Hill, JM; (2016) Mitochondrial calcium uptake pathways as a potential therapeutic target. Doctoral thesis , UCL (University College London).

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Abstract

It has been known since the 1960’s that mitochondria have a huge capacity to take up and accumulate calcium from the cytosol, regulating energy production, autophagy, and mitochondrial morphology. Dysfunctional mitochondrial calcium homeostasis is also involved in the pathology of many disease states. Therefore, mitochondrial calcium handling represents an interesting therapeutic target. In this thesis I aimed to investigate related therapeutic avenues and further understand the role of mitochondrial calcium handling machinery. When mitochondrial calcium uptake exceeds the calcium buffering capacity of the mitochondrial matrix, the mitochondrial permeability transition pore (mPTP) opens in the inner mitochondrial membrane. Pore opening causes mitochondrial depolarisation, energetic collapse, swelling, rupture, and cell death. Using isolated mitochondria, I screened a range of novel mitochondrial targeted derivatives of the canonical mPTP inhibitor, cyclosporin A (CsA), to assess their efficacy as mPTP inhibitors, and as potential therapeutics for neurological disease. Two compounds were found to be more effective than CsA at low concentrations, and one was selected for further study. The compound’s target was investigated and found to be Cyclophilin D, and it’s effect on various mitochondrial parameters was assessed, and it was found to be non-toxic ex vivo and in vitro. The molecular identity of the mitochondrial calcium uniporter (MCU) was only recently discovered, followed by discovery of several associated proteins – MCUb, MICU1, MICU2, MICU3, EMRE, and MCUR1. I developed a screen using the bioluminescent protein aequorin to identify mitochondrial specific inhibitors of the MCU, and I developed a stable cell line in which the MCU protein was knocked down, in order to further examine the role of MCU and investigate the effect of reduced mitochondrial calcium uptake on mitochondrial and cellular properties. While mitochondrial calcium uptake was significantly reduced upon stimulation in these cells, cytosolic calcium, oxygen consumption, cell growth and mitochondrial morphology were not affected. I also investigated the protein expression level of the components of the MCU complex in the rat brain throughout development.

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