The start of the amyloidogenic pathway in Alzheimer’s disease (AD) begins with the deposition of the Aβ₁₋₄₂ peptide surrounded by astrocytes. High levels of arginine and low amounts of neuronal nitric oxide synthase (nNOS) are associated with AD. These astrocytes store reserve arginine that is eventually metabolized by nNOS, within the vicinity of the Aβ₁₋₄₂ peptide. We propose the existence of an association vs. dissociation equilibrium between Aβ and nNOS such that nNOS is an amyloidogenic catalyst for fibrils. When Aβ binds to nNOS, it inhibits the activity of the enzyme (association phase). However when the amyloid peptide dissociates into a form that can no longer bind, later deduced as a fibril, the activity is restored. Thus, the interaction of Aβ with nNOS could serve to regulate the interaction between nNOS and arginine by restoring activity of the enzyme but at the same time promoting fibrillogenesis. Given this event occurring with the neuron, both nNOS and amyloid can serve as a biomarker for the early onset of AD. The enzyme nNOS catalyzed the formation of fibrils in the presence of Aβ peptides, while Ag nps were shown to reverse the fibril formation from Aβ peptides more so than Au and curcumin either through electrostatic or π-π stacking (aromatic) influences. Our studies have shown that the fragments of Aβ₁₋₄₂ i.e. the pentapeptide (Aβ₁₇₋₂₁) and the three glycine zipper peptides (Aβ₂₅₋₂₉, Aβ₂₉₋₃₃, Aβ₃₃₋₃₇) and the full length glycine zipper stretch (Aβ₂₅₋₃₇) all inhibited nNOS activity to varying degrees. The peptides Aβ₁₇₋₂₁ and Aβ₂₉₋₃₃ with their respective Ki values of 5.1 μM and 7.5 μM inhibited the enzyme the most. The Ki values for reversed sequenced peptides (Aβ₁₇₋₂₁r and Aβ₂₉₋₃₃r) were two fold greater than that of the original peptides while the Ki values for the polar forms (Aβ₁₇₋₂₁p and Aβ₂₉₋₃₃p) were between 3-4 fold greater than that of the original peptides. It was also found that Ag nps (Ki = 0.12 μM) inhibited the activity of nNOS the most compared to Au nps; (Ki = 0.15 μM) and curcumin (Ki = 0.25 μM). At 298K, all the ligands bound at a single site on the enzyme (n=1) and a single Trp residue (θ =1), (later identified as Trp678) was made available on the enzyme surface for quenching by the ligands. Increasing the temperature from 298K-313K, increased the value of Ksv and pointed to a dynamic quenching mechanism for Aβ peptides, nps and curcumin interaction with nNOS. The positive signs for entropy and enthalpy for all Aβ peptides nps and curcumin pointed to hydrophobic–hydrophobic interaction with the enzyme. The fact that Kd increased with temperature emphasized the endothermic nature of the binding reaction and the requirement of thermal energy to aid in diffusion of the ligand to the active site. It was concluded that the binding reaction between the ligands and nNOS was non-spontaneous and endothermic at low temperatures (+ΔG) but spontaneous at high temperatures (-ΔG). The two amino acids Tyr706 and Trp678 moved from their original positions, subject to ligand binding. Trp678 moved a minimum distance of 5 Å toward the heme while Tyr706 moved a maximum distance of 14 Å away from the heme. AutoDock 4.2 was a valuable tool in monitoring the distance of Trp678 within the enzyme interior and fluorescence resonance energy transfer (FRET) was efficient in monitoring the distance moved by Trp residues on the enzyme surface.

Solid-state NMR (ssNMR) has matured into a versatile method to provide structural information, probe protein dynamics and detect small molecule binding and -protein interaction of a variety of biomolecular assemblies including amyloid fibrils, viral particles and membrane proteins. Membrane proteins embedded in liposomes are natural targets for ssNMR as their native states are solids. Magic angle spinning (MAS) ssNMR studies using moderate spinning frequencies provide detailed structural information and probe subtle conformational change. Development of fast magic angle spinning ssNMR enables proton-detection which increases sensitivity and facilitates protein dynamics measurements. In this dissertation, we applied moderate and fast MAS ssNMR to study potassium ion channel and protein dynamics Chapter 1 will introduce concepts and theory of solid-state NMR pulse sequences and experiments. Chapter 2 will discuss the application and perspectives of solid-state NMR to membrane protein systems. In Chapter 3, we test an allostery mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery. In Chapter 4, I optimize protein expression, purification and reconstitution into native environment protocols of a bacterial potassium transporter, KtrB. In chapter 5, methods and experimental details of setting up 60 and 40 kHz fast MAS ssNMR are discussed. With fast MAS ssNMR setup, multidimensional NMR experiments with higher sensitivity could be collected on a perdeuterated sample with less sample mass required. In Chapter 6, we employ fast MAS ssNMR to measure bulk and residue site-specific 15N and carbonyl 13C relaxation of microcrystalline ubiquitin. Carbonyl R1ρ relaxation profiles provide additional information on protein backbone dynamics.

