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.

The zinc metalloprotease neprilysin (NEP) has been shown to degrade small bioactive peptides. Crystal structures of seven NEP-inhibitor complexes and biochemical characterization of NEP activity have highlighted amino acid interactions that are crucial to ligand binding. Studies also indicate that NEP is one of a select group of metalloenzymes that degrade the amyloid-beta peptide (Aß) in vivo and in situ. Accumulation of neurotoxic Aß aggregates in the brain appears to be a causative agent in the pathophysiology of Alzheimer's Disease (AD). For this reason the enzymatic degradation of Aß has been studied extensively, but little is known about specific binding interactions underlying NEP degradation of Aß. Using known crystal structures of NEP, we have conducted comparative computational studies of ligand binding that predict NEP residues Arg 102 and 110 form binding interactions specific to Aß. These interactions may provide insight for using NEP degradation of Aß in AD therapy. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry; / MS; / Thesis;

Apertureless near-field scanning infrared microscopy is a technique in which an impinging infrared beam is scattered by a sharp atomic force microscopy (AFM) tip oscillating at the resonant frequency of the cantilever in close proximity to a sample. Several advantages offered by near-field imaging include nanoscale imaging with high spatial resolution (near-field imaging is not restricted by the diffraction limit of light) and the ability to differentiate between chemical properties of distinct compounds present in the sample under study due to differences in the scattered field. An overview of the assembly, tuning, and implementation of the near-field instrumentation is provided, as well as detailed descriptions about the samples probed and other instrumentation used. A description of the near-field phenomena, a comparison between aperture and apertureless-type near-field microscopy, and the coupled dipoles model explaining the origin of the chemical contrast present in near-field infrared imaging was discussed. Simultaneous topographic and chemical contrast images were collected at different wavelengths for the block copolymer thin film, polystyrene-b-poly(methyl ethacrylate) (PS-b-PMMA) and for amyloid fibrils synthesized from the #21-31 peptide of β2-microglobulin. In both cases it was observed that the experimental scattered field spectrum correlates strongly with that calculated using the far-field absorption spectrum, and using near-field microscopy, nanoscale structural and/or compositional variations were observed, which would not have been possible using ensemble FTIR measurements. Lastly, tip-enhanced Raman spectra of the #21-31 and #16-22 peptide fragments from the β2-microglobulin and Aβ(1-40) peptide were collected, examined, and an outline of the optimization conditions described.

The p38 mitogen-activated protein kinase [p38(MAPK)] mediates responses to extracellular stressors. An increase in the phosphorylated form of p38(MAPK) [p-p38(MAPK)] has been associated with early events in Alzheimer disease (AD). Although most often associated with processes including apoptosis, inflammation and oxidative stress, p-p38(MAPK) also mediates beneficial physiological functions, such as cell growth, survival and phagocytosis of cellular pathogens. Amyloid plaques [β-amyloid aggregates] are a hallmark of AD-related pathology. As p38(MAPK) has been detected in the vicinity of senile plaques, we combined immunohistochemistry and stereological sampling to quantify the distribution of plaques and p-p38(MAPK)-immunoreactive (IR) cells in the sensorimotor cortex of 3-, 6- and 10-month-old TgCRND8 mice. This animal model expresses an aggressive nature of the AD-related human amyloid-β protein precursor (APP). It was confirmed by the appearance of both dense-core (thioflavin-S-positive) and diffuse plaques, even in the youngest mice. p-p38(MAPK)-IR cells were associated with both dense-core and diffuse plaques, but the expected age-dependent increase in the density of plaque-associated p-p38(MAPK)-IR cells was restricted to dense-core plaques. Furthermore, the density of dense-core plaque-associated p-p38(MAPK)-IR cells was inversely correlated with the size of the core within the given plaque, which supports a role for these microglia in restricting core growth. p-p38(MAPK)-IR cells were also observed throughout wildtype and TgCRND8 mouse cortical parenchyma, but the density of these non-plaque-associated cells remained constant, regardless of age or genotype. We conclude that the constitutive presence of p-p38(MAPK)-IR microglia in aging mouse brain is indicative of a longitudinal role for this kinase in normal brain physiology. Additionally, the majority of p-p38(MAPK)-IR cells were predominantly co-immunoreactive for the Macrophage-1 (CD11b/CD18) microglial marker, regardless of whether they were associated with plaques or localized to the parenchyma. We suggest that the facts that a pool of p-p38(MAPK)-IR microglia appears to restrict b-amyloid plaque core development and the non-pathological role of p-p38(MAPK) in parenchyma, needs to be considered when anticipating targeted p38(MAPK) therapeutics in the context of clinical AD.

