Investigating the role of cardiolipin metabolism in mitochondrial DNA replication and mitochondrial division

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Mitochondria provide the major source of energy in human cells and control numerous metabolic pathways. Thirteen subunits of the energy producing machinery are encoded by DNA present in the mitochondria (mitochondrial DNA, mtDNA); while the vast majority of the mitochondrial proteome (>1,500 predicted proteins) are encoded by the nuclear genome and are actively imported into the organelles from the cytosol. Mitochondrial diseases, inherited conditions caused by mutations in nuclear- and mtDNA-encoded mitochondrial genes which impair mitochondrial function, are among the most common genetic neurological disorders, affecting 1 in 4,300 individuals. They often cause devastating illness associated with severe disability and shortened lifespan in children and adults. Unfortunately, there are currently no effective treatments that halt or reverse progression of the disease.One emerging, but poorly-characterised, category of mitochondrial diseases relates to impaired phospholipid metabolism. Cardiolipin (CL) is a phospholipid (PL) found only in mitochondrial membranes with numerous essential mitochondrial functions. CL biosynthesis is a complex process, involving the endoplasmic reticulum (ER), a network of membranous tubules within the cytoplasm of the cell, continuous with the nuclear membrane, and the mitochondria. However, it is ultimately synthesised from phosphatidic acid (PA) within the inner mitochondrial membrane (IMM). Crucial for the transfer of PA from the ER to the IMM is the TRIAP1-PRELID1 complex. Further evidence for the intrinsic connection between the ER and mitochondria has recently emerged with evidence that mtDNA replication occurs at ER-mitochondria contact sites, thus coupling mtDNA synthesis and mitochondrial division. However, the mechanism that links mtDNA synthesis to mitochondrial division, and the impact of perturbed ER-mitochondria contact sites on mtDNA replication, remains poorly-understood.I have identified a patient, in the NHS England Highly Specialised Services for Mitochondrial Disorders that I help run at the National Hospital for Neurology and Neurosurgery, with the first pathogenic mutations (resulting in frameshift of the protein) in the human TRIAP1 gene, the protein product of which is crucial to CL biosynthesis. I have undertaken detailed investigations that reveal impaired CL metabolism and aberrant mtDNA replication in both patient-derived fibroblasts and muscle tissue. This new discovery supports the importance of phospholipid homeostasis in mtDNA maintenance and presents a significant opportunity to advance understanding of fundamental aspects of mitochondrial biology.The overarching research aim of my fellowship is to gain a deeper understanding of how CL metabolism influences mtDNA synthesis and mitochondrial division. The key objectives are to: 1) characterise how perturbed CL metabolism influences mtDNA replication and mitochondrial division; 2) define the TRIAP1 interactome and identifying novel proteins required for CL biosynthesis and PL trafficking; and 3) investigate muscle-specific CL biosynthesis and PL trafficking pathways. These objectives will be achieved by studying CL metabolism and PL trafficking pathways in the following cell models: mutant and knockout (KO) TRIAP1 fibroblasts; mutant TRIAP1 human induced pluripotent stem cell (hiPSC)-derived myoblasts, using a combined cell biology and proteomics approach. The research will be undertaken at the UCL Institute of Neurology, a world-leading neuroscience centre, in collaboration with: the MRC-Mitochondrial Biology Unit, University of Cambridge (Professor Massimo Zeviani), an internationally-recognised centre of excellence for the study of mitochondrial biology and medicine; and the Cluster of Excellence in Cellular Stress Responses in Aging-associated Diseases, University of Cologne (Professor Thomas Langer), a leading European institution for mitochondrial biology and aging.