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1.
Virus Res ; 207: 136-45, 2015 Sep 02.
Article in English | MEDLINE | ID: mdl-25683509

ABSTRACT

The cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein that is most abundant in the central nervous system. It is thought to play a role in many cellular processes, including neuroprotection, but may also contribute to Alzheimer's disease and some cancers. However, it is best known for its central role in the prion diseases, such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. These protein misfolding diseases can be sporadic, acquired, or genetic and are caused by refolding of endogenous PrP(C) into a beta sheet-rich, pathogenic form, PrP(Sc). Once prions are present in the central nervous system, they increase and spread during a long incubation period that is followed by a relatively short clinical disease phase, ending in death. PrP molecules can be broadly categorized as either 'good' (cellular) PrP(C) or 'bad' (scrapie prion-type) PrP(Sc), but both populations are heterogeneous and different forms of PrP(C) may influence various cellular activities. Both PrP(C) and PrP(Sc) are localized predominantly at the cell surface, with the C-terminus attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor and both can exist in cleaved forms. PrP(C) also has cytosolic and transmembrane forms, and PrP(Sc) is known to exist in a variety of conformations and aggregation states. Here, we discuss the roles of different PrP isoforms in sickness and in health, and show the subcellular distributions of several forms of PrP that are particularly relevant for PrP(C) to PrP(Sc) conversion and prion-induced pathology in the hippocampus.


Subject(s)
Intracellular Space/metabolism , PrPC Proteins/metabolism , PrPSc Proteins/metabolism , Animals , Humans , Intracellular Space/genetics , PrPC Proteins/genetics , PrPSc Proteins/genetics , Protein Transport
2.
J Cell Biol ; 204(3): 423-41, 2014 Feb 03.
Article in English | MEDLINE | ID: mdl-24493590

ABSTRACT

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrP(C) into ß-sheet-rich PrP(Sc). PrP(Sc) is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP(Sc) rather than on its truncated PrP27-30 product. We show that N-terminal PrP(Sc) epitopes are exposed in their physiological context and visualize, for the first time, PrP(Sc) in living cells. PrP(Sc) resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrP(Sc) amyloids.


Subject(s)
Amyloid/metabolism , Imaging, Three-Dimensional , Membrane Microdomains/metabolism , PrPSc Proteins/metabolism , Actins/metabolism , Amyloid/chemistry , Amyloid/ultrastructure , Animals , Antibodies/metabolism , Benzothiazoles , Cell Survival , Endocytosis , Hippocampus/metabolism , Mice , Models, Biological , Phosphoinositide Phospholipase C/metabolism , Polysaccharides/metabolism , PrPSc Proteins/chemistry , Protein Binding , Protein Denaturation , Staining and Labeling , Thiazoles/metabolism
3.
Neurobiol Aging ; 34(6): 1621-31, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23481568

ABSTRACT

During prion disease, cellular prion protein (PrP(C)) is refolded into a pathogenic isoform (PrP(Sc)) that accumulates in the central nervous system and causes neurodegeneration and death. We used immunofluorescence, quantitative cryo-immunogold EM, and tomography to detect nascent, full-length PrP(Sc) in the hippocampus of prion-infected mice from early preclinical disease stages onward. Comparison of uninfected and infected brains showed that sites containing full-length PrP(Sc) could be recognized in the neuropil by bright spots and streaks of immunofluorescence on semi-thin (200-nm) sections, and by clusters of cryo-immunogold EM labeling. PrP(Sc) was found mainly on neuronal plasma membranes, most strikingly on membrane invaginations and sites of cell-to-cell contact, and was evident by 65 days postinoculation, or 54% of the incubation period to terminal disease. Both axons and dendrites in the neuropil were affected. We hypothesize that closely apposed plasma membranes provide a favorable environment for prion conversion and intercellular prion transfer. Only a small proportion of clustered PrP immunogold labeling was found at synapses, indicating that synapses are not targeted specifically in prion disease.


