The effects of nutrient deprivation on macroautophagic flux and chaperone-mediated autophagy in a model of alzheimer's disease

Ntsapi, Matlakala Claudia (2018-12)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Introduction: Alzheimer’s disease (AD) is a devastating neurodegenerative disease characterized by progressive cognitive impairment, particularly in brain regions crucial for learning and memory. These symptoms are caused by neuronal death resulting from two pathological features: extracellular senile plaques composed of aggregated amyloid-beta (Aβ) peptides, and intracellular neurofibrillary tangles generated by the hyperphosphorylation of tau protein. Although AD is a multifactorial disease, much of the AD research continues to be guided by the amyloid cascade hypothesis, which posits that Aβ aggregation is the key initiate in AD pathogenesis. Aβ is generated from the proteolytic cleavage of the amyloid precursor protein (APP) by β - and γ-secretase. Accordingly, research efforts to modulate APP processing, and better clarify the mechanisms that regulate intracellular Aβ metabolism and clearance during AD progression have been explored in the treatment of AD. Autophagy, a lysosome-based proteolytic pathway that plays a crucial role in intracellular protein quality control, has been implicated in both the production and clearance of Aβ peptide. Cumulative evidence shows that AD-related autophagic dysfunction coincides with the detection of Aβ within autophagic vacuoles (AVs) that accumulate within dystrophic neurites with the initial increase in Aβ neurotoxicity. Therefore, autophagy dysfunction may exacerbate Aβ pathology and further augment disease progression; however, when in this context autophagy becomes dysfunctional remains unclear. Moreover, although it is known that Aβ levels themselves may induce autophagy, how long autophagy remains upregulated and functional in this process is unclear. It also remains unclear whether autophagy plays a causative, or protective role in Aβ neurotoxicity; or whether autophagy dysfunction is a consequence of the disease process itself. Therefore, the aims of this study were (i) to characterize the expression profile of key amyloidogenic pathway proteins, both macroautophagy and chaperone-mediated autophagy (CMA) proteins as well as the extent of neuronal toxicity using a unique APP overexpression model, (ii) to dissect the interplay between proteolytic pathways and cell death markers in the context of APP overexpression using a proteomics approach, (iii) to assess macroautophagic flux in the context of APP overexpression and to unravel the extent of autophagy dysfunction, (iv) to assess the contribution of macroautophagy and CMA in Aβ clearance and neuronal toxicity by modulating each pathway, and (v) to assess the effects of prolonged intermittent fasting (IF) on the modulation of macroautophagy and CMA in a paraquat (PQ)-induced in vivo brain injury model. Methods: A unique AD overexpression model, the N2a mouse neuroblastoma cell line stably overexpressing the human Swedish double mutation was utilized. APP overexpression was characterized, and the induction of macroautophagy, CMA, and apoptosis was assessed over time using a combination of cell viability assays, western blot analysis, fluorescence microscopy, transmission electron microscopy (TEM), and correlative light and electron microscopy techniques. Moreover, the effect of APP overexpression on a global proteome level was quantified using high resolution liquid-chromatography coupled to tandem mass spectrometry. Finally, a PQ – induced brain injury model was established and utilized to assess the effects of prolonged IF on macroautophagy and CMA using GFP–LC3 transgenic mice. Mice were injected twice weekly with 10 mg/kg PQ for a duration of 3 weeks. A prolonged IF protocol of 48 hrs fasting, followed by 24 hrs refeeding was implemented for a duration of 3 weeks. Modulation of macroautophagy and CMA following chronic oxidative stress exposure, and prolonged IF was evaluated in selected brain regions by western blot analysis, fluorescence microscopy, comparative haematoxylin and eosin staining, and TEM analysis. Results: The results indicate that APP overexpression leads to prominent apoptosis induction after 48 hrs and activates the autophagy machinery in a time-dependent manner. To our surprise, macroautophagic flux analysis reveals that autophagy is upregulated upon APP overexpression but remains elevated in the presence of apoptosis induction. Our CMA analysis indicates that APP overexpression activates the CMA machinery, particularly during the 48 hrs time point. However, induction of apoptosis proceeded despite elevated levels of CMA activity. Next, our proteome analysis reveals a time-dependent increase in APP proteinprotein interaction partners over time. Cumulatively, the in vitro results suggest that the modulation of macroautophagy and CMA augments Aβ clearance and mitigates neuronal toxicity. In vivo, a significant decrease in cytochrome c, and 4HNE expression were observed with prolonged IF intervention in selective brain regions. These changes were associated with elevated levels of macroautophagy and CMA induction, as evidenced by the significant increase in LC3II and LAMP2A protein expression. Therefore, suggesting that protection was brought about by the prolonged IF intervention through the modulation of macroautophagy and CMA. Conclusion: Our findings indicate that autophagy is upregulated in the presence of high levels of APP and Aβ, and to our surprise, remains upregulated even in the presence of apoptosis induction, suggesting an insufficient autophagy response in the mitigation of Aβ neurotoxicity. However, enhanced Aβ clearance was observed with a sufficiently high autophagy response even during 48 hrs APP overexpression, suggesting that autophagy modulation may be a viable treatment approach long into disease progression. These findings were also confirmed with prolonged IF intervention, where markers of apoptosis, and lipid peroxidation were notably decreased in brain regions associated with neurodegeneration. Further studies, specifically using in vivo APP overexpression models are warranted to further verify the clinical use of autophagy control.

