Creation of cybrid cultures containing the mitochondrial genome mutation m.1555A>G (MT-RNR1 gene), which has a protective effect in atherosclerosis

Введение. В настоящее время все больший интерес ученых мира вызывают цибридные клеточные модели, которые являются одним из лучших объектов для изучения патологических процессов в организме человека. Например, сотрудниками нашей лаборатории были впервые созданы подобные модели для изучения протективного эффекта некоторых мутаций митохондриального генома, защищающих организм человека от дисфункции митохондрий и атеросклеротических поражений. Цель: исследования - создание цибридных культур с высоким уровнем гетероплазмии по мутации митохондриального генома m.1555A>G, локализованной в кодирующем регионе митохондриального генома человека в гене MT-RNR1. В наших предварительных исследованиях было установлено, что пороговый уровень гетероплазмии мутации m.1555A>G имеет при атеросклерозе протективный эффект. Методика. Цибридные культуры были созданы путем слияния rho0(безмитохондриальных)-клеток и митохондрий из тромбоцитов с высоким уровнем гетероплазмии исследуемых мутаций. Для получения безмитохондриальных клеток была использована культура моноцитарного происхождения THP-1. Результаты. Получены 4 цибридные клеточные линии, содержащие мутацию m.1555A>G с уровнем гетероплазмии выше порогового значения. Заключение. В данной работе были созданы 4 цибридные культуры с высоким уровнем гетероплазмии по мутации мтДНК m.1555A>G, имеющей при атеросклерозе протективный эффект. Полученные цибридные клеточные линии могут служить моделями для отработки методов генотерапии у пациентов с атеросклерозом. Кроме того, с помощью данных цибридных клеточных моделей можно будет изучать молекулярно-клеточные механизмы, защищающие клетки от митохондриальной дисфункции. Introduction. Cybrid cell models are one of the best objects for studying pathological processes in the human body, and they are of increasing interest to scientists worldwide. Our laboratory was the first to create such models for studying the protective effect of mutations in the mitochondrial genome that protect the human body from mitochondrial dysfunction and atherosclerotic lesions. Aim: To create cybrid cultures with a high heteroplasmy level for the mitochondrial genome mutation m.1555A>G localized within the coding region of the human mitochondrial genome in the MT-RNR1 gene. Preliminary studies showed that the threshold heteroplasmy level for the m.1555A>G mutation has a protective effect in atherosclerosis. Methods. Cybrid cultures were created by fusion of rho0 (mtDNA-depleted) cells and mitochondria from platelets with a high heteroplasmy level for the studied mutations. To obtain mtDNA-free cells, a culture of monocytic origin, THP-1, was used. Results. We obtained four cybrid cell lines containing the m.1555A>G mutation with a heteroplasmy level above the threshold value. Conclusion. Four cybrid cultures with a high heteroplasmy level for the mtDNA mutation m.1555A>G were created. These cybrid cell lines can serve as models for developing methods of gene therapy for patients with atherosclerosis. In addition, using these cybrid cell models, it will be possible to study molecular and cellular mechanisms that protect cells from mitochondrial dysfunction.

The mtDNA mutation spectrum in the PolG mutator mouse reveals germline and somatic selection

Background Mitochondrial DNA (mtDNA) codes for products necessary for electron transport and mitochondrial gene translation. mtDNA mutations can lead to human disease and influence organismal fitness. The PolG mutator mouse lacks mtDNA proofreading function and rapidly accumulates mtDNA mutations, making it a model for examining the causes and consequences of mitochondrial mutations. Premature aging in PolG mice and their physiology have been examined in depth, but the location, frequency, and diversity of their mtDNA mutations remain understudied. Identifying the locations and spectra of mtDNA mutations in PolG mice can shed light on how selection shapes mtDNA, both within and across organisms. Results Here, we characterized somatic and germline mtDNA mutations in brain and liver tissue of PolG mice to quantify mutation count (number of unique mutations) and frequency (mutation prevalence). Overall, mtDNA mutation count and frequency were the lowest in the D-loop, where an mtDNA origin of replication is located, but otherwise uniform across the mitochondrial genome. Somatic mtDNA mutations have a higher mutation count than germline mutations. However, germline mutations maintain a higher frequency and were also more likely to be silent. Cytosine to thymine mutations characteristic of replication errors were the plurality of basepair changes, and missense C to T mutations primarily resulted in increased protein hydrophobicity. Unlike wild type mice, PolG mice do not appear to show strand asymmetry in mtDNA mutations. Indel mutations had a lower count and frequency than point mutations and tended to be short, frameshift deletions. Conclusions Our results provide strong evidence that purifying selection plays a major role in the mtDNA of PolG mice. Missense mutations were less likely to be passed down in the germline, and they were less likely to spread to high frequencies. The D-loop appears to have resistance to mutations, either through selection or as a by-product of replication processes. Missense mutations that decrease hydrophobicity also tend to be selected against, reflecting the membrane-bound nature of mtDNA-encoded proteins. The abundance of mutations from polymerase errors compared with reactive oxygen species (ROS) damage supports previous studies suggesting ROS plays a minimal role in exacerbating the PolG phenotype, but our findings on strand asymmetry provide discussion for the role of polymerase errors in wild type organisms. Our results provide further insight on how selection shapes mtDNA mutations and on the aging mechanisms in PolG mice.

Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation

Mutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy – tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient-derived cells carrying the most prevalent disease related mtDNA mutation, the m.3243 A > G. These studies reveal that the mutation promotes changes in metabolites which are associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 reduced mtDNA mutant load and partially rescued cellular bioenergetic function. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243 A > G mutation.

Mitochondrial dysfunction, UPRmt signaling, and targeted therapy in metastasis tumor

In modern research, mitochondria are considered a more crucial energy plant in cells. Mitochondrial dysfunction, including mitochondrial DNA (mtDNA) mutation and denatured protein accumulation, is a common feature of tumors. The dysfunctional mitochondria reprogram molecular metabolism and allow tumor cells to proliferate in the hostile microenvironment. One of the crucial signaling pathways of the mitochondrial dysfunction activation in the tumor cells is the retrograde signaling of mitochondria-nucleus interaction, mitochondrial unfolded protein response (UPRmt), which is initiated by accumulation of denatured protein and excess ROS production. In the process of UPRmt, various components are activitated to enhance the mitochondria-nucleus retrograde signaling to promote carcinoma progression, including hypoxia-inducible factor (HIF), activating transcription factor ATF-4, ATF-5, CHOP, AKT, AMPK. The retrograde signaling molecules of overexpression ATF-5, SIRT3, CREB, SOD1, SOD2, early growth response protein 1 (EGR1), ATF2, CCAAT/enhancer-binding protein-d, and CHOP also involved in the process. Targeted blockage of the UPRmt pathway could obviously inhibit tumor proliferation and metastasis. This review indicates the UPRmt pathways and its crucial role in targeted therapy of metastasis tumors.

Growth Retardation in the Course of Fanconi Syndrome Caused by the 4977-bp Mitochondrial DNA Deletion: A Case Report

Fanconi syndrome is one of the primary renal manifestations of mitochondrial cytopathies caused by mitochondrial DNA (mtDNA) mutation. The common 4977-bp mtDNA deletion has been reported to be associated with aging and diseases involving multiple extrarenal organs. Cases of Fanconi syndrome caused by the 4977-bp deletion were rarely reported previously. Here, we report a 6-year-old girl with growth retardation in the course of Fanconi syndrome. She had mild ptosis and pigmented retinopathy. Abnormal biochemical findings included low-molecular-weight proteinuria, normoglycemic glycosuria, increased urine phosphorus excretion, metabolic acidosis, and hypophosphatemia. Growth records showed that her body weight and height were normal in the first year and failed to thrive after the age of three. Using a highly sensitive mtDNA analysis methodology, she was identified to possess the common 4977-bp mtDNA deletion. The mutation rate was 84.7% in the urine exfoliated cells, 78.67% in the oral mucosal cells, and 23.99% in the blood sample. After three months of oral coenzyme Q10 and levocarnitine treatment in combination with standard electrolyte supplement, her condition was improved. This is a report of growth retardation as the initial major clinical presentation of Fanconi syndrome caused by the deletion of the 4977-bp fragment. Renal tubular abnormality without any other extrarenal dysfunction may be an initial clinical sign of mitochondrial disorders. Moreover, considering the heterogeneity of the phenotypes associated with mtDNA mutations, the risk of developing Kearns–Sayre syndrome (KSS) with age in this patient should be noted because she had ptosis, retinal involvement, and changes in the brain and skeletal muscle.

