Advertisement

Prion-induced Activation of Cholesterogenic Gene Expression by Srebp2 in Neuronal Cells*

  • Christian Bach
    Affiliations
    Institute of Virology, Technische Universität München, Trogerstrasse 30, 81675 Munich, Germany

    Institute of Virology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
    Search for articles by this author
  • Sabine Gilch
    Affiliations
    Institute of Virology, Technische Universität München, Trogerstrasse 30, 81675 Munich, Germany
    Search for articles by this author
  • Romina Rost
    Affiliations
    Institute of Virology, Technische Universität München, Trogerstrasse 30, 81675 Munich, Germany
    Search for articles by this author
  • Alex D. Greenwood
    Footnotes
    Affiliations
    Institute of Virology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
    Search for articles by this author
  • Marion Horsch
    Affiliations
    Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environment and Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
    Search for articles by this author
  • Glaucia N.M. Hajj
    Affiliations
    Ludwig Institute for Cancer Research, 01323–903 Sao Paulo SP, Brazil
    Search for articles by this author
  • Susanne Brodesser
    Footnotes
    Affiliations
    LIMES, Membrane Biology and Lipid Biochemistry Unit, University of Bonn, c/o Kekulé-Institute for Organic Chemistry and Biochemistry, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany
    Search for articles by this author
  • Axel Facius
    Footnotes
    Affiliations
    Institute for Bioinformatics, Helmholtz Zentrum München, German Research Center for Environment and Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
    Search for articles by this author
  • Sandra Schädler
    Affiliations
    Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environment and Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
    Search for articles by this author
  • Konrad Sandhoff
    Affiliations
    LIMES, Membrane Biology and Lipid Biochemistry Unit, University of Bonn, c/o Kekulé-Institute for Organic Chemistry and Biochemistry, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany
    Search for articles by this author
  • Johannes Beckers
    Affiliations
    Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environment and Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany

    Institute of Experimental Genetics, Technische Universität München, 85350 Freising-Weihenstephan, Germany
    Search for articles by this author
  • Christine Leib-Mösch
    Affiliations
    Institute of Virology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany

    Medical Clinic III, Medical Faculty Mannheim, University of Heidelberg, 68135 Mannheim, Germany
    Search for articles by this author
  • Hermann M. Schätzl
    Correspondence
    To whom correspondence may be addressed: Institute of Virology, Technische Universität München, Trogerstr. 30, 81675 Munich, Germany. Fax: 49-89-4140-6823
    Affiliations
    Institute of Virology, Technische Universität München, Trogerstrasse 30, 81675 Munich, Germany
    Search for articles by this author
  • Ina Vorberg
    Correspondence
    To whom correspondence may be addressed: Institute of Virology, Technische Universität München, Trogerstr. 30, 81675 Munich, Germany. Fax: 49-89-4140-6823
    Affiliations
    Institute of Virology, Technische Universität München, Trogerstrasse 30, 81675 Munich, Germany
    Search for articles by this author
  • Author Footnotes
    * This work was supported by Deutsche Forschungsgemeinschaft Grants VO 1277/1-2, SFB 596 (Projects A8 and B14), by Bayerischer Forschungsverbund FORPRION (TUM 7), and by the European Commission Grant TSEUR LSHB-CT-2005-018805. This study was performed within the framework of EU FP6 Network of Excellence Neuroprion.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3 and Tables 1 and 2.
    1 Present address: Leibnitz Instítute for zoo- and Wildlife Research, Alfred-Kowalke Str. 17, 10315 Berlin, Germany.
    2 Present address: CECAD Cologne, Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany.
    3 Present address: Nycomed GmbH, Pharmacometrics, Byk Gulden Str. 2, 78467 Konstanz, Germany.
Open AccessPublished:September 11, 2009DOI:https://doi.org/10.1074/jbc.M109.004382
      Prion diseases are neurodegenerative diseases associated with the accumulation of a pathogenic isoform of the host-encoded prion protein. The cellular responses to prion infection are not well defined. By performing microarray analysis on cultured neuronal cells infected with prion strain 22L, in the group of up-regulated genes we observed predominantly genes of the cholesterol pathway. Increased transcript levels of at least nine enzymes involved in cholesterol synthesis, including the gene for the rate-limiting hydroxymethylglutaryl-CoA reductase, were detected. Up-regulation of cholesterogenic genes was attributable to a prion-dependent increase in the amount and activity of the sterol regulatory element-binding protein Srebp2, resulting in elevated levels of total and free cellular cholesterol. The up-regulation of cholesterol biosynthesis appeared to be a characteristic response of neurons to prion challenge, as cholesterogenic transcripts were also elevated in persistently infected GT-1 cells and prion-exposed primary hippocampal neurons but not in microglial cells and primary astrocytes. These results convincingly demonstrate that prion propagation not only depends on the availability of cholesterol but that neuronal cells themselves respond to prions with specific up-regulation of cholesterol biosynthesis.

