<p>Reactions and interactions of iron and oxygen can be both beneficial and detrimental to cells and tissues. Iron is mainly found in our blood where it functions as a mediator in the transport of oxygen to the cells and is further vital for the cellular respiration reducing the oxygen to water. The flexible redox state of iron makes it ideal to contribute in single electron transfers, but may also catalyze reactions with oxygen resulting in cell damaging reactive oxygen species (ROS). Normally the cells are protected against iron toxicity by controlling iron uptake and storage. When the intracellular demand for iron increases; the iron uptake is promoted by increasing the expression of transferrin receptor (TfR) and by decreasing the expression of the iron storage protein ferritin. Ferritin has a central role in the cellular iron detoxification by keeping it in a non reactive but still bioavailable form. However, in neurodegenerative diseases like in Alzheimer’s and Parkinson’s disease the iron storage capacity is disturbed and iron induced oxidative stress adds to the pathology of the diseases. The role of iron and its possible contribution to the pathology of prion diseases, like Creutzfeldt-Jakob disease, is less explored. In the first three studies of this thesis, the iron metabolism and the mutual relation between iron and oxygen are studied in scrapie-infected mouse neuroblastoma cells (ScN2a) as compared to control cells (N2a). In the fourth study we have analyzed the expression of ferritin and TfR in response to inflammation by treating the cells with the bacterial endotoxin lipopolysaccharide (LPS). LPS promotes the expression of inducible nitric oxide synthase (iNOS), a producer of nitric oxide (NO), a well known regulator of the iron metabolism.</p><p>In the first study, the scrapie infection was found to reduce the iron levels, to reduce the mRNA and protein levels of ferritin and the TfR. In addition, reduced levels and activities of the iron regulatory proteins 1 and 2 were observed as compared to the uninfected N2a cells.</p><p>In the second study, the addition of iron to the cell medium strongly increased the level of ROS and decreased the cell viability of the ScN2a cells, whereas the N2a cells were unaffected. The ferritin expression in N2a cells in response to the iron treatment was strongly increased and the concomitant measurement of the labile iron pool (LIP) revealed the LIP to be normalized within four hours. In the ScN2a cells the induction of ferritin expression was lower resulting in elevations in LIP that lasted up to 16 h, indicating that the increased ROS levels were iron catalyzed.</p><p>In the third study, the cells were challenged with hydrogen peroxide (H₂O₂) to elevate the oxidative stress and to analyze the effects on the LIP and cell viability. The ScN2a cells were sensitive to the increased oxidative stress according to the cell viability test, and responded to the treatment with marked increase in the LIP levels, probably derived from an intra-cellular source. The cell viability could be reset by the co-addition of an iron chelator to the cell media. The N2a cells did not elevate the LIP and resisted higher concentrations of H₂O₂ than the ScN2a cells, according to the cell viability assay.</p><p>In the fourth study, the LPS treatment resulted in increased mRNA levels of the heavy chain of ferritin, increased the protein levels of ferritin light chain and decreased the protein levels of the TfR in N2a cells, but no effects were observed in the ScN2a cells. Co-treatment with LPS and the iNOS inhibitor aminoguanidine did not affect the LPS induced decrease of TfR in N2a cells, whereas the free radical scavenger N-acetyl-L-cysteine reversed the effect of LPS on TfR expression, indicating that the changes were mediated by an oxidative rather than a nitric oxide mechanism in the N2a cells.</p>

We show that the prion protein (PrP) is expressed on the surface of bone marrow cell populations enriched in long-term repopulating hematopoietic stem cells. Affinity purification of the PrP-positive and PrP-negative fractions from these populations, followed by competitive reconstitution assays, show that all long-term repopulating hematopoietic stem cells express PrP. Hematopoietic stem cells from PrP null bone marrow exhibit impaired self-renewal in serial competitive transplantation experiments, and premature exhaustion when exposed to cell cycle-specific myelotoxic injury. Therefore, PrP is a novel marker for hematopoietic stem cells and regulates their self-renewal. / Singapore-MIT Alliance (SMA)

