Histone deacetylase (HDAC) inhibitors (HDACIs) have long been studied and shown promises in the treatment of various neurodegenerative disorders including Huntington’s disease (HD). Based on many demonstrated potentials of HDACIs in mitigating various diseases, we evaluated the utility of [18F]FAHA, a radiolabeled derivative of suberoylanilide hydroxamic acid (SAHA), as a PET imaging agent for characterizing HDAC activity in a non-human primate model and a R6/2 transgenic mouse model of HD. We were aiming at HD as a potential first application, and therefore also examined the expression of HDAC and acetyl histone (AH) in brains of HD patients. This thesis describes that [18F]FAHA was metabolized rapidly to [18F]FACE in both blood plasma and brain. Kinetic analysis indicated that peripherally generated [18F]FACE contributed to the total brain activity. We therefore used a dual-input function model to analyze the kinetics of tracer accumulation and inhibition by SAHA in rhesus monkeys. Parametric images demonstrated the inhibition of HDAC activity in the brain by SAHA in a dose-dependent manner. Huntington’s mice (R6/2) showed a gradual increase of [18F]FAHA accumulation in all organs including the brain with age. In human tissue we found significant losses of acetyl histons expression from cells in the caudate nucleus and Purkinje cells of the cerebellum in HD, while the level of HDAC 5 was increased in these cells. The data obtained in rhesus monkeys indicated that PET imaging with [18F]FAHA could be used as a pharmacodynamic biomarker of the inhibition of class IIa HDACs by HDACIs in the brain and facilitate the development and clinical translation of novel class-IIa HDACIs.

Mitochondria not only supply the energy for cell function, but also take part in cell signaling. This review describes the dysfunctions of mitochondria in aging and neurodegenerative diseases, and the signaling pathways leading to mitochondrial biogenesis (including PGC-1 family proteins, SIRT1, AMPK) and mitophagy (parkin-Pink1 pathway). Understanding the regulation of these mitochondrial pathways may be beneficial in finding pharmacological approaches or lifestyle changes (caloric restrict or exercise) to modulate mitochondrial biogenesis and/or to activate mitophagy for the removal of damaged mitochondria, thus reducing the onset and/or severity of neurodegenerative diseases.

Advancements in Deep Neural Network Architectures have enabledit to be used in the medical imaging and diagnostics domain, in termsof assisting doctors in making diagnoses. Although Neurodegenera-tive diseases such as Alzheimer’s disease remains a diagnosis basedon clinical grounds, there has been great development and recogni-tion that Deep Neural Networks can address the complexity and vol-ume of imaging data.The purpose of our Master Thesis is to address this problem of theclassification of brain PET scans with 18F-FDG radioactive tracer andtheir interpretability. For the classification, four pre-trained networkswere picked. These networks were trained on ADNI data and theirresults were compared to find the best model. To address the prob-lem of interpretability a hybrid interpretability model was developed;a model based on the combination of t-SNE plots and Class Activa-tion Maps. The hybrid model gave an insight into both the atomisticand holistic working of the network. In the end, the interpretabilitymodel was reviewed by a radiologist to check its effectiveness in thepractical world.Among the four models, the best performing model was the Incep-tionResNetV2 as it highlighted the hypometabolism activity in thebrain regions that are crucial to the diagnosis of Alzheimer’s Diseaseand explained the cluster formation of the correctly classified cases. Itwas concluded that the hybrid explainability model, provided betterinterpretability as compared to the presented individual interpretabil-ity methods.

Dopamine (DA) is a catecholamine that is involved in several neural functions such as modulation of locomotor behaviors, arousal states to appetitive aversive learning and memory. The relationships between DA, stress response and aging are unclear. This thesis examines numerous physiological, behavioral and biochemical parameters following perturbations in DA synthesis and transport in the Drosophila melanogaster model system. Intriguingly, elevated DA pools appear to confer protection, while depleted DA levels or transport increase susceptibility to oxidative insult. Resistance to oxidative stress in mutants with elevated DA levels was attributed to a significant up-regulation of glutathione S-transferase Omega-1. A sexually dimorphic response in aging and senescence characteristics was also recorded among the mutants tested, but no discernable role of DA in these characteristics was observed. Taken together, these results point to a key role played by DA in stress response, which might have implications to age-related neurodegenerative diseases.