      Introduction

      The pathogenesis of prion diseases is typically associated with an abnormal accumulation of a misfolded protein (PrPSc),
      The abbreviations used are: PrPSc
      abnormal prion protein associated with prion disease
      PrPC
      cellular prion protein
      FCS
      fetal calf serum
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      SRE
      sterol regulatory element
      BSA
      bovine serum albumin
      siRNA
      small interfering RNA
      Ldlr
      low density lipoprotein receptor.
      derived from the host-encoded prion protein (PrPC), in the nervous system of affected individuals (
      • Prusiner S.B.
      ,
      • Nunziante M.
      • Gilch S.
      • Schätzl H.M.
      ,
      • Wadsworth J.D.
      • Hill A.F.
      • Beck J.A.
      • Collinge J.
      ,
      • Aguzzi A.
      • Polymenidou M.
      ). Although the pathology of the infected cells in the central nervous system is well documented (
      • Budka H.
      ), the host response to infection with prions and the changes within the cell caused by prions remain obscure. Transcriptome analysis of prion-infected animals and cell cultures suggested alterations in several cellular pathways (
      • Doh-ura K.
      • Perryman S.
      • Race R.
      • Chesebro B.
      ,
      • Riemer C.
      • Queck I.
      • Simon D.
      • Kurth R.
      • Baier M.
      ,
      • Baker C.A.
      • Manuelidis L.
      ,
      • Riemer C.
      • Neidhold S.
      • Burwinkel M.
      • Schwarz A.
      • Schultz J.
      • Krätzschmar J.
      • Mönning U.
      • Baier M.
      ,
      • Xiang W.
      • Windl O.
      • Wünsch G.
      • Dugas M.
      • Kohlmann A.
      • Dierkes N.
      • Westner I.M.
      • Kretzschmar H.A.
      ,
      • Brown A.R.
      • Rebus S.
      • McKimmie C.S.
      • Robertson K.
      • Williams A.
      • Fazakerley J.K.
      ,
      • Greenwood A.D.
      • Horsch M.
      • Stengel A.
      • Vorberg I.
      • Lutzny G.
      • Maas E.
      • Schädler S.
      • Erfle V.
      • Beckers J.
      • Schätzl H.
      • Leib-Mösch C.
      ,
      • Sorensen G.
      • Medina S.
      • Parchaliuk D.
      • Phillipson C.
      • Robertson C.
      • Booth S.A.
      ,
      • Hwang D.
      • Lee I.Y.
      • Yoo H.
      • Gehlenborg N.
      • Cho J.H.
      • Petritis B.
      • Baxter D.
      • Pitstick R.
      • Young R.
      • Spicer D.
      • Price N.D.
      • Hohmann J.G.
      • Dearmond S.J.
      • Carlson G.A.
      • Hood L.E.
      ,
      • Tang Y.
      • Xiang W.
      • Hawkins S.A.
      • Kretzschmar H.A.
      • Windl O.
      ), except for one study (
      • Julius C.
      • Hutter G.
      • Wagner U.
      • Seeger H.
      • Kana V.
      • Kranich J.
      • Klöhn P.C.
      • Klöhn P.
      • Weissmann C.
      • Miele G.
      • Aguzzi A.
      ). However, no conclusive interpretation of the role of specific pathways altered by prion infection could be drawn. This might at least in part be due to the following: (a) different microarray platforms; (b) different prion strains; (c) different sampling time points during the course of infection; or (d) mixed cell populations. To better understand the neuronal response to prion infection, we performed a highly controlled genome-wide microarray analysis of paired populations of infected and mock-infected N2a cells, using the mouse-adapted scrapie strain 22L. Our statistical transcriptome analysis identified over 100 significantly and differentially expressed genes with putative function. Within the group of up-regulated genes, genes involved in cholesterol biosynthesis and uptake predominated in prion-infected cells. Transcription of genes associated with cholesterol biosynthesis and cholesterol uptake is tightly controlled by transcription factor Srebp2 that induces expression upon binding to the sterol regulatory element (SRE) in the promoter region of relevant genes (
      • Horton J.D.
      • Shimomura I.
      ,
      • Horton J.D.
      • Shah N.A.
      • Warrington J.A.
      • Anderson N.N.
      • Park S.W.
      • Brown M.S.
      • Goldstein J.L.
      ). Upon cholesterol depletion, Srebp2 is proteolytically cleaved to its active form and translocates to the nucleus where it binds to SRE in promoters of target genes participating in cholesterol metabolism. This way, Srebp2 activates cholesterol biosynthesis by increasing gene expression of individual target genes at every step of the pathway (
      • Horton J.D.
