KR101556439B1 - A Role and Use of RXR related to mitochondrial retrograde signaling pathways - Google Patents

Abstract

The present invention relates to the association of retinoid X receptor alpha (RXRα) with the mitochondrial retrograde signaling pathway according to the A3243G mutation (mt3243) in mitochondrial tRNALeu, and more specifically, in the mechanism of mitochondrial dysfunction caused by mitochondrial mt3243 mutation Lt; RTI ID = 0.0 > RXRa-PGC1a < / RTI > interaction and thus the molecular mechanism.

Description

A Role and Use of RXRα Related to Mitochondrial Retrograde Signaling Pathways Associated with the Mitochondrial Retrograde Signal Pathway [

The present invention relates to the association of retinoid X receptor alpha (RXRα) with the mitochondrial retrograde signaling pathway according to the A3243G mutation (mt3243) in mitochondrial tRNALeu, and more specifically, in the mechanism of mitochondrial dysfunction caused by mitochondrial mt3243 mutation Lt; RTI ID = 0.0 > RXRa-PGC1a < / RTI > interaction and thus the molecular mechanism.

In general, mitochondria are organelles present in cells and are known as "cell power plants". Mitochondria produce not only ATP in cells but also cells such as apoptosis, ion homeostasis, intermediary metabolism, pyrimidine / urea / heme synthesis, / It performs functions essential for maintaining life of an individual (Schatz, 2007).

In animal cells, mitochondria are the only intracellular organelles that contain genes in addition to the nucleus, and there are dozens to hundreds of copies of mitochondrial DNA (mtDNA) in a single cell. However, most (more than 99%) of the proteins present in the mitochondria migrate into the cytoplasm of the mRNA synthesized in the nucleus, and the post-translational transport of the mature protein into the mitochondria after synthesis of the precursor protein in the cytoplasm (Neupert and Herrmann, 2007). Nucleotide-encoded proteins are a family of proteins, Mitochondrial DNA (mtDNA), on the other hand, is 16,569 bp in size and has a circular structure. mtDNA has an open reading frame (ORF) for 13 proteins, produces 12S and 16S rRNA, and 22 tRNAs. The 13 proteins made by mtDNA are the subunit proteins of complexes I, III, IV and V except the complex II of the electron transport chain complexes involved in oxidative phosphorylation subunit proteins. These 13 mtDNA-encoded proteins make mRNAs with mtDNA as a template in mitochondria and synthesize proteins using tRNA and rRNA in mitochondria (Wallace, 2007). Because the synthesis of 13 proteins synthesized by this mtDNA is similar to the protein synthesis principle of prokaryotes, the origin of mitochondria is explained by the intracellular symbiosis of prokaryotes. Thus, mitochondria can be defined as a single intracellular organelle created by two genes, which is a basic concept for understanding the biogenesis and function of mitochondria.

The production of mitochondria can be defined as a complex process involving the synthesis, transport, and binding of proteins and lipids present in the mitochondria. The abundance, morphology, and functional properties of mitochondria are finely tuned to meet cell-specific energy, metabolism, and signaling requirements. For this, it is essential that the transcription and expression of a large number of nucleotide-encoded mitochondrial genes and the factors that regulate mtDNA replication and transcription co-ordinate (Ryan and Hoogenraad, 2007). However, regulation of mitochondrial production is thought to be an adaptive response over a longer period of time rather than to respond to a temporarily increasing energy demand. In addition, since about 50% of the mitochondrial proteins are expressed in a tissue-specific manner, it is considered that the mitochondrial proteome has a tissue-specific function.

Mitochondrial impairment correlates with aging-related degenerative diseases such as obesity, diabetes, metabolic syndrome, insulin resistance, cardiovascular disease (Kim et al., 2008), Parkinson's disease, Huntington's disease, dementia (Lin and Beal, 2006) The relationship is investigated. In other words, electron microscopic photographs of mitochondria in these disease models show structural changes such as swelling or disappearance of the cristae structure compared to normal, and the number of mitochondrial DNA (mtDNA) per cell may be decreased, Mutation of mtDNA was also observed. Attention has been paid to the effects of aging on mtDNA replication and mtDNA degradation as aging progresses (MtDNA) and mtRNA decrease (Barazzoni, 2004) T414G and T408A mutations accumulate in the D-loop, the regulatory region of transcription (Michikawa et al., 1999; Wang et al., 2001). Recently, it has been reported that the substances identified as factors causing the neurodegenerative diseases are present in the mitochondria or are closely related to the mitochondrial function impairment (Beal, 1995, Chen & Yan, 2007; Scheffler, 2008; Wallace, 2005).

Mitochondrial DNA (mtDNA) epidemiology is an important diagnostic tool. Mutations in mtDNA are often a preliminary indicator of developing disease and serve as biomarkers that point to risk factors associated with the onset of the disease.

In particular, the present inventors have found that by profiling gene expression according to mitochondrial DNA (mtDNA) mutations with the A3243G mutation (mt3243) in tRNALeu, which reduces the amount of protein involved in oxidative phosphorylation encoded by the mitochondrial genome, (Retinoid X receptor alpha), and confirmed its involvement in the pathway involved in the mitochondrial retrograde signal, thus completing the present invention.

U.S. Published Patent Application 2013-0072462 Korean Patent Laid-Open No. 2001-0085746

The present invention is based on the association of certain mitochondrial DNA mutations with RXRa (retinoid X receptor alpha) and mitochondrial retrograde signaling pathways

The main object of the present invention is to provide various uses depending on the relationship of A3243G mutation (mt3243) and RXRa in tRNALeu of mitochondria.

Another object of the present invention is to provide various information on mitochondrial dysfunction according to the mt3243 mutation.

It is another object of the present invention to provide diagnostic and therapeutic uses for diseases involving mt3243-related mitochondrial dysfunction.

In order to solve the above problems, the present invention provides various uses of RXRa-PGC1a interaction and thus molecular mechanism in the mechanism of mitochondrial dysfunction caused by mitochondrial mt3243 mutation.

In one specific embodiment,

An expression or activity promoter of RXRa; Or a cell into which an expression or activity promoter of RXR? Has been introduced as an active ingredient. The present invention also provides a composition for treating or preventing a mitochondrial dysfunction-related disease.

The RXRa regulates the amount and expression of mRNA of oxidative phosphorylation and translation-related genes by interacting with PGC1a (peroxisome proliferator-activated receptor g, coactivator 1 a) transcription factor in mitochondria. In particular, since the amount of mRNA and expression of such oxidative phosphorylation and translation-related genes can be increased by the addition of retinoic acid (RA), the composition may further include retinoic acid.

In the present invention, mitochondrial dysfunction is caused by a mutation (mt3243) from the nucleotide sequence A to G at position 3243 of mitochondrial DNA, and mitochondrial dysfunction-related diseases are also caused by the nucleotide sequence A to G at position 3243 of mitochondrial DNA It is a disease caused by mutation.

For example, the present invention provides a method of treating a patient suffering from a condition selected from the group consisting of obesity, insulin resistance, body fat increase, fatty liver, liver fibrosis, metabolic syndrome, lipid metabolism disorder, hypertension, neurodegenerative disease, bipolar disorder, diabetes, arteriosclerosis, ischemia, Cancer, aging, cardiovascular disease, Parkinson's disease, Huntington's disease, dementia, and the like.

In another specific embodiment of the present invention, there is provided a method for restoring mitochondrial dysfunction through promotion of expression or activity of RXR [alpha].

In this method, expression or activation of RXR [alpha] may be carried out through the addition of retinoic acid (RA).

As described above, the expression or activity promotion of RXR [alpha] increases the interaction between RXR [alpha] -PGC1a, thereby promoting the expression of oxidative phosphorylation and translation-related genes, thereby restoring (restoring) mitochondrial dysfunction.

As such, the present invention provides a method of treating a subject suffering from (i) mitochondrial dysfunction which reduces the amount of RXRA, (ii) RXRA reduction reduces the interaction between RXRA and PGC1a, (iii) (Iv) OXPHOS (oxidative phosphorylation) and translation-related genes are based on a diminishing molecular mechanism, and the usefulness of RXRa as an important therapeutic target in the event of mitochondrial dysfunction due to mitochondrial gene mutations .

Meanwhile, in one embodiment of the present invention, the RXR < alpha > transcription factor is typically tested,

The following 72 transcription factors AHR, AR, ARNT, ATF3, CEBPA, CEBPB, CREB1, ATF2, E2F1, E2F4, E2F6, EGR1, ELF1, ELK1, EP300, ESR1, ESRRA, ETS1, FOS, FOSB, GABPA, GATA1, NFYC, NFYA, NFYB, NFYC, NRF1, PAX4, POU2F1, NFATC1, HIF1A, HNF4A, HSF1, JUN, JUNB, JUND, MAX, MAZ, FOXO4, MYB, MYC, MYOD1, NFATC1, NFATC2, NFATC3, NFATC4, RARA, REL, RELA, RELB, RXRA, SP1, SP3, SPI1, SREBF1, SRF, STAT6, TAF1, TCF3, TCF7, ZEB1, TFAP2A, TFAP4, NR2F1, NR2F2, TP53, USF1, YY1, MZF1, FOSL1 and LEF1 One or more transcription factors selected from the group of constructs may be used.

That is, it is possible to provide a composition for treating or preventing mitochondrial dysfunction related diseases using the 72 transcription factors, or to provide a method for restoring mitochondrial dysfunction. Also provided is a method for screening a therapeutic agent for mitochondrial dysfunction-related diseases, which comprises screening a substance that promotes the expression or activity of the transcription factor.

Information on such transcription factors can be obtained by reference to Figure 2D and www.sciencesignaling.org/cgi/content/full/6/264/rs4/DC1 (table S6A).

Thus, the present invention encompasses a variety of uses that utilize the relationship between intracellular transcription factors through mitochondrial DNA (mtDNA) gene expression profiling with mt3243 mutations and pathways associated with mitochondrial retrograde signals.