Neuronal culture systems have many applications, such as basic research into neuronal structure, function, and connectivity as well as research into diseases, conditions, and injuries affecting the brain and its components. In vitro dissociated neuronal systems have typically been derived from embryonic brain tissue, most commonly from the hippocampus of E18 rats. This practice has been motivated by difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. The overall focus of this dissertation research was to develop a dissociated neuronal culture system from human and animal adult brain tissue, one more functionally and developmentally correlative to the mature brain. To that end, this work was divided into five interrelated topics: development of an adult in vitro neuronal culture system comprised of electrically functional, mitotically stable, developmentally mature neurons from the hippocampus of adult rats; creation of stable two-cell neuronal networks for the study of synaptic communication in vitro; coupling of electrically active adult neurons to microelectrode arrays for high-throughput data collection and analysis; identification of inadequacies in embryonic neuronal culture systems and proving that adult neuronal culture systems were not deficient in similar areas; augmentation of the rat hippocampal culture system to allow for the culture and maintenance of electrically active human neurons for months in vitro. The overall hypothesis for this dissertation project was that tissue engineered in vitro systems comprised of neurons dissociated from mature adult brain tissue could be developed using microfabrication, defined medium formulations, optimized culture and maintenance parameters, and cell-cycle control. Mature differentiated glutamatergic neurons were extracted from hippocampal brain tissue and processed to purify neurons and remove tissue debris. Terminally differentiated rat hippocampal neurons recovered in vitro and displayed mature neuronal morphology. Extracellular glutamate in the culture medium promoted neuronal recovery of electrical function and activity. After recovery, essential growth factors in the culture medium caused adult neurons to reenter the cell cycle and divide multiple times. Only after reaching confluence did some neurons stop dividing. Strategies for inhibition of neuronal mitotic division were investigated, and manipulation of the cdk5 pathway was ultimately found to prevent division in vitro. Prevention of mitotic division as well as optimization of culture and maintenance parameters resulted in a neuronal culture system derived from adult rats in which the neuronal morphology, cytoskeleton and surface protein expression patterns, and electrical activity closely mirrored mature, terminally differentiated adult neurons in vivo. Improvements were also made to the growth surfaces on which neurons attached, regenerated, and survived long-term. Culture surfaces, in this case glass cover slips, were modified with the chemical substrate N-1 (3-(trimethoxysilyl) propyl)-diethylenetriamine (DETA) to create a covalently modified interface with exposed cell-adhesive triamine groups. DETA chemical surfaces were also further modified to create high-resolution patterns, useful in creating engineered two-cell networks of adult hippocampal neurons. Adult hippocampal neurons were also coupled to microelectrode array systems (MEAs) and recovered functionally, fired spontaneously, and reacted to synaptic antagonists in a manner consistent to adult neurons in vivo. Last, neurons from the brains of deceased Alzheimer's disease (AD) patients and from brain tissue excised during surgery for Parkinson's disease (PD), Essential Tremor (ET), and brain tumor were isolated and cultured, with these neurons morphological regenerating and electrically recovering in vitro.