      • Shimomura I.
      ,
      • Horton J.D.
      • Shah N.A.
      • Warrington J.A.
      • Anderson N.N.
      • Park S.W.
      • Brown M.S.
      • Goldstein J.L.
      ,
      • Sakakura Y.
      • Shimano H.
      • Sone H.
      • Takahashi A.
      • Inoue N.
      • Toyoshima H.
      • Suzuki S.
      • Yamada N.
      • Inoue K.
      ). Srebp2 is able to control its own synthesis by a positive feedback mechanism (
      • Sato R.
      • Inoue J.
      • Kawabe Y.
      • Kodama T.
      • Takano T.
      • Maeda M.
      ) and also regulates expression of the low density lipoprotein receptor (Ldlr), which is the main receptor for extracellular cholesterol acquisition in mammalian cells (
      • Horton J.D.
      • Shimomura I.
      ). Real time PCR and reporter gene assays confirmed up-regulation of cholesterogenic genes in prion-infected neuronal cells, including the gene for Srebp2, which was also reflected by increased cholesterol levels. Importantly, primary hippocampal neurons also exhibited elevated cholesterol biosynthesis transcripts upon exposure to prions. Up-regulation of cholesterol biosynthesis appeared to be a characteristic neuronal response, as a microglial cell line and primary astrocytes did not show increased levels of cholesterogenic transcripts in response to prion exposure. Thus, our results suggest that prions differentially alter cholesterol homeostasis in a cell type-specific manner.

      DISCUSSION

      Neuronal integrity is critically dependent on cholesterol, and cholesterol imbalance plays a pivotal role in several neurodegenerative diseases, including Alzheimer disease (
      • Burns M.
      • Duff K.
      ), Niemann Pick disease type C (
      • Ohm T.G.
      • Treiber-Held S.
      • Distl R.
      • Glöckner F.
      • Schönheit B.
      • Tamanai M.
      • Meske V.
      ), and Huntington disease (
      • Valenza M.
      • Rigamonti D.
      • Goffredo D.
      • Zuccato C.
      • Fenu S.
      • Jamot L.
      • Strand A.
      • Tarditi A.
      • Woodman B.
      • Racchi M.
      • Mariotti C.
      • Di Donato S.
      • Corsini A.
      • Bates G.
      • Pruss R.
      • Olson J.M.
      • Sipione S.
      • Tartari M.
      • Cattaneo E.
      ,
      • Valenza M.
      • Cattaneo E.
      ). Up-regulation of cholesterol and lipid metabolism has been shown to cause neurological disease (
      • Ntambi J.M.
      • Miyazaki M.
      ). Thus, imbalances in neuronal cholesterol homeostasis might also contribute to the pathology of prion diseases. The exact cellular location of prion conversion is unknown, but it appears to involve localization of PrPC to so-called lipid rafts, microdomains rich in cholesterol and sphingomyelin (
      • Taraboulos A.
      • Scott M.
      • Semenov A.
      • Avrahami D.
      • Laszlo L.
      • Prusiner S.B.
      • Avraham D.
      ,
      • Vey M.
      • Pilkuhn S.
      • Wille H.
      • Nixon R.
      • DeArmond S.J.
      • Smart E.J.
      • Anderson R.G.
      • Taraboulos A.
      • Prusiner S.B.
      ,
      • Kaneko K.
      • Vey M.
      • Scott M.
      • Pilkuhn S.
      • Cohen F.E.
      • Prusiner S.B.
      ,
      • Marijanovic Z.
      • Caputo A.
      • Campana V.
      • Zurzolo C.
      ). Depletion of cholesterol from membranes abrogates correct PrPC cell surface localization and inhibits PrPSc formation (
      • Taraboulos A.
      • Scott M.
      • Semenov A.
      • Avrahami D.
      • Laszlo L.
      • Prusiner S.B.
      • Avraham D.
      ,
      • Gilch S.
      • Kehler C.
      • Schätzl H.M.
      ,
      • Mangé A.
      • Nishida N.
      • Milhavet O.
      • McMahon H.E.
      • Casanova D.
      • Lehmann S.
      ,
      • Bate C.
      • Salmona M.
      • Diomede L.
      • Williams A.
      ). Furthermore, drugs known to re-distribute cellular cholesterol (
      • Klingenstein R.
      • Löber S.
      • Kujala P.
      • Godsave S.
      • Leliveld S.R.
      • Gmeiner P.
      • Peters P.J.
      • Korth C.
      ) or to lower cholesterol biosynthesis (
      • Bate C.
      • Salmona M.
      • Diomede L.
      • Williams A.
