Creation of Cybrid Cultures Containing mtDNA Mutations m.12315G>A and m.1555G>A, Associated with Atherosclerosis

In the present work, a pilot creation of four cybrid cultures with high heteroplasmy level was performed using mitochondrial genome mutations m.12315G>A and m.1555G>A. According to data of our preliminary studies, the threshold heteroplasmy level of mutation m.12315G>A is associated with atherosclerosis. At the same time, for a mutation m.1555G>A, such a heteroplasmy level is associated with the absence of atherosclerosis. Cybrid cultures were created by fusion of rho0-cells and mitochondria from platelets with a high heteroplasmy level of the investigated mutations. To create rho0-cells, THP-1 culture of monocytic origin was taken. According to the results of the study, two cybrid cell lines containing mutation m.12315G>A with the heteroplasmy level above the threshold value (25% and 44%, respectively) were obtained. In addition, two cybrid cell lines containing mutation m.1555G>A with a high heteroplasmy level (24%) were obtained. Cybrid cultures with mtDNA mutation m.12315G>A can be used to model both the occurrence and development of atherosclerosis in cells and the titration of drug therapy for patients with atherosclerosis. With the help of cybrid cultures containing single nucleotide replacement of mitochondrial genome m.1555G>A, it is possible to develop approaches to the gene therapy of atherosclerosis.

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

ZHurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia» ◽

10.25557/0031-2991.2021.04.121-127 ◽

2021 ◽

pp. 121-127

Author(s):

М.А. Сазонова ◽

В.В. Синёв ◽

А.И. Рыжкова ◽

М.Д. Сазонова ◽

Н.А. Дорощук ◽

...

Keyword(s):

Mitochondrial Genome ◽

Protective Effect ◽

Mitochondrial Dysfunction ◽

Cell Lines ◽

Human Body ◽

Cell Models ◽

Coding Region ◽

Mtdna Mutation ◽

Cellular Mechanisms ◽

Cybrid Cell

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.

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Unbiased PCR-free spatio-temporal mapping of the mtDNA mutation spectrum reveals brain region-specific responses to replication instability

BMC Biology ◽

10.1186/s12915-020-00890-5 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Emilie Kristine Bagge ◽

Noriko Fujimori-Tonou ◽

Mie Kubota-Sakashita ◽

Takaoki Kasahara ◽

Tadafumi Kato

Keyword(s):

Pcr Amplification ◽

Brain Regions ◽

Mutation Spectrum ◽

Mtdna Mutation ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Mtdna Mutations ◽

Proof Reading ◽

Spatio Temporal ◽

Mitochondrial Dna Polymerase

Abstract Background The accumulation of mtDNA mutations in different tissues from various mouse models has been widely studied especially in the context of mtDNA mutation-driven ageing but has been confounded by the inherent limitations of the most widely used approaches. By implementing a method to sequence mtDNA without PCR amplification prior to library preparation, we map the full unbiased mtDNA mutation spectrum across six distinct brain regions from mice. Results We demonstrate that ageing-induced levels of mtDNA mutations (single nucleotide variants and deletions) reach stable levels at 50 weeks of age but can be further elevated specifically in the cortex, nucleus accumbens (NAc), and paraventricular thalamic nucleus (PVT) by expression of a proof-reading-deficient mitochondrial DNA polymerase, PolgD181A. The increase in single nucleotide variants increases the fraction of shared SNVs as well as their frequency, while characteristics of deletions remain largely unaffected. In addition, PolgD181A also induces an ageing-dependent accumulation of non-coding control-region multimers in NAc and PVT, a feature that appears almost non-existent in wild-type mice. Conclusions Our data provide a novel view of the spatio-temporal accumulation of mtDNA mutations using very limited tissue input. The differential response of brain regions to a state of replication instability provides insight into a possible heterogenic mitochondrial landscape across the brain that may be involved in the ageing phenotype and mitochondria-associated disorders.

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Cell and animal models of mtDNA biology: progress and prospects

AJP Cell Physiology ◽

10.1152/ajpcell.00224.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. C658-C669 ◽

Cited By ~ 28

Author(s):

Shaharyar M. Khan ◽

Rafal M. Smigrodzki ◽

Russell H. Swerdlow

Keyword(s):

Gene Therapy ◽

Animal Models ◽

Human Disease ◽

Mitochondrial Genetics ◽

Mtdna Mutation ◽

Mtdna Mutations ◽

The Past ◽

The Future ◽

Basic Biology ◽

Over Time

The past two decades have witnessed an evolving understanding of the mitochondrial genome’s (mtDNA) role in basic biology and disease. From the recognition that mutations in mtDNA can be responsible for human disease to recent efforts showing that mtDNA mutations accumulate over time and may be responsible for some phenotypes of aging, the field of mitochondrial genetics has greatly benefited from the creation of cell and animal models of mtDNA mutation. In this review, we critically discuss the past two decades of efforts and insights gained from cell and animal models of mtDNA mutation. We attempt to reconcile the varied and at times contradictory findings by highlighting the various methodologies employed and using human mtDNA disease as a guide to better understanding of cell and animal mtDNA models. We end with a discussion of scientific and therapeutic challenges and prospects for the future of mtDNA transfection and gene therapy.

