Intracellular pathways involved in formation and degradation of prions

Prions cause invariably fatal neurodegenerative diseases, in which a misfolded host-encoded protein appears to be the main, if not the only, component of the infectious agent. During disease, a normal cellular protein, PrPC, is converted to a disease-related isoform, PrPSc, by a post-translational process that might require auxiliary cellular cofactors. The physiological function of PrPC 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 PrPSc 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 GT1-1 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 PrPSc in the cells. Conversely, inhibition of the MEK/ERK pathway using specific inhibitors of MEK cleared the cells from PrPSc, 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 PrPSc 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 PrPC to PrPSc. 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 PrPSc 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 PrPSc in GT1-1 cells, suggesting that the MEK/ERK and the mTOR pathways might converge to exert a translational regulation of prions. By using PrPC-specific siRNA we found evidence of cellular degradation of PrPSc and by using specific inhibitors as well as RNAi directed to cathepsin B and L, we identified these lysosomal proteases as important mediators of PrPSc degradation. In conclusion, we identified five intracellular signaling pathways, including three MAP kinase cascades, to be involved in the cellular accumulation of PrPSc of different prion strains and, in addition, we identified lysosomal cathepsins as important for degradation of PrPSc. 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