MRC TS Award: 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 most of the mitochondrial proteome (>1,500 predicted proteins) is 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 that 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 (PL) metabolism. Cardiolipin (CL) is a PL found only in mitochondria 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. ER provides an important precursor of CL biosynthesis known as phosphatidic acid (PA). 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.In my original proposal, I reported the first, homozygous pathogenic mutation in TRIAP1, a gene involved in CL biosynthesis. Multiple mtDNA deletions were detected in the patient's muscle, implicating TRIAP1 in mtDNA replication. One exciting development during the 2nd year of my fellowship was the identification of a 2nd patient with different, novel homozygous TRIAP1 variant. Importantly, multiple mtDNA deletions were again present in muscle, as observed in the first case, thus supporting my initial hypothesis that TRIAP1 is a novel regulator of mtDNA maintenance.Identification of a 2nd TRIAP1 case was pivotal, given it: 1) confirmed the biological and medical importance of this pathway for human pathology; and 2) provided unrelated, biological material for functional work to complement the previously available cell line. This represents a unique opportunity to advance fundamental understanding of the role of CL metabolism in mtDNA replication and mitochondrial division and introduces TRIAP1 as a novel regulator of mtDNA replication and segregation. Despite the 2nd TRIAP1 case representing significant "added value" to my intermediate fellowship, time and resource have been redirected away from my original application to adapt the study design and account for this development. In addition, experimental work at UCL Queen Square Institute of Neurology, and at collaborator laboratories, was delayed due to temporary closures of the laboratories (April to July 2020) and stricter social distancing rules preventing two researchers using lab space at the same time (July 2020 to April 2021) caused by COVID-19 restrictions. Transition Support would therefore enable me to complete experiments necessary to fully address and build on my original aim - to determine how CL metabolism influences mtDNA replication and mitochondrial division - and the new models and additional data generated will strongly support my future application for an MRC Senior Fellowship.