      ) were shown to have anti-prion capacity in N2a cells or to increase incubation times in prion-infected mice (
      • Kempster S.
      • Bate C.
      • Williams A.
      ,
      • Mok S.W.
      • Thelen K.M.
      • Riemer C.
      • Bamme T.
      • Gültner S.
      • Lütjohann D.
      • Baier M.
      ), suggesting that prion biogenesis is highly sensitive to changes in cholesterol homeostasis. In this study we now provide strong evidence that cholesterol homeostasis is directly altered upon prion infection of neuronal cells. Microarray analysis revealed that within the group of up-regulated genes, the predominant change observed was in the cholesterol biosynthetic pathway. Interestingly, several genes that are involved in cholesterol biosynthesis (Sc4mol and Cyp51) or harbor the SRE in their promoter region (e.g. Diazepam-binding inhibitor (Dbi) and stearoyl-coenzyme A desaturase 2 (Scd2)) were also found up-regulated in our previous gene expression studies utilizing N2a cells persistently infected with RML/Chandler mouse-adapted scrapie (
      • Greenwood A.D.
      • Horsch M.
      • Stengel A.
      • Vorberg I.
      • Lutzny G.
      • Maas E.
      • Schädler S.
      • Erfle V.
      • Beckers J.
      • Schätzl H.
      • Leib-Mösch C.
      ). Analysis of GT-1 cells persistently infected with RML scrapie confirmed increased levels of Srebf2 and Sc4mol transcripts. Thin layer chromatography analysis of N2a cells further revealed elevated total and free cholesterol levels upon prion infection. Intriguingly, up-regulation of cholesterogenic genes was not restricted to neuronal cell lines, as primary hippocampal neurons also strongly up-regulated cholesterol synthesis transcripts upon exposure to prions. Thus, our experiments suggest that induction of cholesterol biosynthesis and uptake might be a general consequence to a prion challenge of neuronal cells.
      Of note, our data demonstrate that cholesterol biosynthesis was up-regulated as a consequence of prion infection even in the presence of external sources of FCS. We demonstrate by siRNA experiments that endogenous cholesterol biosynthesis is not crucial for PrPSc accumulation in case cholesterol can be replenished from external sources, indicating that prion-infected N2a cells are still capable of taking up exogenous cholesterol. However, as cholesterol deficiency is sensed in the endoplasmic reticulum, one possible explanation for our findings is that prion infection changes the cellular cholesterol distribution, leading to a decrease in endoplasmic reticulum cholesterol levels and thus to an activation of cholesterol biosynthesis. Interestingly, a recent study on neuronal cells now suggests an imbalance between free cholesterol and cholesterol esters in prion-infected cells (
      • Bate C.
      • Tayebi M.
      • Williams A.
      ).
      It was recently reported that infection of N2a cells with RML scrapie did not lead to significant transcriptional changes (
      • Julius C.
      • Hutter G.
      • Wagner U.
      • Seeger H.
      • Kana V.
      • Kranich J.
      • Klöhn P.C.
      • Klöhn P.
      • Weissmann C.
      • Miele G.
      • Aguzzi A.
      ), contrasting with microarray data on RML prion-infected GT1 and N2a cells (
      • Greenwood A.D.
      • Horsch M.
      • Stengel A.
      • Vorberg I.
      • Lutzny G.
      • Maas E.
      • Schädler S.
      • Erfle V.
      • Beckers J.
      • Schätzl H.
      • Leib-Mösch C.
      ) and data from this study. Although differences in experimental design, prion strains, and the use of different microarray platforms can certainly account for these results, the lack of differential gene expression of N2a cells reported by Julius et al. (
      • Julius C.
      • Hutter G.
      • Wagner U.
      • Seeger H.
      • Kana V.
      • Kranich J.
      • Klöhn P.C.
      • Klöhn P.
      • Weissmann C.
      • Miele G.
      • Aguzzi A.
      ) could also be due to the specific cell clone PK1 that was used for the experiment. As this clone had been retrieved by several rounds of subcloning (as opposed to one round in our study) for drastically increased prion susceptibility (
      • Klöhn P.C.
      • Stoltze L.
      • Flechsig E.
      • Enari M.
      • Weissmann C.
      ), it is possible that this procedure led to the selection of a clone in which prion replication minimally interferes with normal cellular metabolism. Our findings are in line with microarray experiments of other groups that also showed a differential expression pattern of some genes belonging to the lipid or cholesterol pathway (
      • Riemer C.
      • Neidhold S.
      • Burwinkel M.
      • Schwarz A.
      • Schultz J.
      • Krätzschmar J.
      • Mönning U.
      • Baier M.
      ,
      • Brown A.R.
      • Rebus S.
      • McKimmie C.S.
      • Robertson K.
      • Williams A.
