Browsing by Author "Du Toit, Andre"

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    Dietary impact on neuronal autophagy control and brain health
    (IntechOpen, 2019) Ntsapi, Claudia; Du Toit, Andre; Loos, Ben
    Autophagy is the major intracellular system which is critical for the removal of harmful protein aggregates and malfunctioning organelles. Dysfunctional autophagy is associated with a multitude of human diseases, such as protein aggregation in Alzheimer’s disease and non-successful aging. Major interest exists in the dietary manipulation of the autophagy pathway activity, so as to tune the cell’s protein degradation capabilities and to prevent cell death onset. It has recently become clear that the machinery required to degrade protein cargo has a distinct activity level which can be altered through specific dietary modulation. Moreover, this activity may differ from that of the proteinaceous cargo. Overall, brain health and successful aging are characterized by limited protein aggregation, with a distinct molecular signature of maintained autophagy function. However, it is largely unclear how to control autophagy through dietary interventions with a precision that would allow to maintain minimal levels of toxic proteins, preserving neuronal cell viability and proteostasis. In this chapter, we carefully dissect the relationship between autophagy- modulating drugs, including caloric restriction mimetics and their impact on neuronal autophagy, in the context of preserving brain health.
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    Measuring and modelling autophagic flux
    (Stellenbosch : Stellenbosch University, 2016-03) Du Toit, Andre; Loos, Ben; Hofmeyr, J.-H. S.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Introduction. Autophagy is a dynamic process that is responsible for cellular protein degradation, which involves sequestering of bulk cytoplasm and its delivery to lysosomes where degradation and recycling occurs. Autophagy is vital for cellular function and can be induced during periods of nutrient deprivation for the recycling of proteins and for removing potentially harmful proteins and organelles. A reduction in the autophagic degradative capacity has been linked to several diseases such as those associated with neurodegeneration. These attributes make autophagy an attractive therapeutic target; clinical trials using autophagy inducers have already shown promising results. In order to successfully exploit autophagy, it is crucial to determine whether the autophagic flux is too high or too low, and adjust it accordingly. However, the accurate measurement of autophagic flux still remains a challenge. Aims. The aim of this project was therefore, rst, to develop a novel method to accurately measure autophagic ux. Second, to assess autophagy using conventional techniques and compare it with the new approach. Our third aim was to construct a kinetic model of the autophagic system that could simulate our experimentally generated data and thereby help us understand the contribution of the different processes involved in autophagy and its dynamic behaviour. Methods. We made use of uorescent-based imaging to acquire z-stack images of mouse embryonic broblasts that stably express GFP-LC3. Images were processed and the total autophagic vesicles pool size was measured using ImageJ with the WatershedCounting3D plugin. Cells were cultured in the presence of an acidotrophic uorescent dye that allows (in-combination with GFP-LC3) the visualisation of autophagosomes, autophagolysosomes and lysosomes. Cells were encased in a humidi ed atmosphere in the presence of 5% CO2 at 37 C in a microscope slide of the IX81 Olympus microscope. First we determined the concentration of ba lomycin A1 required for the complete inhibition of the autophagosome and lysosome fusion process. We calculated the autophagic ux as the initial rate of increase in the number of autophagosomes after inhibition of fusion. Second, we increased autophagosomal synthesis through induction with 25 nM rapamycin and again calculated the autophagic ux from the initial rate of increase in autophagosomes after fusion inhibition. In parallel, we assessed changes in the autophagic markers LC3-II and p62 with Western blot analysis and in the morphology of autophagic vesicles with electron microscopy at time points suggested by the uorescent experimental data. A kinetic model of the autophagic system was constructed and parameterised so as to t the experimental data. Computational modelling was done with the Python Simulator for Cellular Systems (PySCeS). Results. Although we found that 100 nM ba lomycin A1 was su cient to inhibit the fusion of autophagosomes and lysosomes, we chose to use 400 nM ba lomycin A1 in order to be absolutely sure the inhibition was complete. Induction of autophagosomal synthesis with 25 nM rapamycin increased the autophagic ux in MEF cells from its basal value of 25.4 autophagosomes/cell/hr to 105.4 autophagosomes/cell/hr. The transition time, i.e., the time required to clear the autophagosomal pool, decreased from its basal value of 0.53 hr to 0.16 hr after induction. Similarly the transition times for the basal and induced autophagolysosomal pools were 6.7 hr and 2.4 hr. Whereas with our uorescence microscopy method we measured a four-fold increase in autophagic ux from the basal to the induced state, traditional approaches such as Western blot analysis measure only a two-fold increase; electron microscopy proved to be inadequate for assessing autophagic vesicles. Autophagosomes constituted a small percentage of the total GFP-LC3-positive vacuoles. Upon induction with rapamycin the number of autophagosomes/cell increased slightly from 13 to 17, whereas the number of autophagolysosomes/cell increased considerably from 165 to 251. Autophagosomal size was about four times smaller than autophagolysosomal size. Simulating the autophagic system with our kinetic model provided an excellent t to the experimental data. Conclusion. Our novel approach quanti es autophagic variables such as the ux and the number of the di erent types of autophagic vesices accurately at single cell level, and, used in combination with kinetic modelling of the dynamics of autophagy, hold promise for future therapeutic application.