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Respiratory complex and tissue lineage drive mutational patterns in the tumor mitochondrial genome

10.1101/2020.08.18.256362 ◽

2020 ◽

Author(s):

Alexander N. Gorelick ◽

Minsoo Kim ◽

Walid K. Chatila ◽

Konnor La ◽

A. Ari Hakimi ◽

...

Keyword(s):

Mitochondrial Genome ◽

Atp Synthesis ◽

Loss Of Function ◽

Sequencing Data ◽

Mtdna Mutation ◽

Driver Genes ◽

Cancer Driver ◽

Translational Machinery ◽

Synonymous Mutations ◽

Mtdna Mutations

AbstractMitochondrial DNA (mtDNA) encodes essential protein subunits and translational machinery for four distinct complexes of oxidative phosphorylation (OXPHOS). Using repurposed whole-exome sequencing data, we demonstrate that pathogenic mtDNA mutations arise in tumors at a rate comparable to the most common cancer driver genes. We identify OXPHOS complexes as critical determinants shaping somatic mtDNA mutation patterns across tumor lineages. Loss-of-function mutations accumulate at an elevated rate specifically in Complex I, and often arise at specific homopolymeric hotspots. In contrast, Complex V is depleted of all non-synonymous mutations, suggesting that mutations directly impacting ATP synthesis are under negative selection. Both common truncating mutations and rarer missense alleles are associated with a pan-lineage transcriptional program, even in cancer types where mtDNA mutations are comparatively rare. Pathogenic mutations of mtDNA are associated with substantial increases in overall survival of colorectal adenocarcinoma patients, demonstrating a clear functional relationship between genotype and phenotype. The mitochondrial genome is therefore frequently and functionally disrupted across many cancers, with significant implications for patient stratification, prognosis and therapeutic development.

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Maladaptive nutrient signalling sustains the m.3243A>G mtDNA mutation

10.1101/2020.06.18.159103 ◽

2020 ◽

Author(s):

Chih-Yao Chung ◽

Kritarth Singh ◽

Vassilios N Kotiadis ◽

Jee Hwan Ahn ◽

Lida Kabir ◽

...

Keyword(s):

Mitochondrial Genome ◽

Lactic Acidosis ◽

Treatment Options ◽

Disease Phenotype ◽

Wild Type ◽

Mtdna Mutation ◽

Potential Therapeutic Target ◽

Mtdna Mutations ◽

Cellular Bioenergetics ◽

Clinical Conditions

ABSTRACTMutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are limited. Most mtDNA diseases show heteroplasmy - tissues express both wild-type and mutant mtDNA. The relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have extensively characterised changes in bioenergetic, metabolomic, lipidomic and RNAseq profiles in heteroplasmic patient-derived cells carrying the m.3243A>G mtDNA mutation, the cause of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). These studies reveal that the mutation promotes upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 activated mitophagy, reduced mtDNA mutant load and rescued cellular bioenergetics cell-autonomously. The rescue was prevented by inhibition of mitophagy. These findings suggest that activation of the PI3K-Akt-mTORC1 axis is maladaptive and represents a potential therapeutic target for people suffering from the consequences of the m.3243A>G mutation.

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Mitochondrial Genome (mtDNA) and Human Diseases

Science Insights ◽

10.15354/si.21.re201 ◽

2021 ◽

Vol 38 (4) ◽

pp. 317-325

Author(s):

Ronnie L. Davidson

Keyword(s):

Mitochondrial Genome ◽

Genetic Diseases ◽

Human Diseases ◽

Repair System ◽

Technological Advancement ◽

Mtdna Mutation ◽

Maternal Line ◽

Mtdna Mutations ◽

Multiple Phenotypes ◽

Critical Components

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.