      • Fazakerley J.K.
      ,
      • Sorensen G.
      • Medina S.
      • Parchaliuk D.
      • Phillipson C.
      • Robertson C.
      • Booth S.A.
      ,
      • Hwang D.
      • Lee I.Y.
      • Yoo H.
      • Gehlenborg N.
      • Cho J.H.
      • Petritis B.
      • Baxter D.
      • Pitstick R.
      • Young R.
      • Spicer D.
      • Price N.D.
      • Hohmann J.G.
      • Dearmond S.J.
      • Carlson G.A.
      • Hood L.E.
      ,
      • Tang Y.
      • Xiang W.
      • Hawkins S.A.
      • Kretzschmar H.A.
      • Windl O.
      ,
      • Xiang W.
      • Hummel M.
      • Mitteregger G.
      • Pace C.
      • Windl O.
      • Mansmann U.
      • Kretzschmar H.A.
      ,
      • Booth S.
      • Bowman C.
      • Baumgartner R.
      • Sorensen G.
      • Robertson C.
      • Coulthart M.
      • Phillipson C.
      • Somorjai R.L.
      ,
      • Skinner P.J.
      • Abbassi H.
      • Chesebro B.
      • Race R.E.
      • Reilly C.
      • Haase A.T.
      ). However, conflicting results exist as to whether prion infection leads to up-regulation (
      • Riemer C.
      • Neidhold S.
      • Burwinkel M.
      • Schwarz A.
      • Schultz J.
      • Krätzschmar J.
      • Mönning U.
      • Baier M.
      ,
      • Brown A.R.
      • Rebus S.
      • McKimmie C.S.
      • Robertson K.
      • Williams A.
      • Fazakerley J.K.
      ) or down-regulation (
      • Xiang W.
      • Hummel M.
      • Mitteregger G.
      • Pace C.
      • Windl O.
      • Mansmann U.
      • Kretzschmar H.A.
      ) of cholesterol biosynthesis genes. Interestingly, up- or down-regulation appeared to be at least partially dependent on the time point of sampling during the infection process. In one study, the pre-clinical stage of prion infection had only marginal influence on cholesterogenic gene expression, whereas at terminal stages of disease, cholesterogenic transcripts decreased (
      • Xiang W.
      • Hummel M.
      • Mitteregger G.
      • Pace C.
      • Windl O.
      • Mansmann U.
      • Kretzschmar H.A.
      ). In another study, pre-clinical animals infected with mouse-adapted ME7 scrapie prion strain displayed increased cholesterol biosynthesis gene expression in the central nervous system that decreased at terminal stages of the disease (
      • Brown A.R.
      • Rebus S.
      • McKimmie C.S.
      • Robertson K.
      • Williams A.
      • Fazakerley J.K.
      ). Thus, cholesterol metabolism in the brain following prion infection appears to underlie dynamic changes that correlate with the disease state.
      Brain cholesterol is almost exclusively synthesized locally, as the blood-brain barrier restricts import of plasma lipoproteins from peripheral circulation (
      • Turley S.D.
      • Burns D.K.
      • Dietschy J.M.
      ,
      • Turley S.D.
      • Burns D.K.
      • Rosenfeld C.R.
      • Dietschy J.M.
      ). Cholesterol synthesis is mainly accomplished by astroglia, but neuronal cells also synthesize cholesterol at basal levels. Thus, the detected changes in cholesterol biosynthesis genes upon prion infection in vivo might reflect any of the following: (a) cell type-dependent differential expression; (b) disease progression-dependent differential expression; (c) neuronal cell loss-dependent differential expression; or (d) any combination thereof. Because microglial activation, mainly during clinical stages of disease, prominently influences transcript levels in mouse models of prion diseases (
      • Baker C.A.
      • Manuelidis L.
      ,
      • Xiang W.
      • Windl O.
      • Wünsch G.
      • Dugas M.
      • Kohlmann A.
      • Dierkes N.
      • Westner I.M.
      • Kretzschmar H.A.
      ,
      • Xiang W.
      • Hummel M.
      • Mitteregger G.
      • Pace C.
      • Windl O.
      • Mansmann U.
      • Kretzschmar H.A.
      ,
      • Booth S.
      • Bowman C.
      • Baumgartner R.
      • Sorensen G.
      • Robertson C.
      • Coulthart M.
      • Phillipson C.
      • Somorjai R.L.
      ), it is possible that down-regulation of cholesterol biosynthesis during clinical prion disease reflects a glial response that masks neuronal up-regulation. This hypothesis is in agreement with our finding that exposure of microglial cells and primary astrocytes to prions in vitro either caused down-regulation of genes involved in cholesterol biosynthesis, e.g. Sc4mol and Srebf2, or left transcript levels relatively unaffected. Alternatively, neuronal cells in vivo might respond to prion infection dependent on the disease progression. Recent evidence shows that neuronal down-regulation of cholesterol biosynthesis can correlate with apoptosis (
      • Koh C.H.