The present invention relates to the use of RXRa capable of restoring mitochondrial dysfunction and its various uses. By modulating the mitochondrial retrograde signaling pathway activity by the mt3243 mutation by promoting RXRa expression and activity, degenerative changes due to mitochondrial dysfunction It is possible not only to provide specific information on the disease but also to provide an effective treatment and prevention method.

FIG. 1 shows the results of confirming the reduction of the oxidative phosphorylation (OXPHOS) complex by mt3243 mutation in the Sibrid cells.
Figure 2 shows the results of confirming different modulation of 72 transcription factors (TFs) and genes involved in the mitochondrial retrograde signal [A: the relationship of DEGs in three types of siblide cells, B: Different gene expression, C: GO Biological Precesses enrichment, D: target distribution of 72 TFs]
Figure 3 shows the association of DEGs with cellular processes and genes known to be influenced by the mt3243 mutation.
Figure 4 is a graph showing the differential expression of TFs involved in the mitochondrial retrograde signal.
FIG. 5 is a graph showing the network model (A) and the relative amount of mRNA (B and C) connecting the relationship of the PGC1a phases interacting with TFs.
Figure 6 shows the results of confirming the recovery of oxidative phosphorylation gene expression by retinoic acid (RA) treatment. [A: qRT-PCR analysis and Western Blotting analysis, B: relative oxygen consumption and ATP content, C: OXPHOS complex I- Western blotting analysis of V]
FIG. 7 shows results of 163 gene expression patterns (A) and GO Biological Processes enrichment (B) after retinoic acid (RA) treatment and qRT-PCR analysis (C) according to gene silencing.
Figure 8 is a knockdown result of RXRA using siRNA.
Figure 9 shows the results of PLA analysis according to RXRA-PGC1a interaction.
Figure 10 shows the results of qRT-PCR analysis of the OXPHOS gene in FKBP-RXRA and FRB expressing cells after treatment with retinoic acid (RA).
Fig. 11 shows the relative amount (A) of ROS and the results of qRT-PCR analysis and Western blotting analysis (B and C), confirming reduction of RXRA amount through JNK activated by ROS.
Figure 12 shows the amount of RXRA protein and modulation of mRNA through JNK activated by ROS.
Figure 13 relates to the effect of OXPHOS inhibitor (A) on the amount of RXRA and the results of qRT-PCR analysis of translation related genes.
14 is a schematic diagram of a model for a retrograde signal pathway that regulates the amount of mRNA for OXPHOS and translation related genes.

The terms used in the present invention are defined as follows.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included. In one embodiment of the present invention, the present invention was applied to humans.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

"Expression" refers to the biological production of a product encoded by a coding sequence. In most cases, DNA sequences, including coding sequences, are transcribed to form messenger RNA (mRNA). The messenger RNA is translated to form a polypeptide having biological activity. In some cases, however, the RNA product may be considered as a gene product since it may have an associated activity. Expression may include additional processing steps of the transcriptional RNA product, such as splicing for intron removal and / or post-translational processing of the polypeptide product.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Coding region" or "coding sequence" refers to a nucleic acid sequence, a complement thereof, or a portion thereof, which encodes a particular gene product or fragment thereof that is required to be expressed, according to a common base pairing and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are joined together by a biochemical machinery of cells to provide mature mRNA. The antisense strand is a complement of the nucleic acid, and the coding sequence can be deduced therefrom. The coding sequence is placed in the relationship of transcriptional regulatory elements and translation initiation and termination codons such that transcripts of appropriate length are generated and translated in the appropriate reading frame to produce the desired functional product

The term "polynucleotide" or "nucleic acid" refers to nucleotide polymers of any length, including ribonucleotides as well as deoxyribonucleotides. This term refers only to the primary structure of the molecule, and thus to DNA or RNA of a double or single chain. This also applies to known types of modifications, for example, labeling, methylation, "caps", nucleotide substitutions of one or more naturally occurring nucleotides, debonding (eg, methyl phosphonate, phosphotriester, phosphoamidate, carbarnate (Eg, phosphorothioate, phosphorodithioate, etc.), proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-Llysine, etc.) Having an alkyl compound, a modified bond (e.g., alpha (alpha), beta (alpha) anomeric nucleic acid, etc.), as well as inter-nucleotide modification including non-modification of the polynucleotide. Generally, the nucleic acid moieties provided by the present invention may comprise fragments of a genome and a short oligonucleotide linker or series of oligonucleotides, microorganisms or viral operons, or regulatory elements derived from eukaryotic genes, Lt; RTI ID = 0.0 > nucleotides < / RTI > that can be expressed as recombinant transcription units

The term "functional equivalent" refers to, for example, one or more substitutions, deletions or additions from a reference sequence, an actual effect that does not result in various functional dissimilarities between the reference and subject sequences < / RTI > net effect) and the nucleotide sequence of the mutated mutant sequence. Typically, such a substantial equivalent sequence is only about 35% (i.e., the number of each residue substitution, addition, and deletion in a substantially equivalent sequence is compared to the corresponding reference sequence and the total number of residues in the substantially equivalent sequence Which is about 0.35 or less). Such sequences have 65% sequence identity with the listed sequences. A substantial equivalent, e. G. Mutant, amino acid sequence according to the invention has preferably at least 80% sequence identity, more preferably at least 90% sequence identity with the listed amino acid sequence. The nucleotide sequence of the present invention, which is a substantial equivalent, may have a lower percentage of sequence identity, for example, when considering the redundancy or degeneracy of the genetic code. Preferably, the nucleotide sequence should have at least about 65% identity, more preferably at least about 75% identity, and most preferably about 95% identity. For the purposes of the present invention, sequences with substantially equivalent biological activities and substantially equivalent synthetic features are treated as substantial equivalents. To determine the equivalence, the truncation of the maturation sequence (e.g. through a mutation to produce a spurious termination codon) should be ignored.

"Accelerator" means a substance that promotes, enhances or increases the expression or activity of a gene of interest. The activation mechanism of the promoter is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds. For example, 'RXRα promoter' may include a substance that promotes, enhances or increases the expression or activity of an RXRα protein.

"Inhibitor or inhibitor" means a substance that inhibits, blocks or reduces the expression or activity of a gene of interest. The mechanism of activation of the inhibitor is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds.

"Antibody" is used in its broadest sense and specifically includes intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (e. G. Bispecific antibodies) formed from at least two intact antibodies, and And antibody fragments exhibiting the desired biological activity.

"RNAi" refers to RNA Interference. In Korean, it means RNA interference. RNA interference is a specific gene suppression phenomenon that is well conserved among most organisms. siRNA is a small RNA that is artificially introduced into a cell, and binds to a specific mRNA having a complementary sequence to decompose the mRNA.

An "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment.

Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

The term " disorder "is any condition that can benefit from treatment with a molecule identified using the transgenic animal model of the invention. This includes chronic and acute diseases or diseases, including pathological conditions that make mammals susceptible to suspicious diseases. Diseases to be addressed herein are degenerative diseases associated with mitochondrial dysfunction, particularly mitochondrial retrograde signaling pathways according to the A3243G mutation (mt3243) at tRNALeu.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to mitochondrial DNA mutations.

Since the discovery that mitochondria have their own genetic material (mitochondrial DNA, mtDNA) in 1963, the nucleotide sequence of human mitochondrial DNA has been reported in 1981. Since the late 1980s, mtDNA mutations have been reported to cause rare diseases. Recently, it has been reported that diseases that are frequently encountered are related to mitochondrial dysfunction.

The human mitochondrial gene is a double stranded 16,569 bp molecule that is replicated and transcribed in a mitochondrial matrix. Mitochondrial DNA contains 37 genes, 24 of which encode the RNA necessary for protein synthesis (22 tRNA, 2 rRNA) and the remaining 13 are the major proteins of the respiratory chain that play an important role in oxidative phosphorylation ≪ / RTI >

Mitochondrial dysfunction or disease is mostly caused by point mutation or deletion of mitochondrial DNA (mt DNA). The present invention relates to the case where the nucleotide sequence of the 3243 region of DNA in the mitochondrial tRNA Leu mutated from A to G (hereinafter referred to as mt3243).

The present invention encompasses all of the various uses of information obtained through profiling of mitochondrial DNA (mtDNA) gene expression having the mt3243 mutation.

In particular, the present invention provides a variety of uses by using the relationship between intracellular transcription factors and pathways involved in mitochondrial retrograde signals. In one embodiment of the present invention, RXR [alpha] transcription factors are typically tested, but not limited thereto , 72 transcription factors AHR, AR, ARNT, ATF3, CEBPA, CEBPB, CREB1, ATF2, E2F1, E2F4, E2F6, EGR1, ELF1, ELK1, EP300, ESR1, ESRRA, ETS1, FOS, FOSB, GABPA, GATA1, NR3C1 , HIF1A, HNF4A, HSF1, JUN, JUNB, JUND, MAX, MAZ, FOXO4, MYB, MYC, MYOD1, NFATC1, NFATC2, NFATC3, NFATC4, NFKB1, NFKB2, NFYA, NFYB, NFYC, NRF1, PAX4, POU2F1, , REL, RELA, RELB, RXRA, SP1, SP3, SPI1, SREBF1, SRF, STAT6, TAF1, TCF3, TCF7, ZEB1, TFAP2A, TFAP4, NR2F1, NR2F2, TP53, USF1, YY1, MZF1, FOSL1 and LEF1 One or more transcription factors selected from the group can be used.

That is, it is possible to provide a composition for treating or preventing mitochondrial dysfunction related diseases using the 72 transcription factors, or to provide a method for restoring mitochondrial dysfunction. Also provided is a method for screening a therapeutic agent for mitochondrial dysfunction-related diseases, which comprises screening a substance that promotes the expression or activity of the transcription factor.

Information on such transcription factors can be obtained by reference to Figure 2D and www.sciencesignaling.org/cgi/content/full/6/264/rs4/DC1 (table S6A).

