Mitochondrial DNA genetics and inheritance

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Project 1. An ER-anchored transcription factor mediates a novel mitochondrial stress response Given the essential role of mtDNA in energy metabolism, eukaryotic cells must closely monitor mtDNA abundance and integrity and respond accordingly to preserve mitochondrial function and meet cellular metabolic demands. To comprehensively understand the cellular responses to mtDNA deficiency, we expressed a mitochondrially targeted restriction enzyme, MitoXhoI to deplete mtDNA in Drosophila S2 cells and analyzed mRNA profiles at series of time points after the induction of MitoXhoI. At 16 hours, the expression of most nuclear mitochondrial genes was increased. These increases became more evident at 24 hours and reached the highest level at 48 hours . Notably, all nuclear factors involved in mtDNA maintenance and gene expression including mtDNA replication factors, transcription regulators, and mitochondrial ribosomal proteins were upregulated, indicating a compensatory response for mtDNA deficiency. Accordingly, all nuclear encoded ETC subunits were also upregulated. Together, these transcriptional regulations would increase ETC biogenesis to boost energy metabolism. We previously reported a complex transcriptional network, regulating the dual genomic coordination of mitochondrial biogenesis. In the network, CG15011, the fly homolog of human NFXL1 that binds to X-box like sequence and is involved in ER stress responses, emerged as a top-layer TFs. CG15011 RNAi alone had no phenotype, but the combination of CG15011 inhibition with mtDNA deficiency caused severe growth and developmental defect. These observations suggest that CG15011 may regulate mitochondrial biogenesis in responses to mtDNA deficiency specifically. Consistent with this notion, CG15011 was required for compensatory overexpression of nuclear encoded ETC genes induced by mtDNA depletion. CG15011 has a single transmembrane helix. The full length CG15011 protein localized on ER, while the truncated protein lacking the transmembrane helix mainly localized to nucleus. Importantly, overexpression of this truncated form of CG15011 greatly boosted mitochondrial biogenesis. CG15011 protein translocated to the nucleus in response mtDNA depletion induced by MitoXhoI. CG15011 underwent a photocleavage that releases it from ER in response to MitoXhoI. Additional genetic analyses suggest that the calcium pathway and calpains might be involved in this cleavage. Importantly, deletion of CG15011 exacerbated defects caused by mt:CoIT300I, a temperature-sensitive lethal mtDNA mutation, whereas the overexpression of CG15011 or its human ortholog markedly suppressed these defects. These results suggest that CG15011 might be released from ER through a proteo-cleavage process under a mitochondrial stress triggered by mtDNA depletion and subsequently imported into the nucleus to boost mitochondrial biogenesis. Our findings uncover a novel mitochondrial stress responses mediated by an ER-anchored transcription factor, CG15011, while inactive at normal condition, monitors mitochondrial fitness and improves mitochondrial function in response to mtDNA deficiency. Project 2. Mitochondrial DNA removal is essential for sperm development and activity The mitochondrial genome is transmitted exclusively through the maternal lineage in most sexually reproduced organisms. Active mtDNA elimination during spermatogenesis has emerged as a conserved mechanism ensuring the uniparental mitochondrial inheritance in animals. However, given the existence of post-fertilization processes degrading sperm mitochondria, the physiological significance of sperm mtDNA removal is not clear. We uncover a novel mitochondrial exonuclease, Poldip2 that is specially expressed in late spermatogenesis and exclusively required for mtDNA elimination. Loss of Poldip2 impairs mtDNA clearance in elongated spermatids and impedes the progression of individualization complexes that strip away cytoplasmic materials and organelles. Additionally, persistent mtDNA in mature sperm causes marked fragmentation of nuclear genome and complete sterility of polidp2 mutant male flies. All these defects can be suppressed by expressing a mitochondrially targeted bacterial exonuclease to ectopically remove mtDNA. During the reproduction of multicellular organisms, the egg is furnished with maternal-derived organelles and macromolecules, which are deposited with defined polarity and spatial pattern to support the rapid early embryonic cycles and subsequent pattern formation. In contrast, the mature sperm is stripped of all cytoplasmic macromolecules and organelles except for mitochondria and axoneme. In Drosophila, the cytoplasm removal is coupled to the sperm individualization carried out by individualization complexes (ICs). However, mitochondria are known to physically interact with other organelles. Particularly, mtDNA replisomes are concentrated within two membrane-spanning structures and tethered to