Pharmacy

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Browsing Pharmacy by Author "Beazely, Michael"

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    Endocannabinoids as Amyloid-Beta (Aβ) Aggregation Inhibitors
    (University of Waterloo, 2022-05-13) Khavandi, Marzie; Beazely, Michael; Nekkar Rao, Praveen
    The endocannabinoid system, including endogenous cannabinoids and their corresponding receptors, has received extensive attention in the last few years for their neuroprotective effect in the central nervous system. The regulation and metabolism of these molecules are potential therapeutic targets for neurodegenerative diseases such as Alzheimer's disease, which is characterized by Aβ aggregation-induced cell toxicity, inflammation, tau phosphorylation, disruption of neurotransmitters pathways, mitochondrial dysfunction, and oxidative stress. The endocannabinoids, such as 2-AG, AEA, NADA, noladin, OAE, and their main metabolite, arachidonic acid, may be involved in the multiple neuroprotective effects, including excitotoxicity attenuation, oxidative stress reduction, and inflammation prevention through CB1, CB2 receptors as well as other possible pathways, including inhibition of Aβ oligomer formation via interactions with these toxic peptides. However, the interactions of endocannabinoids with Aβ species and their mechanisms have not been fully explored. Therefore, we hypothesized that endocannabinoids might reduce amyloid β-protein deposition and inhibit neuronal cell death through CB1 or other possible pathways. In vitro experiments, including cell studies using two cell lines (HTT22 and CB1-CHO), ThT based kinetic assay, and TEM studies were used to determine the effects of above mentioned five endocannabinoids, arachidonic acid, and a CB1 antagonist to understand the role of endocannabinoid ligands and pathways involved in Aβ-induced neurotoxicity. The results of this study on HT22 cells showed that some, but not all of the endocannabinoids were able to exhibit neuroprotective effects against Aβ-induced toxicity. However, AM251, as a CB1 receptor antagonist, could not reverse this neuroprotection. On the other hand, AM251 was able to inhibit the protective effects of some, but not all, of the endocannabinoids in CB1-CHO cells.
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    GPCR and RTK Regulation in Neurons: The Impact of Stress on GPCR and RTK signalling and Crosstalk
    (University of Waterloo, 2020-04-14) Gondora, Nyasha; Beazely, Michael; Mielke, John
    Crosstalk between receptors allows for the integration of diverse and complex signalling pathways. Transactivation is a form of crosstalk between G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). RTKs are transactivated by GPCRs through two main mechanisms: ligand- independent intracellular pathways and triple membrane passing mechanisms involving GPCR mediated growth factor signaling. Transactivation has neuroprotective potential; however, the physiological relevance of this pathway is not known. Interestingly, some stressors can up- or downregulate GPCR and RTK activity, as well as initiate certain transactivation pathways, leading to the question: could transactivation be a stress response? Given the impact of stress on GPCRs and RTKs, this thesis directly explored the impact of stress, more specifically acute chemical stress, in the form of the stress hormone corticosterone and chronic stress, in the form of Chronic Early Life Social Isolation (CELSI), on the 5HT7-TrkB transactivation pathway. Another aim of this thesis was to analyze the effect of social isolation (CELSI) on the expression of proteins that are implicated in neuroplasticity and to explore if social isolation stress differentially primes the brain's response to stress or other stimuli. Coupling cell line based experiments with ex vivo tissue work, the overall aim of this thesis research was to gain a better understanding of some molecular mechanisms underlying the impact of stress on the brain.
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    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.