Role of the mitochondrial Translocator Protein (mTSPO) in cell homeostasis: a molecular pathway in signalling and self-conservation mechanisms

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In search of the molecules that may set the pace of cell metabolism and define its healthy homeostasis, we are now interested in elucidating the cell biological role of the mTSPO. Although associated with many pathophysiological conditions, its contribution to processes guarding cell integrity such as autophagy and apoptosis is obscure, and its role in mitochondrial performance ill-defined. In order to shed light on these two fundamental processes, it will be instructive to ascertain how mTSPO ratio of expression with that of the VDAC, and its pharmacological modulation, defines the signalling of Ca2+, dynamics of the ROS, ATP rationing and Redox State. The physiology of cellular Ca2+, the ATP balance, the programmed apoptotic cell death as well as the Autophagy all depend on the efficient handling of Ca2+ by the mitochondrion. Despite some minor attempts, however, the contribution of mTSPO to this pathway of signalling has been largely ignored, and we now aim to explore and elucidate it, considering this as the hub in the mTSPO contribution to mitochondrial coupling by which ATP synthesis, ROS generation and network remodelling, via targeted autophagy, originate. However, modifications in ROS signalling would impinge also on downstream effectors: the kinases which promote the unphysiological phosphorylation of VDAC. This indirect pathway, as well as the direct physical interaction between mTSPO and VDAC, will therefore be addressed as potential underlying mechanisms. The following experiments will clarify the above points using Mouse Embryonic Fibroblasts (MEF) cell lines: 1. Live imaging, luminescent and fluorescence based methods will be used to assess mTSPO role in cell autopagy and signalling. a) The mTSPO protein will be transiently manipulated to increase (+) or decrease (-) its expression via conventional techniques of molecular biology; the outcome of this manipulation will then be ascertained on b) Autophagy, c) Ca2+ signalling d) ROS generation and e) Autophagy in conditions of buffered Ca2+ and ROS. 2. Confocal Microscopy, fluorescence and biochemical based methods will be used to determine the role of mTSPO in mitochondrial coupling efficiency and remodelling. After transient modulation of mTSPO or pharmacological binding the following will be addressed: a) The mitochondrial redox state, b) ATP generation, and c) Mitochondrial membrane potential, will be evaluated; d) The volume of the mitochondrial network will be calculated; e) The expression of genes associated with the organelle's genesis will be profiled, and f) The activity of mitochondria-triggered autophagy will be investigated. 3. Live cell imaging and standard assays will test the kinases' activity, VDAC phosphorylation which this generates, and the outcome in mitochondrial regulated apoptotic demise. Following molecular and pharmacological modulation of mTSPO: a) The efficiency of translocation and activity of PKA and PKCepsilon will be evaluated; b) The phosphorylation state of VDAC revealed; and c) The efficiency of mitochondrial-dependent apoptosis assessed. This object will also see the utilization of MEF cell lines from PKCepsilon KO mice. 4. Immunohistochemical, biochemical and yeast based methods will be employed to identify the molecular interplay between mTSPO and VDAC: a) Cellular co-localization between mTSPO and VDAC isoforms will be monitored; b) The interaction and its type studied via two-hybrid strategy and co-immunoprecipitation; and c) The homo-oligomers of VDAC evaluated. All this will reveal the molecular and cellular physiology of an important but so far neglected regulatory pathway. The results of our studies will form a conceptual and experimental framework for future investigations aiming to identify in mTSPO a novel bio-marker and target for pharmacological intervention of conditions associated with remodelling of metabolism and proliferation.