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    Measuring and Modelling Autophagic Flux
    (Stellenbosch : Stellenbosch University, 2019-04) Du Toit, Andre; Loos, Benjamin; Hofmeyr, Jan-Hendrik S.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Introduction. Autophagy is an evolutionarily-conserved cellular process of self-digestion, wherein cytoplasm and organelles are sequestered and delivered to lysosomes for degradation. Autophagy plays a vital role in maintaining cellular function and proteostasis through the recycling of cellular components; it generates the building blocks for de novo synthesis and substrates for energy generation during periods of nutrient deprivation. It also serves to protect cells against diverse pathologies by removing potentially harmful proteins and organelles. Autophagy has been linked to the progression of several diseases; the loss of autophagy function leads to the build up of toxic compounds, such as those associated with neurodegeneration, while enhanced autophagy activity contributes to the resistance of cancer against chemotherapeutic drugs. The role of autophagy in disease has made it an attractive therapeutic target for the treatment of several diseases. Clinical trials using autophagy modulators have already shown promising results. The success of autophagy-targeting therapies depends, however, on our ability to accurately measure autophagy and characterise autophagy modulators, as well as unravelling its role in disease both on a mechanistic (single cell) and a global (whole organ) level. We previously developed a fluorescence-based microscopy technique for accurately measuring autophagosome flux that showed great promise. Aims. The first aim of this project was to validate the reliability and applicability of our approach to accurately measure autophagosome flux and autophagy intermediates. The second aim was to use this technique for the screening of several autophagy modulating-drugs and then, as our third aim, to identify novel biomarkers that could serve as indicators of autophagosome flux in a clinical setting. The fourth aim was to use our approach to investigate the underlying mechanism of autophagy that could provide context for understanding the dynamic nature of autophagy. Our fifth aim, as an extension of our previous modelling efforts, was to perform supply and demand analysis to characterise the distribution of flux and concentration control of the autophagic steady state. Our final aim was to bridge the gap between in vitro and ex vivo by characterising the three-dimensional spatial organisation of autophagy pathway intermediates in brain tissue. Methods. A fluorescence-based imaging approach was employed to measure autophagy variables in mouse embryonic fibroblasts (MEF) and HeLa cells that stably express GFP-LC3. Cells were cultured in the presence of an acidotrophic fluorescent dye that allows, in combination with GFP-LC3, the visualisation of autophagosomes, autolysosomes and lysosomes. We calculated the autophagosome flux as the initial rate of increase in the number of autophagosomes after inhibition of fusion between autophagosomes and lysosomes using bafilomycin A1. We validated the reliability and applicability of this approach through a series of experiments: measuring autophagosome flux in autophagy-silenced cells, evaluating alternative probes, and comparing our approach to a recently-developed flux probe. Then we used this approach to screen several clinically relevant autophagy modulators and characterised their dose-response and time-response curves. Proteomic analysis was performed on MEF cells with autophagy being induced by 25% and 75% using rapamycin and spermidine to identify novel biomarkers of autophagosome ux. A partial-inhibition-of-fusion group was included to filter false positive markers. To investigate the underlying mechanism of autophagy we used our approach to (i) assess cytoplasmic cargo (volume) turnover by measuring autophagosome size and flux before and after induction with rapamycin, spermidine and FBS, (ii) determine changes in the tubulin-associated translocation rate and displacement of autophagosomes in response to induction with rapamycin and spermidine, and in response to partial inhibition with bafilomycin A1, by acquiring an image series of MEF GFP-LC3 cells to analyse autophagosomal movement with TrackMate, and (iii) assess whether autophagosome flux changed in relation to cell size. For the supply and demand analysis the autophagic response to rapamycin induction was used to determine the demand elasticity coefficient and rate characteristics, while the supply elasticity coefficient and rate characteristics were determined by incrementally decreasing the rate of fusion of autophagosomes and lysosomes using bafilomycin A1 and measuring the autophagy variables. Finally, brains were harvested from mice and polymerised in an acrylamide and paraformaldehyde solution. The hydrogel-tissue matrix was made transparent using a 3D-printed clearing station and stained for autophagy markers (LC3, p62 and LAMP2A). Tissue was imaged using a light-sheet and a confocal microscope. Results. Our method quantified the autophagosome flux and the autophagy intermediates in a reliable and robust manner, and was able to detect small changes in autophagy activity, something that was not possible with the other flux probes. It proved to be suitable for high-throughput platforms and was flexible enough to accommodate a range of probes. Drug screening allowed for the characterisation of the dose response curve with high precision, demonstrating that it is possible to finely modulate autophagy. We identified several autophagosome flux markers, the majority