<p>Alzheimer’s disease (AD) is the most common cause of dementia. A characteristic feature of AD is the presence of amyloid plaques in the cortex and hippocampus of the brain. The principal component of these plaques is the amyloid-β (Aβ) peptide, a cleavage product from proteolytic processing of amyloid precursor protein (APP). A central event in AD pathogenesis is the ability of Aβ monomers to aggregate into amyloid fibrils. This process involves the formation of various Aβ intermediates, including protofibrils. Protofibrils have been implicated in familial AD, as the Arctic APP mutation is associated with enhanced rate of protofibril formation <i>in vitro.</i></p><p>This thesis focuses on Aβ aggregation and neurotoxicity <i>in vitro</i>, with special emphasis on protofibril formation. Using synthetic Aβ peptides with and without the Arctic mutation, we demonstrated that the Arctic mutation accelerated both Aβ1-42 protofibril- and fibril formation, and that these processes were affected by changes in the physiochemical environment. </p><p>Oxidation of Aβ methionine delayed trimer and protofibril formation <i>in vitro</i>. Interestingly, these oxidized peptides did not have the neurotoxic potential of their un-oxidized counterparts, suggesting that formation of trimers and further aggregation into protofibrils is necessary for the neurotoxic actions of Aβ. In agreement, stabilization of Aβ wild type protofibrils with the omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) sustained Aβ induced neurotoxicity; whereas in absence of DHA, neurotoxicity was reduced as Aβ fibrils were formed. These results suggest that the neurotoxic potential of Aβ is mainly confined to soluble aggregated forms of Aβ, not Aβ monomer/dimers or fibrillar Aβ. </p><p>Stabilization of Aβ protofibrils with DHA might seem contradictory, as ω3 fatty acids generally are considered beneficial for cognition. However, we also demonstrated that DHA supplementation reduced Aβ levels in cell models of AD, providing a possible mechanism for the reported beneficial effects of DHA on cognitive measures <i>in vivo</i>.</p>

The secondary structure of peptides is of pivotal importance for their biological function. The introduction of photoswitchable moieties into the backbones of peptides provides a unique way of regulating their conformation using an external stimulus, i.e., light. This thesis addresses the design, synthesis, and conformational analysis of photoswitchable peptidomimetics (PSPM). Examples of photomodulation of their functional properties are given. PSPM were prepared by incorporation of stilbene and thioaurone chromophores (switches) into dipeptides. Synthetic schemes for preparing these chromophores have been developed. Their suitability for incorporation into peptidomimetics has been demonstrated, and the resulting PSPM have been subjected to photoisomerization as well as computational and spectroscopic conformational analysis. The chromophore’s potential as a β-hairpin inducer was particularly interesting. To investigate the factors that govern the formation of β-hairpins, a series of decapeptides were prepared. Turn regions consisting of amino acids or chromophores were combined with antiparallel peptide strands with hydrophobic side chains. Linear tryptophan zipper peptidomimetics and cyclic peptidomimetics with a second, hairpin-inducing turn region were particularly promising. Comparison between switches revealed that the more flexible stilbene is a better choice for upholding the β-hairpin conformation than the thioaurone. The catalytic properties of an artificial hydrolase with a helix-loop-helix structure can be improved by introducing a stilbene photoswitch into the loop region. Photoisomerization regulates the catalytic activity of this peptidomimetic, and provides a means to control its aggregation state. The activity of the enzyme Mycobacterium tuberculosis ribonucleotide reductase was realized by incorporating a stilbene moiety into a linear peptide. Here, one photoisomer proved to be an inhibitor at nM concentrations. A significantly lower effect was observed for the other isomer. Finally, the decomposition of thioaurones, mainly to thioflavonols and thiaindenes, under conditions used for solid-phase peptide synthesis has been mapped. These findings are expected to have implications for future use of this chromophore.