Hao, Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 98-115). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / 摘要 --- p.v / List of Abbreviations --- p.vii / List of Figures --- p.x / List of Tables --- p.xi / List of Primers --- p.xii / Publications arising from this study --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's disease --- p.1 / Chapter 1.2 --- APP and its functions --- p.4 / Chapter 1.2.1 --- APP processing --- p.7 / Chapter 1.3 --- APPc-interacting proteins --- p.10 / Chapter 1.3.1 --- FE65 --- p.10 / Chapter 1.3.2 --- Xllα and Xl1β --- p.12 / Chapter 1.3.3 --- JIP-1 --- p.13 / Chapter 1.3.4 --- Dabl and Dab2 --- p.15 / Chapter 1.3.5 --- SNX17 --- p.15 / Chapter 1.3.6 --- Numb --- p.15 / Chapter 1.3.7 --- AIDA-1 --- p.16 / Chapter 1.4 --- Objectives of the project --- p.18 / Chapter 1.4.1 --- Engulfment adaptor protein 1 (GULP1) --- p.19 / Chapter 1.4.2 --- Specific aims of my study --- p.20 / Chapter Chapter 2 --- General Methodology --- p.22 / Chapter 2.1 --- Bacterial culture --- p.22 / Chapter 2.2 --- Mini-preparation/Midi-preparation of plasmid DNA --- p.22 / Chapter 2.3 --- Spectrophotometric analysis of DNA --- p.22 / Chapter 2.4 --- Agarose gel electrophoresis for DNA --- p.23 / Chapter 2.5 --- Preparation of competent E. coli --- p.23 / Chapter 2.6 --- Transformation of competent E. coli --- p.24 / Chapter 2.7 --- Molecular cloning --- p.24 / Chapter 2.7.1 --- Preparation of the cloning vector and insert --- p.25 / Chapter 2.7.2 --- Isolation of DNA from agarose gel --- p.25 / Chapter 2.7.3 --- DNA ligation and transformation --- p.25 / Chapter 2.7.4 --- Rapid screening for ligated plasmid --- p.26 / Chapter 2.8 --- Site-directed mutagenesis --- p.26 / Chapter 2.9 --- Cell culture and transfection --- p.27 / Chapter 2.10 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) --- p.28 / Chapter 2.11 --- Western blotting --- p.29 / Chapter Chapter 3 --- Investigation of the GULP1-APP interaction and the effect of GULP1 on APP processing --- p.31 / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.34 / Chapter 3.2.1 --- DNA constructs --- p.34 / Chapter 3.2.2 --- Antibodies --- p.34 / Chapter 3.2.3 --- GST pull-down assays --- p.35 / Chapter 3.2.4 --- Rat tissues preparation --- p.36 / Chapter 3.2.5 --- Immunostaining --- p.36 / Chapter 3.2.6 --- "siRNA knockdown of GULPl in CHO, HEK293 and SHSY5Y cells" --- p.37 / Chapter 3.2.7 --- Luciferase assays --- p.37 / Chapter 3.2.9 --- Tricine-SDS/PAGE analysis for APP CTFs --- p.38 / Chapter 3.2.9 --- Aβ enzyme-linked immunosorbent assay (ELISA) --- p.39 / Chapter 3.2.10 --- Statistical analysis --- p.40 / Chapter 3.3 --- Results --- p.40 / Chapter 3.3.1 --- GULP1 F145V mutant abandons the GULP1-APP interaction --- p.40 / Chapter 3.3.2 --- GULP1 and APP colocalize in neurons --- p.45 / Chapter 3.3.3 --- "siRNA mediated knockdown of GULPl in CHO, HEK293 and SHSY5Y cells" --- p.48 / Chapter 3.3.4 --- GULP1 enhances the cleavage of APP in APP-GAL4 cleavage system --- p.49 / Chapter 3.3.5 --- GULP1 alters APP processing by increasing the secretion of APP CTFs --- p.52 / Chapter 3.3.6 --- GULP1 stimulates Aβ secretion --- p.55 / Chapter 3.4 --- Discussion --- p.57 / Chapter Chapter 4 --- Identification and characterization of GULPl phosphorylation sites --- p.60 / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Materials and Methods --- p.60 / Chapter 4.2.1 --- DNA constructs --- p.61 / Chapter 4.2.2 --- Antibodies --- p.61 / Chapter 4.2.3 --- Expression and purification of GST fusion proteins --- p.61 / Chapter 4.2.4 --- In vitro phosphorylation of GULP1 by cdk5/p35 --- p.62 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- GULP1 Ser223 can be phosphorylated by cdk5/p35 in vivo --- p.62 / Chapter 4.3.2 --- The phosphorylation ofGULPl Thr35 completely abolished the GULP1-APP interaction --- p.67 / Chapter 4.4 --- Discussion --- p.70 / Chapter Chapter 5 --- Crystallization of the PTB domains of GULPl and GULP1t35d…… --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and