However, based on the relationship between the transcription factor RXRα (RXRA), the most representative form of the present invention, and the mutation mt3243 of the mitochondrial DNA, the following description will focus on this. As described above, it is possible to use other transcription factors other than RXR? (RXRA) among the 72 transcription factors.

[Molecular mechanism according to mt3243 mutation]

The mitochondrial gene 3243 mutation is the most common genetic abnormality that accounts for about 1% of domestic diabetics, with the nucleotide sequence of the 3243 region of mitochondrial DNA mutated from A to G. However, this mutation has not clarified the detailed mechanism of diabetes mellitus as well as the treatment method

The inventors of the present invention for the first time confirmed the involvement of the intracellular transcription factor RXRα (retinoid X receptor alpha) in the mechanism of mitochondrial dysfunction caused by the mutation of the gene 3243 by mitochondria.

In the fusion cell with the mitochondrial gene 3243 mutation, the intracellular reactive oxygen increases and the amount of RXRa is reduced by 50-75%, resulting in a decrease in mitochondrial protein expression and a reduction in mitochondrial function by 45-65%. On the other hand, mitochondrial function was restored by treatment with RXRα active substance. In other words, the treatment of RXRα active substance revealed that the interaction between RXRα and another transcription factor, PGC1a (Peroxisome proliferator activated receptor-coactivating factor-1 a) was increased and mitochondrial function was recovered by about 40%.

Mitochondrial dysfunction by mt3243 is caused by mitochondrial retrograde signaling pathways including retinoid X receptor a, reactive oxygen species, c-JUN N-terminal kinase (JNK), and the globally competent PGC1a. . In one embodiment of the present invention, analysis of the mutation tRNALeu and related gene expression profiles confirmed that 72 transcription factors were potentially involved in mitochondrial retrograde signals. And this RXR pathway exacerbates dysfunction in oxidative phosphorylation by a reduced amount of mitochondria-coded enzyme of oxidative phosphorylation by decreasing the amount of mRNA of oxidative phosphorylase encoded in the nuclear genome.

As such, the inventors first identified the molecular mechanism by which the RXRa-PGC1a interaction diminished when mt3243 caused an abnormality in mitochondrial function, thus first confirming that RXRa may be an important therapeutic target for mitochondrial dysfunction . Thus, the present invention includes all information relating to the mt3243 mutation and the molecular mechanism associated with RXRa-PGC1a.

Typically, it has the following molecular mechanism.

(i) mitochondrial dysfunction reduces the amount of RXRA

(ii) RXRA reduction reduces the interaction between RXRA and PGC1a

(iii) the reduced interaction results in a decrease in mRNA level and translation-related gene, and

(iv) Oxidative phosphorylation (OXPHOS) and translation-related gene reduction reverses all of the above reductions by the addition of retinoic acid (RA).

In an embodiment of the present invention, the mt3243 mutation has been shown to be involved in the production of retinoid X receptor a (RXRA), reactive oxygen species (ROS), kinase JNK (c-JUN N-terminal kinase), and transcriptional activator PGC1a (peroxisome proliferator- g, and coactivator 1 a).

That is, the genes affected by the mt3243 mutation are mitochondrial processes: mitochondrial migration, mitochondrial organization, oxidative phosphorylation (OXPHOS), reaction to oxidative stress and ROS, nitrogen compound biosynthesis processes; Protein homeostasis - related processes: translation, ribosome biogenesis, protein transport and protein ubiquitination regulation; And cell proliferation-related processes: cell proliferation regulation, DNA repair, regulated apoptosis transcription, chromatin assembly and degradation, and RNA processing processes. This implies that mitochondrial dysfunction by a single point (mt3243) mutation affects a broad spectrum of cellular processes that may reflect the activity of various retrograde signaling pathways that alter these cellular processes.

Specifically, the amount of mRNA of the transcription factors was reduced by the mutation (Example 3), and the reduced amount of mRNA resulted in a decrease in the amount of the protein of the nuclear-coded OXPHOS enzymes, which was provided as a positive feedback loop, Induced OXPHOS dysfunction causing a decrease in the amount of mitochondria-coded OXPHOS enzyme (Example 4).

In addition, O ₂ Both consumption and ATP content were reduced by the mt3243 mutation, but it was confirmed that RA treatment contributes to the RXRA-PGC1a complex increase and restoration of OXPHOS gene expression (Examples 5 and 6). It was also confirmed that the amount of mRNA of RXRA was negatively regulated by JNK activated by ROS (Example 7), and that RXRA-PGC1a regulated nuclear-coded genes involved in cytosol and mitochondrial translation 8)

Therefore, it can be seen that mitochondria mainly involve the intracellular transcription factor RXRα (retinoid X receptor alpha) in the mechanism of mutation of mitochondrial function by gene 3243 mutation, and thus mitochondrial function is restored by RXRα active substance treatment .

[Usage]

In one aspect, the present invention relates to a method for restoring mitochondrial dysfunction caused by mt3243 mutation through promotion of expression or activity of RXRα, and a system using the same.

In order to promote the expression or activity of RXR alpha, it is preferable to use a substance selected from the group consisting of a substance promoting transcription of RXR alpha gene, a substance promoting translation of transcribed RXR alpha, or a substance promoting the function of RXR alpha protein, It does not.

As a specific example, a recombinant polynucleotide construct such as an expression or activity promoter of RXRα gene, for example, a recombinant expression vector containing RXRα gene (collectively referred to as "expression vector"), a microorganism transformed with the expression vector , And a mitochondrial dysfunctional recovery system that can be practiced as a composition for treating a mt3243 mutation-related disease comprising any one of the above.

For overexpression of RXRa, a suitable promoter may be operably linked to a DNA / polynucleotide encoding it, for example, a promoter for expression in a mammalian cell system may include a viral promoter, such as a polyoma, fowlpox and adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus (especially, immediate early gene promoter), retrovirus, hepatitis B virus , An actin, a rous sarcoma virus (RSV) promoter and an RNA polymerase II promoter comprising early or late Simian virus 40 and a nonviral promoter such as EF-1 alpha (Mizushima and Nagata Nucleic Acids Res 1990 18 (17): 5322). The selection of such promoters may be based on the proper compatibility with the host cell used for expression.

Further, if desired, additional ' enhancer elements ' may be included in addition to or in addition to the elements present at the promoter positions described above to facilitate expression or activity of RXR [alpha].

In addition, in one embodiment of the present invention, according to such a mechanism, a vector encoded so that RXR [alpha] is overexpressed, and thus transformed cells, may be provided.

Therefore, the present invention provides the use of the above-mentioned molecular mechanism capable of treating a degenerative disease that is caused by abnormal mitochondrial function using a method of promoting RXRa activity from a different viewpoint.

Mitochondria are constantly in contact with reactive oxygen species and are less capable of repairing gene mutations, resulting in gene mutations that are 5 to 10 times more likely to occur than nuclear DNA. Such mitochondrial gene mutations accumulate in somatic cells, resulting in diminished mitochondrial functions, It is considered to be one of the main causes of the disease.

In particular, the abnormality of the mitochondria of the present invention is closely related to the onset of diabetes. One of the genetic causes of diabetes is diabetes mellitus caused by point mutations in mitochondrial DNA (3243A> G). This mutation causes a dysfunction of leucine tRNA in mitochondria as a single point mutation. It is known as the most common variation among all the mutations that cause it. Mitochondrial DNA mutations are inherited as a maternal strain, and patients have diminished mitochondrial function, which results in decreased insulin secretion and function, and is often very skinny.

Therefore, the present invention relates to a therapeutic use for restoring mitochondrial function by promoting RXRα activity based on the relationship of the intracellular transcription factor RXRα (retinoid X receptor alpha) to the mechanism of mitochondrial dysfunction caused by the mutation of mitochondrial gene 3243 . That is, to promote RXR [alpha] activity to improve mitochondrial function, thereby treating and preventing diabetes.

The disease caused by mitochondrial dysfunction in the present invention is a disease caused by a decrease in mitochondrial function, which is selected from the group consisting of obesity, insulin resistance, body fat increase, fatty liver, liver fibrosis, metabolic syndrome, lipid metabolism abnormality, hypertension, neurodegenerative disease, bipolar disorder, , Ischemia, ischemia, heart disease, liver disease, pancreatic disease, cancer, aging, cardiovascular disease, Parkinson's disease, Huntington's disease and dementia.

In one embodiment of the present invention, there is provided a method for treating a disease caused by mitochondrial function impairment by promoting RXRa expression or enhancing activity by transfecting with a genetic material that activates RXRa.

"Transfection or transfection" means introducing foreign material into eukaryotic cells using viral vectors or other delivery means. Transfection of animal cells typically involves opening transient survival or "holes " in the cell plasma membrane to allow the material to be absorbed. A gene substance or a protein such as an antibody. In addition to the electroporation method, transfection can be carried out by mixing cationic lipids with these materials, fusing them with the cytoplasmic membrane, and producing liposomes that deposit cargo therein.

For example, calcium phosphate may be used. HEPES-buffered saline (HeBS) containing phosphate ions is mixed with a calcium chloride solution containing the substance to be transfected. When the two are mixed, a fine precipitate of positively charged calcium and negatively charged phosphate is formed to bind the substance to be transfected to the surface. Next, a suspension of the precipitate is added to the cells to be transfected. The cell absorbs the substance to be transfected together with a part of the precipitate. In another example, a highly branched organic compound (e.g., a dendrimer) is used to bind the gene material (miRNA) of the present invention and enter the cell. Other methods include the incorporation of liposomes, the genetic material to be transfected into a small membrane-like body that is similar in some respects to the structure of the cell and can actually fuse with the cell membrane, and the export of the genetic material into the cell. In the case of eukaryotic cells, lipid-cation based transfection is more typically used because the cells are more sensitive. As another example, a cationic polymer such as DEAE-dextran or polyethyleneimine is used. Negatively charged genetic material binds to polycation, and the complex is absorbed by cells through endocytosis.

