Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5'-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5'-to-3' or 3'-to-5' direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5'-DNA flaps, providing insights into MGME1-mediated 5'-end processing of nascent mtDNA. Our study builds on previously solved MGME1-DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.

NAD+ dependent UPRmt activation underlies intestinal aging caused by mitochondrial DNA mutations.

Aging in mammals is accompanied by an imbalance of intestinal homeostasis and accumulation of mitochondrial DNA (mtDNA) mutations. However, little is known about how accumulated mtDNA mutations modulate intestinal homeostasis. We observe the accumulation of mtDNA mutations in the small intestine of aged male mice, suggesting an association with physiological intestinal aging. Using polymerase gamma (POLG) mutator mice and wild-type mice, we generate male mice with progressive mtDNA mutation burdens. Investigation utilizing organoid technology and in vivo intestinal stem cell labeling reveals decreased colony formation efficiency of intestinal crypts and LGR5-expressing intestinal stem cells in response to a threshold mtDNA mutation burden. Mechanistically, increased mtDNA mutation burden exacerbates the aging phenotype of the small intestine through ATF5 dependent mitochondrial unfolded protein response (UPRmt) activation. This aging phenotype is reversed by supplementation with the NAD+ precursor, NMN. Thus, we uncover a NAD+ dependent UPRmt triggered by mtDNA mutations that regulates the intestinal aging.

Living donor liver transplantation for myocerebrohepatopathy spectrum due to POLG mutations.

BACKGROUND: POLG is one of several nuclear genes associated with mitochondrial DNA maintenance defects and is a group of diseases caused by mitochondrial DNA deficiency that results in impaired adenosine triphosphate production and organ dysfunction. Myocerebrohepatopathy spectrum (MCHS) is the most severe and earliest presentation of POLG mutations, and liver transplantation (LT) for MCHS has never been reported. CASE PRESENTATION: The patient was a 3-month-old boy with acute liver failure and no neurological manifestations (e.g., seizures). We performed a living donor LT using a left lateral segment graft from his father. The postoperative course was uneventful. Subsequently, a homozygous POLG mutation (c.2890C>T, p. R964C) was identified by multigene analysis of neonatal/infantile intrahepatic cholestasis. Moreover, respiratory chain complex I, II, and III enzyme activities and the ratio of mtDNA to nuclear DNA in the liver were reduced. Therefore, we considered that these clinical manifestations and examination findings met the definition for MCHS. During meticulous follow-up, the patient had shown satisfactory physical growth and mental development until the time of writing this report. CONCLUSION: We presumed that the absence of remarkable neurologic manifestations prior to LT in patients with MCHS is a good indication for LT and contributes to a better prognosis in the present case.

The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.

[Status epilepticus in a pregnant patient with a previously unrecognized POLG-associated disease]. / Epilepticheskii status u beremennoi patsientki s vpervye vyyavlennym POLG-assotsiirovannym zabolevaniem.

POLG-associated diseases are rare causes of pharmacoresistant epilepsy and status epilepticus, especially in adult patients. Phenotypic and genotypic variability in these conditions causes the complexity of their diagnosis. In the study, we report a case of a 33-year-old female patient who developed recurrent convulsive status epilepticus with focal clonic onset at the week 22/23 of pregnancy. Intensive anti-seizure therapy was administered, including the use of valproic acid, as well as the treatment of somatic complications. Given the acute onset, the semiology of seizures, the presence of psychopathological symptoms, autoimmune etiology of the disease was initially suspected. A month after the withdrawal of valproic acid, the patient began to show signs of toxic hepatitis, which eventually led to death. According to the results of whole-exome sequencing obtained later, the patient was a carrier of a pathogenic homozygous variant c.2243G>C (p.W748S) in the POLG gene. The presented case highlights the importance of molecular genetic testing and the risk associated with valproic acid hepatotoxicity in patients with cryptogenic epileptic status.

Structure-specific roles for PolG2-DNA complexes in maintenance and replication of mitochondrial DNA.

