Browsing by Author "Leonenko, Zoya"

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Antimicrobial Peptide Daptomycin and its Inhibition by Pulmonary Surfactant: Biophysical Studies using Model Membrane Systems
(University of Waterloo, 2017-04-25) Lee, Brenda; Leonenko, Zoya
Daptomycin is a lipopeptide antibiotic that is clinically used to treat severe infections caused by Gram-positive bacteria. It is highly potent against resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus. However, in cases of community-acquired pneumonia (a leading cause of death worldwide), daptomycin is somehow inhibited by lung surfactant and therefore unable to exert its bactericidal activity against Streptococcus pneumoniae, the primary cause of this disease. This thesis presents the successful development of lipid model systems to mimic the lipid composition of S. pneumoniae bacterial membranes, human cell membranes, and both synthetic and natural lung surfactant. Experiments were performed that help to elucidate the basis for daptomycin’s inhibition by lung surfactant, culminating in a new, detailed model of daptomycin sequestration that summarizes the findings from these studies. Daptomycin is believed to be sequestered by lung surfactant and has been shown to insert into this surfactant. Fluorescence spectroscopy experiments were used to test the interaction of daptomycin with different lipid model membranes in the presence of calcium. The results provided strong evidence that daptomycin is sequestered by lung surfactant and that daptomycin has a similar affinity for both lung surfactant and bacterial membrane, suggesting these two entities play a competitive role in the binding of daptomycin. Increased emission spectra for daptomycin and bacterial membranes at higher concentrations of calcium suggest that calcium may remove an inhibited late step of daptomycin pore formation that has previously been shown. Using Langmuir-Blodgett monolayer techniques, studies were performed on how daptomycin affects monolayer properties. Compression isotherms provided data on monolayer compressibility, and it was found that daptomycin and calcium reduce the compressibility of lung surfactant monolayers, possibly improving its function. Constant-area insertion assays provided additional data that verified daptomycin’s avid binding to lung surfactant at low calcium concentrations. Scanning probe microscopy techniques were employed to obtain atomic force microscopy and Kelvin probe force microscopy images for monolayers in air. In the presence of daptomycin and calcium, the lung surfactant monolayers exhibited multilayer formation and increased electrical surface potential. Atomic force microscopy images taken of model lipid bilayers in liquid show multi-bilayer formation for the lung surfactant bilayers in the presence of daptomycin and calcium. This provides further evidence that daptomycin and calcium induce multilayer formation in lung surfactant. These findings allowed for the development of a novel model of daptomycin inhibition by lung surfactant. In the presence of physiological levels of calcium, daptomycin binds to lung surfactant and is sequestered. This binding causes a decrease in lung surfactant compressibility, allowing it to easily form multilayers that effectively reinforce the sequestration of daptomycin. The lipid models, methods, and experimental protocols developed in this thesis will help foster future studies in the field of membrane biophysics.
The Compared Effects of Lithium Isotopes ⁶Li and ⁷Li on GSK-3-β Activity and the Biochemistry of HT22 Neuronal Cells
(University of Waterloo, 2020-12-09) Livingstone, James David; Leonenko, Zoya
The use of lithium in treatment of mental illnesses like bipolar disorder, major depressive disorder, and schizophrenia dates to the mid 20th century. Some research also indicates that the symptoms of Alzheimer’s Disease can be lessened by lithium treatment. But despite widespread use, the complete mechanisms through which lithium works are largely unknown. The “isotopically impure” lithium used to treat these disorders is comprised of approximately 7.59% ⁶Li and 92.41% ⁷Li, about the normal distribution of the lithium isotopes in nature. Pharmacologically, no distinction has been made between the two isotopes. However, preliminary animal model research suggests that these isotopes may have differential effects on the maternal behaviours of rats, while other cellular research has found a difference in the sustained concentration of each isotope in both neural cells and erythrocytes. The research presented here investigates the possibility that the isotopes ⁶Li and ⁷Li may have different effects on neuronal physiology and molecular processes. No significant difference was found on the rate of glycogen synthase kinase-3-β (GSK-3-β) activity, GSK-3-β phosphorylation rates in HT22 cells, or HT22 cell viability when treated with ⁶Li and ⁷Li. However, a possible difference has been found in the sustained lithium isotope concentrations across the HT22 plasma membrane. More research is needed to confirm and elucidate this global set of data, but the results presented here indicate a reasonable possibility that lithium isotopes are differentially fractionated across the HT22 plasma membrane.
