Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: Mitochondria in AD & PD models. Includes bibliographical references (p. 146-159). Also available online through Digital Dissertations.

>Magister Scientiae - MSc / The pathology of neurodegenerative disorders involves multiple steps, and it is probably for this reason that targeting one particular step in a multi-step process has only yielded limited results. Nitric oxide (NO) is synthesised from L-Arginine by an enzyme known as nitric oxide synthase (NOS). Three isoforms of NOS exist, including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). In the central nervous system (CNS), nNOS is involved in the synthesis of NO, which is involved in various neurological functions. NO is a free radical and this probably explains why an excess amount of it has been implicated in the development of neurodegenerative disorders. In the CNS, the Nmethyl- D-aspartate (NMDA) receptor in its active state allows the influx of calcium ions which activate nNOS thus increasing the amount of NO and other detrimental reactive nitrogen species within neuronal cells. Calcium entry through voltage-gated calcium channels (VGCC) may also contribute to this. Although calcium ions are important for physiological functioning, an excess is responsible for excitotoxicity, which can ultimately lead to neurodegeneration. Our aim was to synthesise a series of adamantane-derived compounds that act at multiple target sites in the neurodegenerative pathway. By conjugating benzyl and phenylethyl moieties with different functional groups (-H, -NO2, -NH2, -NHC(NH)NH2, -OCH3) to the amantadine structure, we aimed to synthesise compounds that display calcium channel and NMDA receptor (NMDAR) channel inhibition, as well as selective inhibition of nNOS. A series of compounds (-H, -NO2, -NH2, -OCH3) were obtained in yields that varied between 16.5 % and 90.25 %. These novel compounds were tested for calcium influx through VGCC and NMDAR inhibition using synaptoneurosomes isolated from rat brain homogenate against the reference compounds MK-801, NGP1-01, amantadine, memantine and nimodipine. A lack of success with the synthesis of the guanidine compounds prevented the use of the oxyhemoglobin capture assay for the determination of nNOS inhibitory activity of these compounds. The novel synthesised compounds display inhibitory activity towards VGCC and the NMDAR in the micromolar range. Further tests are recommended on compounds SE-1, SE-4, SE-11 and SE-12 as they displayed good inhibitory activity against both NMDAR- as well as ii KCl-mediated calcium influx. These novel compounds may be better therapeutic options than amantadine and memantine as they inhibit both NMDAR and VGCC-mediated calcium influx, whereas amantadine and memantine only inhibit NMDA-mediated calcium influx. These novel adamantane derived compounds may possibly serve as novel leads or potential therapeutic agents for the treatment of neurodegenerative disorders.

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L'âge, les facteurs de risque vasculaire, l'inactivité physique sont connus comme facteurs de risque des syndromes démentiels.Le sommeil joue un rôle majeur dans la physiologie cérébrale. Le syndrome d'apnée du sommeil (SAS) est une pathologie fréquente chez les sujets âgés, surtout chez ceux souffrant de pathologie démentielle. Les études chez les patients atteints d'hypertension, et/ou de fibrillation atriale montrent une prévalence 2 à 3 fois plus élevée de SAS. Le cerveau et sa vascularisation forment un ensemble avec le coeur et les poumons qui fournissent l'apport en énergie et en oxygène. Une dynamique harmonieuse entre ces trois organes est nécessaire au fonctionnement physiologique du cerveau et un dysfonctionnement de cet ensemble pourrait engendrer une altération cognitive. Ce travail a pour but d'analyser les liens entre l'altération du débit vasculaire de la macro-circulation témoin de l'activité cardiaque, la pulsatilité du liquide céphalospinal reflétant l'hydrodynamique cérébrale, les paramètres respiratoires en lien avec les apnées du sommeil, et le statut cognitif du sujet âgé. En nous basant sur les résultats des bilans clinique, biologique, neuropsychologique, de l'imagerie par résonnance magnétique (morphologie et flux), des holters tensionnel et rythmique dans une population de 95 patients âgés cognitivement altérés, nous avons analysé les relations entre les paramètres concernant le cerveau, le coeur et les poumons. Cette approche révèle une relation entre ces trois systèmes et la cognition. La découverte d'une prévalence de plus de 70% de SAS dans cette population associée à d’autres résultats inattendus devrait faire l'objet de travaux ultérieurs / Age, vascular disorders, and lack of physical activity are known risk factors for dementia syndromes. Sleep has an important role in cerebral physiology. Sleep apnea syndrome (SAS) is common in elderly patients, especially in those with dementia. It was reported that the prevalence of SAS is 2 to 3 times higher in patients with arterial hypertension and/or atrial fibrillation. The brain and its vascular system cannot be considered separately from the heart and the lungs, which provide energy and oxygen supply. Cognitive alterations do not reflect the function of the brain only, and balanced dynamics between all these organs is necessary to maintain neurological functions. Therefore, the aim of this dissertation was to analyze the impact of altered cerebral blood flow in macrocirculation reflecting cardiac activity, pulsatility of the cerebrospinal fluid reflecting cerebral hydrodynamics, and SAS reflecting respiratory function on the cognitive status of elderly patients. Based on a clinical examination, geriatric and neuropsychological assessment, blood tests, structural magnetic resonance imaging, phase-contrast magnetic resonance imaging, 24-hour ambulatory blood pressure measurement, and 24-hour electrocardiogram in the population of 95 elderly patients aged 75 years and older, and suffering from neurodegenerative diseases, we analyzed the correlations between the neurological, cardiac, and respiratory parameters. This approach allowed an identification of associations between the abnormalities in these 3 systems and cognitive function. An unexpected finding, among some other abnormalities, was the prevalence of SAS exceeding 70%, which will be the subject of future research