A direct approach to transfection is a gene gun in which the genetic material is coupled to an inert solid (usually gold) nanoparticle and then "shot" directly into the nucleus of the target cell. Genetic material can also be introduced into cells using the virus as a mediator. In this case, the technique is called viral transduction, and cells are said to be transduced. Other methods of transfection include, but are not limited to, nucleofection, electroporation, heat shock, magnetofection and registered traits such as Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene or DreamFect Infection reagent.

In another embodiment of the therapeutic use of the present invention, there is provided a composition or pharmaceutical composition for treating or preventing mitochondrial dysfunction-related diseases, comprising an RXRa expression or activity promoter or a cell into which an expression or activity promoter of RXRa has been introduced as an active ingredient Can be provided. And the composition may further comprise retinoic acid (RA).

"Accelerator" means a substance that promotes, enhances or increases the expression or activity of a gene of interest. The activation mechanism of the promoter is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds. As will be apparent to those skilled in the art, it may be chemically or biochemically modified, or it may be formulated into a composition and administered to the target cells by any of a number of means or through an expression construct.

A number of suitable methods are known in the art. Generally, the cells expressing the target gene are transfected. For the transfection, various methods such as, for example, electroporation, use of a cationic lipid or a cationic polymer as a helper for transfection are used . The cells are then cultured under suitable conditions to allow expression of the target gene. The expression of the target gene is then determined using suitable techniques such as, for example, RT-PCR or determination of the amount of reporter gene. Basically, although any type of cell can be used for transfection, in preferred embodiments the cell is a eukaryotic cell, preferably an animal cell, more preferably a mammalian cell, most preferably a human cell. A therapeutically effective amount of a composition according to the present invention may be provided by a known route of administration.

Such a therapeutic composition or cell therapy agent of the present invention can be prepared into a suitable preparation including an acceptable carrier depending on the administration mode. Formulations suitable for the mode of administration are known and may include formulations that facilitate passage, typically through the membrane.

In addition, the therapeutic composition of the present invention can be used in the form of a general pharmaceutical preparation. The parenteral preparation may be in the form of a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion or a lyophilized preparation, or a tablet, troch, capsule, elixir, suspension, syrup or wafer in the case of oral administration. May be formulated in unit dosage ampoules or multiple doses. In addition, the therapeutic composition of the present invention may be administered together with a pharmaceutically acceptable carrier. For oral administration, for example, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring matters or fragrances may be used. In the case of injections, buffering agents, preservatives, An isotonic agent, an isotonic agent, a stabilizer, etc. may be used in combination. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used.

In addition, the method for treating a mitochondrial dysfunction-related disorder using the therapeutic composition of the present invention may include administration through a general route in which a predetermined substance is introduced into a patient by an appropriate method. Such administration methods include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration. However, upon oral administration, the cells may be digested, so that the oral composition is preferably formulated to coat the active agent or protect it from being decomposed from above.

In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell. Preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular or drip injectable. The injectable solution may be a non-aqueous solvent such as an aqueous solvent such as a physiological saline solution or a ring gel solution, a vegetable oil, a higher fatty acid ester (for example, oleic acid), an alcohol (for example, ethanol, benzyl alcohol, propylene glycol or glycerin) (For example, ascorbic acid, sodium hydrogen sulfite, sodium pyrophosphate, BHA, tocopherol, EDTA and the like), an emulsifier, a buffer for pH control, a microbial growth inhibitor Preservatives (for example, mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.). Preferably, the method for treating a brain or neurological disease using the therapeutic composition of the present invention comprises administering a therapeutically effective amount of the therapeutic composition of the present invention. The pharmaceutically effective amount may be appropriately selected depending on the kind of the disease, the age, body weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, administration frequency, And can be readily determined by those skilled in the art depending on the factors.

The use of the present invention extends to a genetic approach associated with the regulation of RXR [alpha] expression by upregulating RXR [alpha].

In general, the general technique used to up-regulate expression may be to increase the number of gene copies or to antagonize the inhibitor of gene expression, for example, to improve the translation of RXR [alpha] Regulatory (post-transciptional gene silencing), etc., thereby enhancing expression of RXR [alpha] mRNA, thereby promoting RXR [alpha] expression.

In another aspect, the present invention includes a method for screening a substance for treating diseases according to mitochondrial dysfunction, which promotes expression of RXRα, based on the facts described above.

In this method, measuring the expression of RXR [alpha] does not limit the form of the biomarker target, such as RNA, DNA, protein, etc. associated therewith.

The expression of a variety of biomarkers in the sample may be determined by immunohistochemical and / or Western analysis, quantitative blood-based analysis (e.g., serum ELISA) (e.g. to investigate protein expression levels), biochemical enzyme activity analysis, Any of the wide variety of assays that can be performed by hybridization, Northern blot analysis and / or PCR analysis, and genome western blot analysis (for example, to investigate gene deletion or amplification), and gene and / or tissue array analysis And can be analyzed by a number of methods including, but not limited to, one of the methods well known in the art and understood by those skilled in the art.

For example, the method can include a protocol for examining mRNA expression in a tissue or cell sample. Methods for evaluating mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes and various nucleic acid amplification assays (such as RT-PCR using complementary primers, etc.). The method may also include a protocol to probe or detect mRNA in a tissue or cell sample by microarray technology. Microarray technology uses nucleic acid hybridization and computer techniques to evaluate the mRNA expression profiles of thousands of genes in a single experiment

As described above, the present invention relates to the use of the mt3243 mutation in the production of retinoid X receptor a, reactive oxygen species (ROS), c-JUN N-terminal kinase (JNK), and peroxisome proliferator- Coactivator 1 a), which is based on the fact that the key signaling RXRa factor is used based on the fact that it induces a retrograde signaling pathway involving mt3243 mutation associated with mitochondrial dysfunction and consequent degenerative diseases It can be used as a target.

<Examples>

Hereinafter, the present invention will be described in more detail with reference to Examples.

It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Experimental Preparation and Experimental Methods

1. Human mtDNA - deficient cells ( lacking cells ) And platelet-mediated transformation (platelet- mediated transformation )

Human 143B osteosarcoma cell-derived rho0 cells, which had been exposed to EtBr (ethidium bromide) for a prolonged period of thymidine kinase activity, were obtained from Y.-H. Wei of National Yang-Ming University, Thailand.

The cells were treated with 5-bromo-2'-deoxyuridine (100 mg / ml) (Sigma-Aldrich), uridine (50 mg / bovine serum) supplemented with DMEM (Dulbeccos modified Eagles medium). Southern blot analysis of the mtDNA target sequence and PCR amplification confirmed the absence of residual mtDNA.

Platelets were used as mtDNA donors to generate cybrid cells.

Briefly, platelet-rich fractions were isolated by centrifugation from blood samples from diabetic patients with sensorineural hearing loss and harboring mutated mitochondria tRNALeu (A3243G), and 143B rho0 cells were added to this fraction. Fusion was performed in the presence of 42% polyethylene glycol 1500 (Sigma-Aldrich).

By limiting the dilution of the cellular fusion product, the sibrid clones were isolated with 3243A homoplasmy (W), 3243G homoplasm (M), and 3243A / G heteroplasmy (H). These clones were confirmed by PCR-RFLP analysis and direct sequencing.

To confirm complete repopulation of mtDNA, functional evaluation of selected clones was performed after 3 months of continuous subculture. For consistent analysis, Sibrid cells were used in 16-18 passages.

To determine the amount of mutated DNA in the sibrid cells, wild type and mutated DNA were mixed to produce 0, 20, 40, 60, 80, and 100% of the heteroplasmic samples; The intensity of the bands in the individual heteroplasma samples was quantitatively analyzed with a densitometer and standard curves were generated with mutation DNA percentages and their intensities.

The standard curve was used to quantify the mutation DNA percentage for the intensities measured in the siblide cells.

2. Measurement of oxygen consumption

A suspension of 1 × 10 ⁵ cells in DMEM were added in a 96-well BD Oxygen Biosensor System plate (BD Biosciences ). The plates were incubated in a 5% CO ₂ incubator at 37 ° C for 30 minutes. Oxygen consumption was measured by a VICTOR3 Multilabel Plate Reader (PerkinElmer) with an excitation wavelength of 485 nm and an emission wavelength of 630 nm.

3. ATP  Generation and Cytochrome  c Oxidase assay

1 x 10 &lt; ⁴ &gt; cells were seeded in microplates and incubated overnight.

ATPLite kit (PerkinElmer) was used to measure the amount of ATP. The activity of cytochrome c oxidase is reported in a well-known document [EA Madden, B. Storrie, The preparative isolation of mitochondria from Chinese hamster ovary cells. Anal. Biochem. 163, 350-357 (1987)). The activity was measured by Beckman DU 650 spectrophotometer.

4. Microarray  Experiment

The gene expression profile of the siblide cells was generated with human HT-12 v3 BeadChip (Illumina) containing 49,896 probes corresponding to 25,202 added genes.

According to the Illumina protocol, three biological triple kites of each type of sibrid cells were analyzed. Total RNA (500 ng) was isolated from siburide cells using the RNeasy Mini Kit (Qiagen GmbH). When measured with the Agilent 2100 Bioanalyzer, the RNA integrity number (RIN) ranged from 9.2 to 10. RNA was reverse transcribed and amplified with the Illumina TotalPrep RNA Amplification Kit (Ambion).

In vitro transcription was performed to produce complementary RNA (cRNA). cRNA was hybridized to the array and labeled with Cy3-streptavidin (Amersham Bioscience). Fluorescence signals were measured on the BeadStation 500 System (Illumina) on the array.

5. Statistical analysis of gene expression data

The probe intensity from the array was converted to a log2 value. The log2 values (intensities) were standardized by a quantile normalization method. The probes were labeled with Lumi 1.8.3.

To identify DEGs, integrative statistical hypothesis testing was performed. Students t test and log2 median ratio test were performed on all genes to calculate T value and log2 median ratios; The empirical distribution of the null hypothesis was evaluated by performing all possible combinations of random permutations of samples. The Gaussian kernel density estimation method was then applied to the T value and the log2 median ratio obtained from the arbitrary substitution group.