Effect of surfaces on amyloid fibril formation
(Public Library of Science (PLOS), 2011) Moores, Bradley; Drolle, Elizabeth; Attwood, Simon J.; Simons, Janet; Leonenko, Zoya
Using atomic force microscopy (AFM) we investigated the interaction of amyloid beta (Aβ) (1–42) peptide with chemically modified surfaces in order to better understand the mechanism of amyloid toxicity, which involves interaction of amyloid with cell membrane surfaces. We compared the structure and density of Aβ fibrils on positively and negatively charged as well as hydrophobic chemically-modified surfaces at physiologically relevant conditions. We report that due to the complex distribution of charge and hydrophobicity amyloid oligomers bind to all types of surfaces investigated (CH3, COOH, and NH2) although the charge and hydrophobicity of surfaces affected the structure and size of amyloid deposits as well as surface coverage. Hydrophobic surfaces promote formation of spherical amorphous clusters, while charged surfaces promote protofibril formation. We used the nonlinear Poisson-Boltzmann equation (PBE) approach to analyze the electrostatic interactions of amyloid monomers and oligomers with modified surfaces to complement our AFM data.
EFFECT OF TREHALOSE AND LITHIUM IN MOLECULAR MECHANISM OF NEUROPROTECTION IN ALZHEIMER’S DISEASE
(University of Waterloo, 2024-08-23) Xu, Yue; Leonenko, Zoya
Alzheimer's Disease (AD) is still a challenging issue for humans since its first case was identified by Alois Alzheimer over one hundred years ago. Approximately thirty years ago, the "Amyloid cascade hypothesis" was proposed, which is a milestone that began to reveal the mystery of AD. The aggregation and deposition of endogenous amyloid-beta (A-beta) proteins in brains are known to be one of the main pathogenic factors of AD. One of the pathways to neurodegeneration driven by A-beta proteins involves A-beta damage to neuronal membranes, which may result in neuron impairment and death. On the other hand, A-beta proteins have antimicrobial properties, suggesting they may serve functionally in the brain. This could be one of the reasons to explain the severe side effects seen in clinical anti-A-beta treatment for AD. Instead of focusing on anti-A-beta, I aim to explore a therapeutic strategy that focuses on membrane protection. The goal of my work is to investigate the potential of membrane-targeted agents, trehalose and lithium, to protect lipid membranes against A-beta toxicity. Trehalose, a natural-derived sugar, is explored as a potential treatment for neurodegenerative Parkinson's Disease. Lithium, as a mood stabilizer, is commonly used for treating bipolar disorder. Both of the agents are investigated for neurological disorders and can interact with cellular membranes with distinct mechanisms. In this thesis, I ask whether their interaction with lipid membranes can protect membranes from A-beta-induced damage, thereby lowering A-beta neurotoxicity. Hence, the entire thesis addresses two main questions. 1. How does trehalose/lithium affect membrane properties? 2. Can trehalose/lithium protect membranes from A-beta toxicity? To explore the two questions for trehalose and lithium, respectively, the thesis is divided into two parts: Part 1 - trehalose (Chapters 3-8) and Part 2 - lithium (Chapters 9-11). In Part 1, I used Langmuir-Blodgett (LB) Trough, atomic force microscopy (AFM), and Kelvin probe force Microscopy (KPFM) to explore the influence of trehalose on the mechanical and electrostatic properties of model lipid monolayers composed of DPPC, POPC lipids, and cholesterol. The study found that trehalose can enhance the fluidity and alter the electrostatic properties of lipid monolayers, with modulation by NaCl. To assess whether trehalose can protect lipid membranes from A-beta damage, I utilized black lipid membrane (BLM) electrophysiological techniques to evaluate the quality and permeability of membranes exposed to trehalose and A-beta. Results from BLM experiments demonstrated trehalose alleviates A-beta-induced membrane disruption. Furthermore, I explored the binding of A-beta to lipid membranes in the presence of trehalose solutions by localized surface plasmon resonance (LSPR) spectroscopy and found that trehalose can reduce A-beta binding to lipid membranes. Finally, I confirmed the unique neuroprotection of trehalose in cell studies, where trehalose decreased the cell mortality rate caused by toxic A-beta proteins. Part 2 explored the potential of lithium in mitigating A-beta toxicity on lipid membranes. Similarly, I used LB trough, AFM, and KFPM to compare the influence of LiCl and KCl on lipid membranes. The results demonstrated the distinct contribution of Li+ and K+ on the mechanical and electrostatic properties of DPPC-POPC-Chol lipid monolayers. Li+ has a pronounced effect on reducing the lipid molecular area, increasing monolayer fluidity, and strongly competing with K+ in interacting with lipid monolayers. Lastly, BLM was employed to evaluate the membrane permeability in exposure to A-beta and LiCl. The membrane conductance results obtained by BLM suggested that the modulation of LiCl at the therapeutic level enhances membrane resilience to A-beta-induced damage. This research exposes the modulation of membrane-active trehalose and lithium on lipid membrane properties and their protective role in AD. It contributes to exploring a new therapeutic approach against A-beta toxicity that focuses on membrane protection, which may aid in developing prevention and treatment strategies for AD.
The Interplay Between the Neuronal Plasma Membrane and Cell Signaling in Alzheimer’s Disease
(University of Waterloo, 2023-03-10) Robinson, Morgan; Beazely, Michael; Leonenko, Zoya
The lack of understanding in the molecular and cellular mechanisms of Alzheimer’s disease (AD) has hindered efforts towards finding treatments that effectively modify disease trajectory. Therapeutic development for AD has focused on targeting amyloid-β (Aβ) pathology, long thought to be the cause of AD pathogenesis, but these have failed in clinical trials. Aβ is a sticky aggregation-prone protein that disrupts membrane structure and interferes with specific receptors in the brain, impairing synaptic plasticity, an important process for learning and memory, and eventually causes cell death. The interplay between disruption of the neuronal membrane and neuronal receptors in AD overlaps with inflammation and oxidative stress in a feedback loop that makes it difficult to ascertain the causes and effects of AD. More recent genetic and epidemiology data indicates that lipid metabolism is critical in AD pathogenesis, underscoring the need to understand how brain lipid composition (especially cholesterol) in brain affects amyloid toxicity. In the first part of this work the relevant background literature of lipid mediated mechanisms of AD is discussed and an overview of methods used herein are provided. In the second part, the results of biomedical nanotechnology experiments where atomic force microscopy (AFM) was used to study interactions of supported lipid bilayers (SLBs) with melatonin and Aβ at the molecular level. Chapter 3 shows the characterization of biophysical changes that melatonin induces in SLBs of DOPC/DPPC/Cholesterol by AFM and atomic force spectroscopy (AFS). Overall, AFM imaging revealed that melatonin increases disordered domain coverage, reduces bilayer thickness and indentation depth, increases membrane fluidity, and decreases membrane adhesion, though large variability was observed. In Chapter 4, for the first-time contact mode high-speed AFM (HS-AFM) was shown to be able to image lipid membranes of different compositions. HS-AFM was used to capture large areas of membranes comparing the effects of Aβ monomers and oligomers to different phase separated lipid bilayers composed of low and high cholesterol showing different interaction mechanisms. In the third part of this thesis the influence of membrane composition and amyloid toxicity on HT22 neuronal cell viability, cholesterol metabolism, morphology, and receptor tyrosine kinase (RTK) signaling pathways was elucidated. Beginning in Chapter 4, cholesterol oxidase assays and AFM verify cell cholesterol content reduction and Aβ structure, respectively. There was no effect of Aβ on cholesterol recovery and cell viability studies show that cholesterol depletion was modestly protective against both Aβ monomers and oligomers. In Chapter 5, the cholesterol-dependent effects of Aβ monomers and oligomers on HT22 cell morphology by phase contrast optical microscopy and atomic force microscopy (AFM) reveal apoptotic and necrotic populations of HT22 cells exposed to Aβ and that that membrane cholesterol depletion prevents these changes in morphology. In Chapter 7, the effects of cholesterol and Aβ on baseline Tyrosine Receptor Kinase B (TrkB) receptors and PDGF receptor-α (PDGFRα) signaling, reveal that RTK signaling is cholesterol-dependent and that high concentration Aβ oligomers increase the likelihood of RTK impairment, but there was no statistically significant effect of Aβ on PDGFRα signaling. This work provides experimental evidence that membrane cholesterol is not strongly involved in the mechanisms of Aβ toxicity in HT22 cells, but its reductions may be mildly protective. RTK signaling in HT22 cells is impaired by Aβ but is not involved in the protective mechanisms of cholesterol depletion. Aβ disrupts membrane biophysical structure and receptor signaling pathways triggering metabolic dysfunction and both apoptotic and necrotic cell death mechanisms.