Many neurodegenerative disorders are characterised by protein misfolding and subsequent amyloid fibrillisation and deposition. Amyloid-beta peptide (Aβ) was found to be the main constituent of the extracellular amyloid plaques of Alzheimer’s disease (AD) in the 1980s. What triggers amyloid formation or inhibits it are of particular interest. This thesis focuses on the effect of endogenous proteins and molecules on amyloid fibrillisation. In Chapter 3, I show that at physiological micromolar levels found in the cerebrospinal fluid, human serum albumin inhibits the rate of Aβ fibrillisation. Indeed in vitro the amount of amyloid fibres generated directly correlates to the proportion of Aβ not competitively bound to albumin. Albumin binds cholesterol and fatty acids in vivo, both of which have been linked with an increased risk of developing AD. In Chapter 4, I show Aβ competes with these molecules for albumin binding, so disrupting albumin’s ability to inhibit Aβ fibrillisation. My observations suggest a significant role for albumin regulating Aβ fibril growth. Albumin also binds Cu2+ in vivo with a tight picomolar affinity. Animal models suggest disrupted Cu2+ homeostasis potentiate AD phenotype. In Chapter 5, I show that regardless of Aβ alloform or fibrillisation stage, the affinity for Cu2+ is in the ~20 picomolar range but weaker than albumin. Circular Dichroism spectroscopy in the visible region (Vis-CD) is a powerful technique to study metal-protein interactions. In Chapter 6, I develop a set of empirical rules that relates the appearance of particular Vis-CD spectral features to the conformation of the complex. These rules are used to gain insight into Cu2+-protein complexes in Prion disease and Parkinson’s disease. I show the N-terminal amino group of cellular prion protein (PrPC) has a tighter affinity for Cu2+ than the individual octa-repeat binding sites present within PrPC and show for the first time that Cu2+ loads on to the N-terminal amino group before the single octa-repeat binding sites. I determine the affinity of Cu2+ for model N-terminal peptides of alpha-synuclein of Parkinson’s disease and show that side- chain coordination stabilises the complex and increases the affinity for copper compared to main-chain coordination only. This thesis highlights the importance of overlapping interactions with endogenous proteins and molecules in the development of neurodegenerative disease. Indeed, the amyloid protein binding partners studied here are all co-localised at the synapse thus future in vitro studies of neurodegenerative disease should consider the complex nature of interactions possible in situ.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. It is characterized by the presence of intracellular inclusions termed Lewy bodies (LBs) and Lewy neuritis (LNs) in the brain, in which α-Syn aggregates constitute the main component. Therefore, α-Syn aggregation was implicated in the pathogenesis of PD. Structurally α-Syn is a disordered protein with little ordered structure under physiological conditions. However, research of α-Syn has provided substantial information about its structural properties. The precise function of α-Syn is still under investigation. Research has also shown that metals, such as copper and iron, accelerate α-Syn aggregation and fibrillation in vitro and are proposed to play an important role in vitro. In this study, isothermal titration calorimetry was used to determine iron binding properties to α-Syn revealing the presence of two binding sites for iron with an affinity of 1.06 x 105 M-1 and a dissociation constant of ~ 10μM which is physiologically relevant to iron content in the brain. In addition, α-Syn was found to reduce iron in the presence of copper. This property was demonstrated via ferrozine based assay. In vitro, thoflavin-T fluorescence assay was used to investigate the mechanism by which metals induce α-Syn aggregation and whether it is related to metal binding. Metals, mainly copper and iron, caused 2-fold increase in the aggregation rate of WT α-Syn and its metal binding mutants. Linking that to the increased metal content in the brain, α-Syn aggregation can cause changes in tissue composition, thus altering the normal functional environment in the brain. Moreover, western blotting analysis showed that copper increases the aggregate formation in mammalian dopaminergic cells over-expressing α-Syn.

"Oxidative stress is a type of cellular stress that can damage and kill cells. While it is naturally occurring, many non-natural substances found in our environment can also induce the formation of reactive oxygen species (ROS), which then cause oxidative stress within the cell. Oxidative stress has been shown to be involved in the death of neurons in a number of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The primary causes for these diseases are still unknown; however, we do know oxidative stress plays a primary role in their development. In conditions where oxidative stress is present, adenosine receptor expression has been upregulated and has played a cytoprotective role, but the specific mechanism of action is unknown. In this thesis, oxidative stress was studied in a model eukaryote, Saccharomyces cerevisiae, and the effects of the expression of the human A1 and A2A receptors upon stress response was examined. Oxidative stress was induced by the addition of hydrogen peroxide at concentrations of .5 mM, 1 mM, and 2 mM. The growth of cells expressing either A1-GFP R or A2A-GFP R at the varying hydrogen peroxide concentrations were compared to the parental cells. Confocal microscopy was performed to determine the receptor expression levels, and to confirm the expression of the receptors via their GFP tag. Immunoblots were also performed to assess the receptor expression level at the differing hydrogen peroxide concentrations. A ROS assay was also performed to show the presence of ROS and oxidative stress in the cells. No significant increase in receptor level expression or localization for either A1 R or A2A R at the varying hydrogen peroxide concentrations was found. The data did show trends indicating that A2A receptors may help process the oxidative stress better than A1 receptors and that A2A receptor containing cells had a shorter doubling time. However, more research on this subject should be performed in the future. However, the concentration of hydrogen peroxide should be greatly increased for further experiments in S. cerevisiae in order to better differentiate the trends observed." / 1 / Bryan Goldman

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Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "May 1998." Typescript. Vita. Includes bibliographical references (leaves 176-210). Also available on the Internet.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 142 pages. Includes Vita. Includes bibliographical references.