Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med 1998, 4:1157-1165[Medline]
  3. Kovács GG, Head MW, Hegyi I, Bunn TJ, Flicker H, Hainfellner JA, McCardle L, Laszlo L, Jarius C, Ironside JW, Budka H: Immunohistochemistry for the prion protein: comparison of different monoclonal antibodies in human prion disease subtypes. Brain Pathol 2002, 12:1-11[Medline]
  4. Fournier JG, Escaig-Haye F, Grigoriev V: Ultrastructural localization of prion proteins: physiological and pathological implications. Microsc Res Tech 2000, 50:76-88[Medline]
  5. Mironov AJ, Latawiec D, Wille H, Bouzamondo-Bernstein E, Legname G, Williamson RA, Burton D, DeArmond SJ, Prusiner SB, Peters PJ: Cytosolic prion protein in neurons. J Neurosci 2003, 23:7183-7193[Abstract/Free Full Text]
  6. Vey M, Pilkuhn S, Wille H, Nixon R, DeArmond SJ, Smart EJ, Anderson RG, Taraboulos A, Prusiner SB: Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. Proc Natl Acad Sci USA 1996, 93:14945-14949[Abstract/Free Full Text]
  7. Grigoriev V, Escaig-Haye F, Streichenberger N, Kopp N, Langeveld J, Brown P, Fournier JG: Submicroscopic immunodetection of PrP in the brain of a patient with a new-variant of Creutzfeldt-Jakob disease. Neurosci Lett 1999, 264:57-60[Medline]
  8. Kitamoto T, Shin RW, Doh-ura K, Tomokane N, Miyazono M, Muramoto T, Tateishi J: Abnormal isoform of prion proteins accumulates in the synaptic structures of the central nervous system in patients with Creutzfeldt-Jakob disease. Am J Pathol 1992, 140:1285-1294[Abstract]
  9. Budka H: Histopathology and immunohistochemistry of human transmissible spongiform encephalopathies (TSEs). Arch Virol Suppl 2000, 16:135-142
  10. Brown P, Kenney K, Little B, Ironside JW, Will R, Cervenakova L, Bjork RJ, San Martin RA, Safar J, Roos R, Haltia M, Gibbs CJ, Gajdusek DC: Intracerebral distribution of infectious amyloid protein in spongiform encephalopathy. Ann Neurol 1995, 38:245-253[Medline]
  11. Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl O, Zerr I, Budka H, Kopp N, Piccardo P, Poser S, Rojiani A, Streichemberger N, Julien J, Vital C, Ghetti B, Gambetti P, Kretzschmar H: Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol 1999, 46:224-233[Medline]
  12. Hill AF, Joiner S, Wadsworth JD, Sidle KC, Bell JE, Budka H, Ironside JW, Collinge J: Molecular classification of sporadic Creutzfeldt-Jakob disease. Brain 2003, 126:1333-1346[Abstract/Free Full Text]
  13. Bennett MV, Barrio LC, Bargiello TA, Spray DC, Hertzberg E, Saez JC: Gap junctions: new tools, new answers, new questions. Neuron 1991, 6:305-320[Medline]
  14. Rouach N, Avignone E, Meme W, Koulakoff A, Venance L, Blomstrand F, Giaume C: Gap junctions and connexin expression in the normal and pathological central nervous system. Biol Cell 2002, 94:457-475[Medline]
  15. Guentchev M, Hainfellner JA, Trabattoni GR, Budka H: Distribution of parvalbumin-immunoreactive neurons in brain correlates with hippocampal and temporal cortical pathology in Creutzfeldt-Jakob disease. J Neuropathol Exp Neurol 1997, 56:1119-1124[Medline]
  16. Guentchev M, Groschup MH, Kordek R, Liberski PP, Budka H: Severe, early and selective loss of a subpopulation of GABAergic inhibitory neurons in experimental transmissible spongiform encephalopathies. Brain Pathol 1998, 8:615-623[Medline]
  17. Jeffrey M, Goodsir CM, Bruce ME, McBride PA, Scott JR, Halliday WG: Infection-specific prion protein (PrP) accumulates on neuronal plasmalemma in scrapie-infected mice. Neurosci Lett 1992, 147:106-109[Medline]
  18. Jeffrey M, Goodsir CM, Bruce ME, McBride PA, Fowler N, Scott JR: Murine scrapie-infected neurons in vivo release excess prion protein into the extracellular space. Neurosci Lett 1994, 174:39-42[Medline]
  19. Arnold JE, Tipler C, László L, Hope J, Landon M, Mayer RJ: The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain. J Pathol 1995, 176:403-411[Medline]
  20. Kunzi V, Glatzel M, Nakano MY, Greber UF, Van Leuven F, Aguzzi A: Unhampered prion neuroinvasion despite impaired fast axonal transport in transgenic mice overexpressing four-repeat tau. J Neurosci 2002, 22:7471-7477[Abstract/Free Full Text]
  21. Ironside JW: Pathology of variant Creutzfeldt-Jakob disease. Arch Virol Suppl 2000, 16:143-151
  22. Kovács GG, Head MW, Bunn T, Laszlo L, Will RG, Ironside JW: Clinicopathological phenotype of codon 129 valine homozygote sporadic Creutzfeldt-Jakob disease. Neuropathol Appl Neurobiol 2000, 26:463-472[Medline]
  23. Rodolfo K, Hassig R, Moya KL, Frobert Y, Grassi J, Di Giamberardino L: A novel cellular prion protein isoform present in rapid anterograde axonal transport. Neuroreport 1999, 10:3639-3644[Medline]
  24. Kovács GG, Voigtlander T, Hainfellner JA, Budka H: Distribution of intraneuronal immunoreactivity for the prion protein in human prion diseases. Acta Neuropathol 2002, 104:320-326[Medline]
  25. McKinley MP, Taraboulos A, Kenaga L, Serban D, Stieber A, DeArmond SJ, Prusiner SB, Gonatas N: Ultrastructural localization of scrapie prion proteins in cytoplasmic vesicles of infected cultured cells. Lab Invest 1991, 65:622-630[Medline]
  26. László L, Lowe J, Self T, Kenward N, Landon M, McBride T, Farquhar C, McConnell I, Brown J, Hope J, Mayer RJ: Lysosomes as key organelles in the pathogenesis of prion encephalopathies. J Pathol 1992, 166:333-341[Medline]
  27. Beringue V, Demoy M, Lasmezas CI, Gouritin B, Weingarten C, Des JP, Andreux JP, Couvreur P, Domont D: Role of spleen macrophages in the clearance of scrapie agent early in pathogenesis. J Pathol 2000, 190:495-502[Medline]
  28. Jeffrey M, Goodsir CM, Bruce ME, McBride PA, Farquhar C: Morphogenesis of amyloid plaques in 87V murine scrapie. Neuropathol Appl Neurobiol 1994, 20:535-542[Medline]
  29. Perry VH, Cunningham C, Boche D: Atypical inflammation in the central nervous system in prion disease. Curr Opin Neurol 2002, 15:349-354[Medline]
  30. Forloni G, Del Bo R, Angeretti N, Chiesa R, Smiroldo S, Doni R, Ghibaudi E, Salmona M, Porro M, Verga L, Giaccone G, Bugiani O, Tagliavini F: A neurotoxic prion protein fragment induces rat astroglial proliferation and hypertrophy. Eur J Neurosci 1994, 6:1415-1422[Medline]
  31. Ironside JW, McCardle L, Hayward PA, Bell JE: Ubiquitin immunocytochemistry in human spongiform encephalopathies. Neuropathol Appl Neurobiol 1993, 19:134-140[Medline]
  32. Kang S, Brown DR, Whiteman M, Li R, Pan T, Perry G, Wisniewski T, Sy M, Wong B: Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice. J Pathol 2003, 203:603-608