      • Peng Z.F.
      • Ou K.
      • Melendez A.
      • Manikandan J.
      • Qi R.Z.
      • Cheung N.S.
      ). Neuronal loss is a hallmark of prion diseases, and several lines of evidence suggest that neurons infected with prions can undergo programmed cell death (
      • Schätzl H.M.
      • Laszlo L.
      • Holtzman D.M.
      • Tatzelt J.
      • DeArmond S.J.
      • Weiner R.I.
      • Mobley W.C.
      • Prusiner S.B.
      ). Interestingly, recent studies on neuronal cultures demonstrated that de-regulation of intracellular cholesterol transport induced apoptotic cell death and was co-incident with decreased cholesterol biosynthesis transcripts. Notably, at earlier time points when no apoptosis was apparent, most cholesterol transcripts were increased, suggesting that damaged neurons might initially up-regulate cholesterol biosynthesis, potentially to compensate for cholesterol imbalances (
      • Koh C.H.
      • Peng Z.F.
      • Ou K.
      • Melendez A.
      • Manikandan J.
      • Qi R.Z.
      • Cheung N.S.
      ). Importantly, N2a cells can be persistently infected with prions and do not appear to undergo apoptosis, which could explain why N2a cells do not demonstrate decreased cholesterol biosynthesis transcripts upon prion infection. Similarly, primary hippocampal neurons exposed to 22L scrapie brain homogenate did not show decreased viability compared with primary neurons exposed to normal brain homogenate, at least not during the course of the experiment (data not shown). In summary, our results show that prions have the potential to alter the cholesterol homeostasis of cells in a cell type-specific manner. Our data provide new insights into the cellular and molecular biology and pathology of prion infection and might delineate new targets that can be used for selective interference in cellular prion propagation.

      Acknowledgments

      We are grateful to Dr. Osborne (Department of Molecular Biology and Biochemistry, University of California, Irvine) for providing the psynSRE plasmid and Dr. Martin Groschup (Friedrich-Loeffler-Institut, Germany) for providing 22L brain.

      REFERENCES

        • Prusiner S.B.
        Science. 1991; 252: 1515-1522
        • Nunziante M.
        • Gilch S.
        • Schätzl H.M.
        ChemBioChem. 2003; 4: 1268-1284
        • Wadsworth J.D.
        • Hill A.F.
        • Beck J.A.
        • Collinge J.
        Br. Med. Bull. 2003; 66: 241-254
        • Aguzzi A.
        • Polymenidou M.
        Cell. 2004; 116: 313-327
        • Budka H.
        Br. Med. Bull. 2003; 66: 121-130
        • Doh-ura K.
        • Perryman S.
        • Race R.
        • Chesebro B.
        Microb. Pathog. 1995; 18: 1-9
        • Riemer C.
        • Queck I.
        • Simon D.
        • Kurth R.
        • Baier M.
        J. Virol. 2000; 74: 10245-10248
        • Baker C.A.
        • Manuelidis L.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 675-679
        • Riemer C.
        • Neidhold S.
        • Burwinkel M.
        • Schwarz A.
        • Schultz J.
        • Krätzschmar J.
        • Mönning U.
        • Baier M.
        Biochem. Biophys. Res. Commun. 2004; 323: 556-564
        • Xiang W.
        • Windl O.
        • Wünsch G.
        • Dugas M.
        • Kohlmann A.
        • Dierkes N.
        • Westner I.M.
        • Kretzschmar H.A.
        J. Virol. 2004; 78: 11051-11060
        • Brown A.R.
        • Rebus S.
        • McKimmie C.S.
        • Robertson K.
        • Williams A.
        • Fazakerley J.K.
        Biochem. Biophys. Res. Commun. 2005; 334: 86-95
        • Greenwood A.D.
        • Horsch M.
        • Stengel A.
        • Vorberg I.
        • Lutzny G.
        • Maas E.
        • Schädler S.
        • Erfle V.
        • Beckers J.
        • Schätzl H.
        • Leib-Mösch C.
        J. Mol. Biol. 2005; 349: 487-500
        • Sorensen G.
        • Medina S.
        • Parchaliuk D.
        • Phillipson C.
        • Robertson C.
        • Booth S.A.
        BMC Genomics. 2008; 9: 114
        • Hwang D.
        • Lee I.Y.
        • Yoo H.
        • Gehlenborg N.
        • Cho J.H.
        • Petritis B.
        • Baxter D.
        • Pitstick R.
        • Young R.
        • Spicer D.
