Study of the effect of the introduction of mitochondrial import determinants into the gRNA structure on the activity of the gRNA/SpCas9 complex in vitro.

It has long been known that defects in the structure of the mitochondrial genome can cause various neuromuscular and neurodegenerative diseases. Nevertheless, at present there is no effective method for treating mitochondrial diseases. The major problem with the treatment of such diseases is associated with mitochondrial DNA (mtDNA) heteroplasmy. It means that due to a high copy number of the mitochondrial genome, mutant copies of mtDNA coexist with wild-type molecules in the same organelle. The clinical symptoms of mitochondrial diseases and the degree of their manifestation directly depend on the number of mutant mtDNA molecules in the cell. The possible way to reduce adverse effects of the mutation is by shifting the level of heteroplasmy towards the wild-type mtDNA molecules. Using this idea, several gene therapeutic approaches based on TALE and ZF nucleases have been developed for this purpose. However, the construction of protein domains of such systems is rather long and laborious process. Meanwhile, the CRISPR/Cas9 system is fundamentally different from protein systems in that it is easy to use, highly efficiency and has a different mechanism of action. All the characteristics and capabilities of the CRISPR/Cas9 system make it a promising tool in mitochondrial genetic engineering. In this article, we demonstrate for the first time that the modification of gRNA by integration of specific mitochondrial import determinants in the gRNA scaffold does not affect the activity of the gRNA/Cas9 complex in vitro.

Import of Proteins and Nucleic Acids into Mitochondria.

Many mitochondrial genes have been transferred to the nucleus in course of evolution. The products of expression of these genes, being still necessary for organelle function, are imported there from the cytosol. Molecular mechanisms of protein import are studied much deeper than those of nucleic acids. The latter, it seems to us, retards the development of mitochondrial genome editing technologies. In this review, we describe mechanisms of DNA, RNA, and protein import into mitochondria of different eukaryotes. The description is given for the natural processes, as well as for artificial targeting of macromolecules into mitochondria for therapy. Also, we discuss different approaches to introduce changes into the mitochondrial DNA sequence.

[Leber hereditary optic neuropathy]. / Nasledstvennaia opticheskaia neiropatiia Lebera.

Leber hereditary optic neuropathy is characterized by bilateral, painless loss of vision in children and young adults (generally up to 25 years old). Since its first description in 1871, the understanding of its etiology and pathogenesis has improved considerably. The article considers Leber neuropathy from the points of view of ophthalmology, neurology and molecular genetics, and presents data on experimental treatment methods, one of which is undergoing clinical trial.

[The association of the mitochondrial DNA oriB variants with metabolic syndrome]. / Rol' polimorfizma saita oriB mitokhondrial'noi DNK v metabolicheskom sindrome.

Different genes are involved in the development of pathology and formation the metabolic syndrome (MS) phenotype. In the literature, there is a data connection to the site oriB polymorphisms of mitochondrial DNA (mtDNA), known as 16184-16193 polycytosine tract, with insulin resistance, type 2 diabetes (T2DM) and other metabolic abnormalities in different ethnic populations. It is supposed that for certain polymorphisms at this site decreases mtDNA copy number in the cells. In this study, we have identified different allelic variants of the mtDNA oriB site in MS patients (n=106) and healthy individuals (n=71) using capillary sequencing, and determined the amount of mtDNA copy blood leukocytes by droplet digital polymerase chain reaction (ddPCR). The continuous polycytosine tract was significantly more common in MS patients, and such a link was particularly strong in MS patients with type 2 diabetes (p<0.01). No significant correlation has been found between mtDNA copy number and the oriB site variants, but in general there is a tendency to decreased mtDNA copy number in MS patients.

Detection of Mutations in Mitochondrial DNA by Droplet Digital PCR.

Mutations in mitochondrial DNA (mtDNA) may result in various pathological processes. Detection of mutant mtDNAs is a problem for diagnostic practice that is complicated by heteroplasmy - a phenomenon of the inferring presence of at least two allelic variants of the mitochondrial genome. Also, the level of heteroplasmy largely determines the profile and severity of clinical manifestations. Here we discuss detection of mutations in heteroplasmic mtDNA using up-to-date methods that have not yet been introduced as routine clinical assays. These methods can be used for detecting mutations in mtDNA to verify diagnosis of "mitochondrial disease", studying dynamics of mutant mtDNA in body tissues of patients, as well as investigating structural features of mtDNAs. Original data on allele-specific discrimination of m.11778G>A mutation by droplet digital PCR are presented, which demonstrate an opportunity for simultaneous detection and quantitative assessment of mutations in mtDNAs.

Mitochondrial Matrix Processes.

Mitochondria possess their own genome that, despite its small size, is critically important for their functioning, as it encodes several dozens of RNAs and proteins. All biochemical processes typical for bacterial and nuclear DNA are described in mitochondrial matrix: replication, repair, recombination, and transcription. Commonly, their mechanisms are similar to those found in bacteria, but they are characterized by several unique features. In this review, we provide an overall description of mitochondrial matrix processes paying special attention to the typical features of such mechanisms.

Mitochondrial Fission and Fusion.

Mitochondria are key cellular organelles responsible for many different functions. The molecular biology of mitochondria is continuously subject to comprehensive studies. However, detailed mechanisms of mitochondrial biogenesis are still unclear. Fusion and fission are among the most enigmatic processes connected with mitochondria. On the other hand, it has been shown that these events are of great biological importance for functioning of living cells. In this review, we summarize existing molecular data on mitochondrial dynamics and discuss possible biological functions of fusion and fission of these organelles.

Effects of Immunoregulatory Cytokines (IL-2, IL-7, and IL-15) on Expression of Gfi1 and U2afll4 Genes in T Cells at Different Stages of Differentiation.

