Intracellular pathways involved in formation and degradation of prions

Prions cause invariably fatal neurodegenerative diseases, in which a misfolded hostencoded protein appears to be the main, if not the only, component of the infectious agent. During disease, a normal cellular protein, PrP, is converted to a disease-related isoform, PrPP, by a post-translational process that might require auxiliary cellular cofactors. The physiological function of PrP P is not completely characterized, but the protein has been implicated in synaptic function, neuroprotection and signal transduction. The studies in this thesis were undertaken with the aim to investigate whether intracellular signaling could affect the cellular accumulation of PrP and to examine whether a molecular cross-talk between prions and key intracellular signaling pathways occur in the infected cells. We found that treatment of scrapie-infected hypothalamic gonadotropin-releasing GT11 cells with brain-derived neurotrophic factor (BDNF), or depolarization of the cells using high [KCl], activated the MEK/ERK MAP kinase cascade and unexpectedly also stimulated prion formation, resulting in increased levels of PrPP in the cells. Conversely, inhibition of the MEK/ERK pathway using specific inhibitors of MEK cleared the cells from PrP P , seemingly by blocking its formation. Exposure of GT1-1 cells to prions of the RML and 22L strains of scrapie caused a transient ERK activation. We, thus, demonstrated a bidirectional interaction between the MEK/ERK cascade and prions in infected cells: On the one hand, prion exposure induces ERK activation and on the other hand, ERK activation leads to increased prion formation. Inhibition of the p38 and JNK MAP kinase pathways using specific inhibitors, led to increased accumulation of PrP in scrapie-infected GT1-1 cells. We demonstrate that the MEK/ERK and the p38 pathways exert opposing effects on prion formation, whereby MEK/ERK stimulates and p38 inhibits the conversion of PrP to PrPP. This suggests that the dynamic balance between these signaling cascades may regulate the replication of prions. Activation of not only the MEK/ERK but also the cAMP/PKA cascade stimulated cellular accumulation of PrPP and was accompanied by phosphorylation of the cytoplasmatic S6 ribosomal protein. This protein is involved in regulation of protein translation, and is a main target for the mTOR pathway. Further studies revealed that also the mTOR signaling pathway influences the accumulation of PrP P in GT1-1 cells, suggesting that the MEK/ERK and the mTOR pathways might converge to exert a translational regulation of prions. By using PrPP-specific siRNA we found evidence of cellular degradation of PrP and by using specific inhibitors as well as RNAi directed to cathepsin B and L, we identified these lysosomal proteases as important mediators of PrP degradation. In conclusion, we identified five intracellular signaling pathways, including three MAP kinase cascades, to be involved in the cellular accumulation of PrP of different prion strains and, in addition, we identified lysosomal cathepsins as important for degradation of PrP. These observations might disclose novel therapeutic strategies in prion diseases, based on manipulation of intracellular signaling. Furthermore, the findings described in this thesis indicate a dual function of ERK activation in neurons during prion infections. Thus, activation of ERK, which is normally beneficial to the cell by inducing protective mechanisms, can at the same time promote formation of the pathogen. LIST OF PUBLICATIONS This thesis is based on the following original articles, which will be referred to in the text by their roman numerals: I. Katarina M. Luhr, Elin K. Nordstrom, Peter Low, Hans-Gustaf Ljunggren, Albert Taraboulos and Krister Kristensson, Scrapie protein degradation by cysteine proteases in CD11c+ dendritic cells and GT1-1 neuronal cells. Journal of Virology, 2004, 78(9): 4776-4782. II. Katarina M. Luhr*, Elin K. Nordstrom*, Peter Low and Krister Kristensson, Cathepsin B and L are involved in degradation of prions in GT1-1 neuronal cells. Neuroreport, 2004, 15: 1663-1667. III. Elin K. Nordstrom, Katarina M. Luhr, Carlos Ibanez and Krister Kristensson, Inhibitors of the Mitogen-Activated Protein Kinase Kinase 1/2 Signaling Pathway Clear Prion-infected Cells from PrP. Journal of Neuroscience, 2005, 25(37): 8451-8456. IV. Elin K. Nordstrom, Gilberto Fisone and Krister Kristensson, Opposing effects of ERK and p38-JNK MAP kinase pathways on prion formation in GT1-1 cells. FASEB Journal, 2009, 23(2): 613-622. V. Elin K. Allard, Mirjana Grujic, Gilberto Fisone and Krister Kristensson, Translational control of prions by MAP kinase and mTOR signaling in GT1-1 cells. Manuscript. LIST OF ABBREVIATIONS 4E-BP1 eukaryotic initiation factor 4E binding protein 1 BDNF brain-derived neurotrophic factor cAMP cyclic adenosine monophosphate CJD Creutzfeldt-Jakob disease CNS central nervous system eIF eukaryotic translation initiation factor ERK extracellular signal-regulated kinase GABA gamma-aminobutyric acid GAGs endogenous glycosaminoglycans GT1-1 cells hypothalamic gonadotropin-releasing hormone neurons GTP guanosine triphosphate JNK stress-activated protein kinase 1 MAP Mitogen-activated protein MEK mitogen-activated protein kinase kinase N2a neuroblastoma cells NCAM neural cell adhesion molecule NGF nerve growth factor NICD Notch-1 intracellular domain p38 stress-activated protein kinase 2-4 PC12 cells cell line derived from rat adrenal medulla PKA protein kinase A PrP normal cellular prion protein PrP scrapie prion protein RML Rocky Mountain Laboratory RNAi RNA interference S6Ks ribosomal S6 kinases S6rp S6 ribosomal protein ScGT1-1 scrapie-infected GT1-1 cells ScN2a scrapie-infected N2a cells siRNA short interfering RNA STI1 stress-inducible protein-1 Trk tropomycin receptor kinase TSE transmissible spongiform encephalopathies