        • Price N.D.
        • Hohmann J.G.
        • Dearmond S.J.
        • Carlson G.A.
        • Hood L.E.
        Mol. Syst. Biol. 2009; 5: 252
        • Tang Y.
        • Xiang W.
        • Hawkins S.A.
        • Kretzschmar H.A.
        • Windl O.
        J. Virol. 2009; 83: 9464-9473
        • Julius C.
        • Hutter G.
        • Wagner U.
        • Seeger H.
        • Kana V.
        • Kranich J.
        • Klöhn P.C.
        • Klöhn P.
        • Weissmann C.
        • Miele G.
        • Aguzzi A.
        J. Mol. Biol. 2008; 375: 1222-1233
        • Horton J.D.
        • Shimomura I.
        Curr. Opin. Lipidol. 1999; 10: 143-150
        • Horton J.D.
        • Shah N.A.
        • Warrington J.A.
        • Anderson N.N.
        • Park S.W.
        • Brown M.S.
        • Goldstein J.L.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12027-12032
        • Sakakura Y.
        • Shimano H.
        • Sone H.
        • Takahashi A.
        • Inoue N.
        • Toyoshima H.
        • Suzuki S.
        • Yamada N.
        • Inoue K.
        Biochem. Biophys. Res. Commun. 2001; 286: 176-183
        • Sato R.
        • Inoue J.
        • Kawabe Y.
        • Kodama T.
        • Takano T.
        • Maeda M.
        J. Biol. Chem. 1996; 271: 26461-26464
        • Ertmer A.
        • Gilch S.
        • Yun S.W.
        • Flechsig E.
        • Klebl B.
        • Stein-Gerlach M.
        • Klein M.A.
        • Schätzl H.M.
        J. Biol. Chem. 2004; 279: 41918-41927
        • Maas E.
        • Geissen M.
        • Groschup M.H.
        • Rost R.
        • Onodera T.
        • Schätzl H.
        • Vorberg I.M.
        J. Biol. Chem. 2007; 282: 18702-18710
        • Vorberg I.
        • Raines A.
        • Story B.
        • Priola S.A.
        J. Infect. Dis. 2004; 189: 431-439
        • Gilch S.
        • Schmitz F.
        • Aguib Y.
        • Kehler C.
        • Bülow S.
        • Bauer S.
        • Kremmer E.
        • Schätzl H.M.
        FEBS J. 2007; 274: 5834-5844
        • Lopes M.H.
        • Hajj G.N.
        • Muras A.G.
        • Mancini G.L.
        • Castro R.M.
        • Ribeiro K.C.
        • Brentani R.R.
        • Linden R.
        • Martins V.R.
        J. Neurosci. 2005; 25: 11330-11339
        • Cronier S.
        • Beringue V.
        • Bellon A.
        • Peyrin J.M.
        • Laude H.
        J. Virol. 2007; 81: 13794-13800
        • Horsch M.
        • Schädler S.
        • Gailus-Durner V.
        • Fuchs H.
        • Meyer H.
        • de Angelis M.H.
        • Beckers J.
        Proteomics. 2008; 8: 1248-1256
        • Livak K.J.
        • Schmittgen T.D.
        Methods. 2001; 25: 402-408
        • Radonic A.
        • Thulke S.
        • Mackay I.M.
        • Landt O.
        • Siegert W.
        • Nitsche A.
        Biochem. Biophys. Res. Commun. 2004; 313: 856-862
        • Bubner B.
        • Gase K.
        • Baldwin I.T.
        BMC Biotechnol. 2004; 4: 14
        • Dooley K.A.
        • Millinder S.
        • Osborne T.F.
        J. Biol. Chem. 1998; 273: 1349-1356
        • Smith P.K.
        • Krohn R.I.
        • Hermanson G.T.
        • Mallia A.K.
        • Gartner F.H.
        • Provenzano M.D.
        • Fujimoto E.K.
        • Goeke N.M.
        • Olson B.J.
        • Klenk D.C.
        Anal. Biochem. 1985; 150: 76-85
        • Williams M.A.
        • McCluer R.H.
        J. Neurochem. 1980; 35: 266-269
        • Momoi T.
        • Ando S.
        • Magai Y.
        Biochim. Biophys. Acta. 1976; 441: 488-497
        • Yao J.K.
        • Rastetter G.M.
        Anal. Biochem. 1985; 150: 111-116
        • Xiang W.
        • Hummel M.
        • Mitteregger G.
        • Pace C.
        • Windl O.
        • Mansmann U.
        • Kretzschmar H.A.
        J. Neurochem. 2007; 102: 834-847
        • Priller J.
        • Prinz M.
        • Heikenwalder M.
        • Zeller N.
        • Schwarz P.
        • Heppner F.L.
