Thesis (Ph.D.)--University of South Florida, 2004. / Includes vita. Includes bibliographical references (leaves 183-193). Also available online.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 196-228).

Type II diabetes mellitus (T2D) and Alzheimer's disease (AD) share aetiology and have a high incidence of co-morbidity. Evidence suggests that both diseases are caused by the pathogenic aggregation of an intrinsically disordered native amyloid peptide. Furthermore, T2D and AD share risk factors such as age, obesity and vascular health. Recent studies demonstrate that amylin, an amyloidogenic pancreatic hormone deposited in the pancreas in T2D, is also deposited in the brain in AD. We hypothesised that amylin directly contributes to AD through deposition in the brain and activation of pathogenic signalling cascades. We provide evidence to validate that amylin is deposited in the brain parenchyma and vasculature. Furthermore, we present data demonstrating amylin (IAPP) expression in the brain is significantly elevated in AD; and that amylin treatment increases amyloid-beta (AB) secretion in neuronal culture. Soluble oligomeric species of AB cause AD by initiation of downstream signalling cascades that dysregulate kinase activity, promote tau phosphorylation and result in neuronal death. One such pathway involves AB oligomer activation of the Src-family kinase Fyn, through binding to the cellular prion protein (PrPC) receptor complex. We provide evidence that amylin activates Fyn in neuroblastoma and stem cell derived neurons, this activation is possibly mediated through PrPC. Together the data presented in this thesis demonstrate multiple modes of action whereby amylin may directly propagate or indirectly exacerbate AD-associated processes. Amylin aggregation, deposition, up-regulation and signalling should be considered one of several links between T2D and AD. The pathogenic actions of AB and amylin are mediated by oligomer species. Therefore therapeutics which prevent oligomerisation or oligomer action may be valuable in AD and T2D. One such class of therapeutic are flavonoids. Our collaborators have recently demonstrated the flavonoids rutin and quercetin reduce amylin aggregation and extend lifespan in diabetic animal models. As a result of this we investigated the anti-amyloidogenic and anti-oligomeric properties of the flavonoid quercetin against AB. Quercetin treatment prevented AB oligomerisation, cell binding of pre-formed AB oligomers and also reduced APP processing in cell models. These data suggest quercetin is a multimodal therapeutic with potential utility in AD and T2D and should be explored for further drug development.

7Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by accumulation and deposition of extracellular beta-amyloid peptides (Aβ) and intra-neuronal hyperphosphorylated tau in the brain. The insulin signalling pathway begins upstream at the insulin receptor (IR), where the intracellular insulin receptor substrate 1 (IRS1) is phosphorylated, thus propagating the signal downstream to the PI3K/Akt signalling pathway, which affects both the glycogen synthase kinase 3 beta (GSK3β), which is a tau kinase, and mTOR, which is a critical part of the mTORC1 and mTORC2 complexes that not only mediate a wide range of cell functions, but also feed back upstream to regulate Akt. Increasing evidence builds a strong case for the role of soluble Aβ oligomers (AβOs) in the impairment of insulin signalling in AD. Our in vitro studies with neuroblastoma 2a (N2a) cells stably transfected with human APP695 gene (N2a-APP), which secrete excess Aβ, show that the phosphorylation and expression of several but not all critical signalling proteins along the insulin signalling pathway are dysregulated in the cells in comparison to the parental N2a cells. N2a-APP cells were also found to be phenotypically insulin resistant. Subsequently, N2a-APP cells were treated with the Aβ binding peptide (ABP), which binds Aβ oligomers. The ABP treatment was observed to enhance insulin signalling response compared to untreated controls. The results suggest that Aβ may be responsible for inducing the insulin resistant phenotype in N2a-APP cells, and that the removal of Aβ oligomers is a potential treatment consideration for dysfunctional insulin signalling involved in Alzheimer’s disease.