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Maneuvering Mitochondria for Better Understanding of Therapeutic Potential of mtDNA Mutation

10.5772/intechopen.96915 ◽

2021 ◽

Author(s):

Sanket Tembe

Keyword(s):

Mitochondrial Genome ◽

Ethical Issues ◽

Therapeutic Potential ◽

Genetic Manipulation ◽

Mitochondrial Diseases ◽

Mitochondrial Genetics ◽

Mtdna Mutation ◽

Genetic Etiology ◽

Mtdna Mutations ◽

Good Potential

Heterogeneity of mitochondrial diseases in terms of genetic etiology and clinical management makes their diagnosis challenging. Mitochondrial genome, basic mitochondrial genetics, common mutations, and their correlation with human diseases is well-established now and advances in sequencing is accelerating the molecular diagnostics of mitochondrial diseases. Major research focus now is on development of mtDNA intervention techniques like mtDNA gene editing, transfer of exogenous genes (sometimes even entire mtDNA) that would compensate for mtDNA mutations responsible for mitochondrial dysfunction. Although these genetic manipulation techniques have good potential for treatment of mtDNA diseases, research on such mitochondrial manipulation fosters ethical issues. The present chapter starts with an introduction to the factors that influence the clinical features of mitochondrial diseases. Advancement in treatments for mitochondrial diseases are then discussed followed by a note on methods for preventing transmission of these diseases.

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Creation of cybrid cell cultures containing a single-nucleotide substitution associated with atherosclerosis in the MT-TL2 gene

ZHurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia» ◽

10.25557/0031-2991.2020.04.140-147 ◽

2020 ◽

pp. 140-147

Author(s):

М.А. Сазонова ◽

В.В. Синёв ◽

А.И. Рыжкова ◽

М.Д. Сазонова ◽

Н.А. Дорощук ◽

...

Keyword(s):

Mitochondrial Genome ◽

Mitochondrial Dysfunction ◽

Cell Lines ◽

Cell Cultures ◽

Gradient Centrifugation ◽

Sybr Green ◽

Sybr Green I ◽

Cellular Mechanisms ◽

Study Participants ◽

Cybrid Cell

Введение. Цибридные клеточные модели наиболее перспективны для изучения патогенеза различных заболеваний. Авторами статьи впервые были созданы такие модели для изучения митохондриальной дисфункции и патологических процессов, развивающихся при атеросклерозе. Цель работы - создание цибридных культур с высоким уровнем гетероплазмии по мутации митохондриального генома m.12315G>A. В предварительных исследованиях авторами статьи было установлено, что пороговый уровень гетероплазмии мутации m.12315G>A ассоциирован с атеросклерозом. Методика. Цибридные культуры создавали путем слияния безмитохондриальных клеток (rho0) и митохондрий из тромбоцитов участников исследования с высоким уровнем гетероплазмии исследуемых мутаций. Для создания rho0-клеток была взята культура моноцитарного происхождения THP-1. Безмитохондриальные клетки были получены с помощью метода M. Kинга и Г. Аттарди. Тромбоциты выделяли из цельной крови участников исследования. Для этого был применен метод центрифугирования в градиенте плотности фиколла-урографина. Для получения цибридных культур клеток была использована методика «ПЭГ-слияния». В созданных безмитохондриальных и цибридных клеточных культурах был проведен количественный анализ копий митохондриального генома. Согласно результатам данного анализа было подтверждено либо отсутствие митохондрий (rho0-клетки), либо их наличие (цибриды). Количество копий мтДНК детектировалось с помощью реал-тайм ПЦР в присутствии красителя SYBR Green I. Результаты. Получены 4 цибридные клеточные линии, содержащие мутацию m.12315G>A с уровнем гетероплазмии выше порогового значения. Заключение. Созданы 4 цибридные культуры с высоким уровнем гетероплазмии по мутации митохондриального генома m.12315G>A. Полученные цибридные клеточные линии могут служить моделями для изучения молекулярно-клеточных механизмов митохондриальной дисфункции при атеросклерозе и других сердечно-сосудистых заболеваниях. Цибридные культуры можно использовать для моделирования атерогенеза, а также для подбора патогенетически обоснованной лекарственной терапии при атеросклерозе. Introduction. Cybrid cell models are most promising for studying pathological mechanisms in different diseases. The authors for the first time created such models for studying mitochondrial dysfunction and pathological processes underlying atherosclerosis. Aim. Creation of cybrid cultures with a high heteroplasmy level for mitochondrial genome mutation m.12315G>A. A preliminary study by the authors showed that the heteroplasmy level of mutation m.12315G>A was associated with atherosclerosis. Methods. Cybrid cultures were created by fusing non-mitochondrial cells (rho0) and mitochondria from platelets of study participants with a high heteroplasmy level of the mutations under study. A THP-1 culture of monocytic origin was used to create rho0 cells. Non-mitochondrial cells were obtained using the M. King and G. Attardi method. Platelets were extracted from whole blood of study participants with Ficoll-Urografin density gradient centrifugation. Cybrid cell cultures were obtained by the PEG-mediated fusion method. In the created non-mitochondrial and cybrid cell cultures, quantitative analysis of mitochondrial genome copies was performed. This analysis confirmed either the absence of mitochondria (rho0-cells) or their presence (cybrids). The mtDNA copies were quantified using real-time PCR in the presence of the SYBR Green I stain. Results. Four cybrid cell lines were obtained, which contained the m.12315G>A mutation with heteroplasmy levels higher than the threshold level. Conclusion. Four cybrid cultures were created with a high heteroplasmy level for the mitochondrial genome mutation m.12315G>A. The obtained cell lines can be used as models for studying molecular cellular mechanisms of mitochondrial dysfunction in atherosclerosis and cardiovascular diseases. In addition, they may be useful for modeling atherogenesis in cells and for selecting therapy for patients with atherosclerosis.

