Masters Degrees (Physiological Sciences)

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

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    Dissection of autophagy machinery and protein cargo flux
    (Stellenbosch : Stellenbosch University, 2020-04) De Wet, Sholto; Loos, Ben; Stellenbosch University. Faculty Science. Dept. of Physiology.
    ENGLISH ABSTRACT: Introduction: Neurodegenerative diseases, such as Alzheimer’s disease, are characterised by the increased histological expression of insoluble protein aggregates. Evidence has demonstrated that the soluble protein oligomers which constitute these insoluble protein aggregates, are a major source of neurotoxicity. Autophagy is known to be a major protein degradative pathway and has been shown to be active during neurodegenerative diseases. In the past, macroautophagy has been described as a non-specific means of degrading long-lived cytoplasmic proteins. However, recent evidence has demonstrated that various subtypes of macroautophagy (hereafter autophagy) exist, distinguished from one another by their preferred cargo. Of particular interest in the context of neurodegenerative diseases is regulating and controlling protein degradation through autophagy. p62 has been shown to be the preferred autophagy cargo receptor during protein degradation. In addition to p62, NBR1 has been shown to be another autophagy cargo receptor of proteins and has been shown to compensate for a loss of p62 expression levels. How these two receptors behave with respect to one another to support effective degradation over a 24 hour period of autophagy induction is unknown. Additionally, it is unknown to what extent these receptors interact with autophagosomes to contribute toward effective autophagy flux. Aims and methods: The aim of this study was to investigate the changes that occur to components of the autophagy system within an autophagy model system established in mouse embryonic fibroblasts (MEFs) not expressing any disease symptoms. MEFs were micropatterned as a means of standardising cell shape and size. Autophagy changes were induced using Rapamycin and Spermidine, respectively at relatively high and low concentrations. Studies were conducted over 24 hours to understand what impact time had on autophagy and its components. Western blotting was used to measure the abundance changes of LC3-II, p62, NBR1 and LAMP2a. Additionally, fluorescence microscopy was used to observe GFP-LC3, p62 and NBR1 puncta counts. Furthermore, studies were done in the presence and absence of Bafilomycin A1, an inhibitor of autophagosome/ lysosome fusion, to better understand the clearance activities of each protein. Results and discussion: Initial investigations using western blotting techniques demonstrated that Rapamycin caused an increase in LC3-II abundance levels but does not change receptor levels. Additionally, Spermidine treatment caused an increase in autophagosome clearance and receptor abundance but does not change receptor clearance levels. Fluorescence microscopy imaging revealed that autophagy induction with 1 μM Rapamycin caused an increase in GFP-LC3 and receptor puncta count 2 hours after incubation. However, no change was seen in the receptor clearance as was shown by the lack of co-localised puncta clearance. 10 nM Rapamycin on the other hand demonstrated fewer autophagosomes, however; effective receptor turnover, was demonstrated, especially of p62. Spermidine results demonstrated different behaviours, as 20 nM Spermidine showed a slower increase in GFP-LC3 than 1 μM Rapamycin, but demonstrated highly effective p62 clearance at 2 hours, followed by effective NBR1 clearance at the same time and at 8 hours, where p62 turnover was found to be at its lowest. 5 nM Spermidine did not induce the system in the same way as 20 nM Spermidine as was seen by less effective GFP-LC3 turnover. However, 5 nM Spermidine did demonstrate effective p62 clearance at 8 hours as well as effective co-localised puncta clearance at 2 hours and 8 hours of treatment. Conclusion: The means by which autophagy is induced, either by mTOR-dependent or –independent inducers, has an impact on autophagy components expression levels. Furthermore, treatment with higher concentrations of drugs demonstrated a more robust and immediate response of the autophagy components measured as well as their clearance. Conversely, lower drug treatment concentrations demonstrate different times of effectiveness. Taken together this study has shown that the effectiveness of autophagy flux is multifactorial and should be adjusted according to the autophagosome as well as receptor involvement for future research to be successful.
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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.