Masters Degrees (Physiological Sciences)

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Browsing Masters Degrees (Physiological Sciences) by Subject "Alzheimer's disease -- Treatment"

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    Investigating Tau pathology in an in vitro model for Alzheimer’s disease
    (Stellenbosch : Stellenbosch University, 2016-12) Powrie, Yigael Samuel Louis; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction Alzheimer’s disease is a neurodegenerative disease of the brain and the leading cause of dementia globally. Severe cognitive and short term memory deficits are commonly associated with this disease. The pathology is characterised by two molecular hallmarks that manifest in brain tissue, which are intercellular plaques composed of β-amyloid, and intracellular protein aggregates known as neurofibrillary tangles (NFTs) composed of phosphorylated Tau, a microtubule (MT) associated protein (MAP). Under homeostatic conditions Tau facilitates the dynamic polymerisation of the microtubule network, which acts as part of the cytoskeleton and platform for vesicular transport. Tau is generally phosphorylated to modulate its binding affinity to the network. However, under pathological conditions it becomes hyperphosphorylated, leading to dissociation from the MT. Dissociated Tau is thought to form NFT aggregates, which causes the MT to become susceptible to cleavage by the severing proteins, Katanin p60 and Spastin. However, it has not been determined when this process occurs in disease progression and whether it is indeed a confounding factor leading to the onset of neuronal cell death. Moreover, although dysfunction of the autophagic lysosomal pathway, an inherent proteolytic process for long-lived proteins and organelles, has been shown to be implicated in the onset of protein aggregation, its role in the context of MT dysfunction remains unclear. Aims and Methods The aims of this study were to assess microtubulin and Tau dynamics in an in vitro model of autophagic dysfunction that is similar to the Alzheimer’s disease pathology. It was hypothesized that a disruption in the autophagy process would lead to maladaptive changes in the microtubulin and Tau dynamics prior to the onset of cell death. GT1-7 neuronal cells were cultured under standard conditions and treated with Chloroquine diphosphate (CQ), a lysosome deacidifying agent, to induce an autophagic dysfunctional state. Two time points of exposure to CQ were established using a WST-1 assay to assess the molecular changes occurring prior to and during the onset of cell death. Western blot analysis was utilised to quantify protein levels of acetylated α-tubulin, Tau, pTau, Katanin p60 and Spastin in response to CQ-induced autophagy dysfunction. Furthermore, cells were transfected with a GFP-Tau DNA construct, using the Neon® transfection system. Additionally, cells were fixed and stained post-transfection with fluorescent Alexa® Fluor secondary antibodies against primary antibodies recognising acetylated α-tubulin, pTau, Katanin p60 and Spastin. Fluorescent microscopy analysis was performed using Super Resolution Structured Illumination Microscopy (SR-SIM), Stochastic Optical Reconstruction Microscopy (STORM), Correlative Light and Electron Microscopy (CLEM) and confocal microscopy techniques on the LSM-780 Elyra PS.1 system to assess protein localisation in response to CQ treatment. Moreover, co-localisation was assessed between acetylated α-tubulin and Tau, pTau, Spastin and Katanin p60 respectively. Results Fluorescent microscopy analysis revealed that CQ-induced autophagy dysfunction caused acetylated α-tubulin protein structure to become progressively impacted, which manifested as breakages in the network. Tau protein levels decreased non-significantly, but fluorescent microscopy revealed the formation intracellular Tau aggregates. In addition, Tau co-localised with acetylated α-tubulin under control conditions and remained co-localised in response to CQ treatment. Phosphorylated Tau protein levels did increase non-significantly, but fluorescent microscopy revealed no aggregate formation. Katanin p60 protein levels significantly increased, however, the protein did not co-localise with acetylated α-tubulin under control conditions or in response to CQ-induced autophagy dysfunction. Spastin protein levels increased non-significantly, however, Spastin co-localised with acetylated α-tubulin under control conditions, which significantly increased in response to autophagy dysfunction. Discussion and Conclusion Our results indicate that CQ-induced autophagy dysfunction causes Tau aggregation, but no dissociation from the microtubule network. Furthermore, the microtubulin network becomes unstable, despite its continuous association with Tau, which may be caused by increased Spastin-mediated severing. To conclude, the data clearly demonstrate that these pathological perturbations occur prior to the onset of cell death, which not only highlights novel therapeutic targets, but also the lack of optimal timing in the therapeutic interventions utilised in Alzheimer’s disease treatment.