Dissection of autophagy machinery and protein cargo flux
| dc.contributor.advisor | Loos, Ben | en_ZA |
| dc.contributor.author | De Wet, Sholto | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty Science. Dept. of Physiology. | en_ZA |
| dc.date.accessioned | 2020-02-26T15:36:01Z | |
| dc.date.accessioned | 2020-04-28T15:13:52Z | |
| dc.date.available | 2020-02-26T15:36:01Z | |
| dc.date.available | 2020-04-28T15:13:52Z | |
| dc.date.issued | 2020-04 | |
| dc.description | Thesis (MSc)--Stellenbosch University, 2020. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Inleiding: Neurodegeneratiewe siektes, soos byvoorbeeld Alzhiemer’s siekte, word gekenmerk deur verhoogte histologiese vlakke van onoplosbare proteïn aggregate. Studies het al gewys dat die oplosbare proteïn oligomere wat die onoplosbare proteïn aggregate opmaak, ‘n hoof bron is van neurologiese toksisiteit. Autofagie word gekenmerk as ‘n hoof bron van proteïn afbraak en word waargeneem gedurende neurodegeneratiewe siektes. In die verlede is makro-autofagie omskryf as ‘n nie- spesifieke manier om lang-lewende sitoplasmiese proteïne af te breek. Onlangse bewyse demonstreer egter dat verskeie sub-tipes van makro-autofagie (hierna autofagie) bestaan, en dat dit van mekaar onderskei word deur hul verskillende ladings. Van spesifieke belang in die konteks van neurodegeneratiewe siektes is die regulering en beheer van proteïn afbraak deur autofagie. Daar is bewyse wat toon dat p62 die verkieslike autofagie lading ontvanger gedurende proteïn afbraak is. Bydraend tot p62 het NBR1 ook resultate gelewer as nog ‘n bydraede autofagie lading onvanger van proteïne en het getoon dat dit kan kompenseer vir die verlies aan p62 vlakke. Hoe hierdie twee ontvangers se gedrag deur mekaar beïnvloed word om effektiewe afbraak tydens ‘n 24 uur periode van autofagiese induksie, is onbekend. Bygese is dit onbekend tot watter vlakke hierdie ontvangers met autofagosome reageer om by te dra tot ‘n effektiewe autofagiese afbreek tempo. Doel en metodes: Die doel van die studie was om ondersoek in te stel na die veranderinge wat voorkom in komponente van die autofagiesie stelsel in ‘n gevestige autofagiese model sisteem van muis embrioniese fibroblaste (MEFs) wat nie siektes simptome demonstreer nie. MEFs was ge-mikrovorm om grootte en vorm te standaardiseer. Autofagiese veranderinge was deur die gebruik van Rapamycin en Spermidine geïnduseer teen relatief hoë en lae konsentrasies, respektiewelik. Studies was uitgevoer oor ‘n periode van 24 uur om te bepaal wat die impak van tyd was op autofagie en die verskeie komponente daarvan is. Westelike klad analise is gebruik om metings uit te voer om die oorvloedige veranderings van LC3-II, p62, NBR1 en LAMP2a te bepaal. Bydraend was fluorescene mikroskopie gebruik om GFP-LC3, p62 en NBR1 punt tellings uit te voer. Verder was alle studies gedoen in die teenwoordigheid en afwesigheid van Bafilomycin A1, ‘n inhibeerder van autofagosoom/ lisosome vorming, om sodoende die opruimings aktiwiteite van elke proteïn beter te verstaan. Resultate en bespreking: Vroeë ondersoeke, deur die gebruik van westelike klad analise, dui daarop dat Rapamycin verhoogde vlakke van LC3-II oorvloedigheid veroorsaak, maar het nie die ontvangers se vlakke verander nie. Spermidine behandeling het egter ‘n verhoogde autofagosomiese verwydering en ontvanger oorvloed, maar het nie die ontvanger se opruimings vlakke verander nie. Fluorisente mikroskopie beelde het gewys dat induksie met 1 μM Rapamycin wel ‘n verhoogde GFP-LC3 en ontvanger punt telling na twee ure vanaf inkubasie toon. Daar was egter geen veranderinge in die ontvanger se opruimings vlakke soos gedemonstreer is deur die gebrek van saam-gelokaliseerde punt verwyderings. Aan die ander kant het 10 nM Rapamycin minder autofagosome gedemonstreer, maar daar was beter ontvanger opruiming, veral van p62. Spermidine resultate toon egter ‘n verskillende gedrag deurdat 20 nM Spermidine ‘n stadiger verhoging van GFP-LC3 getoon het as 1 uM Rapamycin, maar demonstreer hoogs effektiewe p62 opruiming teen 2 ure, gevolg deur effektiewe NBR1 opruiming teen dieselfde tyd as gemeet op 8 ure; die tyd wanneer p62 verwyderings op sy laagste vlakke was. 5 nM Spermidine het nie induksie van die stelsel in dieselfde manier beïnvloed as 20 nM Spermidine nie, soos gesien deur die minder effektiewe GFP-LC3 verwydering. 5 nM Spermidine het egter wel effektiewe p62 verwydering gedemonstreer teen 8 ure se gebruik sowel as effektiewe GFP-LC3/p62 saam-gelokaliseerde punt opruimings na 2 ure en 8 ure van behandeling. Gevolgtrekkings: Die metode waarvolgens autofagie geïnduseer word, deur mTOR-afhanklik of –onafhanklike inisieerders, het wel ‘n impak op autofagiese komponente se uitdrukkings vlakke. Verder toon behandeling van hoër konsentrasies van die middels ‘n meer robuste en onmiddellike respons van autofagiese komponente soos gemeet deur hulle verwydering. Aan die ander kant toon laer middel konsentrasies verkillende tye van effektiwiteit. Hierdie studie het gevolglik bewys dat die effektiwiteit van autofagiese tempo multifaktoriaal en is behoort vervolgens aangepas te word om autofagosome sovel as ontvangers se bydraende invloed te bepaal in toekomstige navorsing. | af_ZA |
| dc.description.version | Masters | en_ZA |
| dc.format.extent | vii, 115 pages : illustrations (some color) | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/108437 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Autophagy -- Analysis | en_ZA |
| dc.subject | Alzheimer's disease | en_ZA |
| dc.subject | Neurodegenerative diseases | en_ZA |
| dc.subject | Proteins -- Physiological transport | en_ZA |
| dc.subject | Autophagic vacuoles -- Mechanism of action | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | Dissection of autophagy machinery and protein cargo flux | en_ZA |
| dc.type | Thesis | en_ZA |