The P value of each gene for each experiment was calculated by two-tailed test with an empirical null distribution, and the P values from both experiments were combined by the Stouffers method. Calculate the FDRs for the combined P values in the Storey method; The first set of DEGs was identified as a gene (FDR &lt; 0.05).

Of the first set of DEGs with FDR ≤ 0.05, the DEGs with an absolute log2 expression variation greater than the nodal distribution of cutoff (0.55; 1.46-fold change), log2 expression changes of mean 0.5th and 99.5th percentiles The final set was selected.

6. GO  Enrichment of biological processes ( Enrichment ) analysis

For GO enrichment analysis on the gene list, a list of genes used in each GO biological process analysis was obtained. For OXPHOS (oxidative phosphorylation), genes obtained from both the oxidative phosphorylation cycle terms (0006119) and the oxidative phosphorylated KEGG pathway (hsa00190) were used.

For other GO biological process analyzes, only the genes obtained from the corresponding GO periods were used. Using the lists of genes for GO biological process analysis, the P values were calculated as described in DAVID in individual clusters (FIG. 2B). We confirmed abundant GO biological processes in the genes in each cluster (P <0.05).

7. Potential retrograde signals TFs Confirmation of

To identify potential retrograde signal TFs, the retrograde signal TFs, 223,665 TF target interactions were collected.

Using the TF-target information for each TF, the number of targets in 12 clusters was counted and a statistically significant number of 72 targets were selected in the 12 DEG cluster (FDR <0.01): For TF, first, arbitrarily sampled as many as 100,000 times the same number of genes as DEGs in each cluster from a pool of genes spotted on the array, and the target number of TFs among the sample genes was counted; In each cluster, Gaussian kernel density estimation was used to evaluate the empirical distribution of the target number of TFs from any 100,000 samplings; Through empirical distributions, FDRs were calculated for the target number of TFs observed in the corresponding clusters using the Storey method.

This process yielded FDRs results for each TF in 12 clusters. The potential retrograde signal TF candidates were selected according to the following criteria: the number of DEGs in cluster i divided by the total number of 2404 DEGs in four different clusters (25% of 12 clusters) In cluster i (excluding 0), Ii is 1 if FDR <0.01. With this stringent criterion, reliable TF candidates were obtained resulting in a reduced false positives ratio.

8. siRNA injection

After siRNA synthesis, cells were transiently transfected using the OneDrop Microporator MP Kit (Invitrogen).

1 (RXRA; Thermo Scientific Dharmacon RNAi Technologies, L-003443-00-0005) × 10 5 cell suspension was re-suspended again in 100 ml of buffer and 100 nM siRNA was transfected into. After transfection, the cells were transferred to 6 well plates (Nunc) containing warm pre-warmed DMEM and maintained at 37 ° C and 5% CO ₂ for 72 hours. The amount of mRNA for specific transcription was monitored by qRT-PCR.

9. Immunocytochemistry ( Immunocytochemistry )

Cells were grown on a Lab-Tek II Chamber Slide (Nunc) and treated with RA for 24 hours. The cells were rinsed with phosphorylated-buffered saline (PBS), fixed in methanol for 20 minutes, and rinsed again with PBS. Cells were incubated with antibodies against RXRA (rabbit, Santa Cruz, sc-553) or PGC1a (mouse, Santa Cruz, sc-13067) and mitochondrial markers (mouse, Abcam, ab3298) [www.sciencesignaling.org/cgi/content/ full / 6/264 / rs4 / DC1 reference (table S11)] at 4 ° C overnight. Cells were rinsed with PBS and incubated for 1 hour at room temperature with secondary antibody: Alexa Fluor 555 donkey anti-rabbit immunoglobulin G (IgG) (Invitrogen, A31572) and IgG (Invitrogen, A21427). The concentration information is described in the following table.

For counter-staining of nuclei, cells were incubated with DAPI (1 mg / ml; Sigma-Aldrich) for 40 seconds. After washing with PBS, cover slips were mounted on glass slides with VECTASHIELD mounting medium (Vector Laboratories) and analyzed with LSM 710 confocal microscope (Carl Zeiss).

10. PLA ( Proximity ligation assay )

PLA was performed on each type of siburid cell without RA treatment and treatment to visualize the RXRA-PGC1a interaction. Cells were washed with cold PBS and incubated overnight at 4 ° C with antibodies against RXRA (rabbit, Santa Cruz, sc-553) or PGC1a (mouse, Santa Cruz, sc-13067) (Table 2). Proximity ligation was performed with Duolink detection kit (Olink Bioscience). During the detection reaction, Hoechst staining was performed to visualize the nucleus.

Samples were mounted with a VECTASHIELD mounting medium (Vector Laboratories) and analyzed with an LSM 710 confocal microscope. The number of in situ PLA signals per cell was counted by semi-automated imaging with BlobFinderV3.0.

11. FRB  analysis

After plasmid DNA synthesis, cells were transiently transfected using the OneDrop Microporator MP Kit (Invitrogen). For qRT-PCR, illustration was 5 × 10 ⁵ cells re-suspended in 100 ml of resuspension buffer, and transfected with plasmids containing 10mg FKBP and FRB-RXRA.

For PLA analysis, 1 × 10 ⁴ resuspended glass with fresh DMEM pre-warmed in 10 ml of resuspension buffer, the cells placed in a well of the bottom dish (glass-bottomed dish (Nunc) ) containing FKBP-RXRA and FRB after transfection with plasmids each of which 1 mg was maintained at 37 ° C and 5% CO ₂ for 72 hours. The cells were then incubated with 1 nM rapamycin for 20 minutes at room temperature, and the interaction between RXRA and PGC1a was measured by PLA analysis. The mRNA abundance of the specific transcriptional activity was monitored by qRT-PCR.

12. RA , Ascorbic acid, JNK  Exposure of cells to inhibitor or mitochondrial toxin

Cells were grown in high glucose DMEM supplemented with 10% FBS and incubated at 37 ° C and 5% CO ₂ . The cells were treated with 10 mM 9-cis-RA (Sigma, R4643) for 24 hours.

The cells were then exposed to 5 mM ascorbic acid (Sigma-Aldrich, A4403) or 10 mM SP600125 (JNK inhibitor, Sigma-Aldrich, S5567) for 1 hour. Cells exposed to ascorbic acid or SP600125 and mitochondrial toxin were treated with 5 mM ascorbic acid or 10 mM SP600125 for 1 hour and then treated with 3 mM rotenone (Sigma-Aldrich, R8875), 15 mM antimycin A (Sigma-Aldrich, A8674), or 12 mM oligomycin (Sigma-Aldrich, O4876) were added and the cells were incubated at 37 ° C and 5% CO ₂ for 18 hours. The cells were harvested and subjected to Western blot and qRT-PCR

13. Quantitative RT -PCR ( Quantitative RT - PCR )

RNA was isolated with TRIzol reagent (Invitrogen Corp.), pooled, and first strand complementary DNA synthesis was performed using a Reverse Transcription System (Takara) . The qPCR primer sequences were designed by Primer Express (Applied Biosystems; Table 3).

QRT-PCR was performed with the 7900HT Fast Real-Time PCR System (Applied Biosystems). The threshold cycle number and reaction efficiency were determined using software on a GeneAmp 7900HT Sequence Detection System (PerkinElmer Corp.).

The following profiles were used: 10 min at 95 ° C, then 15 cycles at 95 ° C and 40 cycles at 60 ° C for 60 s. Expression was normalized by GAPDH.

Western blot analysis Cells lysates (50 mg) were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies. After washing, the blots were incubated with HRP (horseradish peroxidase) -binding secondary antibodies.

The blots were developed into SuperSignal West Pico Chemiluminescent Substrate (Pierce). A list of primary antibodies is set forth in Table 2 above. Measurement of intracellular ROS cells was performed by treating 2 ', 7'-dichlorohydrofluorescein diacetate (Invitrogen) in DMEM medium supplemented with 10% FBS for 15 minutes. Fluorescent materials were detected using a VICTOR3 Multilabel Plate Reader (PerkinElmer) at excitation wavelength 485 nm and emission wavelength 535 nm.

14. Statistical analysis

All data were normalized to mean values of replica data in untreated W cells (control) and expressed as mean +/- SD. Two-tailed Students t test was used for comparison of two groups (eg, siRNA treatment and untreated samples). Also, one-way of Bonferroni correction as a post hoc test for comparison between multiple groups of one variable (e.g., O2 consumption in W, H and M cells, ATP content, mRNA, and RXRA protein content) ANOVA was used.

Finally, a two-way ANOVA of Bonferroni correction was used as a post hoc test to compare multiple groups of two variables (for example, load and processing). In all cases, a criterion of significance was set at P < 0.05.

Example 1: Identification of mitochondrial dysfunctions in sibrid cells with mt3243 mutation

To identify the retrograde signaling pathway induced by mitochondrial dysfunction by the mt3243 mutation, three sibridate cells were generated using mitochondria-mediated transformation.

mtDNA deficient cells were fused with mtDNAs in the mt3243 mutation, which was isolated from platelets in diabetic patients with sensorineural hearing loss. The ratio of mutant mtDNA and wild type of the sibridate cells was determined by restriction fragment length polymorphism (PCR) analysis using an mtDNA probe (Fig. 1A).

Three types of Sibrid cells were isolated: W cells had wild-type mtDNA (3243A homoplasm), H cells were mutated and wild type mtDNA (3243A / G heteroplasmy) with 70% mtDNA containing 3243G ), And M cells have only mutated mtDNA (3243G homoplasmy).

In addition, O2 consumption and relative adenosine 5'-triphosphate (ATP) content were measured in three types of siburide cells to confirm mitochondrial dysfunction due to mt3243 mutation.

In H and M cells, both O2 consumption and ATP content were significantly reduced compared to W cells (Fig. 1, B and C), which is consistent with previous studies.