Nanoscale physics of surfactant gene delivery
(University of Waterloo, 2016-01-18) Henderson, Robert Douglas Evert; Leonenko, Zoya; Wettig, Shawn
Medicine has met a revolution in the expansion of possibilities for therapy based upon synthetic gene delivery. Imagine the ability to correct problems of a genetic origin with a simple drug -- such science fiction fantasies are becoming future's reality. Still in relative infancy, synthetic gene therapy has such potential for so many medical issues that it is a very high priority area of research on a global level. Equally revolutionary is the growth in the use of nanotechnology, now that instruments have been developed to probe most any physical system on the nanometre scale. The relatively new appearance of these technologies leaves research rife with fruit for the picking, and forming new interdisciplinary connections only multiplies the possibilities. Nature has developed excellent nanoscale machines -- viruses -- for gene delivery. Unfortunately for human beings, the end result is often detrimental rather than beneficial. Despite this, in typical human fashion we seek to adapt nature's solutions for our own purposes. Such endeavours are extremely difficult undertakings, but we persist for the benefit of all. So far, researchers have figured out how we can pack DNA with nanoscale carriers rather well using surfactants and lipids of various structures, and that these systems do an `okay' job of transfecting genes. However, we do not really know at all, let alone for certain, why some lipid or surfactant structures are better transfecting agents than others, or really how these carriers enter living cells and become expressed. The answers to these questions can never, ever be solved by observing these systems from the perspective of a single field of study, for in order to understand why, and thus to predict a better how, we must use the entire spectrum of science from the most fundamental to the most clinical. Over the decades of fairly clinical and in vitro studies that have characterised gene delivery research, precious little literature exists at the extreme end of fundamental physics. And yet, so much depends on the physical interactions of the gene delivery systems and their targets that an understanding of the physics of gene delivery could lead to more focussed and efficient clinical research. For this reason, the present work aims to bring together clinical research into gene delivery with state-of-the-art nanotechnology, observed through the lens of physics. Our primary instrument in this work is the atomic force microscope, which is a type of scanning probe microscope capable of imaging surfaces on nanometre scales using a micromachined cantilever tip. Our particular instrument is one of the most advanced that is presently available to implement recent developments in Kelvin Probe Force Microscopy (KPFM), a variant of atomic force microscopy (AFM) that is designed to image electrical surface potentials on the nanoscale. In the present work, we utilise an advanced method of frequency modulation KPFM, which allows surface potential imaging with nanometre resolution. This thesis begins with some of the most promising building blocks of surfactant gene delivery, gemini surfactants, and explores their nanoscale behaviour and interactions with other critical ingredients: lipids and DNA. Gemini surfactants have shown to achieve superior transfection efficiency, while maintaining a high level of versatility and flexibility yet requiring less material. These surfactants are also fairly inexpensive to manufacture. Such benefits make gemini surfactants an attractive candidate for synthetic gene therapy solutions. Further enhancements to transfection efficiency are made with the addition of `helper' lipids, an issue which we also explore. A fundamental aspect of the physics of gene delivery is how the systems interact with their targets: a living cell. By constructing a model monolayer of a cell using lipids commonly found in most cell membranes, we compared the structure of a `plain' model cell monolayer with one which has been infused with gemini surfactant. We found that the gemini surfactant exhibited strong interactions with the gel-phase lipid present in the model monolayer, and that the resulting domains had a more positive surface potential. Using the unique capabilities of KPFM, we were able to show the presence of cationic surfactant in the monolayer from its electrical signal. As an extension of the above, we added DNA into our monolayers to explore the effects of DNA binding. This binding behaviour is important to understand for the purposes of gene therapy. Our mixture of two cell membrane phospholipids (DOPC and DPPC), with gemini surfactant, showed three distinct domains, which we deduce to be DOPC, DPPC+gemini