The p38 mitogen-activated protein kinase [p38(MAPK)] mediates responses to extracellular stressors. An increase in the phosphorylated form of p38(MAPK) [p-p38(MAPK)] has been associated with early events in Alzheimer disease (AD). Although most often associated with processes including apoptosis, inflammation and oxidative stress, p-p38(MAPK) also mediates beneficial physiological functions, such as cell growth, survival and phagocytosis of cellular pathogens. Amyloid plaques [β-amyloid aggregates] are a hallmark of AD-related pathology. As p38(MAPK) has been detected in the vicinity of senile plaques, we combined immunohistochemistry and stereological sampling to quantify the distribution of plaques and p-p38(MAPK)-immunoreactive (IR) cells in the sensorimotor cortex of 3-, 6- and 10-month-old TgCRND8 mice. This animal model expresses an aggressive nature of the AD-related human amyloid-β protein precursor (APP). It was confirmed by the appearance of both dense-core (thioflavin-S-positive) and diffuse plaques, even in the youngest mice. p-p38(MAPK)-IR cells were associated with both dense-core and diffuse plaques, but the expected age-dependent increase in the density of plaque-associated p-p38(MAPK)-IR cells was restricted to dense-core plaques. Furthermore, the density of dense-core plaque-associated p-p38(MAPK)-IR cells was inversely correlated with the size of the core within the given plaque, which supports a role for these microglia in restricting core growth. p-p38(MAPK)-IR cells were also observed throughout wildtype and TgCRND8 mouse cortical parenchyma, but the density of these non-plaque-associated cells remained constant, regardless of age or genotype. We conclude that the constitutive presence of p-p38(MAPK)-IR microglia in aging mouse brain is indicative of a longitudinal role for this kinase in normal brain physiology. Additionally, the majority of p-p38(MAPK)-IR cells were predominantly co-immunoreactive for the Macrophage-1 (CD11b/CD18) microglial marker, regardless of whether they were associated with plaques or localized to the parenchyma. We suggest that the facts that a pool of p-p38(MAPK)-IR microglia appears to restrict b-amyloid plaque core development and the non-pathological role of p-p38(MAPK) in parenchyma, needs to be considered when anticipating targeted p38(MAPK) therapeutics in the context of clinical AD.

Age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. At present, there are an estimated one million people in Canada with some form of AMD and this number is expected to double to two million by 2031. These estimates are sobering, and it is predicted that costs for treatment and care of individuals who suffer vision loss from AMD will have significant impact on the social and public health systems in Canada in the next two decades. There are treatments to slow the progression of vision loss, but unfortunately, there are currently no cures available for AMD. In order to develop effective second generation therapies and cures, further insights into how and why AMD develops are greatly needed. Recent studies have provided novel insights into the role of inflammation in the pathogenesis of AMD. Inflammation, or swelling of the retinal tissues, causes harmful processes that promote macular degeneration. The proposed studies will focus on the triggers of inflammation in the retina. It is hypothesized that macular degeneration may be slowed or stopped by eliminating the molecules that cause inflammation in the retina. This study will focus on amyloid beta (Aβ), a toxic molecule that has been implicated in retinal inflammation, and the role that it may play in gene expression of the retinal pigment epithelial cell. Amyloid beta is a well studied peptide in another age related disorder, Alzheimer’s disease. It is the major extracellular deposit in Alzheimer’s disease plaques, and has recently been discovered as a component of drusen, the hallmark extracellular deposits in the retina of patients with the ‘dry’ form of AMD. These studies will allow the development of new treatment regimens that target retinal inflammation and thus minimize the processes that ‘trigger’ the onset of macular degeneration.

Alzheimer’s disease (AD) is a highly complex and rapidly progressive neurodegenerative disorder characterized by the systemic collapse of cognitive function and formation of dense amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs). AD pathology is derived from the cholinergic, amyloid and tau hypotheses, respectively. Current pharmacotherapy with known anti-cholinesterases, such as Aricept ® and Exelon ®, only offer symptomatic relief without any disease-modifying effects (DMEs). It is now clear that in order to prevent the rapid progression of AD, new therapeutic treatments should target multiple AD pathways as opposed to the traditional “one drug, one target” approach. This research project employed medicinal chemistry tools to develop multifunctional small organic molecules against three key targets of AD pathology – the cholinesterases (AChE and BuChE), AChE-induced and self-induced Aβ1-40 aggregation and generation (β-secretase). A chemical library composed of 112 derivatives was generated to gather structure-activity relationship (SAR) data. The derivatives were based on a novel, non-fused, 2,4-disubstituted pyrimidine ring (2,4-DPR) template with substituents at the C-2 and C-4 position varying in size, steric and electronic properties. Molecular modeling was utilized to investigate their binding modes within the target enzymes and along with the acquired SAR, the chemical library was screened to identify lead multifunctional candidates.