Alzheimer-Demenz ist eine Erkrankung, die durch den demografischen Wandel immer mehr an Bedeutung gewinnt. Eine effektive und frühzeitige Diagnostik ist daher entschei-dend. Da die neuropsychiatrische Testung mit einer diagnostischen Unsicherheit von 10% bis 30% zu ungenau ist und auch erst bei Ausbruch der Symptomatik eine Alzheimer-Demenz diagnostiziert werden kann, wurde auf Parameter wie Beta-Amyloid zurückgegrif-fen. Beta-Amyloid stellt einen Hauptbestandteil der Alzheimer-Demenz Pathologie dar und ist bereits vor Ausbruch der Symptome nachweisbar. Da die visuelle Analyse, welche die Beta-Amyloid Hirn-PET-Daten auswertet, durch ihren hohen Zeitaufwand im Alltag nicht einsetzbar ist, wurden automatische Auswerteverfahren entwickelt. Das BRASS zeigt sich mit einer Sensitivität von 78,4% und einer Spezifität von 80,5% im Bezug zur visuellen Analyse als gut geeignet in der Differenzierung zwischen Probanden mit und ohne Anreicherung von Beta-Amyloid. Eine weitere Verbesserung der Ergebnisse ist durch eine ROC-Analyse im Bezug zu den histopathologischen Befunden vorstellbar, welches in Phase 3 der Studie überprüft wird. Innerhalb des BRASS sind bei der Testung einige Schwierigkeiten aufgetreten, die bei der Weiterentwicklung der Software berücksichtigt und verbessert werden müssen. Auch dadurch ist mit einer weiteren Erhöhung der Sensitivität und Spezifität zu rechnen, sodass der Einsatz des BRASS als ein Standardver-fahren in der Alzheimer-Demenz Diagnostik realistisch ist.

The thesis deals with the testing of amyloidogenicity of various carbon nanoparticles and polymers. The first part of the thesis provides the theoretical background of amyloidoses, a group of diseases in which proteins are stored in the insoluble form of amyloid. In addition, the theoretical part also deals with a general overview of nanomaterials and the most important methods. Several types of nanomaterials were tested within the thesis, so the part Results and Discussion was divided into two subchapters: 1) Carbon nanospecies and amyloid fibril formation, and 2) Polysaccharides, glycogen modifications and amyloid fibril formation. The first subchapter concerns the testing of four types of carbon nanoparticles (single-walled carbon nanotubes (SWNT), fullerenes (C60), carbon quantum dots (CDs) and nanodiamonds (NDs)). These materials were tested on a model system hen egg white lysozyme (HEWL). Using fluorescence measurements and transmission electron microscopy (TEM), the nanoparticles were ranked from the most to the least amyloidogenic as follows: NDs> control> C60> CDs> SWNT. The second subchapter deals with the effect of selected polysaccharides (glycogen (GG), mannan (MAN), phytoglycogen (PG)) and modified GG on amyloid fibril formation. These materials were tested on the HEWL model system,...

Le Syndrome de Détresse Respiratoire Aiguë (SDRA) est la forme la plus sévère d’agression alvéolaire aiguë. Il estcaractérisé par un dommage alvéolaire diffus, suivi d’une phase de réparation nécessaire à la guérison. Bien queles monocytes/macrophages soient des acteurs incontournables de la pathogénie et de la résolution du SDRA, leurpolarisation et leur rôle dans la réparation alvéolaire restent mal connus chez l’Homme. L’hypothèse défendue parcette thèse est que l’environnement alvéolaire module la différenciation et la polarisation desmonocytes/macrophages au cours du SDRA, et que les macrophages alvéolaires ainsi polarisés participentactivement à la réparation et à sa régulation. Les principaux résultats de nos travaux ont permis d’établir que : 1)l’environnement alvéolaire de SDRA inhibe la différenciation des monocytes en fibrocytes (précurseursmésenchymateux associés à la fibroprolifération et à pronostic péjoratif). L’inhibition est majoritairement due à laSerum Amyloid protein P (SAP), provenant en partie du relargage de ses stocks matriciels pulmonaires à la faveurde la lésion alvéolaire ; 2) l’environnement alvéolaire de SDRA induit une polarisation anti-inflammatoire desmacrophages se rapprochant de la polarisation induite in vitro par l’IL-10 ; 3) les macrophages anti-inflammatoirespolarisés par le lavage broncho-alvéolaire (LBA) de patients SDRA favorisent la réparation alvéolaire épithéliale viala production polarisation-dépendante d’Hepatocyte Growth Factor (HGF). Cette production macrophagique d’HGFest amplifiée via une boucle autocrine PTGS2/PGE2 chez l’Homme ; 4) ces résultats semblent étayés par lesdonnées obtenues sur une cohorte clinique qui montrent que les concentrations de sCD163 (marqueur depolarisation anti-inflammatoire) et d’HGF rapportées au nombre de macrophages alvéolaires sont plus élevéeschez les patients survivants que chez les patients décédés de SDRA. L’ensemble de nos travaux démontrent pour lapremière fois chez l’Homme le rôle bénéfique de l’environnement alvéolaire sur les monocytes/macrophages,d’une part en inhibant leur différenciation en fibrocytes contribuant ainsi à limiter la fibroprolifération pulmonaire,et d’autre part en induisant un phénotype macrophagique anti-inflammatoire, contribuant à limiter l’inflammationengendrée par la lésion alvéolaire initiale et favorisant la réparation épithéliale via la production d’HGF. Lesdonnées physiopathologiques obtenues pourraient permettre d’envisager la reprogrammation anti-inflammatoiredes macrophages comme une cible thérapeutique du SDRA en cas d’excès d’inflammation ou de fibro-proliférationavec réparation aberrante. / Acute Respiratory Distress Syndrome (ARDS) is the most severe form of acute lung injury. ARDS is characterized bydiffuse alveolar damage followed by a phase of alveolar repair necessary to recovery. Althoughmonocytes/macrophages are key actors of pathogenicity and resolution of ARDS, little is known about theirpolarization and role on alveolar repair during human ARDS. The hypothesis of our studies was that ARDS alveolarenvironment modulates differentiation and polarization of monocytes and macrophages, and that polarizedmacrophages are involved in alveolar repair and its regulation. The main results of our work have shown that: 1)ARDS alveolar environment inhibited monocytes differentiation into fibrocytes (mesenchymal progenitorsassociated with fibroprolifération and a poor prognosis), mainly through its Serum Amyloid P (SAP) content,originating, at least in part, from the release of SAP associated with lung connective tissue during ARDS; 2) ARDSalveolar environment drove an anti-inflammatory macrophage polarization, close to that induced by IL-10 in vitro;3) anti-inflammatory macrophages polarized by broncho-alveolar lavage (BAL) from ARDS patients favored alveolarepithelial repair through a polarization-dependent production of Hepatocyte Growth Factor (HGF). This HGFproduction is amplified by an autocrine PTGS2/PGE2 dependent loop in human macrophages; 4) these results mayhave clinical relevance, since sCD163 (a marker of anti-inflammatory polarization) and HGF concentrations,expressed relatively to BAL macrophage count, were higher in ARDS survivors than non-survivors. Taken together,our works demonstrate for the first time the beneficial role of the ARDS alveolar environment onmonocytes/macrophages, inhibiting their differentiation into fibrocytes, thus limiting excessive lungfibroproliferation, and inducing an anti-inflammatory macrophage polarization, thus limiting the inflammationgenerated by the initial alveolar damage and favoring epithelial repair through HGF production. The datapresented in this thesis may allow considering anti-inflammatory macrophage repolarization as a potential newtherapeutic target of ARDS with excessive inflammation or fibro-proliferation with aberrant repair.