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PINK1 and parkin shape the organism-wide distribution of a deleterious mitochondrial genome

10.1101/576165 ◽

2019 ◽

Cited By ~ 1

Author(s):

Arnaud Ahier ◽

Nadia Cummins ◽

Chuan-Yang Dai ◽

Jürgen Götz ◽

Steven Zuryn

Keyword(s):

Nervous System ◽

Caenorhabditis Elegans ◽

Mitochondrial Genome ◽

Wide Distribution ◽

Causal Link ◽

Mtdna Mutation ◽

Ubiquitin Protein Ligase ◽

Mtdna Mutations ◽

Somatic Tissues ◽

Multiple Species

AbstractIn multiple species, certain tissue types are prone to acquiring greater loads of mitochondrial genome (mtDNA) mutations relative to others, however the mechanisms that drive these heteroplasmy differences are unknown. We found that the conserved PTEN-induced putative kinase (PINK1/PINK-1) and the E3 ubiquitin-protein ligase parkin (PDR-1), which are required for mitochondrial autophagy (mitophagy), underlie stereotyped differences in heteroplasmy of a deleterious mitochondrial genome mutation (ΔmtDNA) between major somatic tissues types in Caenorhabditis elegans. We demonstrate that tissues prone to accumulating ΔmtDNA have lower mitophagy responses than those with low mutation levels, such as neurons. Moreover, we show that ΔmtDNA heteroplasmy increases when proteotoxic species that are associated with neurodegenerative disease and mitophagy inhibition are overexpressed in the nervous system. Together, these results suggest that PINK1 and parkin drive organism-wide patterns of heteroplasmy and provide evidence of a causal link between proteotoxicity, mitophagy, and mtDNA mutation levels in neurons.

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The Detection and Partial Localisation of Heteroplasmic Mutations in the Mitochondrial Genome of Patients with Diabetic Retinopathy

International Journal of Molecular Sciences ◽

10.3390/ijms20246259 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6259 ◽

Cited By ~ 1

Author(s):

Afshan N. Malik ◽

Hannah S. Rosa ◽

Eliane S. de Menezes ◽

Priyanka Tamang ◽

Zaidi Hamid ◽

...

Keyword(s):

Diabetic Retinopathy ◽

Mitochondrial Genome ◽

Energy Production ◽

Mtdna Mutation ◽

Mtdna Damage ◽

Mtdna Mutations ◽

High Prevalence ◽

Severity Of The Disease ◽

Surveyor Nuclease ◽

Cellular Organelles

Diabetic retinopathy (DR) is a common complication of diabetes and a major cause of acquired blindness in adults. Mitochondria are cellular organelles involved in energy production which contain mitochondrial DNA (mtDNA). We previously showed that levels of circulating mtDNA were dysregulated in DR patients, and there was some evidence of mtDNA damage. In the current project, our aim was to confirm the presence of, and determine the location and prevalence of, mtDNA mutation in DR. DNA isolated from peripheral blood from diabetes patients (n = 59) with and without DR was used to amplify specific mtDNA regions which were digested with surveyor nuclease S1 to determine the presence and location of heteroplasmic mtDNA mutations were present. An initial screen of the entire mtDNA genome of 6 DR patients detected a higher prevalence of mutations in amplicon P, covering nucleotides 14,443 to 1066 and spanning the control region. Further analysis of 42 subjects showed the presence of putative mutations in amplicon P in 36% (14/39) of DR subjects and in 10% (2/20) non-DR subjects. The prevalence of mutations in DR was not related to the severity of the disease. The detection of a high-prevalence of putative mtDNA mutations within a specific region of the mitochondrial genome supports the view that mtDNA damage contributes to DR. The exact location and functional impact of these mutations remains to be determined.

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