No description available.

Protein aggregation, oligomer and fibril formation is one of the dominant characteristics in the pathogenesis of a number of neurodegenerative diseases, such as Alzheimer’s disease (AD). Inhibition of toxic oligomer and fibril formation is one of the approaches to find potential drug candidates for AD. Additionally, early diagnosis of these amyloid species can provide mechanistic understanding of protein aggregation and thus can pave the way for preventing the onset of AD. The aim of this dissertation was 1) to explore the effects of charged cholesterol derivatives on the aggregation kinetic behavior of Amyloid-β40 (Aβ40), 2) to probe Aβ40 oligomer and amyloid formation in vitro using gold nanoparticles (AuNPs), and 3) to monitor the kinetic effect of various natural product molecules on Aβ40 aggregation in vitro. In the first chapter, a general introduction about AD as an amyloidogenic disease, amyloid cascade hypothesis, and the manipulation of Aβ peptides aggregation kinetics using different approaches was presented. In the second chapter, we studied the effects of oppositely charged cholesterol derivatives on the aggregation kinetics of Aβ. In the third chapter, we developed a gold nanoparticles (AuNPs) assay to probe Aβ40 oligomers and amyloid formation. In chapter IV, we monitored the effects of various small molecules on the aggregation kinetics of Aβ40. In chapter V, we discussed the methods and experimental details. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Wu, Chi Chung. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 86-96). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Chapter 1. --- INTRODUCTION / Chapter 1.1. --- Neurodegenerative disorders 一 a brief overview --- p.1 / Chapter 1.2. --- Polyglutamine diseases --- p.1 / Chapter 1.3. --- Polyglutamine protein conformers and toxicity --- p.5 / Chapter 1.4. --- in vivo modeling of polyglutamine diseases in Drosophila / Chapter 1.4.1. --- GAL4/UAS transgene expression system in Drosophila --- p.13 / Chapter 1.4.2. --- Temporal control of transgene expression systemin Drosophila --- p.15 / Chapter 1.4.3. --- Drosophila as a model to study polyglutamine diseases --- p.16 / Chapter 1.5. --- in vitro polyglutamine diseases models --- p.19 / Chapter 1.6. --- Aim of study --- p.23 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1. --- Drosophila culture and manipulation / Chapter 2.1.1. --- Drosophila culture --- p.25 / Chapter 2.1.2. --- Pseudopupil assay of adult retinal degeneration --- p.25 / Chapter 2.2. --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 2.2.1. --- Protein extraction from adult Drosophila heads --- p.26 / Chapter 2.2.2. --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.27 / Chapter 2.2.3. --- Western blotting --- p.28 / Chapter 2.2.4. --- Immunodetection --- p.29 / Chapter 2.3. --- Solubilization of SDS-insoluble protein --- p.31 / Chapter 2.4. --- Filter retardation assay --- p.31 / Chapter 2.5. --- Immunoprecipitation --- p.32 / Chapter 2.6. --- Nucleocytoplasmic fractionation --- p.33 / Chapter 2.7. --- PCR cloning / Chapter 2.7.1 . --- Drosophila DNA preparation --- p.34 / Chapter 2.7.2. --- Construction of pGEX4T3-MJDflQ27/81 expression plasmid --- p.34 / Chapter 2.8. --- in vitro aggregation assay / Chapter 2.8.1. --- Expression and purification of GST-MJDAQ27/81 protein --- p.36 / Chapter 2.8.2. --- in vitro aggregation --- p.37 / Chapter 2.8.3. --- Native slot-blot --- p.38 / Chapter 2.9. --- Reagents and buffers / Chapter 2.9.1. --- Reagents for Drosophila culture --- p.39 / Chapter 2.9.2. --- Reagents for SDS-PAGE --- p.39 / Chapter 2.9.3. --- Reagents for filter retardation assay --- p.42 / Chapter 2.9.4. --- Reagents for immunoprecipitation --- p.43 / Chapter 2.9.5. --- Reagents for nucleocytoplasmic fractionation --- p.43 / Chapter 2.9.6. --- Reagents for PCR cloning --- p.44 / Chapter 2.9.7. --- Reagents for in vitro aggregation assay --- p.46 / Chapter 3. --- Establishment of a GAL80ts-mediated transgenic Drosophila model of Machado-Joseph Disease (MJD) / Chapter 3.1. --- Introduction --- p.48 / Chapter 3.2. --- Results / Chapter 3.2.1. --- GAL80ts-mediated expression of expanded full-length MJD protein caused progressive neuronal degenerationin Drosophila --- p.49 / Chapter 3.2.2. --- Detection of SDS-insoluble expanded full-length MJD protein and its correlation with neuronal degeneration / Chapter 3.2.2.1. --- Progressive neuronal degeneration is not mediated by progressive accumulation of expanded full-length MJD protein --- p.51 / Chapter 3.2.2.2. --- SDS-soluble expanded full-length MJD protein does not correlate with progressive neuronal degeneration --- p.53 / Chapter 3.2.2.3. --- Progressive accumulation of SDS-insoluble expanded full-length MJD protein correlate with progressive neuronal degeneration --- p.55 / Chapter 3.3. --- Discussion --- p.57 / Chapter 4. --- Detection of conformational changes of expanded full-length MJD protein and its association with neuronal degeneration / Chapter 4.1. --- Introduction --- p.60 / Chapter 4.2. --- Results / Chapter 4.2.1. --- Expanded full-length MJD protein underwent conformational changes from monomer to fibrils and such conformational changes correlated with neuronal degeneration --- p.61 / Chapter 4.2.2. --- Mechanistic studies of how conformational changes of expanded full-length MJD protein triggers neuronal degeneration / Chapter 4.2.2.1. --- Expanded full-length MJD protein gradually accumulated in the nucleus during the course of neurodegeneration --- p.62 / Chapter 4.2.2.2. --- Fibrillar expanded full-length MJD protein caused transcriptional dysregulation of endogenous Hsp70 gene --- p.66 / Chapter 4.2.3. --- Consolidation of the role of fibrillar expanded full-length MJD protein in neuronal degeneration --- p.67 / Chapter 4.3. --- Discussion --- p.72 / Chapter 5. --- Attempts to generate new conformation-specific antibody against recombinant expanded full-length MJD proteins / Chapter 5.1. --- Introduction --- p.75 / Chapter 5.2. --- Results / Chapter 5.2.1. --- Recombinant expanded full-length MJD protein underwent conformational changes during in vitro aggregation --- p.75 / Chapter 5.3. --- Discussion --- p.77 / Chapter 6. --- GENERAL DISCUSSION --- p.81 / Chapter 7. --- CONCLUSION --- p.84 / Chapter 8. --- REFERENCES --- p.86

Mitochondria plays a crucial role in cells by maintaining energy metabolism and directing cell death mechanisms by buffering calcium (Ca2+ )from cytosol. Therefore, the Ca2+ overload of mitochondria due to the upregulated cytosolic Ca2+ , observed in many neurological disorders is hypothesized to be a key pathway leading to mitochondrial dysfunction and cell death. In particular, Ca2+ homeostasis disruptions due to Alzheimer’ s disease (AD)-causing presenilins (PS1/PS2) and oligomeric forms of β-amyloid peptides Aβ commonly found in AD patients are presumed to cause detrimental effects on the mitochondria and its ability to function properly. We begin by showing that Familial Alzheimer’s disease (FAD)-causing PS mutants affect intracellular Ca2+ ([Ca2+]i) homeostasis by enhancing the gating of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ channels on the endoplasmic reticulum (ER), leading to exaggerated Ca2+ release into the cytoplasm. Using experimental IP3R-mediated Ca2+ release data in conjunction with a computational model of mitochondrial bioenergetics, we explore how the differences in mitochondrial Ca2+ uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca2+ ([Ca2+ ]m). We find that as a result of exaggerated [Ca2+]i in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca2+ dependent enzymes that stimulate NADH production. This leads to decreased rates of proton pumping due to diminished membrane potential (Ψm) along with less ATP and enhanced ROS production. These results show that through Ca2+ signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially cell death. Next, the model for the mitochondria is expanded to include the mitochondrial uniporter (MCU) that senses Ca2+ in the microdomain formed by the close proximity of mitochondria and ER. Ca2+ concentration in the microdomain ([Ca2+] mic) depends on the distance between the cluster of IP3R channels (r) on ER and mitochondria, the number of IP3R in the cluster (nIP3R), and open-probability (Po) of IP3R. Using the same experimental results for Ca2+ release though IP3R due to FAD-causing PS mutants, in conjunction with a computational model of mitochondrial bioenergetics, a data-driven