Examining changes in autophagic flux and metabolism of human CLN7-/- neural progenitor cells (NPCs), derived from induced pluripotent stem cells (iPSCs)

Smith, Rhys Alexander (2018) Examining changes in autophagic flux and metabolism of human CLN7-/- neural progenitor cells (NPCs), derived from induced pluripotent stem cells (iPSCs). Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Neuronal ceroid lipofuscinoses make up the most common group of neurodegenerative diseases amongst children. Classified into 13 distinct variants (CLN1-13), extensive research has been undertaken in the previous decade in order to understand the pathogenesis of the variations of the disease, however, very little is still known about the variant, CLN7. The aim of this project was to utilise existing techniques in neural differentiation in order to deduce any differences in metabolism and autophagic flux between neural progenitor cells and further differentiated CLN7 patient cells compared to those of control. A stepwise neural differentiation protocol was implemented in order to differentiate human induced pluripotent stem cells to NPCs and subsequently neurons. A range of qPCR targets relating to pluripotency, metabolism and inflammation were selected in order to deduce changes in gene expression in both NPCs and iPSCs. Quantitative analysis provided insight and demonstrated a clear metabolic shift toward glycolysis in NPCs of CLN7, whereas controls favoured oxidative phosphorylation, evident by an increase in oxidative stress, a consequence of OXPHOS due to increased ROS production, indicating mitochondrial dysfunction. An upregulation in the inflammatory pathway, NFkB1 was evident, indicating an increase in inflammation in CLN7 NPCs. Western blot analysis provided insight into an impairment of autophagy through accumulation of p62 as well as activation of p70, a downstream target of the mTORC1 pathway. The results obtained during the project provide a new insight into the pathogenesis of the CLN7 and potentially the pathways relating to the disease resulting in neurodegeneration.