ER-mitochondrial contacts. We postulated that mtDNA, if not degraded, could be associated with ICs indirectly through these structures in individualizing spermatids, impeding the progression of traveling ICs. This proposition explains the individualization defects in poldip2 mutant flies. Additionally, persistent mtDNA could potentially cause mito-nuclear imbalance. In mature sperm, nuclear genes’ expression is completely shut down. Persistent mtDNA could produce excessive, unassembled mtDNA-encoded electron transport chain subunits, which may impair mitochondrial respiration and lead to the generation of damaging free radicals . Supporting this idea, mature sperm of old poldip2 mutant flies exhibited increased ROS levels, along with markedly fragmented nuclear genomes. Altogether, mtDNA removal appears essential for two key aspects of male reproductive biology: the effective removal of cytoplasm during sperm development and the prevention of potential mito-nuclear imbalance in mature sperm. The stringent uniparental inheritance of the mitochondrial genome, one of the most mysterious genetic phenomena in multicellular organisms, might be a prerequisite for the asymmetry between two gametes in sexual reproduction. Project 3. Replication competition drives the selective mtDNA inheritance in Drosophila ovary Mitochondria are transmitted through maternal lineage exclusively in most metazoan, and hence female germline is tasked to limit the transmission of deleterious mtDNA mutations. Based on studies from our lab and other labs over years, we proposed a model of replication competition that wild type mtDNA or healthy genomes would be replicated more frequently and thereby outcompete deleterious variants. While this model is logically compelling, it has not been empirically demonstrated that deleterious mtDNA variants indeed replicate less than wild-type genome in the same germ cell. Most mtDNA variants in D. melanogaster were generated using a selection scheme based on mitochondrially targeted restriction enzymes. These variants differ from the wild-type genome on merely a single nucleotide or small indels on the corresponding enzyme sites. Distinguishing single nucleotide polymorphisms on mtDNA in situ remains a technical challenge. We developed a highly specific and efficient in situ imaging method capable of visualizing mtDNA variants that differ by only a few nucleotides at single-molecule resolution in Drosophila ovaries. Using this method, we revealed that selection primarily occurs within a narrow developmental window during germline cysts differentiation. At this stage, the proportion of the deleterious mtDNA variant decreases without a reduction in its absolute copy number. Instead, the healthier mtDNA variant replicates more frequently, thereby outcompeting the co-existing deleterious variant. Our findings provide key insights into the mechanisms driving mtDNA selection and inheritance, establishing a powerful framework for future studies on mtDNA dynamics with spatial resolutions across different tissues and model systems. Currently, our assay was specifically designed to detect mtDNA mutations within restriction enzymes’ cleavage sites. Nonetheless, it could be improved by adopting CRISPR, ZFN, or TALEN based DNA editing technologies, potentially detecting any mutations on mtDNA in tissues, and hence would greatly advance studies on mtDNA diseases that are often caused by heteroplasmic point mutations. Project 4. Exploring potential roles of intermediates metabolism in mitonuclear Communication. Mitochondrial metabolic pathways are not only vital for providing energy, structural components, and macromolecules biosynthesis, but also regulate intracellular signaling. We aim to explore potential contribution of mitochondrial metabolic pathways in mitochondria-nucleus communications. Utilizing the previously developed genetics system, we induced mtDNA deficiency and carried out modifier screening targeting genes involved in nucleotide and lipid metabolism. Nurf38, the Drosophila homolog of human inorganic pyrophosphatases, PPA1 and PPA2, which localize to nucleus and mitochondria respectively, emerged as potential regulator of mitochondrial biogenesis. Nurf38 has three isoforms. We found that the isoform A was a mitochondrial protein, isoform B localized to cytoplasm, and isoform C localized to nucleus. Mutations in human PPA2 are associated with sudden cardiac death and cardiomyopathy, while the diseases’ mechanisms remain unknown. We have generated series transgenes that disrupt different Nurf38 isoforms. We found whole body knockout is lethal, while inhibition of Nurf38 in heart specifically caused arrhythmias, demonstrating that Nurf38's indispensable role in heart function and individual survival. Moreover, isoform-specific report knock-in revealed that the mitochondrial form of Nurf38 is predominantly expressed in pericardial nephrocytes, suggesting that nephrocytes contribute to normal heart function. We have introduced the patient-derived PPA2 mutations in mutant background