Alzheimer’s disease (AD) is a neurodegenerative disease with characteristic amyloid, neurofibrillary tangles, and neuronal death in the brain. The major component of amyloid is monomeric amyloid beta (Aβ) protein, as well as its oligomers and large fibrils. Aβ, especially oligomeric Aβ, causes neurotoxicity. The aim of the present study was to investigate how Aβ is metabolized in the presence of microglia cells. We have used a cellular model to study the phagocytosis and degradation of Aβ by BV-2, a murine microglia cell line. We also examined if amylin, a pancreatic peptide, influences the metabolism of Aβ by BV-2 cells. Through Western blot analysis, we observed the phagocytosis of Aβ by BV-2 cells. In our investigation of the role of amylin, we observed a trend in our finding that amylin increased phagocytosis in BV-2 cells and decreased aggregation in the culture medium. Our data demonstrate that amylin may modulate microglia function in the AD brain and has potential to become therapeutic for the disease.

Misfolding and accumulation of insoluble protein aggregates in the form of amyloid fibrils is associated with a number of prevalent and debilitating mammalian disorders. In addition, amyloid-like nanostructures exhibit robust material properties, biological compatibility and replicative properties, making them of particular interest in the development of novel nanomaterials. Understanding fibril formation is essential to the development of strategies to control, manipulate or prevent fibril growth. The amyloid hypothesis is that since amyloid-like fibrils share a common core structure, they also share common formation mechanisms. Utilising a combination of turbidity and extrinsic fluorescence techniques this thesis provides insight into the diagnostic strength of simple, inexpensive kinetic measurements of aggregate growth. These simple techniques are found to be capable of delivering a substantial amount of information about the growth mechanisms controlling aggregation, and the effect of solution and environmental conditions, forming a solid basis for further investigation. Two competing fibrillar pathways are observed for hen egg white lysozyme at low pH in the presence of salt. These two pathways, leading to the formation of either curvilinear, worm-like fibrils or to the more widely recognised rigid, straight fibrils are not particular to hen egg white lysozyme, and similar competition may affect growth curve analysis in many other protein assays, including a-synuclein. Many proteins aggregate in the presence of membranes and detergents, and the kinetics of a-synuclein aggregation in the presence of SDS are strongly influenced by SDS concentration. Most descriptions of amyloid fibril growth currently lack heterogeneous nucleation events, and these may be important for predicting aggregation of membrane-active species in vivo. It is clear that simple analytical solutions to growth models are unable in many cases to capture the complexities of filament growth. Even in relatively simple in vitro experiments different growth processes can dominate growth rate over time, competing fibrillar species can result in composite kinetic growth signals and some growth mechanisms have not yet been sufficiently incorporated into an overall description of fibril growth.

Alzheimer's disease (AD) is the most common cause of dementia; a problem that is growing in size and cost as the population ages. Early soluble aggregates composed misfolded 'amyloid' peptide sequences have been implicated as key to the initiation and onset of AD pathology, although little is definitively known as to when and how these assemblies form or interact to instigate pathology. The primary focus of this study was to evaluate whether L-Tryptophanol (Trol) signal, which has been shown to be induced via soluble amyloid species, increases with AD severity in a range of ex vivo human samples. Testing of this hypothesis was carried out in several stages: Initially synthetic versions of the amyloid beta (Aβ) peptide were tested in vitro to corroborate Trols propensity to associate to amyloid assemblies and allow for method development. Next, a range of brain lysates from several transgenic mouse lines and aged human AD cases and controls were assessed using the reporter. These experiments demonstrated Trols sensitivity to Aβ and tau, and provided compelling evidence that Trol signal tracks disease progression in brain lysates. During the final stage of testing cerebrospinal fluid (CSF) from AD and Parkinson's disease (PD) patients, and blood plasma samples from PD patients was evaluated. Results from this phase of testing indicated that Trol was able to detect differences in sample composition between healthy and diseased individuals, however differences were not clear cut and could have been affected by confounding factors. Overall, the data presented here suggest that Trol may be able to track disease progression in amyloidopathies when implemented in brain lysates. However, further testing is required to completely validate this finding. These findings highlight the potential of simple techniques for amyloid detection to aid within the diagnosis, evaluation of disease progression and study of AD and other neurodegenerative diseases.

abstract: Vitamin D deficiency has been previously associated with a higher Alzheimer’s disease (AD) risk, a condition marked by dependent living and severe cognitive impairment. AD is histologically defined by the presence of brain amyloid beta (Aβ) plaques and neurofibrillary tangles. Ways to enhance Aβ clearance have been examined in order to sustain cognition and delay AD onset. In vitro and in vivo studies suggest that vitamin D might enhance brain Aβ transportation to the periphery by up-regulating P-glycoprotein production. The purpose of this study was to examine the effect of vitamin D supplementation on plasma Aβ in an older population. This study was a parallel-arm, double-blinded, randomized control trial. Participants consumed either a vitamin D supplement or placebo once a week for eight weeks (n=23). Only vitamin D insufficient (serum total 25-OH, D < 30 ng/mL) people were included in the study, and all participants were considered to be cognitively normal (MMSE scores > 27). Serum total 25-OH, D and plasma Aβ1-40 measurements were recorded before and after the eight-week trial. The plasma Aβ1-40 change was compared between the vitamin D group and control group. The vitamin D group experienced a 45% greater change in plasma Aβ1-40 than the control group. The effect size was 0.228 when controlling for baseline plasma Aβ1-40 (p=0.045), 0.197 when controlling for baseline plasma Aβ1-40 and baseline physical activity (p=0.085), and 0.179 when controlling for baseline plasma Aβ1-40, baseline physical activity, and age (p=0.116). In conclusion, vitamin D supplementation might increase brain Aβ clearance in humans, but physical activity and age also appear to modulate Aβ metabolism. / Dissertation/Thesis / Masters Thesis Nutrition 2015