Methods --- p.72 / Chapter 5.2.1 --- DNA constructs --- p.72 / Chapter 5.2.2 --- Small-scale protein expression and purification --- p.73 / Chapter 5.2.3 --- Large-scale protein expression and purification --- p.73 / Chapter 5.2.4 --- Dynamic light scattering measurement --- p.76 / Chapter 5.2.5 --- Crystallization screening GULP1-PTB --- p.76 / Chapter 5.2.6 --- Optimization of GULP1-PTB crystals by grid screen --- p.76 / Chapter 5.2.7 --- Optimization of GULPl -PTB crystals by additive screen and detergent screen --- p.79 / Chapter 5.3 --- Results --- p.79 / Chapter 5.3.1 --- Large-scale expression and purification of GULP 1-PTB --- p.79 / Chapter 5.3.2 --- Small-scale expression and purification of GULP1T35d-PTB --- p.86 / Chapter 5.3.3 --- Crystallization screening and optimization --- p.88 / Chapter 5.4 --- Discussion --- p.91 / Chapter Chapter 6 --- Conclusion and future perspective --- p.94 / Chapter 6.1 --- Conclusion --- p.94 / Chapter 6.2 --- Future perspective --- p.95 / References --- p.98

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Beta-Amyloid (1-40) (Aβ40) is an aggregation prone protein, which undergoes a nucleation-dependent aggregation process causing the pathological neurodegeneration by amyloid plaque formation implicated in Alzheimer’s disease. In this thesis, we investigated the effects of small molecule modulators extracted from the marine invertebrate Pseudopterogorgia elisabethae on the Aβ40 amyloidogenic process using in- vitro ThT fluorescence assay and atomic force microscopy. We also investigated the effects of neutral and anionic phospholipid liposomes on Aβ40 aggregation. Our results show that a marine natural product Pseudopterosin-A and its derivatives can suppress and modulate the Aβ40 aggregation process. Furthermore, our results demonstrate that a neutral phospholipid liposome inhibits Aβ40 fibril formation, whereas the anionic liposomes promote it. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Alzheimer’s disease (AD) is the leading cause of dementia but despite being identified over a century ago, current treatments remain limited. To date, no disease-modifying therapies are available. Soluble, intracellular forms of β-amyloid (Aβ), a protein associated with AD, have been identified and intracellular targets of Aβ are being investigated as potential targets for new drugs. Amyloid binding alcohol dehydrogenase (ABAD) was previously identified as a mitochondrial target of Aβ and is known to be up-regulated in AD. This interaction results in production of reactive oxygen species and cell death. Using a small peptide, known as the “decoy peptide”, disruption of this interaction has been shown to reverse biochemical and behavioural symptoms in an AD mouse model. The work reported in this thesis describes the approaches taken to develop methods for in vitro and ex vivo study of the interaction between ABAD and Aβ. A fluorogenic assay for measuring the intracellular activity of ABAD in living cells was developed and using this technique, the intracellular inhibition of ABAD by Aβ was observed for the first time. Surface plasmon resonance was used to measure binding between ABAD and Aβ and also showed the first quantitative analysis of direct binding of the decoy peptide to Aβ42. In order to synthesise small molecule inhibitors of ABAD activity with the aim of developing a molecular probe of the enzyme’s activity, compounds were identified by screening a fragment-based library. Subsequent optimisation of the compound structure led to a 10-fold improvement in the IC50 and has resulted in a lead compound for future development. A similar screening strategy was employed to identify potential small molecule inhibitors of the ABAD-Aβ interaction. This research has resulted in a range of tools and methods for studying ABAD activity and interactions, which will greatly benefit future work on developing compounds that inhibit the ABAD-Aβ interaction to provide a novel method for treating Alzheimer’s disease.