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
G. G. Kovacs and H. Budka
Prion Diseases: From Protein to Cell Pathology
Am. J. Pathol., March 1, 2008; 172(3): 555 - 565.
[Abstract] [Full Text] [PDF]

Home page
 J. Gen. Virol.Home page
C. Kratzel, D. Kruger, and M. Beekes
Prion propagation in a nerve conduit model containing segments devoid of axons
J. Gen. Virol., December 1, 2007; 88(12): 3479 - 3485.
[Abstract] [Full Text] [PDF]

Home page
 J. Gen. Virol.Home page
G. Marruchella, C. Ligios, V. Albanese, M. G. Cancedda, L. Madau, G. Lalatta-Costerbosa, M. Mazzoni, P. Clavenzani, R. Chiocchetti, G. Sarli, et al.
Enteroglial and neuronal involvement without apparent neuron loss in ileal enteric nervous system plexuses from scrapie-affected sheep
J. Gen. Virol., October 1, 2007; 88(10): 2899 - 2904.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. Priller, M. Prinz, M. Heikenwalder, N. Zeller, P. Schwarz, F. L. Heppner, and A. Aguzzi
Early and Rapid Engraftment of Bone Marrow-Derived Microglia in Scrapie.
J. Neurosci., November 8, 2006; 26(45): 11753 - 11762.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Kristiansen, M. J. Messenger, P.-C. Klohn, S. Brandner, J. D. F. Wadsworth, J. Collinge, and S. J. Tabrizi
Disease-related Prion Protein Forms Aggresomes in Neuronal Cells Leading to Caspase Activation and Apoptosis
J. Biol. Chem., November 18, 2005; 280(46): 38851 - 38861.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
S. J. Barmada and D. A. Harris
Visualization of Prion Infection in Transgenic Mice Expressing Green Fluorescent Protein-Tagged Prion Protein
J. Neurosci., June 15, 2005; 25(24): 5824 - 5832.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kovács, G. G.
Right arrow Articles by Budka, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kovács, G. G.
Right arrow Articles by Budka, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS