mtDNA Copy Number Research Articles

FAM43A coordinates mtDNA replication and mitochondrial biogenesis in response to mtDNA depletion.

Mitochondrial retrograde signaling (MRS) pathways relay the functional status of mitochondria to elicit homeostatic or adaptive changes in nuclear gene expression. Budding yeast have "intergenomic signaling" pathways that sense the amount of mitochondrial DNA (mtDNA) independently of oxidative phosphorylation (OXPHOS), the primary function of genes encoded by mtDNA. However, MRS pathways that sense the amount of mtDNA in mammalian cells remain poorly understood. We found that mtDNA-depleted IMR90 cells can sustain OXPHOS for a significant amount of time, providing a robust model system to interrogate human intergenomic signaling. We identified FAM43A, a largely uncharacterized protein, as a CHK2-dependent early responder to mtDNA depletion. Depletion of FAM43A activates a mitochondrial biogenesis program, resulting in an increase in mitochondrial mass and mtDNA copy number via CHK2-mediated upregulation of the p53R2 form of ribonucleotide reductase. We propose that FAM43A performs a checkpoint-like function to limit mitochondrial biogenesis and turnover under conditions of mtDNA depletion or replication stress.

Oocytes with impaired meiotic maturation contain increased mtDNA deletions.

Induction of meiotic competence is a major goal of the controlled ovarian stimulation used in ART. Do factors intrinsic to the oocyte contribute to oocyte maturation? Deletions in mtDNA accumulate in long-lived post mitotic tissues and are found in human oocytes. If oogenesis cleanses the germline of deleterious deletions in mtDNA, meiotically competent oocytes should contain lower levels of mtDNA deletions vs. meiotically arrested oocytes. We tested this hypothesis using a novel PCR assay for a deletion ratio in human oocytes derived from IVF. A real-time PCR assay was developed to measure total mtDNA copy number (mtDNACN) and mtDNA harboring the 5Kb "common deletion" to enable calculation of the mtDNA deletion ratio (mtDNADR) in 143 cultured oocytes. Kruskal-Wallis test was carried out to compare the total mtDNACN and the mtDNADR among oocytes which matured to metaphase II (MII) vs. oocytes arrested at GV or metaphase I (MI). 51.75% of oocytes reached MII, and 17% remained at MI. Mean mtDNADR in GV, MI and MII oocytes were 27.87%, 31.88% and 20.05%, respectively. The difference in deletion ratios between GV and MII and between MI and MII stages was statistically significant p < 0.001 and p = 0.034, respectively. Additionally, patient age was found to be positively correlated with time to Polar body extrusion (- 0.278 Pearson correlation). Oocytes with impaired meiotic maturation contain an increased load of mtDNA deletions. This is the first report of an association between the mtDNA deletion ratio and human oocyte maturation in vitro.

Epigenetic Threads of Neurodegeneration: TFAM's Intricate Role in Mitochondrial Transcription.

There is a myriad of activities that involve mitochondria that are crucial for maintaining cellular equilibrium and genetic stability. In the pathophysiology of neurodegenerative illnesses, mitochondrial transcription influences mitochondrial equilibrium, which in turn affects their biogenesis and integrity. Among the crucial proteins for keeping the genome in optimal repair is mitochondrial transcription factor A, more commonly termed TFAM. TFAM's non-specific DNA binding activity demonstrates its involvement in the control of mitochondrial DNA (mtDNA) transcription. The role of TFAM in controlling packing, stability, and replication when assessing the quantity of the mitochondrial genome is well recognised. Despite mounting evidence linking lower mtDNA copy numbers to various age-related diseases, the correlation between TFAM abundance and neurodegenerative disease remains insufficient. This review delves into the link between neurodegeneration and mitochondrial dysfunction caused by oxidative stress. Additionally, the article will go into detail about how TFAM controls mitochondrial transcription, which is responsible for encoding key components of the oxidative phosphorylation (OXPHOS) system.

Network pharmacology combined with experimental verification for exploring the potential mechanism of phellodendrine against depression

The anti-inflammatory effect of phellodendrine (PHE), derived from Phellodendri Chinensis Cortex, has been verified in previous studies. Major depressive disorder (MDD) is associated with immune dysregulation and inflammatory processes. This study aimed to explore the therapeutic effects of PHE on MDD through network pharmacology and experimental validation. Multiple databases were used to predict the targets of PHE and MDD. The intersection targets between PHE and MDD were obtained to identify as targets for PHE against MDD, followed by protein–protein interaction network, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Molecular docking was applied to further confirm the anti-MDD effects of PHE. The mitochondrial DNA (mtDNA) copy number, inflammatory cytokines and pathway-related mRNA expressions in PC12 cell were determined via quantitative PCR (qPCR) and enzyme-linked immunosorbent assay to verify our finding. Thirty-eight intersection targets were obtained between PHE and MDD. PHE exerted an anti-MDD effect by regulating SLC6A4, SLC6A3, SLC6A2, MAOA and other targets through serotonergic synapse, salivary secretion, dopaminergic synapse, and cAMP signalling pathway. In vitro, PHE induced an increment in mtDNA copy number compared with the CORT group. PHE affected the levels of IL6 and IL1β with different concentrations. The mRNA levels of CHRM1, HTR1A and key targets of the PI3K/Akt signalling pathway were also influenced. Our research reveals novel mechanisms underlying the anti-MDD effects of PHE through network pharmacology and experiments, which provides a new direction for the development of antidepressants.

Molecular Mechanisms and Therapeutic Potential of Mulberry Fruit Extract in High-Fat Diet-Induced Male Reproductive Dysfunction: A Comprehensive Review.

High-fat diet (HFD)-induced obesity represents a significant challenge to male reproductive health, affecting approximately 13% of the global adult population. This comprehensive review synthesizes current evidence regarding mulberry (Morus alba L.) fruit extract's therapeutic potential for HFD-induced male reproductive dysfunction. Through comprehensive analysis of the peer-reviewed literature from multiple databases (PubMed, Web of Science, Scopus, and Google Scholar; 2005-2024), we evaluated mulberry extract's effects on testicular morphology, spermatogenesis, sperm parameters, and the underlying molecular mechanisms. Mechanistic studies reveal that standardized mulberry extract mediates protective effects through multiple pathways: enhanced antioxidant enzyme activities (SOD: +45%, Catalase: +38%, GPx: +35%), reduced inflammatory markers (TNF-α: -64%, IL-6: -58%), and modulated NF-κB signaling (-42.3%). These effects are facilitated by mulberry's rich phytochemical profile, particularly anthocyanins (2.92-5.35 mg/g dry weight) and polyphenols (4.23-6.38 mg/g). The extract demonstrates particular efficacy in preserving seminiferous tubule integrity and maintaining blood-testis barrier function, with treated groups maintaining up to 85% of normal tubular architecture compared to HFD controls. Key molecular mechanisms include AMPK/SIRT1 pathway activation (2.3-fold increase), enhanced mitochondrial function (67% increase in mtDNA copy number), and epigenetic regulation of metabolic pathways. Temporal analysis indicates optimal therapeutic effects after 28 days of treatment, with initial improvements observable within 14 days. While current evidence is promising, limitations include predominant reliance on rodent models and lack of standardized extraction protocols. Future research priorities include well-designed human clinical trials, standardization of preparation methods, and investigation of potential synergistic effects with other therapeutic agents. This comprehensive review indicates that mulberry extract is a promising therapeutic candidate for obesity-related male infertility, warranting further clinical investigation.

The total extract of Abelmoschus manihot (L.) medic flowers (TEA) mediated Nrf2-TFAM signalling to regulate mitochondrial antioxidant mechanism

Skin, as the first line of defence of the human body, is exposed to dangers such as overheating substances, ultraviolet rays, and environmental pollutants, and the incidence of skin diseases is increasing annually. Oxidative stress plays a dominant role in most skin diseases. Abelmoschus manihot (L.) medic flower (TEA) is a traditional Chinese medicine widely used to treat injuries to the skin such as water and fire scalds. It has been reported that TEA has excellent antioxidant effects. In this study, we aimed to explore the antioxidant and mitochondrial protection effects of TEA in H2O2-mediated HaCaT cell damage. HaCaT cells were incubated with H2O2 to simulate oxidative stress in the skin. The effect of TEA on HaCaT cells was also evaluated. Cell morphology was observed via inverted microscopy, and cell viability was measured via the MTT reagent. The cells were stained with Hoechst 33,324 solution. Reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA) and ATP detection kits were used to detect the corresponding indicators. The mitochondrial membrane potential was detected by JC-1. RT-PCR was used to detect mRNA and mtDNA expression. The expression of the target protein was detected by Western blotting and immunofluorescence. H2O2 triggered oxidative damage in HaCaT cells, which manifested as apoptosis, increased ROS and MDA contents, and decreased SOD activity. H2O2 activates the KEAP1/Nrf2/NQO1 signalling pathway, which decreases the expression of the intracellular KEAP1 protein and slightly increases the expression of the Nrf2 and NQO1 proteins, further causing mitochondrial oxidative stress, resulting in changes in the mitochondrial membrane potential, a reduction in the mtDNA copy number, and decreased expression of the PGC-1α and TFAM proteins. In addition the expression of mitochondrial respiratory chain genes and proteins decreased. TEA promoted the expression of Nrf2 in HaCaT cells, activated the downstream antioxidant response, and alleviated the oxidative stress and mitochondrial damage caused by H2O2. ML385 is an Nrf2 inhibitor, under which the antioxidant and mitochondrial protective effects of TEA are inhibited. When TFAM was knocked down, the protective effect of TEA on mitochondria was also inhibited. TEA protects HaCaT cells from H2O2-induced oxidative damage and mitochondrial oxidative damage through the KEAP1/Nrf2/NQO1/PGC-1α/TFAM pathway.

PGC-1α Promotes Mitochondrial Biosynthesis and Energy Metabolism of Goose Fatty Liver

To investigate the functions of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the goose fatty liver, a total of 30 healthy 63-day-old male Landes geese were selected and randomly assigned to control group and overfeeding group. The overexpression or RNA interference assay of PGC-1α was performed in goose primary hepatocytes. Our data showed that the PGC-1α expression was increased in fatty liver. The abundance of mitochondrial biosynthesis-related and energy metabolism-related genes, including mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M), mitochondrial transcription factor B2 (TFB2M), nuclear respiratory factor 1 (NRF1), DNA topoisomerase I mitochondrial (TOP1MT), peroxisome proliferator-activated receptor gamma coactivator 1-beta (PGC-1β), sirtuin 3 (SIRT3), mitochondrially encoded cytochrome B (CYTB), and AMP-activated protein kinase alpha (AMPKα) were significantly increased in fatty liver. The abundance of TFAM, TFB1M, TFB2M, NRF1, and TOP1MT transcript was induced by PGC-1α overexpression, but inhibited by PGC-1α interference in primary hepatocytes. The mRNA expression levels of PGC-1β, SIRT3, SIRT5, CYTB, and AMPKα were significantly enhanced after PGC-1α overexpression. However, the mRNA expression levels of PGC-1β, SIRT5 and AMPKα were decreased after PGC-1α interference. Furthermore, we observed a significant increase in the mitochondrial DNA (mtDNA) copy number, the activity of mitochondrial respiratory chain complex Ⅳ (MRCC Ⅳ), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and the NAD+/NADH ratio in fatty liver. But the activity of MRCC Ⅴ, as well as the levels of ADP and ATP in fatty liver were reduced. Additionally, the mtDNA copy number, the activity of MRCC Ⅰ, MRCC Ⅲ-Ⅴ, SDH, and MDH, and NAD+/NADH ratio were enhanced by PGC-1α overexpression; Whereas the mtDNA copy number, the activity of MRCC Ⅰ, SDH, and MDH, and the ratio of NAD+/NADH were inhibited by PGC-1α interference. In conclusion, these findings suggest that PGC-1α improves mitochondrial biosynthesis and energy metabolism in goose fatty liver, which may be an adaptive mechanism for goose fatty liver to cope with steatosis.

Carvacrol acetate activated Nrf2 modulates mitophagy for the treatment of neurocyte oxidative stress induced by chlorpyrifos

This study explored the protective effect and potential mechanism of carvacrol acetate (CAA) on the oxidation of chlorpyrifos (CPF). A model of oxidative stimulus damage was established in Sprague–Dawley rats by subcutaneous injection of the CPF poison. PC12 cells were used to construct an oxidative injury model using CPF, and the protective effects and mechanism of action of CAA against CPF-induced oxidative damage were explored in vitro. The key role of Nuclear factor erythroid-2-related factor 2 (Nrf2) in alleviating CPF-induced damage via CAA was further confirmed by administering Nrf2 inhibitors to PC12 cells. Administration of CAA significantly enhanced the locomotor ability of the rats, alleviated neuronal pathological alterations, and increased the number of Nissl bodies, while increasing autophagic bodies. In vitro, CAA promoted cell survival and augmented the mitochondrial membrane potential. It decreased both intra- and extracellular levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), while markedly elevating mitochondrial DNA (mtDNA) copy number. Moreover, PC12 cells treated with Nrf2 inhibitors failed to exhibit any improvement in survival rate when treated with CAA after a toxic insult. Furthermore, ROS and MDA levels were not significantly reduced, SOD enzyme activity did not increase, and mitochondrial membrane potential and mtDNA copy number did not improve. Western blot analysis showed that the expression of Tfam, Beclin1, and LC3II/LC3I proteins in the CAA group decreased significantly after Nrf2 inhibition. These findings suggest that CAA modulates mitochondrial function and autophagy by regulating the Nrf2 signalling pathway to mitigate the toxic damage. Finally, the effect of the autophagy inhibitor, 3-MA, on PC12 cells suggests that CAA promotes mitophagy by participating in the Nrf2 pathway, thereby preventing CPF-induced oxidative stress damage.

Gestational exposure to carbon black nanoparticles triggered fetal growth restriction in mice: The mediation of inactivating autophagy-lysosomal degradation system in placental ferroptosis.

Carbon black nanoparticles (CBNPs) are ubiquitous in our daily ambient environment, either resulting from tobacco combustion or constituting the core of PM2.5. Despite the potential risk of trafficking CBNPs to the fetus, the underlying toxicity of nano-sized carbon black particles in the placenta remains unambiguous. Pregnant C57BL/6 mice received intratracheal instillation of 30nm or 120nm CBNPs. CBNPs deposited in the lungs could infiltrate the red blood cells, further cross into the placenta, and cause fetal growth restriction. Mechanistically, we proposed a two-hit hypothesis in placenta response to CBNPs. The first hit was that CBNPs caused mitochondrial damage, reflected in the reduced mitochondrial matrix, the excessive mitochondrial fission, and the decreased mitochondrial membrane potential and mtDNA copy number. The second hit was that CBNPs disrupted the autophagy-lysosomal degradation system, impeding the removal of dysfunctional mitochondria and resulting in ferroptosis. Ferrestatin-1, a ferroptosis inhibitor, and rapamycin, an autophagy promotor, reversed ferroptosis and further confirm our suspicion. The findings suggested that CBNPs-triggered double-hit evoked placental ferroptosis, leading to fetal growth restriction. The study raised concerns about the potential placental toxicity of CBNPs and its impact on the fetal adverse outcome, which may propose potential targets for interventions in placental damage.

