MRC TS Award: Investigating the role of cardiolipin metabolism in mitochondrial DNA replication and mitochondrial division

线粒体提供人体细胞的主要能量来源，并控制许多代谢途径。能量产生机制的13个亚基是由存在于线粒体中的DNA编码的（线粒体DNA， mtDNA）；而大部分线粒体蛋白质组（约1500种预测蛋白）由核基因组编码，并从细胞质中主动导入细胞器。线粒体疾病是由核和mtdna编码的线粒体基因突变引起的遗传性疾病，损害线粒体功能，是最常见的遗传性神经系统疾病之一，每4300人中就有1人患病。它们常常导致与儿童和成人严重残疾和缩短寿命有关的毁灭性疾病。不幸的是，目前还没有有效的治疗方法可以阻止或逆转这种疾病的进展。一种新兴的，但缺乏特征的线粒体疾病与磷脂（PL）代谢受损有关。心磷脂（CL）是一种仅存在于线粒体中的PL，具有许多线粒体的基本功能。CL生物合成是一个复杂的过程，涉及内质网（ER），细胞质内的膜小管网络，与核膜和线粒体相连。内质网提供CL生物合成的重要前体磷脂酸（PA）。对于PA从内质网转移到内质网至关重要的是TRIAP1-PRELID1复合物。内质网和线粒体之间内在联系的进一步证据最近出现，有证据表明mtDNA复制发生在内质网线粒体接触位点，从而耦合mtDNA合成和线粒体分裂。然而，将mtDNA合成与线粒体分裂联系起来的机制，以及er -线粒体接触位点受到干扰对mtDNA复制的影响，仍然知之甚少。在我最初的提案中，我报告了TRIAP1基因的第一个纯合致病性突变，TRIAP1是一个参与CL生物合成的基因。在患者肌肉中检测到多个mtDNA缺失，暗示TRIAP1参与mtDNA复制。在我担任研究员的第二年，一个令人兴奋的进展是发现了第二个携带不同的新型纯合TRIAP1变异的患者。重要的是，正如在第一个病例中观察到的那样，肌肉中再次出现了多个mtDNA缺失，从而支持了我最初的假设，即TRIAP1是一种新的mtDNA维持调节因子。第二例TRIAP1病例的鉴定至关重要，因为它：1)证实了该途径对人类病理的生物学和医学重要性；2)为功能性工作提供了不相关的生物材料，以补充先前可用的细胞系。这是一个独特的机会，可以促进对CL代谢在mtDNA复制和线粒体分裂中的作用的基本理解，并介绍TRIAP1作为mtDNA复制和分离的新调节剂。尽管第二个TRIAP1案例为我的中级奖学金带来了显著的“附加价值”，但为了适应研究设计和考虑这一发展，我从最初的申请中重新分配了时间和资源。此外，伦敦大学学院女王广场神经病学研究所和合作实验室的实验工作也被推迟，原因是实验室暂时关闭（2020年4月至7月），以及更严格的社交距离规定，禁止两名研究人员同时使用实验室空间（2020年7月至2021年4月）。因此，过渡支持将使我能够完成必要的实验，以充分解决和建立我最初的目标——确定CL代谢如何影响mtDNA复制和线粒体分裂——新模型和额外的数据将有力地支持我未来申请MRC高级奖学金。

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.