In addition, the amount of representative enzymes of the five OXPHOS complexes was tested (Fig. 1D). In H and M cells, the amount of the three enzymes decreased. In addition, the activity of cytochrome c oxidase in OXPHOS complex IV decreased in H and M cells (Fig. 1E). Three data showed that the mt3243 mutation induced OXPHOS dysfunctions by decreasing both the amount and activity of the OXPHOS enzyme.

Example 2: Change in mRNA amount due to mt3243 mutation

OXPHOS anomalies caused by the mt3243 mutation activate the inversed signaling pathway, resulting in changes in nuclear gene expression. Gene expression profiling of three types of Sibrid cells (W, H, and M cells) was performed and the amount of mRNA between different types was compared (H vs W, M vs W, M vs H).

In at least three comparisons, we identified 2404 differentially expressed genes (DEGs) with a false discovery rate (FDR) of less than 0.05 and a multiple of 1.46 or more (Figure 2A).

These DEGs code for proteins involved in cellular processes previously known to be affected by the mt3243 mutation (Figure 3A). Of the 56 genes known to be affected by the mt3243 mutation, 19 genes shared 2404 DEGs (P <10-6; FIG. 3B).

A large number of DEGs were identified from a comparison of M vs W and M vs H (1687 and 1350 DEGs respectively), but only 540 DEGs were confirmed from a comparison of H vs W.

These data indicate that gene expression can be significantly altered by a single point mutation in a mutation load-dependent manner.

The mt3243 mutation primarily affects OXPHOS, allowing subsequent disturbing of multiple cellular processes. To systematically investigate these processes, 2404 DEGs were grouped into 26 clusters based on different expression patterns (Table 4 and S3).

Of the 26 clusters, 12 clusters were more focused (Figure 2B), with each cluster containing at least 25 genes (1% of the total number of DEGs; Table 4).

Cellular processes expressed by genes were identified by performing enrichment analysis of DAVID software using Gene Ontology (GO) Biological Processes in each individual cluster [Fig. 2C and www.sciencesignaling.org/cgi/content/ full / 6/264 / rs4 / DC1 reference (table S4)].

These analyzes indicate that genes affected by the mt3243 mutation are involved in mitochondrial processes (mitochondrial migration, mitochondrial organization, OXPHOS, response to ROS and oxidative stress, nitrogen compound biosynthesis processes), protein homeostasis- It is primarily involved in processes related to biogenesis, protein transport and protein ubiquitination, and cell proliferation-related processes such as cell proliferation regulation, DNA repair, apoptosis regulatory transcription, chromatin assembly and degradation and RNA processing. .

These results have shown that mitochondrial dysfunction by single point (mt3243) mutations affects a broad spectrum of cellular processes that may reflect the activity of various retrograde signaling pathways that alter these cellular processes.

Example  3: mt3243  By mutation mRNA Change of quantity  Potential to regulate TFs

Several signal paths and their downstream TFs are involved in mitochondrial retrograde signal conditioning.

To investigate the retrograde signaling pathways activated by the mt3243 mutation, potential downstream TFs that could contribute to changes in gene expression in the selected 12 clusters were identified (FIG. 2B).

For 799 TFs, 223,665 TF-target interactions from MetaCore and 5 protein-DNA interactome databases (Table 1) were used to count the number of their targets in individual DEG clusters.

Thereafter, 72 TFs with a significant number of targets (FDR < 0.01) in 12 clusters were selected as TF candidates that could participate in the retrograde signal (Fig. 2D and www.sciencesignaling.org/cgi/content/full / 6/264 / rs4 / DC1 reference (table S6A)]. A large number of candidates (72 TFs) suggest that mitochondrial dysfunction due to the mt3243 mutation induces a variety of retrograde signaling pathways. Of the 72 TFs, 26 are involved in known retrograde signaling paths [see www.sciencesignaling.org/cgi/content/full/6/264/rs4/DC1 (table S6B)].

In addition, the amount of mRNA of 15 out of 72 TFs was increased or decreased in cells with higher mutation load (H vs W, M vs W, or M vs H) compared to cells with lower mutation loading: NR2F2 MRNA levels, ETS1, FOSB, FOS, E2F6, and MYC were increased. And the amounts of HSF1, RXRA, MAZ, ELK1, ELF1, CREB1, EGR1, FOSL1, and JUNB are reduced [see www.sciencesignaling.org/cgi/content/full/6/264/rs4/DC1 (table S6B)].

Of the 15 TFs, 7 (FOSB, FOS, MYC, CREB1, EGR1, FOSL1, and JUNB) are reported to be involved in mitochondrial retrograde signals. [See Figure 4 and www.sciencesignaling.org/cgi/content/full/6/264/rs4/DC1 (table S6B)].

Example 4: Reverse-signaling TFs regulating the amount of mRNA of the OXPHOS gene

Changes in mRNA levels of genes encoding OXPHOS enzymes are a well-known feature over mitochondrial function. Depending on the severity and type of mitochondrial function, the amount of mRNA of the OXPHOS gene is increased or decreased.

Of the 46 OXPHOS genes that were altered by the presence of the mt3243 mutation, 44 mRNA levels were reduced.

The reduced amount of mRNA results in a decrease in the amount of nuclear-coded OXPHOS enzyme protein (Fig. 1D), which is provided as a positive feedback loop, resulting in a decrease in the amount of mitochondria-coded OXPHOS enzymes according to the mt3243 mutation Or more.

Therefore, the mitochondrial retrograde signal, which results in a decrease in the amount of mRNA of the nuclear-coded OXPHOS gene causing OXPHOS anomalies, is induced by the presence of the mt3243 mutation.

Enrichment analysis of Gene Ontology Biological Processes (Fig. 2C) showed that OXPHOS-related genes were most prominent in cluster 17 (C17) among 12 clusters, indicating that mRNA (Fig. 2B).

Of the 46 OXPHOS genes (Table S7) that were altered by the presence of the mt3243 mutation, 36 genes belonged to C17. To identify the retrograde signaling pathways that alter the expression of genes in C17, we identified 52 candidate TFs with a significant number of targets at C17 (FDR <0.01; www.sciencesignaling.org/cgi/content/full/6/264/rs4/DC1 reference (table S6A) and Figure 2D].

10 TFs (AR, ESR1, ESRRA, HNF4A, NKFB1, NRF1, REL, RELA, RXRA, and YY1) were identified as PGC1a (PPARGC1A) [www.sciencesignaling.org/cgi/content/full/6/264/rs4 / DC1 reference (table S6B)], major regulators of OXPHOS and mitochondrial production (biogenesis). These data suggest that the PGC1a-TF complex contributes to the reduction of the mRNA level of the gene at C17.

To ascertain whether the processes represented by the genes in C17 were regulated by PGC1a-interacting TFs (Fig. 2C), a network model was constructed using PGC1a-interacting TFs and their target genes in C17 5A). The network showed that these 10 TFs mainly regulate C17 target genes classified as mitochondrial processes and translations, including OXPHOS.

Of the 10 PGC1a-interacting TFs, only genes encoding RXRA, the C17 gene, showed reduced expression in the presence of the mt3243 mutation. Ligand-stimulated RXRA selects PGC1a for the RA response elements in the promoter of the target genes, and PGC1a as a coactivator functions to promote the transcriptional activity of RXRA. 9-cis-RA induces the expression of mitochondrial-associated genes.

Thus, we hypothesized that the reduced amount of RXRA reduced recruitment of PGC1a, leading to decreased expression of OXPHOS genes and enhanced mitochondrial dysfunction (FIG. 5B).

To confirm the gene expression data, qRT-PCR (quantitative reverse transcriptase PCR) analysis was performed on 19 OXPHOS genes and 5 and transcription-and-mitochondrial translation related genes in the network model (Fig. 5) By comparison, it was shown that the amount of their mRNA was decreased in M cells that are not H cells, consistent with different expression patterns of C17.

Example  5: RXRA Of increased transcriptional activity OXPHOS  Restoration of genes

To experimentally test whether the reduced amount of RXRA contributes to the reduction of OXPHOS gene expression, it was confirmed that the amount of RXRA protein and the amount of mRNA decreased in M cells (Fig. 6A).

To investigate the functional role of RXRA in the regulation of mRNA level of OXPHOS gene, treatment of H and M cells with RA reduced both O ₂ consumption and ATP content by the mt3243 mutation (Fig. 1 B and C) Lt; RTI ID = 0.0 &gt; (FIG. 6B). &Lt; / RTI &gt; However, recovery of O ₂ consumption did not reach the control of untreated W cells. In addition, the amount of multiple OXPHOS enzymes was restored to close to W cells (Fig. 6C).

For the 564 genes deduced in the M cells by the mt3243 mutation in C17 (FIG. 2B), the 163 genes recovered the mRNA level by the RA treatment of M cells (FDR <0.05) (Fig. 7A and www.sciencesignaling.org/) cgi / content / full / 6/264 / rs4 / DC1 reference (table S8)]. Concentration analysis of the GO Biological Processes of 163 genes showed that they were mainly associated with OXPHOS, mitochondrial translocation and oxidative stress response (P < 0.05) (Fig. 7B), suggesting that among mitochondrial processes expressed by genes in C17 Which means that these processes are restored (FIG. 5A).

Of the 36 OXPHOS genes at C17, 10 genes were recovered in the amount of mRNA.

To demonstrate that OXPHOS genes are regulated by RXRA, RXRA was knocked down with short interfering RNA (siRNA) in W cells (Figure 8) and the amount of mRNA in 10 OXPHOS genes was measured.

10 genes was reduced by RXRA knockdown, which means that 10 OXPHOS genes are regulated by RXRA (Figure 7C). Therefore, the reduction of RXRA contributed to the decrease of mRNA level of OXPHOS genes.

Example  6: RXRA - PGC1a  Due to increased interaction OXPHOS  Recovery of gene expression

To determine whether reduction of RXRA reduced the amount of RXRAPGC1a complex and inhibited the expression of OXPHOS gene, it was examined whether the induction of RXRA-PGC1a interaction restored the amount of mRNA of OXPHOS gene.