Markov chain model for IP3R gating, and a model for the dynamics of the mitochondrial permeability transition pore (PTP), we explore the difference in mitochondrial Ca2+ uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Ca2+]m due to the gain-of-function enhancement of IP3R channels in the cell expressing PS1-M146L leads to the opening of PTP in high conductance state (PTPh), where the latency of opening is inversely correlated with r and proportional to nIP3R. Furthermore, we observe diminished inner mitochondrial Ψm, [NADH], [Ca2+]m, and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na+/ Ca2+ -exchanger affect the latency of PTP to open in PTPh. Intracellular accumulation of oligomeric forms of Aβ are now believed to play a key role in the early phase of AD as their rise correlates well with the early symptoms of the disease. Extensive evidence points to impaired neuronal Ca2+ homeostasis as a direct consequence of the intracellular Aβ oligomers. To study the effect of intracellular Aβ on Ca2+ signaling and the resulting mitochondrial dysfunction, we employed data-driven modeling in conjunction with total internal reflection fluorescence (TIRF) microscopy (TIRFM). High resolution fluorescence TIRFM together with detailed computational modeling provides a powerful approach towards the understanding of a wide range of Ca2+ signals mediated by the IP3R. Achieving this requires a close agreement between Ca2+ signals from computational models and TIRFM experiments. However, we found that elementary Ca2+ release events, puffs, imaged through TIRFM do not show the rapid single-channel opening and closing during x and between puffs using data-driven single channel models. TIRFM also shows a rapid equilibration of 10 ms after a channel opens or closes which is not achievable in simulation using standard Ca2+ diffusion coefficients and reaction rates between indicator dye and Ca2+. Using the widely used Ca2+ diffusion coefficients and reaction rates, our simulations show equilibration rates that are eight times slower than TIRFM imaging. We show that to get equilibrium rates consistent with observed values, the diffusion coefficients and reaction rates have to be significantly higher than the values reported in the literature. Once a close agreement between experiment and model is achieved, we use multiscale modeling in conjunction with patch-clamp electrophysiology of IP3R and fluorescence imaging of whole-cell Ca2+ response, induced by intracellular Aβ42 oligomers to show that Aβ42 inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP3R, which is then extended to build a model for the whole-cell Ca2+ signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca2+ release from the ER by intracellular Aβ42 oligomers through G-protein-mediated stimulation of IP3 production. The estimated IP3 concentration as a function of intracellular Aβ42 content together with the whole-cell model allows us to show that Aβ42 oligomers impair mitochondrial function through pathological Ca2+ uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and [H2O2]. We further show that mitochondrial function can be restored by the addition of Ca2+ buffer EGTA, in accordance with the observed abrogation of Aβ42 cytotoxicity by EGTA in our live cells experiments. Finally, our modeling study was extended to other pathological phenomena such as epileptic seizures and spreading depolarizations (SD) and their effects on mitochondria by incorporating conservation of particles and charge, and accounting for the energy required to restore ionic gradients to the neuron. By examining the dynamics as a function of potassium and oxygen we can account for a wide range of neuronal hyperactivity from seizures, normoxic SD, and hypoxic SD (HSD) in the model. Together with a detailed model of mitochondria xi and Ca2+ -release through the ER, we determine mitochondrial dysfunction and potential recovery mechanisms from HSD. Our results demonstrate that HSD causes detrimental mitochondrial dysfunction that can only be recovered by restoration of oxygen. Once oxygen is replenished to the neuron, organic phosphate and pH gradients along the mitochondria determine how rapid the neuron recovers from HSD.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.