[Truncated abstract] Alzheimer’s disease (AD) is a progressive neurodegenerative disease which manifests clinically as personality changes and global cognitive decline resulting in a loss of function, ultimately leading to death. Whilst causal genetic mutations have been identified, accounting for a small proportion of familial cases, the vast majority of all AD cases are late onset and idiopathic. However, a number of risk factors have been identified, including age associated changes in the reproductive hormones – estrogen and the gonadotropins. Previous in vitro and in vivo studies have implicated both estrogen and the gonadotropins in the regulation of the neurotoxic beta amyloid (Aß) peptide, accumulation of which is thought to be a key pathogenic event in the development of AD, but the role of these hormones in the etiology and pathogenesis of AD remains contentious. The aim of this thesis was to further understanding of the role of female reproductive hormones in modulating susceptibility to AD. The role of menopausal hormone dysregulation in behavior, cognitive decline and Aß-related neuropathology was examined in vivo in 4 studies using animal models of AD and menopause. The first two studies used a mouse model of AD expressing a human PS1 mutation (PS1KI) to examine the effects of ovariectomy as a model of menopause on cognition and neuropathology. Ovariectomy was found to selectively impair learning on a spatial working memory task without affecting working memory recall or reference memory performance. However, this cognitive impairment was not associated with any changes in Aß accumulation or oxidative stress. ... However, these findings cannot explain the lack of effect of estrogen supplementation on Aß levels. It is possible that supra-physiological doses of estrogen are necessary to yield anti-amyloidogenic and anti-oxidative benefits in ovariectomized sheep. It is becoming clear that the relationship between hormone changes at menopause and risk of AD may be more complicated than previously conceived. This study has begun to tease apart the relative contributions of estrogen and the gonadotropin hormones in the modulation of Aß, accumulation of which may confer susceptibility to AD. The findings presented indicate that the gonadotropins may play an important role in the regulation of AD-related behavior and cognition. The observed functional effects of the gonadotropins may also have implications for our understanding of behavioral and cognitive changes occurring during reproductive events. Based on the evidence presented here, combined with previous literature, it is clear that both estrogen and the gonadotropins are involved in the modulation of Aß accumulation, however, elucidation of the circumstances necessary to elicit these effects and their clinical relevance to humans will require further investigation. These findings contribute to a more sophisticated understanding of the post-menopausal hormonal milieu, recognizing the role of the gonadotropin hormones and that gonadal estrogen depletion does not necessarily result in brain estrogen depletion.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 8 uppsatser.

Amyloid beta (Aβ) has been postulated to be the principle initiator of the pathogenesis of Alzheimer’s disease (AD). Therefore, understanding the underlying mechanisms of Aβ induced neurotoxicity in the early stages of AD would be essential for finding potential therapeutic targets of AD. Aβ-binding alcohol dehydrogenase (ABAD) has been shown to be a mitochondrial binding site for Aβ. Expression of ABAD has been found to be increased in brains of AD sufferers. Two dimensional electrophoresis studies have revealed that endophilin 1 was upregulated in Tg mAPP/ABAD mice brains as compared to Tg mAPP, Tg ABAD and non-Tg mice brains. Increased expression of endophilin 1 has also been found in brains of AD patients as compared to non-demented control brain tissues. Endophilin1 has been reported to regulate c-Jun N-terminal kinase (JNK) activation. In this study, expression of dominant negative forms of endophilin 1 (DN-endophilin 1) in mouse cortical neurons exhibited a significant reduction of Aβ induced JNK activation. Furthermore, using cell counting methods, it was shown that the transfection of DN-endophilin 1 increased neuron survival after Aβ treatment. Aβ has also been proposed to disrupt the interaction of ABAD and Cyclophilin D (CypD), which would trigger mitochondrial permeable transition, thereby leading to neurotoxicity. For fluorescence resonance energy transfer (FRET) analysis of the interaction of ABAD and CypD, a mitochondria targeted, EYFP tagged ABAD plasmid (pMito-ABAD-EYFP) and an ECFP tagged CypD (pCypD-ECFP) plasmid were developed. Positive FRET signals in SK-N-SH cells co-expressing pMito-ABAD-EYFP and pCypD-ECFP indicated that ABAD interacts with CypD in the mitochondria of mammalian cells. RAGE (receptor for advanced glycation end products) has been reported to bind to Aβ and mediate the toxic effects of Aβ peptides on neurons and microglia. It has been shown previously that Tg mAPP/DN-RAGE mice display preserved cognitive function as compared to Tg mAPP mice. To investigate possible mechanisms involved in rescuing cognitive function by RAGE blockage, two dimensional electrophoresis was used to analyze differential protein expression between Tg mAPP and Tg mAPP/DN-RAGE mice cortex. Altered expression of four proteins, including NADH dehydrogenase flavoprotein 2 (NDUFV2), glyoxalase 1 (GLO1), proteasome subunit beta type 4 (PSMB4, or β7 subunit of proteasome) and nitrilase family, member 2 (Nit2) have been observed between Tg mAPP/DN-RAGE mice cortex and Tg mAPP mice cortex. NDUFV2 is a 24kDa subunit of complex 1 which is involved in ATP synthesis. GLO1 is a cytosolic enzyme that plays a role the glutathione-dependent detoxification of α-oxoaldehydes, such as methylglyoxal. PSMB4 is a subunit of the 26s proteosome which is in the degradation of ubiquitinylated proteins. The function of Nit2 is still unclear.