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

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.

By manipulating mTSPO expression or pharmacologically inhibiting its function in Murine Fibroblasts we aim to assess: 1) Cell autophagy and signalling; 2) Mitochondrial homeostasis and remodelling; 3) Kinase-mediated phosphorylation of VDAC and the result on apoptosis, and 4) Nature of mTSPO and VDAC interaction. Seventeen experiments were designed which break down as follows: I. 1. By techniques of molecular biology modify mTSPO up and down regulating its expression with recombinant protein and short hairpin RNA; 2. By combining means of imaging and biochemistry define the outcome of this on cell autophagy; 3. Evaluate the homeostasis of Ca2+ with fluorescence and luminescence tools. Monitor 4. The intracellular level of ROS; 5. Autophagic activity after Ca2+ and ROS neutralization. II. 6. Mitochondrial pools of NAD will be evaluated via confocal analysis. 7. Both cell imaging and luminescent techniques will be used to assess ATP generation. 8. Inner mitochondrial membrane potential will be imaged and 9. Via combined fluorescent probes and dyes the volume fraction of the organelles pondered. 10. The genes responsible for the biogenesis of the mitochondria expansion will be mapped with RT-QPCR and Immunoblotting as well as 11. Targeted autophagosomes investigated. III. 12. Test PKA and PKCepsilon activity and mitochondrial localization following activation in cells manipulated for mTSPO expression in response to PK11195 treatment; in the same cells 13. Analyze by immunoblotting VDAC phosphorylation and 14. Exploit the results of this conformational change on the intrinsic pathway of apoptosis. IV. 15. The co-localization of mTSPO and VDAC will be scrutinized by immunofluorescence and immunoblotting. 16. The interaction between the two proteins profiled by conventional immunoprecipitation and yeast two-hybrid system. Lastly, 17. Clarify via biochemical analysis if VDAC homo-oligomers are affected by mTSPO modified expression or pharmacological modulation.

The cell biology of the mTSPO remains enigmatic and unexplored although the literature provides evidence for its involvement in cholesterol transport, synthesis of steroid hormones, apoptosis, cell proliferation and immunomodulation. Binding of the mTSPO with [11C](R)-PK11195 via positron emission tomography (PET) is routinely employed to diagnose inflammatory states of the Central Nervous System (CNS) as mTSPO level of expression is increased in activated microglia. mTSPO ligands are also used in experimental protocols to mark cancerous cells where mTSPO is remarkably over-expressed. In spite of that, the actual contribution of mTSPO in regulation of metabolism and mechanisms to avoid cellular pathologies remains unexplained. This proposal aims to address these fundamental aspects of cell physiology associated with mTSPO and to inform novel approaches in which mTSPO can be considered in the context of the diagnosis and treatment of pathologies. We have therefore identified the following principal points of impact for this study: I. The growing interest by pharmaceutical and biotechnological companies in the outcomes of our study will be further exploited, and strategies to foster collaborative work established. The first aim is to create a bond with partners for the creation of protocols for clinical purposes in order to consolidate mTSPO as read-out of pathological conditions, as well as functional assays to assess the efficacy of therapeutic agents acting on it. Nevertheless, we are currently working to identify the ideal partner to present a case for a PhD studentship to increase the number of people involved in the subject. II. The data generated will be pubblished by various activities set ad hoc for the project. The relevance of this fundamental research to major diseases means it has general and popular interest, contributing to greater public awareness and understanding of science. III. A thorough understanding of the protein's cell biology will assist specialists developing therapies directed to this protein. The eventual patenting of the intellectual property that may arise from this project will be provided by the appointed organisation of the college. This will be made possible by the close involvement of the Enterprise Team of the Royal Veterinary College, from the earliest stages in research project development, which will provide assistance in impact planning, identification of and liaison with company partners as well as in formalizing commercial contracts. Pro-active market research and marketing are used to identify and develop relationships with potential licensee businesses. Proof-of-concept funding will be accessed, whenever appropriate and available, for market research and technical development to enhance the appeal of new developments for commercial partners. IV. The major findings from this project will be disseminated accordingly and people involved in the research will contribute to that by providing interviews or press quotes as appropriate. To this end, we will also benefit from the Communications Management Team who will advise on the creation of web-based material for publicising the work to a general audience. V. Staff appointed to run this project will benefit from highly qualified training, becoming skilled in cutting-edge techniques for cell-based investigation. In conclusion, the results of this work will lead to a greater understanding of molecular regulators of cell energy and quality. A deeper comprhension of the mTSPO role will enable novel approaches to diagnose and manage diseases related to alterations of pathways associated to malfunctions of autophagy, metabolism and apoptosis. Hence the economy (e.g. Pharmaceutical and Biotechnological companies) may benefit from this study as well as the quality of life of cohorts of patients by the development of novel therapeutics.