A genetic bottleneck of mitochondrial DNA during human lymphocyte development

Mitochondria are essential organelles in eukaryotic cells that provide critical support for energetic and metabolic homeostasis. Mutations that accumulate in mitochondrial DNA (mtDNA) in somatic cells have been implicated in cancer, degenerative diseases, and the aging process. However, the mechanisms used by somatic cells to maintain proper functions despite their mtDNA mutation load are poorly understood. Here, we analyzed somatic mtDNA mutations in more than 30,000 human single peripheral and bone marrow mononuclear cells and observed a significant overrepresentation of homoplastic mtDNA mutations in B, T and NK lymphocytes despite their lower mutational burden than other hematopoietic cells. The characteristic mutational landscape of mtDNA in lymphocytes were validated with data from multiple platforms and individuals. Single-cell RNA-seq and computational modeling demonstrated a stringent mitochondrial bottleneck during lymphocyte development likely caused by lagging mtDNA replication relative to cell proliferation. These results illuminate a potential mechanism used by highly metabolically active immune cells for quality control of their mitochondrial genomes.

Positive selection on mitochondria may eliminate heritable microbes from arthropod populations

Diverse eukaryotic taxa carry facultative heritable symbionts, microbes that are passed from mother to offspring. These symbionts are coinherited with mitochondria, and selection favouring either new symbionts, or new symbiont variants, is known to drive loss of mitochondrial diversity as a correlated response. More recently, evidence has accumulated of episodic directional selection on mitochondria, but with currently unknown consequences for symbiont evolution. We therefore employed a population genetic mean field framework to model the impact of selection on mitochondrial DNA (mtDNA) upon symbiont frequency for three generic scenarios of host–symbiont interaction. Our models predict that direct selection on mtDNA can drive symbionts out of the population where a positively selected mtDNA mutation occurs initially in an individual that is uninfected with the symbiont, and the symbiont is initially at low frequency. When, by contrast, the positively selected mtDNA mutation occurs in a symbiont-infected individual, the mutation becomes fixed and in doing so removes symbiont variation from the population. We conclude that the molecular evolution of symbionts and mitochondria, which has previously been viewed from a perspective of selection on symbionts driving the evolution of a neutral mtDNA marker, should be reappraised in the light of positive selection on mtDNA.

Mitochondrial Genome (mtDNA) and Human Diseases

Mitochondria not only provide necessary energy for cells, but more importantly, they participate in the regulation of various biological functions and activities of cells. As one of the critical components of the body’s genome, mitochondrial genome (mtDNA) is the key to cell bioenergetics and genetics. However, since no protection of histones and a complete self-repair system, mtDNA is extremely prone to mutate. Human diseases caused by mtDNA mutations are only transmitted through the maternal line. The same phenotype can come from multiple mtDNA mutations, and the same mtDNA mutation can lead to multiple phenotypes. This is the major reason that makes the diagnosis and identification of mtDNA genetic diseases difficult. Meanwhile, mtDNA mutations may be the culprit involved in mediating the aging and tumorigenesis. Currently, no effective therapeutics for diseases caused by mtDNA mutations, but with the deepening of research and technological advancement, it is promising that breakthroughs in the diagnosis and treatment of mitochondrial-related diseases in the near future.

The Role of Mitochondrial Genes in Neurodegenerative Disorders

: Mitochondrial disorders are clinically heterogeneous, resulting from nuclear gene and mitochondrial mutations that disturb the mitochondrial functions and dynamics. There is a lack of evidence linking mtDNA mutations to neurodegenerative disorders, mainly due to the absence of noticeable neuropathological lesions in postmortem samples. This review describes various gene mutations in Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis, Multiple Sclerosis, and Stroke. These abnormalities, including PINK1, Parkin, and SOD1 mutations, seem to reveal mitochondrial dysfunctions due to either mtDNA mutation or deletion, the mechanism of which remains unclear in depth.

The Role of Mitochondrial Energetics and Inflammasome NLRP3 in Diabetic Cardiomyopathy

Diabetic Cardiomyopathy is the worldwide leading cause of lethal heart disorders burdening the healthcare systems. Mitochondrion is the key regulator of myocardial metabolism. It fuels the cardiocytes and regulates the pumping activity of heart. People living with diabetes have defected myocardial metabolism which may likely to cause ventricular dysfunction or other heart disorders due to mitochondrial DNA (mtDNA) mutation. Furthermore, the inflammatory injury due to inflammasome activation is a potent contributor to the cardiac injuries. Though the mechanism of inflammation is still poorly known. This review highlights the association of altered mitochondrial energetics and inflammasome activation with cardiomyopathies.