Measuring and Modelling Autophagic Flux

Du Toit, Andre (2019-04)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

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.

AFRIKAANSE OPSOMMING: Inleiding. Autofagie is 'n evolusion^er-bewaarde sellul^ere proses van selfvertering, waarin sitoplasma en organelle in vesikels gesekwestreer word en na lisosome afgelewer word vir degradasie. Autofagie speel 'n belangrike rol in sellul^ere funksie en proteostase deur die herwinning van sellul^ere komponente; dit genereer beide boustene vir de novo sintese van proteiene en substrate vir energieproduksie gedurende periodes van voedingstoftekorte. Dit beskerm ook selle teen diverse siektetoestande deur die verwydering van potensieel skadelike proteiene en organelle. Autofagie is al geassosieer met verskeie siektes: 'n afname in autofagie degradasiekapasiteit lei tot die opbou van skadelike proteiene, soos di_e geassosieer met neurodegenerasie, terwyl verhoogde autofagie-aktiwiteit bydra tot die robuustheid van kanker teenoor chemoterapeutiese middels. Hierdie eienskappe maak autofagie 'n aantreklike terapeutiese teiken vir die behandeling van verskeie siektes. Kliniese proewe wat van autofagiemoduleerders gebruik maak het alreeds goeie resultate getoon. Die sukses van autofagie-geteikende terapiee hang af van ons vermo e om autofagie akkuraat te meet en autofagiemoduleerders te karakteriseer, asook om autofagie se rol in siekte op 'n meganistiese (enkele sel) en 'n globale (hele orgaan) vlak te ontrafel. Ons het voorheen 'n uoressensie-gebaseerde mikroskopiese tegniek om autofagosoomuksie akkuraat te kan meet ontwikkel wat belowende resultate getoon het. Doel. Die doel van hierdie projek was eerstens om die betroubaarheid en toepaslikheid van ons tegniek om die autofagosoomuksie en sy intermedi^ere akkuraat te kan meet te bevestig. Die tweede doel was om dit te gebruik om verskeie autofagiemodulerende-middels te evalueer, en dan, as ons derde doel, om nuwe merkers te identi_seer wat as meetbare aanwysers van autofagosoomuksie in 'n kliniese omgewing te kan dien. Die vierde doel was om ons tegniek te gebruik om van die onderliggende autofagiese meganisme te ondersoek om ons begrip van die dinamiese aard van autofagie te verbeter. Ons vyfde doelwit, as uitbreiding van ons vorige modelleringspogings, was om 'n aanbod-aanvraag analise te doen op autofagie om die verspreiding van uksie- en konsentrasiekontrole te karakteriseer. Laastens, met die doel om die gaping tussen in vitro en ex vivo te oorbrug, het ons 'n weefselverhelderingstegniek ge-evalueer om die 3-dimensionele organisasie van autofagie intermedi^ere in breinweefsel te karakteriseer. Metodes. Fluoressensie-gebaseerde afbeelding is gebruik om autofagie veranderlikes in muis embrioniese fibroblaste (MEF) en HeLa selle wat stabiele GFPLC3 uitdruk te meet. Selle is gekweek in die teenwoordigheid van 'n asidotro_ese uoresserende kleurstof wat in kombinasie met GFP-LC3 die visualisering van autofagosome, autolisosome en lisosome moontlik maak. Die autofagosoomuksie is bereken as die aanvanklike snelheid waarmee autofagosome toeneem na inhibisie van fusie van autofagosome en lisosome met ba_lomisien. Die betroubaarheid en toepaslikheid van hierdie tegniek is bevestig deur autofagosoomuksie te meet in selle waarin autofagie stilgemaak is, alternatiewe probes te evalueer, en ons benadering met 'n onlangs ontwikkelde uksie-probe te evalueer. Daarna het ons die tegniek gebruik om verskeie klinies-relevante autofagiemoduleerders te evalueer en hul dosis- en tyd-responskurwes te karakteriseer. Proteomiese analise is uitgevoer op MEF selle waarvan die autofagosoomuksie met 25% en 75% geinduseer is deur rapamisien en spermidien om nuwe merkers van autofagosoomuksie te identi fiseer. 