AFRIKAANSE OPSOMMING: Inleiding: Alzheimer se siekte (AS) is ‘n vernietigende neuro-degeneratiewe siekte wat gekenmerk word deur ‘n progressiewe verlies in kognitiewe funksie in dele van die brein wat ‘n rol speel in geheue en leervermoë. Hierdie simptome word toegeskryf aan die doodgaan van neurone as gevolg van twee patologiese kenmerke: ekstrasellulêre seniele plake wat bestaan uit geaggregeerde beta-amiloïed (Aβ) peptiede en intrasellulêre neurofibrillêre knope wat ontaard danksê te hiperfosforilasie van die tau proteïen. Alhoewel AS vele oorsake het, volg die meerderheid van huidige navorsing die amiloïed kaskade hipotese waarin Aβ aggregasie as die hoof oorsaak van AS beskou word. Proteolitiese skeiding van die amiloïed voorloper proteïen (AVP) by β - en γ-sekretase veroorsaak die produksie van Aβ. Daar is tans vele navorsing veldtogte geloods wat poog om AVP prosessering te moduleer asook om die regulerende meganismes van intrasellulêre Aβ metabolisme en verwydering te ontbloot, met die hoop dat dit kan bydra tot beter behandeling van AS. Autofagie is ‘n lisosoom gebaseerde proteolitiese sisteem wat ‘n belangrike rol speel in die beheer van intrasellulêre proteïen kwaliteit en is gevind om betrokke te wees in beide die produksie en verwydering van die Aβ peptied. Kumulatiewe bewyse dui aan dat tydens AS verwante autofagiese disfunksie kan Aβ binne autofagiese vakuole (AV) gevind word en dat hierdie AV akkumuleer binne distrofiese neuriete tydens die aanvanklike vermeerdering in Aβ neurotoksisiteit. Daarom kan autofagiese disfunksie die Aβ patologie vererger en bydra tot die vorming van AS. Merendeels, alhoewel dit bewys is dat autofagie aangeskakel kan word namate die Aβ konsentrasie verhoog, is dit nog nie bekend vir hoe lank autofagie aangeskakel en funksioneel bly tydens hierdie proses nie. Dit is ook onduidelik of autofagie ‘n veroorsakende of beskermende rol in Aβ neurotoksisiteit speel en of autofagiese disfunksie nie net ‘n na gevolg van die siektetoestand self is nie. Dus was die doelwitte van hierdie studie om (i) ‘n proteïen uitdrukkings profiel van belangrike amiloïed reguleeringsproteïne te skep, (ii) die wisselwerking tussen proteolitiese prosesse en sel dood reguleerders in die konteks van AVP ooruitdrukking te verduidelik deur gebruik te maak van proteomics, (iii) die spoed van autofagie verwante proteïen degenerasie te assesseer in die konteks van APP ooruitdrukking asook die mate van autofagiese disfunksie, (iv) die bydrae van makroautofagie asook gemedieërde autofagie (GA) in Aβ verwydering en neuron toksisiteit te assesseer deur modulering van elke proses en (v) om die effek van langdurige afwisselende vas tydperke op die modulering van makroautofagie en GA te assesseer in ‘n paraquat (PQ)-geïnduseerde in vivo brein beskadiging model. Metodes: ‘n Unieke AS ooruitdrukking model was geskep deur gebruik te maak van die N2a muis neuroblastoma sellyn wat ‘n dubbele Sweedse mutasie stabiel ooruitgedruk het. AVP ooruitdrukking was sodoende gekarakteriseer en die induksie van makroautofagie, GA en apoptose was met verloop van tyd geassesseer deur gebruik