The homodimeric PolG2 accessory subunit of the mitochondrial DNA polymerase gamma (Pol γ) enhances DNA binding and processive DNA synthesis by the PolG catalytic subunit. PolG2 also directly binds DNA, although the underlying molecular basis and functional significance are unknown. Here, data from Atomic Force Microscopy (AFM) and X-ray structures of PolG2-DNA complexes define dimeric and hexameric PolG2 DNA binding modes. Targeted disruption of PolG2 DNA-binding interfaces impairs processive DNA synthesis without diminishing Pol γ subunit affinities. In addition, a structure-specific DNA-binding role for PolG2 oligomers is supported by X-ray structures and AFM showing that oligomeric PolG2 localizes to DNA crossings and targets forked DNA structures resembling the mitochondrial D-loop. Overall, data indicate that PolG2 DNA binding has both PolG-dependent and -independent functions in mitochondrial DNA replication and maintenance, which provide new insight into molecular defects associated with PolG2 disruption in mitochondrial disease.

Deoxyribonucleoside treatment rescues EtBr-induced mtDNA depletion in iPSC-derived neural stem cells with POLG mutations.

Mutations in POLG, the gene encoding the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (Pol-γ), lead to diseases driven by defective mtDNA maintenance. Despite being the most prevalent cause of mitochondrial disease, treatments for POLG-related disorders remain elusive. In this study, we used POLG patient-induced pluripotent stem cell (iPSC)-derived neural stem cells (iNSCs), one homozygous for the POLG mutation c.2243G>C and one compound heterozygous with c.2243G>C and c.1399G>A, and treated these iNSCs with ethidium bromide (EtBr) to study the rate of depletion and repopulation of mtDNA. In addition, we investigated the effect of deoxyribonucleoside (dNs) supplementation on mtDNA maintenance during EtBr treatment and post-treatment repopulation in the same cells. EtBr-induced mtDNA depletion occurred at a similar rate in both patient and control iNSCs, however, restoration of mtDNA levels was significantly delayed in iNSCs carrying the compound heterozygous POLG mutations. In contrast, iNSC with the homozygous POLG mutation recovered their mtDNA at a rate similar to controls. When we treated cells with dNs, we found that this reduced EtBr-induced mtDNA depletion and significantly increased repopulation rates in both patient iNSCs. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation also within intact neural lineage cells and suggest that those with compound heterozygous mutation have a more severe defect of mtDNA synthesis. Our findings further highlight the potential for dNs to improve mtDNA replication in the presence of POLG mutations, suggesting that this may offer a new therapeutic modality for mitochondrial diseases caused by disturbed mtDNA homeostasis.

The Saccharomyces cerevisiae mitochondrial DNA polymerase and its contribution to the knowledge about human POLG-related disorders.

Most eukaryotes possess a mitochondrial genome, called mtDNA. In animals and fungi, the replication of mtDNA is entrusted by the DNA polymerase γ, or Pol γ. The yeast Pol γ is composed only of a catalytic subunit encoded by MIP1. In humans, Pol γ is a heterotrimer composed of a catalytic subunit homolog to Mip1, encoded by POLG, and two accessory subunits. In the last 25 years, more than 300 pathological mutations in POLG have been identified as the cause of several mitochondrial diseases, called POLG-related disorders, which are characterized by multiple mtDNA deletions and/or depletion in affected tissues. In this review, at first, we summarize the biochemical properties of yeast Mip1, and how mutations, especially those introduced recently in the N-terminal and C-terminal regions of the enzyme, affect the in vitro activity of the enzyme and the in vivo phenotype connected to the mtDNA stability and to the mtDNA extended and point mutability. Then, we focus on the use of yeast harboring Mip1 mutations equivalent to the human ones to confirm their pathogenicity, identify the phenotypic defects caused by these mutations, and find both mechanisms and molecular compounds able to rescue the detrimental phenotype. A closing chapter will be dedicated to other polymerases found in yeast mitochondria, namely Pol ζ, Rev1 and Pol η, and to their genetic interactions with Mip1 necessary to maintain mtDNA stability and to avoid the accumulation of spontaneous or induced point mutations.

Effects of Parkin on the Mitochondrial Genome in the Heart and Brain of Mitochondrial Mutator Mice.