b-amyloid is derived from amyloid precursor protein (APP) and tightly associated with the pathogenesis of Alzheimer¡¦s disease (AD). Structurally, APP belongs to type I transmembrane protein family and is composed of a large glycosylated extracellular component, a single membrane-spanning region, and a short cytoplasmic domain. Although physiological function of APP remains unclear, the proteolytic processing of APP by b secretase and g secretase gives rise to the production and secretion of b-amyloid. The C-terminus of APP is believed to participate in the intracellular trafficking of APP and signal transduction via interacting with adaptors and signaling proteins, respectively. Three phosphorylation sites (Thr654, Ser655 and Thr668, numbering for APP695 isoform) and several functional motifs in the cytoplasmic domain of APP have been identified and demonstrated that the phosphorylation can indeed affect APP metabolism including: the rate of secretion, endocytosis and b-amyloid production. In this study, we focused on how APP binding protein1 and the phosphorylation affect on APP metabolism. The reasons are as following: (1) Among many APP associated proteins, APP binding protein 1 (APPBP1) is involved in S-M checkpoint regulation. (2) Recent evidence indicates that aberrantly activation of mitotic events may play an important role in development of AD. Since progression through mitosis is regulated by Cdc2 that has been demonstrated to phosphorylate APP on Thr668-Pro669, the phosphorylation of APP at Thr668 may play the important role in regulating APP metabolism and may also relate to AD development. (3) Moreover, protein phosphorylation induces the conformational change and affects the protein- protein interaction. Phosphorylation of Ser / Thr-Pro motif is a central mechanism controlling progression of the cell cycle, including mitosis. Proline residues provide a potential backbone switch in the polypeptide chain controlled by the cis / trans isomerization. Pin1 is an important mitotic regulator and a highly specific peptidyl-prolyl cis / trans isomerases (PPIase) that catalyzes the isomerization of phosphorylated Ser / Thr-Pro bonds. Our unpublished data have shown that Pin1 can bind to the phosphorylated Thr668-Pro669 APP peptide with high affinity (20 nM) that suggested that Pin1 may interact and regulate mitotic APP. Taken together, these data suggested that the interaction of APP and APPBP1 or Pin1 may affect the APP metabolism and its physiological function. This study investigated the hypothesis above and revealed includes the following results (i) the subcellular localization of the C-terminus of APP and APPBP1; (ii) the interaction between APPBP1 and the C-terminus of APP in vivo and in vitro; (iii) Thr668 of APP is the Cdc2 phosphorylation site; (iv) the binding of APPBP1 to the C-terminus of APP reduces the phosphorylation of APP by Cdc2; (v) the phosphorylation at Thr668 can abolish the interaction between APPBP1 and the C-terminus of APP; (vi) the C-terminus of APP is one of the caspase 3 targets; (vii) the phosphorylation of APP at Thr668 also reduces the caspase 3 activity forward to the C-terminus of APP cleavage; (viii) both APPBP1 and Pin1 can inhibit the C-terminus of APP cleavage by caspase 3 that suggested two novel mechanisms to regulate APP metabolism.

Alzheimer’s disease (AD) is a neurodegenerative disorder that is one of such diseases associated with protein aggregation. Aβ is the main protein component of senile plaques in AD, and is neurotoxic when aggregated. In particular, soluble oligomeric forms of Aβ are closely related to neurotoxicity. In this dissertation, we examine the differences in Aβ aggregation intermediates, and final structures formed when only a simple modification in Aβ aggregation conditions is made, the presence or absence of mixing during aggregation. We show that intermediates in the aggregation pathway show significantly different structural rearrangements. The protein stabilities of Αβ species show that spherical aggregates corresponding to the most toxic Αβ species change their structure the most rapidly in denaturant, and that in general, increased toxicity correlated with decreased aggregate stability. In Alzheimer’s disease, even delaying Aβ aggregation onset or slowing its progression might be therapeutically useful, as disease onset is late in life. Small heat shock proteins (sHsps) may be useful for prevention of Αβ aggregation, since sHsps can interact with partly folded intermediate states of proteins to prevent incorrect folding and aggregation. In this research, several small heat shock proteins (sHsps) are tested to prevent Aβ aggregation and toxicity. sHsps used in this research are Hsp17.7, Hsp27, and Hsp20. All types of Hsp20, Hsp20-MBP, His-Hsp20 and His-Hsp20 without 11 residues in C-terminus, can prevent Aβ1-40 aggregation. Hsp20 also prevents Aβ toxicity in the same concentration ranges of it aggregation prevention activity. Hsp17.7 and Hsp27, however, can inhibit Αβ1-40 aggregation but not toxicity. A number of experiments to examine the mechanism of Hsp20 suggest that multivalent binding of sHsp to Aβ is necessary for the toxicity prevention activity. Conclusively, different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the morphology, the toxicity and the conformation of intermediates in the aggregation pathway. Hsp20 rather than other sHsps may be a useful molecular model for the drug design of the next generation of Aβ aggregation inhibitors to be used in the treatment of AD.

<p>The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems since huge volumes of the product go to waste.</p><p>In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these kinds of conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster.</p><p>In the following experiment I am going to investigate if the experiments and conclusions done before, where different kinds of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions.</p>