Reactions and interactions of iron and oxygen can be both beneficial and detrimental to cells and tissues. Iron is mainly found in our blood where it functions as a mediator in the transport of oxygen to the cells and is further vital for the cellular respiration reducing the oxygen to water. The flexible redox state of iron makes it ideal to contribute in single electron transfers, but may also catalyze reactions with oxygen resulting in cell damaging reactive oxygen species (ROS). Normally the cells are protected against iron toxicity by controlling iron uptake and storage. When the intracellular demand for iron increases; the iron uptake is promoted by increasing the expression of transferrin receptor (TfR) and by decreasing the expression of the iron storage protein ferritin. Ferritin has a central role in the cellular iron detoxification by keeping it in a non reactive but still bioavailable form. However, in neurodegenerative diseases like in Alzheimer’s and Parkinson’s disease the iron storage capacity is disturbed and iron induced oxidative stress adds to the pathology of the diseases. The role of iron and its possible contribution to the pathology of prion diseases, like Creutzfeldt-Jakob disease, is less explored. In the first three studies of this thesis, the iron metabolism and the mutual relation between iron and oxygen are studied in scrapie-infected mouse neuroblastoma cells (ScN2a) as compared to control cells (N2a). In the fourth study we have analyzed the expression of ferritin and TfR in response to inflammation by treating the cells with the bacterial endotoxin lipopolysaccharide (LPS). LPS promotes the expression of inducible nitric oxide synthase (iNOS), a producer of nitric oxide (NO), a well known regulator of the iron metabolism. In the first study, the scrapie infection was found to reduce the iron levels, to reduce the mRNA and protein levels of ferritin and the TfR. In addition, reduced levels and activities of the iron regulatory proteins 1 and 2 were observed as compared to the uninfected N2a cells. In the second study, the addition of iron to the cell medium strongly increased the level of ROS and decreased the cell viability of the ScN2a cells, whereas the N2a cells were unaffected. The ferritin expression in N2a cells in response to the iron treatment was strongly increased and the concomitant measurement of the labile iron pool (LIP) revealed the LIP to be normalized within four hours. In the ScN2a cells the induction of ferritin expression was lower resulting in elevations in LIP that lasted up to 16 h, indicating that the increased ROS levels were iron catalyzed. In the third study, the cells were challenged with hydrogen peroxide (H2O2) to elevate the oxidative stress and to analyze the effects on the LIP and cell viability. The ScN2a cells were sensitive to the increased oxidative stress according to the cell viability test, and responded to the treatment with marked increase in the LIP levels, probably derived from an intra-cellular source. The cell viability could be reset by the co-addition of an iron chelator to the cell media. The N2a cells did not elevate the LIP and resisted higher concentrations of H2O2 than the ScN2a cells, according to the cell viability assay. In the fourth study, the LPS treatment resulted in increased mRNA levels of the heavy chain of ferritin, increased the protein levels of ferritin light chain and decreased the protein levels of the TfR in N2a cells, but no effects were observed in the ScN2a cells. Co-treatment with LPS and the iNOS inhibitor aminoguanidine did not affect the LPS induced decrease of TfR in N2a cells, whereas the free radical scavenger N-acetyl-L-cysteine reversed the effect of LPS on TfR expression, indicating that the changes were mediated by an oxidative rather than a nitric oxide mechanism in the N2a cells.

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories: Loss-of-function diseases Gain-of-toxic-function diseases Infectious misfolding diseases   Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.   The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease. Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

The conformational conversion of the cellular prion protein (PrPC) to PrP scrapie (PrPSc) is a hallmark of prion diseases [1]. The cellular role of PrP and the mechanism of PrPSc neurotoxicity remain largely elusive. Therefore, the identification of new prion-like proteins can assist in revealing the function of PrP. A recent study identified a sub-branch of ZIP (Zrt-, Irt-like protein) metal transporters, including ZIP5, ZIP6 and ZIP10, to be evolutionarily related to PrP. This thesis attempts to understand the functional relevance of this relationship between PrP and ZIP transporters with regard to PrP pathobiology. Preliminary observations indicated that PrP, ZIP6 and ZIP10 underwent endoproteolysis in scrapie-infected mouse brains. PrP and ZIP10 processing mimicked the proteolysis which occurs in cell culture during zinc-deficient conditions, suggesting that scrapie infection may be associated with zinc deficiency. More work is needed to uncover whether ZIPs can contribute to the propagation of prion diseases.