being cytosolic proteins that decrease with increasing autophagosome flux. Moreover, proteomic analysis revealed that autophagy machinery proteins are not best suited as flux markers. We were able to shed light on the underlying mechanism of autophagy through a series of experiments that showed that (i) mTOR-dependent induction of autophagy modulates both autophagosome flux and autophagosome size, while mTOR-independent induction only modulates autophagosome flux, (ii) autophagosome flux increases in relation to cell size, and (iii) an increase in autophagosome flux leads to a decrease in the rate of autophagosome translocation. Supply and demand analysis revealed that the supply of autophagosomes (synthesis of autophagosomes) determines flux through the autophagy vesicular pathway while the demand of autophagosomes (their fusion with lysosomes) controls the homeostatic maintenance of autophagosomes under normal physiological conditions. Finally, we demonstrated it is possible to assess autophagy pathway intermediates in a three-dimensional neuroanatomical context. Conclusion. Our approach quantifies autophagy variables in a robust and reliable manner and promises to be a technique of choice for characterising autophagy modulators. Biomarkers identified in this study could serve as a starting point for developing assays that can be used to measure autophagosome flux in a clinical setting. The data generated in our study of the autophagy system contributes to our understanding of autophagy as a whole and paves the way to the point where we will be able to finely modulate autophagy.
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    Mitochondrial event localiser (MEL) to quantitativelydescribe fission, fusion and depolarisation in the three-dimensional space
    (Public Library of Science, 2020-12) Theart, Rensu P.; Kriel, Jurgen; Du Toit, Andre; Loos, Ben; Niesler, Thomas R.
    ENGLISH ABSTRACT: Mitochondrial fission and fusion play an important role not only in maintaining mitochondrial homeostasis but also in preserving overall cellular viability. However, quantitative analysis based on the three-dimensional localisation of these highly dynamic mitochondrial events in the cellular context has not yet been accomplished. Moreover, it remains largely uncertain where in the mitochondrial network depolarisation is most likely to occur. We present the mitochondrial event localiser (MEL), a method that allows high-throughput, automated and deterministic localisation and quantification of mitochondrial fission, fusion and depolarisation events in large three-dimensional microscopy time-lapse sequences. In addition, MEL calculates the number of mitochondrial structures as well as their combined and average volume for each image frame in the time-lapse sequence. The mitochondrial event locations can subsequently be visualised by superposition over the fluorescence micrograph z-stack. We apply MEL to both control samples as well as to cells before and after treatment with hydrogen peroxide (H2O2). An average of 9.3/7.2/2.3 fusion/fission/depolarisation events per cell were observed respectively for every 10 sec in the control cells. With peroxide treatment, the rate initially shifted toward fusion with and average of 15/6/3 events per cell, before returning to a new equilibrium not far from that of the control cells, with an average of 6.2/6.4/3.4 events per cell. These MEL results indicate that both pre-treatment and control cells maintain a fission/fusion equilibrium, and that depolarisation is higher in the post-treatment cells. When individually validating mitochondrial events detected with MEL, for a representative cell for the control and treated samples, the true-positive events were 47%/49%/14% respectively for fusion/fission/depolarisation events. We conclude that MEL is a viable method of quantitative mitochondrial event analysis.
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    The precision control of autophagic flux and vesicle dynamics - a micropattern approach
    (MDPI, 2018) Du Toit, Andre; De Wet, Sholto; Hofmeyr, Jan-Hendrik S.; Muller-Nedebock, Kristian K.; Loos, Ben
    Autophagy failure is implicated in age-related human disease. A decrease in the rate of protein degradation through the entire autophagy pathway, i.e., autophagic flux, has been associated with the onset of cellular proteotoxity and cell death. Although the precision control of autophagy as a pharmacological intervention has received major attention, mammalian model systems that enable a dissection of the relationship between autophagic flux and pathway intermediate pool sizes remain largely underexplored. Here, we make use of a micropattern-based fluorescence life cell imaging approach, allowing a high degree of experimental control and cellular geometry constraints. By assessing two autophagy modulators in a system that achieves a similarly raised autophagic flux, we measure their impact on the pathway intermediate pool size, autophagosome velocity, and motion. Our results reveal a differential effect of autophagic flux enhancement on pathway intermediate pool sizes, velocities, and directionality of autophagosome motion, suggesting distinct control over autophagy function. These findings may be of importance for better understanding the fine-tuning autophagic activity and protein degradation proficiency in different cell and tissue types of age-associated pathologies
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    Spermidine and rapamycin reveal distinct autophagy flux response and cargo receptor clearance profile
    (MDPI, 2021-01-07) De Wet, Sholto; Du Toit, Andre; Loos, Ben
    Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance.