Browsing by Author "Ntsapi, Matlakala Claudia"

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    The effects of nutrient deprivation on macroautophagic flux and chaperone-mediated autophagy in a model of alzheimer's disease
    (Stellenbosch : Stellenbosch University, 2018-12) Ntsapi, Matlakala Claudia; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    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.