Alzheimer’s disease (AD) is the most common cause of dementia. A characteristic feature of AD is the presence of amyloid plaques in the cortex and hippocampus of the brain. The principal component of these plaques is the amyloid-β (Aβ) peptide, a cleavage product from proteolytic processing of amyloid precursor protein (APP). A central event in AD pathogenesis is the ability of Aβ monomers to aggregate into amyloid fibrils. This process involves the formation of various Aβ intermediates, including protofibrils. Protofibrils have been implicated in familial AD, as the Arctic APP mutation is associated with enhanced rate of protofibril formation in vitro. This thesis focuses on Aβ aggregation and neurotoxicity in vitro, with special emphasis on protofibril formation. Using synthetic Aβ peptides with and without the Arctic mutation, we demonstrated that the Arctic mutation accelerated both Aβ1-42 protofibril- and fibril formation, and that these processes were affected by changes in the physiochemical environment. Oxidation of Aβ methionine delayed trimer and protofibril formation in vitro. Interestingly, these oxidized peptides did not have the neurotoxic potential of their un-oxidized counterparts, suggesting that formation of trimers and further aggregation into protofibrils is necessary for the neurotoxic actions of Aβ. In agreement, stabilization of Aβ wild type protofibrils with the omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) sustained Aβ induced neurotoxicity; whereas in absence of DHA, neurotoxicity was reduced as Aβ fibrils were formed. These results suggest that the neurotoxic potential of Aβ is mainly confined to soluble aggregated forms of Aβ, not Aβ monomer/dimers or fibrillar Aβ. Stabilization of Aβ protofibrils with DHA might seem contradictory, as ω3 fatty acids generally are considered beneficial for cognition. However, we also demonstrated that DHA supplementation reduced Aβ levels in cell models of AD, providing a possible mechanism for the reported beneficial effects of DHA on cognitive measures in vivo.

No description available.

The AC133 epitope of the pentaspan transmembrane glycoprotein CD133 has been used as a cell-surface marker for normal and cancer stem cells from a broad range of tissue types. Despite the utility of CD133 as a marker, little is known regarding its regulation and biological function. To study these poorly understood aspects of CD133, I took two main experimental approaches: RNA interference (RNAi) screening and affinity purification coupled with mass spectrometry (AP-MS) to identify CD133 regulatory genes and CD133 protein-protein interactions (PPIs), respectively. Both of these experimental approaches relied on a human embryonic kidney (HEK) 293 cell line that exogenously expresses affinity tagged CD133 (HEK293/AC133). This cell line allowed me to perform a large-scale RNAi screen to interrogate 11,248 genes for their involvement in cell-surface AC133 recognition. This resulted in the identification of the N-glycosylation pathway as a direct contributor to CD133 plasma membrane localization and cell-surface AC133 detection. I used the same RNAi screening approach on the colon adenocarcinoma cell line Caco-2, which express CD133 from its native promoter, to identify factors that regulate endogenous CD133 transcription. I was able to demonstrate that AF4 promotes CD133 transcription in a number of cancer cell lines. Furthermore, I showed that CD133 expression in an acute lymphoblastic leukemia (ALL) cell line SEM, which is dependent on the mixed-lineage leukemia (MLL)-AF4 gene fusion, is critical for the viability of these cells. To gain further insight into the function of CD133, I performed AP-MS using HEK293/AC133 cells to identify CD133 PPIs. I identified histone deacetylase 6 (HDAC6) as a CD133 protein interaction partner. I found that HDAC6 negatively regulates CD133 trafficking into the endosomal-lysosomal degradation pathway. CD133 binds HDAC6 to prevent inhibition of HDAC6 deacetylase activity by phosphorylation. Protection of HDAC6 from phosphorylation promotes HDAC6 deacetylation of β-catenin, which results in β-catenin dependent signalling and the suppression of cancer cell differentiation. My thesis provide functional roles for CD133 as a pro-proliferative protein and as a key signalling protein in certain cancer cell lines.