surfactant, and gemini surfactant+DNA. The latter region was the highest, exhibiting a network of thread-like domains. Most intriguingly, only the `middle' region exhibited a positive surface potential signal, a fact which can only be determined with KPFM imaging. We studied mixtures of gemini surfactants, helper lipid and DNA in monolayer form so that we could explore the nanoscale structures that these molecules create. In this way, we were able to create controlled environments in which to study the interactions of components of gene tranfection complexes. In addition, we used a Langmuir trough to gather pressure-area curves for our monolayers to draw further conclusions on the roles of the various components. We found that gemini surfactants play a significant role in compacting DNA, and that this compaction is enhanced by the presence of the helper lipid DOPE. Furthermore, the nanoscale structure of these monolayers was affected by factors such as acidity and the ratio of helper lipid to gemini surfactant. Finally, we used AFM and KPFM to probe gemini surfactant gene transfection complexes (nanoparticles), which were directly deposited onto an atomically flat substrate. We found that their size distributions are broad, ranging from a few tens of nanometres to a few hundred nanometres. This research demonstrates the unique capabilities of AFM and KPFM to probe systems of relevance to gene therapy, and that the nanoscale structure of transfection components is affected by a number of key factors such as the particular surfactant, amount of helper lipid, the presence of DNA, and environmental factors such as acidity. Given that the nature of these interactions is typically electrostatic in origin, it is clear that KPFM has a significant role to play. This thesis provides an introduction to novel methodologies for this purpose, illustrated by applications to gemini surfactant systems.
A preclinical study of SG pseudopeptide amyloid-β inhibitors for the treatment of Alzheimer’s disease: single molecule force spectroscopy and cell viability
(University of Waterloo, 2017-08-23) Robinson, Morgan; Leonenko, Zoya
Alzheimer's disease (AD) is one of the single greatest healthcare challenges facing our society today with no treatments available that cure, prevent or slow the disease. Neurodegeneration in AD is observed alongside pathology featuring amyloid-β (Aβ) deposits in the brain. Aβ monomers themselves have minimal toxicity but misfold into β-sheets and aggregate to form neurotoxic soluble oligomers, than aggregate further into less toxic insoluble fibrils and plaques. To treat or prevent AD one potential strategy has been suggested which involves the use of rationally designed pseudopeptides that bind Aβ with high affinity, inhibit aggregation into toxic Aβ oligomers and allow for natural clearance. A class of pseudopeptide Aβ inhibitors, designated “SG”, have been proposed with the use of computer aided drug design (CADD) by medicinal chemists at the University of Calgary led by Dr. Arvi Rauk. It is the aim of this thesis to verify, experimentally, that SG inhibitors behave as expected, moving these potential AD drug candidates further along the drug development pipeline. In this thesis a brief overview of Aβ pathology and therapeutics directed against Aβ are discussed followed by a review of the literature relevant to SG inhibitor design. The results from two separate experiments evaluating SG inhibitor target engagement and neuroprotection against Aβ are then discussed. In the first experiment, a nanoscale biophysics approach was used to assess the ability of SG inhibitors to bind Aβ and prevent dimerization – the first step in toxic oligomer formation. This single-molecule biophysics assay built on an atomic force microscopy (AFM) platform demonstrated that all the inhibitors engage the target and prevent Aβ dimerization. In the second set of experiments, a series of in vitro cell viability studies with HT-22 murine derived hippocampal cells was performed to assess SG inhibitor toxicity and the ability of SG inhibitor to mitigate Aβ toxicity. Most SG inhibitors exhibited no apparent toxicity to HT-22 cells however myristic acid modification for delivery of inhibitors to the brain caused dose dependent toxicity. Importantly, two of the five inhibitors demonstrated a small but promising effect on preventing Aβ oligomer neurotoxicity as demonstrated by one third increase of HT-22 cell viability in MTT assays. The inhibitor SGA1 may cause a slight increase in the toxicity of Aβ prepared under fibril forming conditions. Overall the work described here presents experimental evidence that indicates SG inhibitors as a potential therapeutic for Aβ toxicity and informs recommendations for SG inhibitor design to improve safety and efficacy for future lead candidates.