and will use this humanized fly model to dissect potential contribution of nephrocyte-heart cross-talk in heart arrhythmias. Project 5. AI-based development of small molecules for mtDNA diseases Challenges associated with mitochondrial DNA (mtDNA)-based gene therapy are renewing the need for small molecule therapeutics. We are leveraging generative artificial intelligence (GenAI) to generate candidate small molecules for treating mtDNA diseases. As a proof of principle, we are currently focusing on Leber’s Hereditary Optic Neuropathy (LHON), one of the most common mitochondrial diseases, primarily caused by the m.11778G>A mutation on mtDNA. This mutation results in the ND4 R340H variant, which impairs mitochondrial complex I activity and confers resistance to the complex I inhibitor rotenone. We are using NIH BIOWULF high-performance computing system, we de novo generated approximately 370,000 small molecules targeting the ND4-H340 site via GenAI and evaluated them using the DENOVO-DOCK scoring method, a docking scoring system we modified specifically for GenAI. Consistent with the biochemical studies, our virtual docking and molecular dynamics (MD) simulations revealed that rotenone binding near ND4-R340 is destabilized in the mutant, and the ND4R340H variant disrupts key hydrogen bond networks essential for proton transfer. This analysis validates the efficacy and accuracy of this AI based approach in generate biochemically active small molecules. Importantly, among top candidates that underwent MD simulations, two molecules successfully restored hydrogen bond connectivity in the ND4R340H proton channel, demonstrating in silico therapeutic potential for LHON. We plan to validate the therapeutic effects of these de novo generated molecules in vitro using patient-derived cells carrying the ND4R340H mutation and to improve mitochondrial penetration and therapeutic efficacy through medicinal chemistry. If successful, this pipeline could be extended to other mtDNA diseases. Project 6. Exploring the molecular basis of mitochondrial differentiation As dynamic organelles, mitochondria undergo continuous cycles of fusion and fission in most eukaryotic cells, enabling equilibration of mitochondrial contents and maintaining function. While this dynamic behavior promotes homogeneity, emerging evidence suggests that mitochondria can form metabolically distinct subpopulations within single cells. Such heterogeneity has been observed under specific metabolic conditions—for instance, when cells rely exclusively on oxidative phosphorylation for ATP production or in response to differential energy demands in tumor microenvironments, such as in non-small cell lung cancer models. However, the molecular mechanisms driving the formation of these functionally distinct mitochondrial subpopulations remain poorly understood. We are leveraging the highly asymmetric mitochondria of Drosophila spermatids as a model to investigate how distinct mitochondrial subpopulations with specialized metabolic activities are formed within a cell. During spermatogenesis, mitochondria aggregate and fuse to form the nebenkern, which then gives rise to two mitochondrial derivatives that elongate alongside the developing spermatid tail. At early stages, the two derivatives are morphologically indistinguishable. However, as elongation proceeds, they diverge: the major mitochondrial derivative accumulates electron-dense paracrystalline material, while the minor derivative loses much of its volume. Although the key molecular components of the paracrystalline material have been identified, the functional and molecular distinctions between the two mitochondrial derivatives remain largely unexplored. A major challenge in studying these structures is the limited resolution of conventional and even super-resolution confocal microscopy, which cannot reliably distinguish the two mitochondrial derivatives. We have tested various imaging methods and found that expansion microscopy—a technique that physically enlarges tissue—allowed clear visualization of the two mitochondrial derivatives in Drosophila spermatids. Using this approach in combination with immunohistochemistry, we examined the localization of a series of mitochondrial proteins. While most tested proteins—including TFAM, Pink1, and Poldip2—showed similar expression levels in both mitochondria throughout spermatogenesis, ATP synthase subunit 5A showed preferential enrichment in one mitochondrial derivative during elongation, despite being equally expressed initially. This asymmetric localization suggests functional specialization, raising the possibility that only one mitochondrial derivative performs ATP production via OXPHOS in mature sperm. We are now employing a range of approaches including Drosophila genetics, high-resolution confocal imaging, and focused ion beam scanning electron microscopy to explore the molecular basis of this divergence. We aim to gain broader insights into mitochondrial heterogeneity and functional specialization in diverse biological contexts.