'n Groep selle waarvan die fusie gedeeltelik met ba_lomisien geinhibeer is is ingesluit om vals positiewes uit te _ltreer. Om die onderliggende autofagiese meganisme te ondersoek het ons ons benadering gebruik om (i) die verandering in sitoplasmiese omset in respons op rapamisien, spermidien en FBS te bepaal deur autofagosoomgrootte en autofagosoomuksie te meet voor en na induksie, (ii) die veranderinge in die tubulien-geassosieerde translokasietempo en verplasing van autofagosome te bepaal in respons op rapamisien, spermidien en ba_lomisien in MEF GFP-LC3 selle deur die verkryging van 'n beeldreeks om autofagosoom beweging met TrackMate te ontleed, en (iii) te bepaal of autofagosoomuksie in verhouding tot selgrootte verander. Vir die aanbod-aanvraaganalise is die autofagie respons op rapamisien gebruik om die aanvraag elastisiteitskoeffisient en die aanvraag snelheidskarakteristiek van autofagosome te bepaal, terwyl die aanbod elastisiteitskoeffisient en aanbod-snelheidskarakteristiek bepaal is deur inkrementele inhibisie van fusie met ba_lomisien en meting van die autofagie veranderlikes. Laastens is geisoleerde muisbreine gepolimeriseer in 'n akrielamied en paraformaldehied-oplossing. Die hidrogel-weefselmatriks is deursigtig gemaak met behulp van 'n 3D-gedrukte skoonmaakstasie en gekleur vir autofagiemerkers (LC3, P62 en LAMP2A). Weefsel is afgebeeld deur gebruik te maak van 'n ligveld en 'n konfokale mikroskoop. Resultate. Ons benadering kon autofagosoomuksie en autofagie-intermediate op 'n betroubare en robuuste wyse kwanti_seer, en was in staat om klein veranderinge in autofagie aktiwiteit te bespeur, iets wat nie moontlik was met die ander uksie-probes was nie. Dit is geskik vir hoe-deurset afbeeldingplatforms en kan 'n reeks probes akkommodeer. Ons kon met ho e akkuraatheid die dosisresponskurwes van autofagiese moduleerders karakteriseer, wat daarop wys dat dit moontlik is om autofagie fyn te kan moduleer. Ons het verskeie autofagosoom- uksiemerkers geidentifiseer, waarvan die meerderheid sitosoliese proteiene was wat afneem met toenemende autofagosoomuksie. Proteoomanalise het ook getoon dat die proteiene van die autofagie-masjinerie nie goeie uksiemerkers is nie. Die autofagiestelsel se onderliggende meganisme is uitgelig deur 'n reeks eksperimente wat gewys het dat (i) mTOR-afhanklike induksie van autofagie beide autofagosoom- uksie en autofagosoomgrootte moduleer, terwyl mTOR-onafhanklike induksie slegs autofagosoomuksie moduleer, (ii) autofagosoomuksie toeneem met 'n toename in selgrootte, en (iii) 'n toename in autofagosoomuksie lei tot 'n afname in die tempo van autofagosoomtranslokasie. Aanbod-aanvraaganalise het aangedui dat die aanbod van autofagosome (sintese van autofagosome) die uksie deur die autofagiese vesikul^ere pad bepaal, terwyl die aanvraag van autofagosome (fusie met lisosome) die homeostatiese instandhouding van autofagosome onder normale _siologiese toestande beheer. Laastens het ons gewys dat dit moontlik is om die autofagie-intermediate in 3-dimensionele neuro-anatomiese konteks te beskryf. Gevolgtrekking. Ons benadering kwanti_seer autofagie veranderlikes op 'n robuuste en betroubare manier en blyk 'n goeie tegniek te wees vir die karakterisering van autofagiemoduleerders. Die autofagiese uksiemerkers wat in hierdie studie geidentifiseer is kan dien as 'n beginpunt vir die ontwikkeling van toetse wat e_ektief in 'n kliniese omgewing gebruik kan word om autofagosoomuksie te assesseer. Die data uit hiedie studie dra by tot ons begrip van autofagie as geheel en stel ons in staat om autofagie fyn te moduleer.

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