L'objectif de ce projet est d'élaborer des composés capables d'interagir avec des processus biologiques impliquant des associations protéines-protéines, soit pour les inhiber, soit pour les marquer. Deux cibles protéiques impliquées dans la maladie d'Alzheimer et formant des fibres amylogènes sont envisagées : la protéine tau et le peptide Aβ40. Notre conception des inhibiteurs consiste à présenter de façon multimérique des composés biologiquement actifs (peptides et molécules organiques) sur un châssis moléculaire, la dicétopiperazine. L'activité biologique de nos composés est évaluée par des tests in vitro et in vivo. Un autre aspect du projet est la conception, la synthèse et la caractérisation d'un peptidomimétique des fibres de la protéine tau. / The aim of our project is to synthesize compounds able to interact with biological processes involved in protein-protein interactions. Two amyloidogenic proteins involved in Alzheimer's disease have been chosen: tau protein and Aβ40 peptide. Our approach is based on the multimeric presentation of biological compounds (peptides and organic compounds) and our inhibitors are designed from a diketopiperazine scaffold. The biological activity of our compounds is evaluated in vivo and in vitro. Other part of the project is the design, synthesis and characterization of compounds able to mimic Tau fibers.

abstract: Alzheimer's Disease (AD) is a progressive neurodegenerative disease accounting for 50-80% of dementia cases in the country. This disease is characterized by the deposition of extracellular plaques occurring in regions of the brain important for cognitive function. A primary component of these plaques is the amyloid-beta protein. While a natively unfolded protein, amyloid-beta can misfold and aggregate generating a variety of different species including numerous different soluble oligomeric species some of which are precursors to the neurofibrillary plaques. Various of the soluble amyloid-beta oligomeric species have been shown to be toxic to cells and their presence may correlate with progression of AD. Current treatment options target the dementia symptoms, but there is no effective cure or alternative to delay the progression of the disease once it occurs. Amyloid-beta aggregates show up many years before symptoms develop, so detection of various amyloid-beta aggregate species has great promise as an early biomarker for AD. Therefore reagents that can selectively identify key early oligomeric amyloid-beta species have value both as potential diagnostics for early detection of AD and as well as therapeutics that selectively target only the toxic amyloid-beta aggregate species. Earlier work in the lab includes development of several different single chain antibody fragments (scFvs) against different oligomeric amyloid-beta species. This includes isolation of C6 scFv against human AD brain derived oligomeric amyloid-beta (Kasturirangan et al., 2013). This thesis furthers research in this direction by improving the yields and investigating the specificity of modified C6 scFv as a diagnostic for AD. It is motivated by experiments reporting low yields of the C6 scFv. We also used the C6T scFv to characterize the variation in concentration of this particular oligomeric amyloid-beta species with age in a triple transgenic AD mouse model. We also show that C6T can be used to differentiate between post-mortem human AD, Parkinson's disease (PD) and healthy human brain samples. These results indicate that C6T has potential value as a diagnostic tool for early detection of AD. / Dissertation/Thesis / M.S. Biological Design 2013

abstract: Alzheimer's disease (AD) is the most common type of dementia, affecting one in nine people age 65 and older. One of the most important neuropathological characteristics of Alzheimer's disease is the aggregation and deposition of the protein beta-amyloid. Beta-amyloid is produced by proteolytic processing of the Amyloid Precursor Protein (APP). Production of beta-amyloid from APP is increased when cells are subject to stress since both APP and beta-secretase are upregulated by stress. An increased beta-amyloid level promotes aggregation of beta-amyloid into toxic species which cause an increase in reactive oxygen species (ROS) and a decrease in cell viability. Therefore reducing beta-amyloid generation is a promising method to control cell damage following stress. The goal of this thesis was to test the effect of inhibiting beta-amyloid production inside stressed AD cell model. Hydrogen peroxide was used as stressing agent. Two treatments were used to inhibit beta-amyloid production, including iBSec1, an scFv designed to block beta-secretase site of APP, and DIA10D, a bispecific tandem scFv engineered to cleave alpha-secretase site of APP and block beta-secretase site of APP. iBSec1 treatment was added extracellularly while DIA10D was stably expressed inside cell using PSECTAG vector. Increase in reactive oxygen species and decrease in cell viability were observed after addition of hydrogen peroxide to AD cell model. The increase in stress induced toxicity caused by addition of hydrogen peroxide was dramatically decreased by simultaneously treating the cells with iBSec1 or DIA10D to block the increase in beta-amyloid levels resulting from the upregulation of APP and beta-secretase. / Dissertation/Thesis / M.S. Chemical Engineering 2014

Septinas fazem parte de uma família de proteínas de ligação ao nucleotídeo guanina que atuam no ciclo de divisão celular e também são amplamente encontradas em doenças neurodegenerativas tais como mal de Parkinson e Alzheimer e em alguns tipos de câncer como leucemia, linfoma e tumores sólidos. Neste trabalho investigamos como a temperatura e a concentração impactam na agregação do domínio GTPase da septina 2 (SEPT2G), podendo levar a formação de bras amilóides, por espalhamento de luz (DLS) e Raios-X a baixos ângulos (SAXS). Resultados de DLS revelaram que a cinética de agregação da proteína é da ordem de segundos para temperaturas maiores que 25ºC. Os dados de SAXS da proteina a 0,5 mg/ml mostraram que a SETP2G é um dímero em solução aquosa a 4ºC e esta conguração se mantém estável por cerca de 1 hora de observação experimental. A 15ºC, os resultados de SAXS revelaram uma coexistência de três populações em solução compostas por 88% de dímeros, 10% de agregados pequenos tipo-cilindros (protobrilas), e 2% de agregados grandes maiores que a resolução da técnica. Após cerca de 30 minutos existe um rearranjo preferencial de dímeros em favor de agregados muito grandes cuja contribuição à curva de espalhamento torna-se 8%. A 25ºC, a porcentagem de dímeros decresce para 70% com uma contribuição de cerca de 30% de agregados grandes já no início das medidas experimentais. Nas temperaturas de 37ºC e 45ºC, dímeros e agregados muito grandes coexistem em solução desde o início das medidas experimentais, cujo equilíbrio se desloca rapidamente tal que após 20 minutos de observação a solução é composta majoritariamente por agregados muito grandes, identicados como estruturas amilóides pela técnica de uorescência da tioavina, que se intercala em estruturas cross-. A 1 mg/mL e temperatura de 4ºC, a proteína permaneceu estável durante cerca de 1 hora de observação sendo que existe um equilíbrio de dímeros (93%) com agregados alongados (contendo cerca de 80 monômeros) em solução. Com o aumento da temperatura para 15ºC, a maioria da população ainda é dimérica. Já a 25ºC, a presença de agregados muito grandes é bem significativa (da ordem de 30% coexistindo com dímeros e oligômeros). A 37ºC e 45ºC existe a formação de grandes agregados similar ao observado para a SEPT2G a 0,5 mg/mL. Em suma, os resultados de SAXS demonstraram que a SEPT2G tem uma cinética muito rápida de agregação a temperatura siológica, acentuada com o aumento de concentração da proteína em solução. / Septins are proteins from the GTP-binding family and participate in cell division cycle performing functions such as secretion and cytoskeletal division. They can also be found in neurodegenerative conditions as Alzheimer\'s and Parkinson\'s diseases and some kinds of cancer as leukemia, lymphoma and solid tumors. In this work, we investigated the influence of temperature and concentration on the septin 2 GTPase domain (SEPT2G) aggregation using dynamic light scattering (DLS) and small angle x-ray scattering (SAXS). DLS results revealed the protein aggregation kinetic is around seconds for temperatures above 25ºC. SAXS data of the protein at 0.5 mg/mL showed that SEPT2G is a dimer in aqueous solution at 4_C and this condition is kept stable for approximately one hour of experimental observation. At 15ºC, SAXS results revealed the coexistence of three populations in solution composed by 88% of dimers, 10% of cylinder-like smaller aggregates (protofibrils) and 2% of aggregates bigger than the technique detection. After 30 minutes there is a preferential rearrangement of dimers into very large aggregates which contribution on the scattering curve becomes 8%. At 25ºC, the dimers percentage decreases to 70% with a contribution of circa 30% of bigger aggregates, even at the beginning of data acquisition. At temperatures of 37ºC and 45ºC, dimers and very large aggregates coexist in solution since the beginning of data acquisition, which equilibrium quickly shifts in such a way that after 20 minutes of observation the solution is mostly composed by very large aggregates, indented as amyloid structures by the thioflavine fluorescence technique, which intercalates in the cross- structures. At 1 mg/mL and 4ºC, the protein was stable over 1 hour of observation where an equilibrium of dimers (93%) and elongated structures (composed by approximately 80 monomers) in solution takes place. Increasing the temperature to 15ºC, most of the protein remains dimeric. On the other hand, at 25ºC the very large aggregates contribution is around 30% coexisting with dimers and oligomers. At 37ºC and 45ºC there is the formation of large aggregates, similar to what was observed at 0.5 mg/mL. In conclusion, our SAXS results indicated that the aggregation process of SEPT2G in solution may follow different pathways depending on concentration and temperature.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第18328号 / 農博第2053号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4835(農学部図書室) / 31186 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 入江 一浩, 教授 河田 照雄, 教授 保川 清 / 学位規則第4条第1項該当

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. / Medicine, Faculty of / Graduate