te maak van lewensvatbaarheid stoetse, Westerse blottering tegnieke, fluoressensie mikroskopie, transmissie elektron mikroskopie (TEM) en korrelatiewe lig en elektron mikroskopie (KLEM). Merendeels was die effek van AVP ooruitdrukking op globale proteoom vlakke gekwantifiseer deur gebruik te maak van hoë resolusie vloeistof chromatografie gekoppel aan tandem massa spektrometrie. Laastens, ‘n PQ geïnduseerde breinbeskadigingsmodel was opgestel en gebruik om die effek van langdurige afwisselende vas tydperke op makroautofagie en GA te toets in GFP-LC3 transgeniese muise. Die muise was twee maal per week ingespuit met 10mg/kg PQ vir ‘n tydperk van 3 weke. Die langdurige afwisselende vas protokol was ook oor 3 weke toegepas en het bestaan uit 48 ure se vas, gevolg deur 24 ure van voeding. Evaluering van makroautofagiese en GA modulasie na blootstelling aan kroniese oksidatiewe stres en langdurige afwisselende vas in geselekteerde brein areas het geskied deur middel van Westerse blottering, fluoressensie mikroskopie, vergelykende haematoxylin en eosien kleuring asook TEM analise. Resultate: Die resultate dui aan dat AVP ooruitdrukking tot prominente apoptose lei na 48 ure en dat die autofagie in ‘n tyd verwante manier aktiveer. ‘n Onverwagte bevinding was dat die spoed van autofagie nie net verhoog het tydens AVP ooruitdrukking nie, maar dat hierdie hoë vlakke behou was selfs tydens apoptose. Dit was ook gevind dat AVP ooruitdrukking GA reguleerders aanskakel, veral tydens die 48ste uur. Desnieteenstaande, die induksie van apoptose het voortgegaan ongeag die verhoogde GA aktiwiteit. Ons proteoom analise dui ook ‘n tydafhanklike verhoging in AVP proteïen tot proteïen interaksies. Gesamentlik dui die in vitro resultate aan dat die modulasie van makroautofagie en GA ‘n rol speel in Aβ verwydering en neuron toksisiteit verlaag. In vivo was daar ‘n beduidende vermindering van sitokroom c en 4HNE uitdrukking was gevind tydens langdurige afwisselende vas intervensies en selektiewe dele van die brein. Dus dui dit aan dat hierdie vas tydperke meegebring was deur modulasie van beide makroautofagie en GA. Gevolgtrekkings: Ons bevindinge dui aan dat autofagie aangeskakel word in die aanwesigheid van hoë hoeveelhede AVP en Aβ, en merkwaardig hierdie toestand behou selfs tydens apoptose aktivering, moontlik as gevolg van ‘n onvoldoende autofagiese reaksie om Aβ neurotoksisiteit teen te werk. Desnieteenstaande, verhoogde Aβ verwydering was gevind met ‘n voldoende autofagiese reaksie selfs tydens 48 ure van AVP ooruitdrukking, wat aandui dat autofagie modulasie ‘n lewensvatbare behandelings alternatief kan wees lank na die siektetoestand bevestig is. Hierdie resultate was ook bevestig deur langdurige afwisselende vas intervensies, waar aanduiders van apoptose en lipied peroksidasie beduidend verlaag was in die areas van die brein wat geassosieer word met neurodegenerasie. Verdere studies, spesifiek dié wat gebruik maak van in vivo AVP ooruitdrukking modelle, word dus benodig om die kliniese belang van autofagie regulering te bevestig.

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