Subject(s)
Brain Chemistry , Brain/pathology , Cell Membrane/chemistry , Cell Membrane/pathology , PrPSc Proteins/metabolism , Prion Diseases/pathology , Animals , Brain/metabolism , Brain/ultrastructure , Cell Communication , Cell Membrane/ultrastructure , Mice , PrPSc Proteins/chemistry , PrPSc Proteins/ultrastructure , Prion Diseases/metabolism
4.
PLoS Pathog ; 7(12): e1002449, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22216002

ABSTRACT

After oral exposure, prions are thought to enter Peyer's patches via M cells and accumulate first upon follicular dendritic cells (FDCs) before spreading to the nervous system. How prions are actually initially acquired from the gut lumen is not known. Using high-resolution immunofluorescence and cryo-immunogold electron microscopy, we report the trafficking of the prion protein (PrP) toward Peyer's patches of wild-type and PrP-deficient mice. PrP was transiently detectable at 1 day post feeding (dpf) within large multivesicular LAMP1-positive endosomes of enterocytes in the follicle-associated epithelium (FAE) and at much lower levels within M cells. Subsequently, PrP was detected on vesicles in the late endosomal compartments of macrophages in the subepithelial dome. At 7-21 dpf, increased PrP labelling was observed on the plasma membranes of FDCs in germinal centres of Peyer's patches from wild-type mice only, identifying FDCs as the first sites of PrP conversion and replication. Detection of PrP on extracellular vesicles displaying FAE enterocyte-derived A33 protein implied transport towards FDCs in association with FAE-derived vesicles. By 21 dpf, PrP was observed on the plasma membranes of neurons within neighbouring myenteric plexi. Together, these data identify a novel potential M cell-independent mechanism for prion transport, mediated by FAE enterocytes, which acts to initiate conversion and replication upon FDCs and subsequent infection of enteric nerves.


Subject(s)
Enteric Nervous System/metabolism , Enterocytes/metabolism , Peyer's Patches/metabolism , Prion Diseases/transmission , Prions/metabolism , Prions/pathogenicity , Animals , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Dendritic Cells, Follicular/metabolism , Dendritic Cells, Follicular/ultrastructure , Endosomes/metabolism , Endosomes/ultrastructure , Enteric Nervous System/ultrastructure , Enterocytes/ultrastructure , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Macrophages/metabolism , Macrophages/ultrastructure , Mice , Mice, Knockout , Peyer's Patches/ultrastructure , Prion Diseases/genetics , Prion Diseases/metabolism , Prion Diseases/pathology , Prions/genetics , Protein Transport , Time Factors
5.
Histochem Cell Biol ; 132(3): 253-62, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19649648

ABSTRACT

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be 'docked' or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as 'high pressure freezing' with 'freeze-substitution followed by room temperature plastic sectioning' or 'cryo-sectioning of unperturbed vitreous fully hydrated samples' have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported.


Subject(s)
Cryoelectron Microscopy/methods , Nanostructures/ultrastructure , Organelles/ultrastructure , Saccharomyces cerevisiae/ultrastructure , Tomography/methods
6.
J Neurosci ; 28(47): 12489-99, 2008 Nov 19.
Article in English | MEDLINE | ID: mdl-19020041

ABSTRACT

Prion diseases are caused by accumulation of an abnormally folded isoform (PrP(Sc)) of the cellular prion protein (PrP(C)). The subcellular distribution of PrP(Sc) and the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrP(C) and PrP(Sc); and F4-31, which only detects PrP(C) in undenatured sections. At a late subclinical stage of prion infection, both PrP(C) and PrP(Sc) were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrP(Sc) may be oligomeric, protease-sensitive PrP(Sc).


Subject(s)
Cryoelectron Microscopy/methods , PrPC Proteins/metabolism , PrPC Proteins/ultrastructure , PrPSc Proteins/metabolism , PrPSc Proteins/ultrastructure , Animals , Dendrites/metabolism , Dendrites/ultrastructure , Disease Models, Animal , Hippocampus/metabolism , Hippocampus/pathology , Mice , Mice, Knockout , Neurons/metabolism , Neurons/pathology , Neurons/ultrastructure , Neuropil/metabolism , PrPSc Proteins/genetics , Prion Diseases/etiology , Prion Diseases/metabolism , Synapses/metabolism , Synapses/ultrastructure
7.
J Neurochem ; 98(3): 748-59, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16749906