The Role of Melatonin in Amyloid Interactions with the Model Neuronal Membranes
(University of Waterloo, 2023-11-29) Mei, Nanqin; Leonenko, Zoya
The amyloid cascade hypothesis has long directed research efforts in understanding the pathogenesis of Alzheimer’s disease (AD), ever since it was first proposed through clinical evidence by Hardy and Higgins in 1992. Currently, amyloid-β (Aβ) species have attracted significant interest, particularly as they demonstrate profound cellular membrane toxic effects. When there is no cure for AD, the current research direction shows there is a need to develop the protective strategies. One natural molecule, melatonin, has been shown to be protective against amyloid toxicity in animal and cellular studies, while the molecular mechanisms are not well understood. One of the hypothesis is that melatonin can alter the membrane structure and thus reduce amyloid toxicity to lipid membranes. In this thesis research, how the incorporation of melatonin to the lipid membrane would change the structure of the membranes and protect the membrane from amyloid toxicity was examined. This thesis focuses on investigations on the neuroprotective effects of melatonin against Aβ toxicity were achieved using various membrane models that mimic cellular membranes at different stages of AD. Utilizing a range of advanced nanotechnology tools including nuclear magnetic resonance (NMR), localized surface plasmon resonance (LSPR), atomic force microscopy (AFM), black lipid membrane (BLM) studies, complemented by neutron scattering, neutron reflectivity, and molecular dynamics (MD) simulations, the study explores the intricate molecular mechanisms behind melatonin’s interaction with Aβ peptides. Different membrane models representing various disease states were employed to assess melatonin’s multifaceted effects. The research uniquely contributes by combining these diverse techniques to offer a comprehensive understanding of the potential therapeutic role of melatonin in AD, paving the way for the development of more targeted and effective treatments. Future exploration into melatonin’s interactions with various forms of Aβ aggregates may further enhance therapeutic strategies. The research employs a range of neuronal membrane models — simple model (SPM) (DPPC/POPC/cholesterol) and complex (DPPC/POPC/cholesterol/SM/GM1) models —to elucidate the protective role of melatonin against Aβ toxicity. Each complex model represents a different disease state of membrane (healthy model (HM), early diseased model (EDM) and late diseased model (LDM)), allowing tests on multifaceted effects of melatonin in varying biological contexts and disease states. The studies demonstrated that melatonin changed the phase separation of lipid membranes, altered the amyloid binding to the membranes, and enhanced the resilience of membranes against amyloid-induced damage. Insights from the NMR study presented in Chapter 2 indicated that melatonin promoted DPPC/POPC phase separation in a simple lipid membrane model. The LSPR study, complemented with AFM in Chapter 3, showed that melatonin altered the amyloid binding to complex lipid membrane models. Interestingly, the intensity of this effect varied depending on the disease state, with the most pronounced protective effects of melatonin observed in EDM. The BLM study in Chapter 4 further investigated melatonin’s effect on different types of amyloid-induced membrane damages. The results clearly demonstrated melatonin’s ability to enhance the longevity of lipid membranes and increase the membrane’s resistance to rupturing. The advancement of knowledge on melatonin’s membrane protective effects can pave the way for the development of new preventive strategies and potentially open doors for innovative therapeutic approaches.