Mitochondrial dysfunction has been implicated in neurodegenerative diseases like Parkinson's disease (PD). This study investigates the role of Parkin, a protein involved in mitochondrial quality control, and strongly linked to PD, in the context of mitochondrial DNA (mtDNA) mutations. Mitochondrial mutator mice (PolgD257A/D257A ) (Polg) are used and bred with Parkin knockout (PKO) mice or mice with disinhibited Parkin (W402A). In the brain, mtDNA mutations are analyzed in synaptosomes, presynaptic neuronal terminals, which are far from neuronal soma, which likely renders mitochondria there more vulnerable compared with brain homogenate. Surprisingly, PKO results in reduced mtDNA mutations in the brain but increased control region multimer (CRM) in synaptosomes. In the heart, both PKO and W402A lead to increased mutations, with W402A showing more mutations in the heart than PKO. Computational analysis reveals many of these mutations are deleterious. These findings suggest that Parkin plays a tissue-dependent role in regulating mtDNA damage response, with differential effects in the brain and heart. Understanding the specific role of Parkin in different tissues may provide insights into the underlying mechanisms of PD and potential therapeutic strategies. Further investigation into these pathways can enhance the understanding of neurodegenerative diseases associated with mitochondrial dysfunction.

The disease-causing mutation p.F907I reveals a novel pathogenic mechanism for POLγ-related diseases.

Mutations in the catalytic domain of mitochondrial DNA polymerase γ (POLγ) cause a broad spectrum of clinical conditions. POLγ mutations impair mitochondrial DNA replication, thereby causing deletions and/or depletion of mitochondrial DNA, which in turn impair biogenesis of the oxidative phosphorylation system. We here identify a patient with a homozygous p.F907I mutation in POLγ, manifesting a severe clinical phenotype with developmental arrest and rapid loss of skills from 18 months of age. Magnetic resonance imaging of the brain revealed extensive white matter abnormalities, Southern blot of muscle mtDNA demonstrated depletion of mtDNA and the patient deceased at 23 months of age. Interestingly, the p.F907I mutation does not affect POLγ activity on single-stranded DNA or its proofreading activity. Instead, the mutation affects unwinding of parental double-stranded DNA at the replication fork, impairing the ability of the POLγ to support leading-strand DNA synthesis with the TWINKLE helicase. Our results thus reveal a novel pathogenic mechanism for POLγ-related diseases.

Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.

POLG genotype influences degree of mitochondrial dysfunction in iPSC derived neural progenitors, but not the parent iPSC or derived glia.

Diseases caused by POLG mutations are the most common form of mitochondrial diseases and associated with phenotypes of varying severity. Clinical studies have shown that patients with compound heterozygous POLG mutations have a lower survival rate than patients with homozygous mutations, but the molecular mechanisms behind this remain unexplored. Using an induced pluripotent stem cell (iPSC) model, we investigate differences between homozygous and compound heterozygous genotypes in different cell types, including patient-specific fibroblasts, iPSCs, and iPSC-derived neural stem cells (NSCs) and astrocytes. We found that compound heterozygous lines exhibited greater impairment of mitochondrial function in NSCs than homozygous NSCs, but not in fibroblasts, iPSCs, or astrocytes. Compared with homozygous NSCs, compound heterozygous NSCs exhibited more severe functional defects, including reduced ATP production, loss of mitochondrial DNA (mtDNA) copy number and complex I expression, disturbance of NAD+ metabolism, and higher ROS levels, which further led to cellular senescence and activation of mitophagy. RNA sequencing analysis revealed greater downregulation of mitochondrial and metabolic pathways, including the citric acid cycle and oxidative phosphorylation, in compound heterozygous NSCs. Our iPSC-based disease model can be widely used to understand the genotype-phenotype relationship of affected brain cells in mitochondrial diseases, and further drug discovery applications.

Mitochondria-Related TFAM and POLG Gene Variants and Associations with Tumor Characteristics and Patient Survival in Head and Neck Cancer.