ABSTRACT

Prion diseases are invariably fatal, neurodegenerative diseases transmitted by an infectious agent, PrPSc, a pathogenic, conformational isoform of the normal prion protein (PrPC). Heterocyclic compounds such as acridine derivatives like quinacrine abolish prion infectivity in a cell culture model of prion disease. Here, we report that these compounds execute their antiprion activity by redistributing cholesterol from the plasma membrane to intracellular compartments, thereby destabilizing membrane domains. Our findings are supported by the fact that structurally unrelated compounds with known cholesterol-redistributing effects - U18666A, amiodarone, and progesterone - also possessed high antiprion potency. We show that tricyclic antidepressants (e.g. desipramine), another class of heterocyclic compounds, displayed structure-dependent antiprion effects and enhanced the antiprion effects of quinacrine, allowing lower doses of both drugs to be used in combination. Treatment of ScN2a cells with quinacrine or desipramine induced different ultrastructural and morphological changes in endosomal compartments. We synthesized a novel drug from quinacrine and desipramine, termed quinpramine, that led to a fivefold increase in antiprion activity compared to quinacrine with an EC50 of 85 nm. Furthermore, simvastatin, an inhibitor of cholesterol biosynthesis, acted synergistically with both heterocyclic compounds to clear PrPSc. Our data suggest that a cocktail of drugs targeting the lipid metabolism that controls PrP conversion may be the most efficient in treating Creutzfeldt-Jakob disease.


Subject(s)
Antidepressive Agents, Tricyclic/pharmacology , Detergents/pharmacokinetics , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Prions/antagonists & inhibitors , Prions/metabolism , Quinacrine/pharmacology , Amiodarone/pharmacology , Androstenes/pharmacology , Animals , Antidepressive Agents, Tricyclic/chemistry , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/genetics , Cell Membrane/metabolism , Chickens , Cholesterol/metabolism , Drug Combinations , Humans , Mice , Progesterone/pharmacology , Quinacrine/chemistry
8.
J Comp Neurol ; 453(1): 57-70, 2002 Nov 04.
Article in English | MEDLINE | ID: mdl-12357432

ABSTRACT

Bird embryos are exposed to maternal androgens deposited in the egg, but the role of these hormones in embryonic development and hatchling survival is unclear. To identify possible target organs, we used in situ hybridization to study the distribution of androgen receptor (AR) RNA in the developing zebra finch brain. The first brain expression domain of AR mRNA is in the hindbrain. From embryonic day 7 (E7) onward, when the hypoglossal motor nucleus (nXII) has just formed, there was AR mRNA expression in both its lingual (nXIIl) and its tracheosyringeal (nXIIts) parts, and this was the major site of hindbrain expression at all embryonic stages and in both sexes. From E8 onward, we also found AR mRNA in the supraspinal motor nucleus (nSSp), which innervates neck muscles. Furthermore, the syrinx, the target of the nXIIts, contained AR mRNA by E10, localized principally in the perichondria. Muscle was first evident in the syringeal region at E9, but no AR was detected in syringeal muscles until after hatching. The expression pattern of AR in the zebra finch embryo suggests that maternal androgens act via AR in the brainstem and syrinx to influence hatching as well as acoustic and visual components of food-begging behavior. Maternal androgens seem unlikely to function in the development of sexual dimorphisms in the zebra finch nXIIts and syrinx, insofar as these are not evident until between 10 and 20 days posthatching.


Subject(s)
Receptors, Androgen/biosynthesis , Rhombencephalon/embryology , Rhombencephalon/metabolism , Animals , Cell Count , Embryo, Nonmammalian/physiology , Female , Hypoglossal Nerve/embryology , Hypoglossal Nerve/growth & development , Hypoglossal Nerve/metabolism , Male , Muscles/embryology , Muscles/metabolism , Neck/embryology , Neck/innervation , Neurons/cytology , Organ Size , Rhombencephalon/growth & development , Sex Characteristics , Songbirds , Trachea/embryology , Trachea/growth & development , Trachea/metabolism
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