Opposite effects of common γ-chain cytokines (IL-2, IL-7, and IL-15) on the expression of Gfi1 and U2af1l4 genes regulating alternative splicing of Ptprc gene in T cells at different stages of differentiation were demonstrated in vitro. Generally, produced a dose-dependent activating effect on T cells, while to the effects of rIL-7 and rIL-15 on T cells at different stages of differentiation were opposite to that of rIL-2: maximum concentrations of recombinant cytokines IL-7 and IL-15 produced the most pronounced inhibitory effect on U2af1l4 and to a lesser extent on Gfi1 gene expression, thus limiting activation of resting cells. This is consistent with their biological effects on T cells. In general, common γ-chain cytokines IL-2, IL-7, and IL-15 prevent differentiation of naïve T cells in vitro and limit activation of primed T cells in the absence of antigenic stimulus, which can contribute to the formation of cytokine imbalance.

[Insulin resistance pathogenesis in metabolic obesity].

In this review we discuss the molecular mechanisms of insulin resistance concomitant with metabolic inflammation. We also analyze the world results of experimental and clinical studies which aimed at identifying the molecular targets for the development of new prevention and treatment of insulin resistance.

Mutations in mitochondrial DNA and approaches for their correction.

Apart from the nucleus, the mitochondrion is the only organelle of an animal cell that contains its own genome. Mitochondrial DNA is much less in size than the nuclear one and codes for only several dozens of biological macromolecules. Nevertheless, mutations in mitochondrial genes often result in the occurrence of serious hereditary neuromuscular diseases. New mitochondrial DNA mutations and their relations to clinical symptoms are continuously reported in the scientific literature. In this review, we summarize existing data about such mutations, and also about contemporary gene therapy approaches that have been developed for their suppression.

[Evaluating the mitochondrial dna copy number in leukocytes and adipocytes from metabolic syndrome patients: pilot study].

The metabolic syndrome is a complex of metabolic, hormonal and clinical disorders. Defects in mitochondrial functions play an important role in the metabolic syndrome pathogenesis. Here, mitochondrial DNA (mtDNA) copy number variations were evaluated different fat tissue and peripheral blood leukocyte samples from metabolic syndrome patients ranked by, body mass indexes. It was elucidated a tendency in decreasing the mtDNA copy number with increasing a body mass index.

[Dose-response effect of steroid hormones on the Gfi1 and U2afil4 gene expression in T lymphocytes at different stages of differentiation].

Alternative splicing of Ptprc gene is a key event in memory T cell differentiation. This gene encodes transmembrane tyrosine phosphatase CD45. One of potential mechanisms of alternative splicing regulation is based on antagonistic effects of auxiliary splicing factor U2AF26 and transcription factor Gfi1. These two proteins regulate antigen-dependent T cell activation. We have shown that steroid hormones have different effects on U2af1l4 and Gfi1 transcription regulation in dissimilar differentiation stage cell culture, subjected to antigen-independent stimulation. Low concentrations of glucocorticoid (Dex) and female sex hormone (Est) can activate expression of U2af1l4 in re-stimulated cells that probably induce terminal receptor CD45 isoforms formation mechanism, whereas high doses of hormones inhibit the process. In the same conditions Dex in a wide range of concentrations (10(-5)-10(-7) M) and Est (10(-6) and 10(-7) M) activate U2af1l4 gene expression that probably leads to "surrogate memory T cells" formation. Dose dependent testosterone (Test) effect is opposite to Est and Dex effect on priming (CD45RO+) and naive (CD45RA+) lymphocytes. The role of steroid hormones in memory T cell differentiation in antigen-independent stimulation conditions is of great interest for the understanding of chronic hormonal and immune disbalance mechanisms.

[Mitochondrial genome and human mitochondrial diseases].

Today there are described more than 400 point mutations and more than hundred of structural rearrangements of mitochondrial DNA associated with characteristic neuromuscular and other mitochondrial syndromes, from lethal in the neonatal period of life to the disease with late onset. The defects of oxidative phosphorylation are the main reasons of mitochondrial disease development. Phenotypic diversity and phenomenon of heteroplasmy are the hallmark of mitochondrial human diseases. It is necessary to assess the amount of mutant mtDNA accurately, since the level of heteroplasmy largely determines the phenotypic manifestation. In spite of better understanding of the processes of phenotypic expression, currently there are no adequate treatments for mitochondrial diseases.

[Modern concepts of the mitochondrial structure and functions].

Recently accumulated data provide insight into some generally accepted models of mitochondrial genome. Here brief review of the new data is presented.

[Role of natural selection in evolution of mitochondrial haplogroups from Northeastern Eurasia].

The role of natural selection in the evolution of human populations from Northeastern Eurasia was studied. Selection for the regions-specific haplogroup C was demonstrated.

[The genetic history of long-term Russian resident populations of polar northeastern Siberia based on mitochondrial DNA variability].

The mtDNA variation has been studied in representatives of the Russkoe Ust'e (n = 30), Kolyma (n = 31), and Markovo (n = 26) ethnic subgroups originating from Russian military men, hunters, and fishers who married local Yukaghir women and settled at the Arctic Ocean coast and on the Anadyr' River more than 350 years ago. The mtDNA haplotypes characteristic of indigenous Siberian peoples have been demonstrated to form the basis of the mitochondrial gene pool of long-term Russian resident populations of the region. Only one of 30 identified haplotypes belonging to 11 haplogroups (H2a) is characteristic of European populations. The C and D haplogroups are the most diverse. The analysis has revealed the characteristics of the population structure of the long-term Russian resident populations and allowed them to be interpreted in terms of recent historical and environmental processes.