In 2020, 878,348 newly reported cases and 444,347 deaths related to head and neck cancer were reported. These numbers suggest that there is still a need for molecular biomarkers for the diagnosis and prognosis of the disease. In this study, we aimed to analyze mitochondria-related mitochondrial transcription factor A (TFAM) and DNA polymerase γ (POLG) single-nucleotide polymorphisms (SNPs) in the head and neck cancer patient group and evaluate associations between SNPs, disease characteristics, and patient outcomes. Genotyping was performed using TaqMan probes with Real-Time polymerase chain reaction. We found associations between TFAM gene SNPs rs11006129 and rs3900887 and patient survival status. We found that patients with the TFAM rs11006129 CC genotype and non-carriers of the T allele had longer survival times than those with the CT genotype or T-allele carriers. Additionally, patients with the TFAM rs3900887 A allele tended to have shorter survival times than non-carriers of the A allele. Our findings suggest that variants in the TFAM gene may play an important role in head and neck cancer patient survival and could be considered and further evaluated as prognostic biomarkers. However, due to the limited sample size (n = 115), further studies in larger and more diverse cohorts are needed to confirm these findings.

[Adult-onset sensory neuropathy and ataxia as a clinical manifestation of POLG gene mutations]. / Ataxia y neuropatía sensitiva de inicio en la edad adulta como manifestación clínica de mutaciones en el gen POLG.

INTRODUCTION: Sensory ataxia is a frequent symptom in numerous neurological pathologies, being a frequent clinical manifestation in diseases related to genes influencing mitochondrial metabolism, such as POLG. The aim is to describe the differential characteristics of four patients with pathogenic variants in the POLG gene with clinical expression in the form of adult-onset ataxia and sensory neuropathy. PATIENTS AND METHODS: We reviewed the clinical features of patients diagnosed with POLG pathogenic variants from a tertiary hospital. RESULTS: Three men and one woman (mean age: 40 years; 27-46) with no family history were studied with symptoms for 10 years. All patients developed a gait disturbance related to sensory ataxia. All patients had oculomotor abnormalities. The neurophysiological study showed a sensory axonal neuropathy. Brain magnetic resonance imaging studies showed atrophy and cerebellar white matter lesion and muscle magnetic resonance imaging showed fatty substitution in thigh and calf muscles without a specific pattern. A molecular study revealed pathogenic variants in the POLG gene. CONCLUSIONS: In cases of adult-onset sensory ataxia, the molecular analysis of the POLG gene should be considered, especially if associated with sensory neuropathy or ophthalmoparesis.

TITLE: Ataxia y neuropatía sensitiva de inicio en la edad adulta como manifestación clínica de mutaciones en el gen POLG.Introducción. La ataxia sensitiva es un síntoma frecuente en numerosas patologías neurológicas con causas múltiples y es una manifestación clínica frecuente en enfermedades relacionadas con genes que influyen en el metabolismo mitocondrial, como POLG. El objetivo del presente trabajo es describir las características diferenciales de cuatro pacientes con variantes patógenas en el gen POLG y expresión clínica común en forma de ataxia y neuropatía sensitiva de inicio en la edad adulta. Pacientes y métodos. Se realizó una revisión de las características clínicas de los pacientes portadores de variantes patógenas en el gen POLG de una consulta de enfermedades neuromusculares en un hospital de tercer nivel. Resultados. Se estudió a tres varones y una mujer de edad adulta (edad media: 40 años; 27-46) sin antecedentes familiares reseñables, con una duración de los síntomas de en torno a 10 años. El síntoma que motivó la consulta fue una alteración de la marcha en relación con ataxia sensitiva. Todos los pacientes presentaban anomalías oculomotoras. El estudio neurofisiológico evidenció una neuropatía sensitiva de predominio axonal. La resonancia magnética cerebral mostró atrofia y lesión de la sustancia blanca cerebelosa. La resonancia magnética muscular mostró sustitución grasa en músculos de muslos y gemelos sin un patrón específico. Todos ellos fueron portadores (homocigotos o heterocigotos compuestos) de variantes patógenas en el gen POLG. Conclusiones. El análisis molecular del gen POLG es una posibilidad diagnóstica prioritaria que se debe considerar en casos de ataxia sensitiva de inicio en la edad adulta, especialmente si se asocia a neuropatía sensitiva u oftalmoparesia.