Alzheimer’s Disease (AD) is a neurodegenerative disease marked by progressive neuronal cell death, leading to dementia. AD is the most common disease that results in dementia and largely affects the elderly, with five million people in the United States diagnosed with the disease as of 2015 and approximately 35 million people worldwide. Diseases resulting in dementia cost the US healthcare system an estimated $172 billion in 2010 and that cost is expected to increase as the population ages and as diagnostic techniques improve so that more people are treated (Holtzman, 2011). The disease was first reported by psychiatrist Alois Alzheimer at the onset of the 20th century, when one of his patients “suffered memory loss, disorientation, hallucinations and delusions and died at the age of 55,” then was found to have severe brain atrophy post-mortem (Cipriani, Dolciotti, Picchi, & Bonuccelli, 2011). There are palliative treatments available that marginally slow disease progression but there is currently no cure for the disease (Awasthi, Singh, Pandey, & Dwivedi, 2016). More research is needed to develop effective therapeutic strategies to combat the disease. Currently, AD cytotoxicity is believed to be caused by increased amyloid β (Aβ) peptide plaque deposition in the brain, as described by the amyloid cascade hypothesis (Barage & Sonawane, 2015). The current understanding is that oligomers of Aβ peptide lead to neuronal death through multiple mechanisms, most notably hyper-phosphorylation of the tau protein. Having a better understanding of the structural changes in the fibrillization process of Aβ will provide a broader insight into mechanisms of cell death and open new possibilities for pharmacological treatments, which is what this research intends to provide.

Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease (AD). Aβ1-42 forms oligomeric structures that undergo further aggregation into protofibrils and fibrils. Oligomeric Aβ1-42 is more toxic than monomers or mature fibrils. In this work, we used two distinct approaches to inhibit Aβ1-42 oligomerization and toxicity. First, seven distinct but overlapping Aβ fragments were used to identify their individual aggregation propensities and their effects on Aβ1-42 oligomerization and cytotoxicity. Studies on suppression of Aβ1-42 cytotoxicity by peptides, including those derived from Aβ1-42, have been conducted before, but peptides encompassing the whole Aβ1-42 sequence have not been systematically analyzed. Aβ1-42 was allowed to aggregate and form oligomeric assemblies in aqueous buffer for 4 h in the absence or presence of 2-fold molar excess of an Aβ fragment. Cytotoxicity analysis then recorded the impact of each fragment on Aβ1-42 cytotoxicity as well as the toxicity of the fragments themselves. An enzyme-linked immunosorbent assay that detects oligomeric Aβwas used to determine the effect of each fragment on Aβ1-42 oligomerization after 4 h of aggregation. Four fragments of Aβ1-42 inhibited the toxicity of oligomeric Aβ1-42 to various degrees, while two others conferred no cellular protection against Aβ1-42 toxicity. Interestingly, one fragment enhanced Aβ1-42 toxicity after 4 h of aggregation. Three of the four fragments that blocked Aβ1-42 toxicity partially disrupted oligomer formation, showing correlation between the inhibition of Aβ1-42 aggregation and the inhibition of cellular toxicity. Second, we examined whether protein disulfide isomerase (PDI), a chaperone mainly found in the endoplasmic reticulum, could reverse the oligomeric state of aggregated Aβ1-42 and thus its toxicity. Previous work has demonstrated that PDI inhibits Aβ1-42 aggregation at sub-stoichiometric concentrations. To assess PDI's effect on Aβ1-42 toxicity, Aβ1-42 was allowed to aggregate for 2 h before the addition of PDI at a 1:10 molar ratio of PDI to Aβ1-42 and then allowed to aggregate for another 2 h. MTS cytotoxicity assays using PC-12 cells showed that adding PDI 2 h after the start of aggregation improves cell survival. Through a differential centrifugation assay followed by Western blot, we qualitatively illustrated that PDI can reverse a 2 h aggregate of Aβ1-42 to the monomeric state. Overall, in this project we have learned that inhibiting the oligomeric assembly of Aβ1-42 directly decreases the effect of Aβ1-42 toxicity. Inhibition of Aβ1-42 toxicity was seen with both fragments derived from Aβ1-42 and PDI, shedding light into two novel approaches as possible therapeutics for AD.