        • Aguzzi A.
        J. Neurosci. 2006; 26: 11753-11762
        • Cronier S.
        • Laude H.
        • Peyrin J.M.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12271-12276
        • Burns M.
        • Duff K.
        Ann. N. Y. Acad. Sci. 2002; 977: 367-375
        • Ohm T.G.
        • Treiber-Held S.
        • Distl R.
        • Glöckner F.
        • Schönheit B.
        • Tamanai M.
        • Meske V.
        Pharmacopsychiatry. 2003; 36: S120-S126
        • Valenza M.
        • Rigamonti D.
        • Goffredo D.
        • Zuccato C.
        • Fenu S.
        • Jamot L.
        • Strand A.
        • Tarditi A.
        • Woodman B.
        • Racchi M.
        • Mariotti C.
        • Di Donato S.
        • Corsini A.
        • Bates G.
        • Pruss R.
        • Olson J.M.
        • Sipione S.
        • Tartari M.
        • Cattaneo E.
        J. Neurosci. 2005; 25: 9932-9939
        • Valenza M.
        • Cattaneo E.
        Prog. Neurobiol. 2006; 80: 165-176
        • Ntambi J.M.
        • Miyazaki M.
        Prog. Lipid. Res. 2004; 43: 91-104
        • Taraboulos A.
        • Scott M.
        • Semenov A.
        • Avrahami D.
        • Laszlo L.
        • Prusiner S.B.
        • Avraham D.
        J. Cell Biol. 1995; 129: 121-132
        • Vey M.
        • Pilkuhn S.
        • Wille H.
        • Nixon R.
        • DeArmond S.J.
        • Smart E.J.
        • Anderson R.G.
        • Taraboulos A.
        • Prusiner S.B.
        Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14945-14949
        • Kaneko K.
        • Vey M.
        • Scott M.
        • Pilkuhn S.
        • Cohen F.E.
        • Prusiner S.B.
        Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 2333-2338
        • Marijanovic Z.
        • Caputo A.
        • Campana V.
        • Zurzolo C.
        PLoS Pathog. 2009; 5: e1000426
        • Gilch S.
        • Kehler C.
        • Schätzl H.M.
        Mol. Cell. Neurosci. 2006; 31: 346-353
        • Mangé A.
        • Nishida N.
        • Milhavet O.
        • McMahon H.E.
        • Casanova D.
        • Lehmann S.
        J. Virol. 2000; 74: 3135-3140
        • Bate C.
        • Salmona M.
        • Diomede L.
        • Williams A.
        J. Biol. Chem. 2004; 279: 14983-14990
        • Klingenstein R.
        • Löber S.
        • Kujala P.
        • Godsave S.
        • Leliveld S.R.
        • Gmeiner P.
        • Peters P.J.
        • Korth C.
        J. Neurochem. 2006; 98: 748-759
        • Kempster S.
        • Bate C.
        • Williams A.
        Neuroreport. 2007; 18: 479-482
        • Mok S.W.
        • Thelen K.M.
        • Riemer C.
        • Bamme T.
        • Gültner S.
        • Lütjohann D.
        • Baier M.
        Biochem. Biophys. Res. Commun. 2006; 348: 697-702
        • Bate C.
        • Tayebi M.
        • Williams A.
        BMC Biol. 2008; 6: 8
        • Klöhn P.C.
        • Stoltze L.
        • Flechsig E.
        • Enari M.
        • Weissmann C.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 11666-11671
        • Booth S.
        • Bowman C.
        • Baumgartner R.
        • Sorensen G.
        • Robertson C.
        • Coulthart M.
        • Phillipson C.
        • Somorjai R.L.
        J. Gen. Virol. 2004; 85: 3459-3471
        • Skinner P.J.
        • Abbassi H.
        • Chesebro B.
        • Race R.E.
        • Reilly C.
        • Haase A.T.
        BMC Genomics. 2006; 7: 114
        • Turley S.D.
        • Burns D.K.
        • Dietschy J.M.
        Am. J. Physiol. 1998; 274: E1099-E1105
        • Turley S.D.
        • Burns D.K.
        • Rosenfeld C.R.
        • Dietschy J.M.
        J. Lipid Res. 1996; 37: 1953-1961
        • Koh C.H.
        • Peng Z.F.
        • Ou K.
        • Melendez A.
        • Manikandan J.
        • Qi R.Z.
        • Cheung N.S.
        J. Cell. Physiol. 2007; 211: 63-87
        • Schätzl H.M.
        • Laszlo L.
        • Holtzman D.M.
        • Tatzelt J.
        • DeArmond S.J.
        • Weiner R.I.
        • Mobley W.C.
        • Prusiner S.B.
        J. Virol. 1997; 71: 8821-8831