RXRA-PGC1a interactions were measured in the siblide cells after RA treatment using PLA (proximity ligation assay) analysis

In M cells, RA treatment significantly increased RXRA-PGC1a interaction in the nucleus (Fig. 9), whereas RA treatment did not significantly change the amount of interaction in W or H cells, indicating that the mRNA level of OXPHOS genes at C17 Lt; / RTI &gt; cells (Fig. 2B). This means that it acts as a sensor above the mitochondrial function by mutation.

FK506 binding protein (FKBP) -RXRA and FKBP-rapamycin binding domain (FRB) after wild type RXRA silencing using siRNA in M cells to test whether RXRA-PGC1a interaction regulates the amount of mRNA of OXPHOS gene ) M cells were transfected with the fusion vector. The FRB was positioned in the plasma membrane. Rapamycin treatment induces an interaction between FKBP-RXRA and FRB, whereby FKBP-RXRA migrates to the plasma membrane and reduces the interaction between FKBP-RXRA and PGC1a at the nucleus.

When the interaction between FKBP-RXRA and PGC1a is reduced by rapamycin treatment, RA treatment inhibits the recovery of the amount of mRNA of the OXPHOS gene in M cells (Fig. 10). RA-derived interaction data and sequestration data were consistent with a model in which RA treatment contributes to RXRA-PGC1a complex increase and recovery of OXPHOS gene expression in M cells.

Example  7: ROS Activated by JNK Through RXRA of mRNA  Positive phonological regulation

These data indicate that a decrease in RXRA reduces the amount of mRNA of OXPHOS genes. Various mitochondrial retrograde signaling pathways initiated by mitochondrial dysfunction include changes in Ca2 +, ROS, and AMP. Among these mediators, ROS reduces both mRNA and protein levels of RXRA in neonatal rat cardiac myocytes.

Thus, we measured the amount of ROS in three types of Sibrid cells. Relative amounts of ROS were increased in H and M cells (Figure 11A). To determine whether increased ROS reduced the amount of RXRA, the amount of mRNA and protein of RXRA was measured after treating the cells with ascorbic acid, an oxidizing agent that reduces the amount of ROS in the cells (Fig. 11B). In M cells, both mRNA and protein amounts of RXRA were restored and the amount of protein exceeded that of control W cells. This means that increased ROS contributes to reducing the amount of RXRA.

In contrast, in H cells that did not differ significantly from the W cells in the amount of RXRA protein (Fig. 6A and Fig. 11B), there was no significant change in the amount of RXRA mRNA in the reaction of ascorbic acid treatment (Figs. 11B and 12A ). This suggests that there may be a limit amount of ROS that affects the amount of RXRA (FIG. 11B).

In neonatal rat cardiac myocytes, the decrease in RXRA amount by ROS is mediated by a pathway involving JNK. Therefore, the effect of the JNK inhibitor, SP600125 (anthrapyrazolone), on the amount of RXRA was investigated.

In M cells, inhibition of JNK activity restored the amount of RXRA mRNA relative to the amount in control W cells and increased the amount of RXRA protein. This means that JNK activity contributes to the reduction of the amount of RXRA (FIG. 11C and FIG. 12B). Consistent with the lack of effect of antioxidants, inhibition of JNK activity did not alter the amount of RXRA mRNA or protein in H cells. Consistent with ROS and JNK participating in the same pathway in the reduction of RXRA, W cells exposed to ROS hydrogen peroxide showed reduced amounts of RXRA transcription and protein, and this reduction was partially alleviated by inhibition of JNK activity 12C).

Three OXPHOS inhibitors that interfere with the activity of OXPHOS complexes I, III, and V, respectively, that cause OXPHOS anomalies to determine if OXPHOS anomalies due to mitochondrial toxicity reduce the amount of RXRA and include the pathway of ROS and JNK - W cells were treated with either rotenone, antimycin A, or oligomycin.

Exposing W cells to the OXPHOS inhibitor results in a marked reduction of RXRA mRNA and decreases the amount of protein. This effect prevents the reduction of ROS or JNK inhibitors by ascorbic acid (Figs. 13A and 12D). Therefore, these data indicate that the amount of RXRA mRNA is negatively regulated by ROS-activated JNK, suggesting that it is induced by mitochondrial dysfunction due to the presence of the mt3243 mutation or the addition of an OXPHOS inhibitor.

Example  8 : RXRA  Regulation of translation-related genes by retrograde signaling pathways

Processes classified as translations are cases affected by the presence of the mt3243 mutation (Figure 2C). Of the 2404 DEGs, 92 genes are classified as being involved in translation.