Amyloidoses comprise a group of gain-of-toxic function protein misfolding diseases, in which normally soluble proteins in their functional state undergo conformational changes into highly organized and generally intractable thread-like aggregates, termed amyloid fibrils. These structures accumulate predominantly in the extracellular space but growing evidence suggests that amyloids may start to form intracellularly. At least 26 different human proteins, intact or in fragmented form, are known to form amyloid, which is linked with many debilitating neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt-Jakob disease, and transthyretin (TTR)-related amyloidosis (ATTR). In this work, we focus on ATTR, which is one of the most frequent systemic amyloid diseases. A functional link was established between hereditary ATTR, a severe and fatal disorder and the enhanced propensity of the human plasma protein transthyretin (TTR) to form aggregates, caused by single point mutations in the TTR gene. The disease is heterogeneous and clinical symptoms vary from cardiomyopathy to progressing sensorimotor polyneuropathy depending on TTR variant involved and the amyloid deposition site. Despite the fact that TTR-derived amyloid accumulates in different organs such as heart, kidney, eyes, and predominantly in the peripheral nerves of ATTR patients, the exact mechanism of the disease development is not understood. In contrast to the case of AD, it has been difficult to generate an animal model for ATTR in transgenic mice that would be useful in understanding TTR aggregation processes and the mechanisms of the associated toxicity as these mice did not develop any neuropathic phenotype besides amyloid deposits. Therefore, we created a disease-model in Drosophila due to its huge repertoire of genetic techniques and easy genotype – phenotype translation, as well as its success in modeling human neurodegeneration. We have generated transgenic flies that over-express the clinical amyloidogenic variant TTRL55P, the engineered variant TTR-A (TTRV14N ⁄ V16E), and the wild-type protein. All TTR variants were found in the secreted form in the hemolymph where misfolding occurred and depending on the pool of toxic species, the fate of the fly was decided. Within a few weeks, both mutants (but not the wild-type TTR) demonstrated a time-dependent aggregation of misfolded molecules in vivo. This was associated with neurodegeneration, change in wing posture, attenuation of locomotor activity including compromised flying ability, and shortened life span. In contrast, expression of the wild-type TTR had no discernible effect on either longevity or fly behavior. In this work, we also addressed the correlation between TTR transgene dosage and thus, protein levels, with the severity of the phenotypes observed in TTR-A flies which developed a “dragged wing” phenotype. Remarkably, we established that degenerative changes such as damage to the retina strictly correlated with increased levels of mutated TTR but inversely with behavioral alterations and the dragged wing phenotype. We characterized formation of aggregates in the form of 20 nm spherules and amyloid filaments intracellularly in the thoracic adipose tissue and brain glia (both tissues that do not express the transgene). Moreover, we detected a fraction of neurotoxic TTR-A in the hemolymph of young but not old flies. We proposed that these animals counteract formation and persistence of toxic TTR-A species by removal from the circulation into intracellular compartments of glial and fat body cells and this is part of a mechanism that neutralizes the toxic effects of TTR. We validated the fly model for ATTR by applying a genetic screen during study of modifier genes. We found Serum amyloid P component (a product of the APCS gene) as a potent modifier of TTR amyloid-induced toxicity that was effective in preventing the apoptotic response in cell culture assay and capable of reducing the dragged wings when co-expressed in TTR-A flies. Finally, we optimized this fly model in order to screen for therapeutic compounds effective against ATTR. Feeding assays showed the effectiveness of several compounds among known native-state kinetic stabilizers of TTR against its aggregation. We described several early endpoints in this model, which can be used as a rapid and cost-effective method for optimizing concentrations and pre-screening of drug candidates. As the proof of principle, by feeding flies with increasing doses of diflunisal analogue (an FDA-approved Non-Steroidal Anti-Inflammatory Drug) a dose-dependent reduction of the dragged wings was observed.