TGF-β negatively affects the maintenance and expansion of hematopoietic stem cells ex vivo and its inhibition has been widely studied as a treatment for numerous hematopoietic disorders and cancers. Current inhibitory strategies (small molecule ATP competitors and neutralizing antibodies) are compared to a novel cell-permeable peptide-based inhibitor of TGF-β RI. Multiple levels of assay from biochemical to functional are utilized with the aim of applying the most successful inhibitor to hematopoietic stem cell culture. The neutralizing antibody proved ineffective in the short-term biochemical assay but was extremely effective at neutralizing TGF-β signalling in proliferation and hematopoietic colony-forming cell assays with no evidence of toxicity. The small molecule inhibitors (SD-208 and Pyrazole TGF-β RI inhibitor) were equally effective at micromolar levels in all forms of assay, with SD-208 being slightly more potent. The novel peptide inhibitor proved ineffective in all assays, which is likely a result of its rapid degradation in live cells.

Type 1 and type 2 diabetes are characterized by an excessive loss of insulin producing β-cells. β-cells are particularly susceptible to increased oxidative stress induced apoptosis due to low expression of major antioxidants. Peroxiredoxin-2 (PRDX2) belongs to a group of antioxidants with antiapoptotic roles. Preliminary data indicate PRDX2 is expressed in the β-cells. Endogenous PRDX2 in the β-cell line MIN6 is found to decrease under oxidative stress conditions. I hypothesize that PRDX2 has a role in protecting β-cells against oxidative stress induced apoptosis. Overexpression or knockdown strategies were used to examine the role of PRDX2 in insulin-secreting MIN6 cells treated with various stimuli (cytokines, palmitate, streptozotocin) to induce apoptosis. Results showed that PRDX2 overexpression decreased oxidative stress induced apoptosis markers and cell death indicators, whereas knockdown of PRDX2 exaggerated oxidative stress induced toxicity. These findings suggest that PRDX2 plays a protective role in pancreatic β-cells under oxidative stress conditions.

Type 1 and type 2 diabetes are characterized by an excessive loss of insulin producing β-cells. β-cells are particularly susceptible to increased oxidative stress induced apoptosis due to low expression of major antioxidants. Peroxiredoxin-2 (PRDX2) belongs to a group of antioxidants with antiapoptotic roles. Preliminary data indicate PRDX2 is expressed in the β-cells. Endogenous PRDX2 in the β-cell line MIN6 is found to decrease under oxidative stress conditions. I hypothesize that PRDX2 has a role in protecting β-cells against oxidative stress induced apoptosis. Overexpression or knockdown strategies were used to examine the role of PRDX2 in insulin-secreting MIN6 cells treated with various stimuli (cytokines, palmitate, streptozotocin) to induce apoptosis. Results showed that PRDX2 overexpression decreased oxidative stress induced apoptosis markers and cell death indicators, whereas knockdown of PRDX2 exaggerated oxidative stress induced toxicity. These findings suggest that PRDX2 plays a protective role in pancreatic β-cells under oxidative stress conditions.

TGF-β negatively affects the maintenance and expansion of hematopoietic stem cells ex vivo and its inhibition has been widely studied as a treatment for numerous hematopoietic disorders and cancers. Current inhibitory strategies (small molecule ATP competitors and neutralizing antibodies) are compared to a novel cell-permeable peptide-based inhibitor of TGF-β RI. Multiple levels of assay from biochemical to functional are utilized with the aim of applying the most successful inhibitor to hematopoietic stem cell culture. The neutralizing antibody proved ineffective in the short-term biochemical assay but was extremely effective at neutralizing TGF-β signalling in proliferation and hematopoietic colony-forming cell assays with no evidence of toxicity. The small molecule inhibitors (SD-208 and Pyrazole TGF-β RI inhibitor) were equally effective at micromolar levels in all forms of assay, with SD-208 being slightly more potent. The novel peptide inhibitor proved ineffective in all assays, which is likely a result of its rapid degradation in live cells.

名古屋大学博士学位論文 学位の種類 : 医学博士(論文) 学位授与年月日:平成4年9月22日 大野元嗣氏の博士論文として提出された