92 translation-related genes 1: increased mRNA abundance,
0: not changed,
-1: decreased mRNA abundance H / W
M / W M / H Entrez
ID Symbol Cluster H
/ W M
/ W M
/ H FDR log2-Fold change FDR log2-Fold change FDR log2-Fold change 9801 MRPL19 2 One One 0 0.004291693 0.662381354 0.031303397 0.593059111 0.76230345 -0.069322243 2107 ETF1 5 One 0 0 0.000131037 0.807148007 0.007232159 0.50691651 0.192000117 -0.300231498 79631 EFTUD1 6 One 0 -One 0.00154101 0.665376057 0.99179007 0.027847224 0.000317706 -0.637528833 6176 RPLP1 9 One -One -One 0.007911044 0.630437527 0.027871178 -0.800275668 0.001604868 -1.430713195 1615 DARS 10 0 One One 0.649594757 -0.078615482 0.005270054 0.794721996 0.003110133 0.873337478 8661 EIF3A 10 0 One One 0.539879713 0.123693326 0.007715468 0.811029282 0.011764703 0.687335956 57129 MRPL47 10 0 One One 0.878952295 0.020729881 2.43E-05 0.9030789 0.000303727 0.882349019 64432 MRPS25 10 0 One One 0.393560659 0.119449476 0.000557404 0.734467932 0.001286852 0.615018457 4677 NARS 10 0 One One 0.799044088 0.099856533 0.004575041 0.830999985 0.003958787 0.731143452 8893 EIF2B5 11 0 One 0 0.206534904 0.151791569 0.001976779 0.558912374 0.011699535 0.407120805 1965 EIF2S1 11 0 One 0 0.025957189 0.428236093 0.001541565 0.669407145 0.044825703 0.241171052 10480 EIF3M 11 0 One 0 0.117183921 0.108283311 0.009552888 0.579868029 0.021792276 0.471584718 51253 MRPL37 11 0 One 0 0.681567556 0.129824245 0.003793238 0.577833788 0.011011404 0.448009543 28977 MRPL42 11 0 One 0 0.249849819 0.105330359 0.004351007 0.571201368 0.007315762 0.465871009 84545 MRPL43 11 0 One 0 0.051037409 0.297977032 0.001133498 0.655136907 0.043185514 0.357159876 51021 MRPS16 11 0 One 0 0.001352794 0.462803227 0.000515331 0.677858872 0.099805748 0.215055645 54516 MTRF1L 11 0 One 0 0.754199755 0.124015499 0.030616016 0.630365952 0.038172723 0.506350452 5859 QARS 11 0 One 0 0.014308488 0.389857378 0.002365371 0.5921183 0.076374648 0.202260922 5935 RBM3 11 0 One 0 0.037039777 0.525491863 0.009168814 0.765509628 0.294092434 0.240017765 6228 RPS23 11 0 One 0 0.020226371 0.377680289 0.005004439 0.628671703 0.023544371 0.250991414 51187 RSL24D1 11 0 One 0 0.004248701 0.488321827 0.0056558 0.642268523 0.31051556 0.153946695 6897 TARS 11 0 One 0 0.206015236 0.186520893 0.019367757 0.764129998 0.052305968 0.577609105 83939 EIF2A 13 0 0 One 0.055678238 -0.204650681 0.072312615 0.459635833 0.014797958 0.664286514 3646 EIF3E 13 0 0 One 0.196106715 -0.12799821 0.024890985 0.49987558 0.007752787 0.62787379 8668 EIF3I 13 0 0 One 0.001419219 -0.358421225 0.421816653 0.19321474 0.013778007 0.551635965 26156 RSL1D1 13 0 0 One 0.002038821 -0.398782109 0.063340069 0.389064008 0.003549494 0.787846117 27335 EIF3K 14 0 0 -One 0.14666588 0.277284094 0.042817084 -0.503383635 0.007373729 -0.780667729 29093 MRPL22 14 0 0 -One 0.171797351 0.137249484 0.063992555 -0.460454839 0.020173124 -0.597704323 51073 MRPL4 14 0 0 -One 0.181108606 0.265534007 0.110020248 -0.386116798 0.030113531 -0.651650805 64963 MRPS11 14 0 0 -One 0.000883284 0.451467419 0.098970941 -0.31889519 0.001293903 -0.770362609 6183 MRPS12 14 0 0 -One 0.128559659 0.151451104 0.089340462 -0.561473152 0.025939992 -0.712924256 51373 MRPS17 14 0 0 -One 0.017529407 0.306204778 0.095114379 -0.57609504 0.008765991 -0.882299818 64951 MRPS24 14 0 0 -One 0.312736583 0.09985579 0.054190052 -0.660302616 0.025140499 -0.760158406 6168 RPL37A 14 0 0 -One 0.149395728 0.222415968 0.072091885 -0.417864162 0.006311695 -0.64028013 6208 RPS14 14 0 0 -One 0.260230076 0.058860909 0.067229229 -0.553635887 0.034618042 -0.612496797 7407 VARS 14 0 0 -One 0.098621523 0.171833536 0.026874399 -0.390316969 0.003450979 -0.562150506 10643 IGF2BP3 16 0 -One 0 0.001960683 -0.357890628 0.000546086 -0.677733365 0.021811942 -0.319842737 64960 MRPS15 16 0 -One 0 0.065941771 -0.233079572 0.007344083 -0.764209761 0.027290861 -0.531130189 6154 RPL26 16 0 -One 0 0.185895595 -0.161020898 0.007354806 -0.617598643 0.024557059 -0.456577745 6155 RPL27 16 0 -One 0 0.350766271 -0.124333399 0.015286028 -0.628458066 0.02181563 -0.504124667 6166 RPL36AL 16 0 -One 0 0.929195148 -0.04332669 0.045381252 -0.552231687 0.040649891 -0.508904997 80325 ABTB1 17 0 -One -One 0.667017958 0.136687003 0.011209338 -0.559014484 0.002174882 -0.695701487 391356 C2orf79 17 0 -One -One 0.219586516 0.136064388 0.025636039 -0.776518714 0.010815402 -0.912583102 1725 DHPS 17 0 -One -One 0.840761913 -0.022616981 0.015866808 -0.621431083 0.019453888 -0.598814101 2197 FAU 17 0 -One -One 0.973243927 -0.006101568 0.018000127 -0.780187449 0.014338339 -0.774085882 440737 LOC440737 17 0 -One -One 0.395564204 0.086490797 0.010218907 -0.980310981 0.00430854 -1.066801778 643949 LOC643949 17 0 -One -One 0.021638236 0.335044129 0.034576266 -0.851432858 0.00619576 -1.186476987 648343 LOC648343 17 0 -One -One 0.999711331 -0.000336264 0.025209697 -0.943687994 0.020696058 -0.94335173 653773 LOC653773 17 0 -One -One 0.546774001 0.08719095 0.027228395 -0.700064052 0.011724517 -0.787255001 6182 MRPL12 17 0 -One -One 0.855876987 0.103120658 0.011146274 -0.643815899 0.006620585 -0.746936557 64928 MRPL14 17 0 -One -One 0.886107512 0.015793659 0.007390047 -1.020553174 0.006233991 -1.036346833 51069 MRPL2 17 0 -One -One 0.66738549 0.101482508 0.018911119 -0.615400572 0.010428578 -0.71688308 219927 MRPL21 17 0 -One -One 0.10371143 0.170713931 0.019190311 -0.664342176 0.004338176 -0.835056108 6150 MRPL23 17 0 -One -One 0.139003942 0.196025437 0.006103751 -0.963835888 0.001855226 -1.159861325 51264 MRPL27 17 0 -One -One 0.412395852 0.106093743 0.02176335 -0.729242463 0.00834398 -0.835336206 9553 MRPL33 17 0 -One -One 0.272300235 -0.104078439 0.00066842 -1.253108571 0.001246402 -1.149030133 64981 MRPL34 17 0 -One -One 0.889732281 0.020344455 0.03028033 -0.738275741 0.019664581 -0.758620196 64979 MRPL36 17 0 -One -One 0.065717081 -0.190506811 0.004760419 -0.954961399 0.010345443 -0.764454588 64975 MRPL41 17 0 -One -One 0.323526324 0.157336625 0.005511898 -1.128958434 0.002361706 -1.286295059 51258 MRPL51 17 0 -One -One 0.067358245 -0.123355847 0.008210309 -0.908103622 0.012589257 -0.784747774 128308 MRPL55 17 0 -One -One 0.925147434 -0.036155348 0.032019253 -0.723237203 0.025562217 -0.687081855 51023 MRPS18C 17 0 -One -One 0.135926826 -0.138802926 0.007519118 -0.96180026 0.011360372 -0.822997334 54460 MRPS21 17 0 -One -One 0.186449281 0.152120414 0.023331572 -0.769515636 0.007867581 -0.92163605 51121 RPL26L1 17 0 -One -One 0.504049107 0.081286476 0.007139124 -0.770070629 0.00315367 -0.851357106 6159 RPL29 17 0 -One -One 0.049518209 -0.245441161 0.004768024 -0.920812366 0.031620273 -0.675371206 6156 RPL30 17 0 -One -One 0.752402254 0.042681235 0.018496284 -0.627454348 0.01021793 -0.670135582 6164 RPL34 17 0 -One -One 0.00059296 -0.530640448 0.000540269 -1.529060978 0.009842611 -0.99842053 6165 RPL35A 17 0 -One -One 0.51932045 0.074387019 0.009510733 -0.816519641 0.004430201 -0.89090666 25873 RPL36 17 0 -One -One 0.47561439 -0.092842937 0.002754811 -1.412947902 0.004320482 -1.320104965 6173 RPL36A 17 0 -One -One 0.933150235 0.046080848 0.008534626 -1.054173027 0.005666599 -1.100253875 6169 RPL38 17 0 -One -One 0.73766522 -0.039467707 0.014129996 -0.664966219 0.01576782 -0.625498511 6170 RPL39 17 0 -One -One 0.9538771 0.029455896 0.022611124 -0.683884901 0.014408384 -0.713340797 116832 RPL39L 17 0 -One -One 0.750328175 0.021467918 0.011018181 -0.643825631 0.027015582 -0.665293549 6171 RPL41 17 0 -One -One 0.831501883 -0.127210563 0.009109837 -0.994182067 0.006967129 -0.866971504 6209 RPS15 17 0 -One -One 0.568362923 -0.122205447 0.006978975 -1.092841057 0.009188527 -0.97063561 6210 RPS15A 17 0 -One -One 0.020160932 0.233485384 0.040148395 -0.705455306 0.006671058 -0.938940689 6218 RPS17 17 0 -One -One 0.000903288 0.546878085 0.022452536 -0.673166043 0.000560228 -1.220044127 6227 RPS21 17 0 -One -One 0.185149677 -0.145545243 0.004488208 -1.103231226 0.007829369 -0.957685983 6229 RPS24 17 0 -One -One 0.790715274 -0.027211373 0.022794958 -0.597332061 0.025573517 -0.570120688 6231 RPS26 17 0 -One -One 0.445236486 -0.045899306 0.003911016 -1.15137663 0.005002532 -1.105477323 441502 RPS26P11 17 0 -One -One 0.353338164 -0.055813461 0.017322851 -0.698281551 0.016503324 -0.642468091 51065 RPS27L 17 0 -One -One 0.019611379 0.290409929 0.005691262 -1.108879167 0.00110383 -1.399289096 6199 RPS6KB2 17 0 -One -One 0.849293979 -0.009210131 0.002883977 -0.561754512 0.00322286 -0.552544381 6238 RRBP1 17 0 -One -One 0.829537828 0.021868913 0.000855651 -1.161215489 0.006148279 -1.183084401 54938 SARS2 17 0 -One -One 0.768887385 -0.058058812 0.009084117 -0.810095909 0.014447916 -0.752037097 833 CARS 22 -One 0 0 0.00174478 -0.581033295 0.059172133 -0.319050468 0.129742438 0.261982827 5188 PET112L 22 -One 0 0 0.002040472 -0.652360394 0.089567576 -0.372441984 0.02088355 0.27991841 6137 RPL13 22 -One 0 0 0.004570414 -0.577080888 0.0094971 -0.532122772 0.885533003 0.044958116 6133 RPL9 22 -One 0 0 0.006264831 -0.562098045 0.134354135 -0.337752315 0.049512197 0.224345729 10667 FARS2 24 -One -One One 1.24E-05 -1.178895769 0.002055403 -0.594126537 0.000259814 0.584769232 65008 MRPL1 25 -One -One 0 0.025952342 -0.572227715 0.04434426 -0.579608037 0.989214096 -0.007380323 51650 MRPS33 25 -One -One 0 8.15E-06 -1.441511468 2.34E-06 -1.929602246 0.005354298 -0.488090778

Of the 92 translation related genes, 44 was included in C17 and expression was decreased in M cells (Fig. 2B). The network model (Figure 5A) showed that translation was also regulated by the RXRA-PGC1a complex. Gene expression analysis in RA-treated M cells showed that 13 of the 44 translation-related genes at C17 were restored at the mRNA level by RA treatment (Fig. 7B).

In order to identify gene expression data, five genes from 13 genes were selected to prevent the induction of these genes by RA in rapamycin-induced screening assays for FKBP-RXRA and FRB expressing cells , Indicating that RXRA-PGC1a interaction is required for RA-mediated expression (Figure 13B). Therefore, all of these data were consistent with a model in which, in addition to the OXPHOS gene, RXRA-PGC1a modulates nuclear-coded genes involved in cytosol and mitochondrial translation. And this was mediated by an activated pathway in response to mitochondrial dysfunction associated with the presence of the mt3243 mutation, including ROS-mediated activity of JNK (Figure 14).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (14)

A composition for preventing and treating a mitochondrial dysfunction-related disorder, which comprises retinoic acid (RA) as an active ingredient as an expression or activity promoter of RXRα,
Characterized in that the mitochondrial dysfunction is caused by a mutation (mt3243) from the nucleotide sequence A to G at position 3243 of mitochondrial DNA,
Wherein said mitochondrial dysfunctional disorder is any disease selected from the group consisting of obesity, insulin resistance, fatty liver, liver fibrosis, hypertension, bipolar disorder, arteriosclerosis, liver disease, cardiovascular disease and dementia A pharmaceutical composition. delete The method according to claim 1,
Wherein the RXRa interacts with a transcription factor (PGC1a) (peroxisome proliferator-activated receptor, coactivator 1 a) in mitochondria. delete delete delete A method for restoring a mitochondrial dysfunction-related disorder through promotion of expression or activity of RXR [alpha]
The expression or activity promotion of RXR [alpha] is performed by treating retinoic acid (RA)
The expression or activation promotion of RXR [alpha] is characterized by increasing the interaction between RXR [alpha] -PGC1a,
Wherein said mitochondrial dysfunctional disorder is any disease selected from the group consisting of obesity, insulin resistance, fatty liver, liver fibrosis, hypertension, bipolar disorder, arteriosclerosis, liver disease, cardiovascular disease and dementia (Except for the human mitochondria). delete delete 8. The method of claim 7,
Wherein the increased interaction between RXR [alpha] -GC1a promotes the expression of oxidative phosphorylation and translation related genes. delete delete delete delete

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