The conformational conversion of the cellular prion protein (PrPC) to PrP scrapie (PrPSc) is a hallmark of prion diseases [1]. The cellular role of PrP and the mechanism of PrPSc neurotoxicity remain largely elusive. Therefore, the identification of new prion-like proteins can assist in revealing the function of PrP. A recent study identified a sub-branch of ZIP (Zrt-, Irt-like protein) metal transporters, including ZIP5, ZIP6 and ZIP10, to be evolutionarily related to PrP. This thesis attempts to understand the functional relevance of this relationship between PrP and ZIP transporters with regard to PrP pathobiology. Preliminary observations indicated that PrP, ZIP6 and ZIP10 underwent endoproteolysis in scrapie-infected mouse brains. PrP and ZIP10 processing mimicked the proteolysis which occurs in cell culture during zinc-deficient conditions, suggesting that scrapie infection may be associated with zinc deficiency. More work is needed to uncover whether ZIPs can contribute to the propagation of prion diseases.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Stanley Prusiner’s proposal that a protein, the prion, was the infectious agent of transmissible spongiform encephalopathies (TSEs) created a great deal of interest, and discussion was not limited to the TSE research field. In fact, Prusiner actively appealed to individuals outside the TSE research field, particularly science writers, corporate funders, and scientists in other research areas. These individuals, in turn, provided Prusiner with the resources, publicity, and expertise to undertake his innovative research program. Prusiner’s emphasis on the revolutionary nature of prion and his claim that prion research could shed light on more common diseases fostered media interest and corporate support. Early newspaper reports stressed Prusiner’s interpretation of experimental results and helped to keep the idea of a protein agent alive until more evidence could be amassed. Corporate funders, with the aid of non-expert scientists, provided Prusiner with the means to carry out expensive, novel experiments and with the opportunity to collaborate with renowned researchers. Prusiner and his supporters were also adept at communicating the protein-only theory and were able to mitigate the impact of specialist criticism. They conceptualized prion protein conversion by using metaphors and analogies and by arguing that parallel phenomena already occurred in Nature. This shifted the discussion away from the problem of replication and recast the search for the TSE agent as a biochemical puzzle. These conceptualizations also enabled Prusiner to engage scientists in other fields, thereby turning his research program into an interdisciplinary enterprise. Moreover, the BSE crisis and Prusiner’s Nobel Prize provided opportunities to further discussion of prions to a wider audience. The engagement of non-experts ultimately created a vast and stable network of interested parties and supporters that was crucial to Prusiner’s success and to the acceptance of the protein-only theory.

Stanley Prusiner’s proposal that a protein, the prion, was the infectious agent of transmissible spongiform encephalopathies (TSEs) created a great deal of interest, and discussion was not limited to the TSE research field. In fact, Prusiner actively appealed to individuals outside the TSE research field, particularly science writers, corporate funders, and scientists in other research areas. These individuals, in turn, provided Prusiner with the resources, publicity, and expertise to undertake his innovative research program. Prusiner’s emphasis on the revolutionary nature of prion and his claim that prion research could shed light on more common diseases fostered media interest and corporate support. Early newspaper reports stressed Prusiner’s interpretation of experimental results and helped to keep the idea of a protein agent alive until more evidence could be amassed. Corporate funders, with the aid of non-expert scientists, provided Prusiner with the means to carry out expensive, novel experiments and with the opportunity to collaborate with renowned researchers. Prusiner and his supporters were also adept at communicating the protein-only theory and were able to mitigate the impact of specialist criticism. They conceptualized prion protein conversion by using metaphors and analogies and by arguing that parallel phenomena already occurred in Nature. This shifted the discussion away from the problem of replication and recast the search for the TSE agent as a biochemical puzzle. These conceptualizations also enabled Prusiner to engage scientists in other fields, thereby turning his research program into an interdisciplinary enterprise. Moreover, the BSE crisis and Prusiner’s Nobel Prize provided opportunities to further discussion of prions to a wider audience. The engagement of non-experts ultimately created a vast and stable network of interested parties and supporters that was crucial to Prusiner’s success and to the acceptance of the protein-only theory.