Ocular post‐mortem analyses with histopathological and molecular assessments in leber hereditary optic neuropathy following AAV2 gene therapy

Aims/Purpose: To perform post‐mortem ocular analyses in two patients with Leber hereditary optic neuropathy (LHON) caused by the m.11778G &gt; A MT‐ND4 mutation who received lenadogene nolparvovec (AAV2‐ND4) gene therapy.Methods: Two patients with MT‐ND4 LHON received an intravitreal injection of lenadogene nolparvovec in one eye and a sham injection in the other during the RESCUE study. Post‐mortem histopathological and molecular (ddPCR) analyses were performed on laser‐captured retinal and optic nerve samples.Results: Within 2 months of gene therapy, both participants developed intraocular inflammation in the treated eye; both events resolved within months. Patient 1 died of acute alcohol toxicity and patient 2 of cardiac arrest. Patient 1 had severe optic atrophy and loss of retinal ganglion cells (RGCs), while patient 2 was a much milder case of LHON, with consistent preservation of RGCs and optic nerve axons, being only the temporal fibers lost. In patient 1, an angiocentric lymphocytic chronic inflammation was noted in the optic nerve head and temporal retina. No persistent inflammation was seen in patient 2. AAV2‐ND4 distribution in the treated eye was asymmetric in both patients, more prevalent in temporal RGCs. A much lower level of AAV2‐ND4 was detected in the retina of the untreated eye in both cases. Finally, mtDNA copy number was upregulated in both eyes of patient 2, possibly reflecting better preservation of RGCs and axons.Conclusions: This is the first postmortem study of human eyes injected unilaterally with AAV2‐based gene therapy. Gene transfection of RGCs was successful in both eyes despite unilateral administration. AAV2‐ND4 transfer seems to be facilitated by axonal preservation and may be confirmed, and further characterized by analysis of the eyes, nerves, optic chiasm and optic tracts from patient 2.

The role of mitochondrial DNA copy number in female infertility: a bidirectional two-sample Mendelian randomization study.

Previous studies on the impact of mitochondrial DNA (mtDNA) copy number on female infertility were limited and inconsistent. The causal relationship between mtDNA copy number and female infertility was evaluated using a bidirectional 2-sample Mendelian randomization (MR) method. Inverse variance weighted (IVW) method was applied for principal analysis, and MR-Egger, weighted median, simple mode, weighted mode method for secondary analyses. Sensitivity analysis was conducted using MR-PRESSO, MR-Egger, Cochran's Q, and leave-one-out tests. Two large-scale GWAS mtDNA copy number datasets were employed for testing and validation to ensure reliable results. According to the forward MR analysis, genetically predicted mtDNA copy number was not associated with premature ovarian failure (POF) (OR = 1.969, 95% CI 0.571-6.789; p = .283), polycystic ovary syndrome (PCOS) (OR = 0.821, 95% CI 0.314-2.142; p = .686), endometriosis (OR = 1.281, 95% CI 0.962-1.704; p = 0.090), or female infertility (OR = 0.966; 95% CI 0.744-1.253; p = .794) but was associated with intestinal endometriosis (OR = 7.528; 95% CI 1.654-34.262; p = .009) and adenomyosis (OR = 1.710; 95% CI 1.118-2.616; p = .013). Reverse MR studies did not reveal a correlation between female infertility and mtDNA copy number. Similar results were observed in the validation data. Our study suggested that there is no causal relationship between mtDNA copy number and female infertility, but there is a causal relationship between mtDNA copy number and intestinal endometriosis and adenomyosis. The genetic evidence provided by this study provides a new perspective for studying the impact of mtDNA copy number on female infertility.

Liver transplantation in a boy with TFAM mutation associated mtDNA depletion syndrome

AbstractMitochondrial transcription factor A (TFAM) deficiency may cause mtDNA depletion syndrome, which manifests as neonatal liver failure or primary ovarian insufficiency, hearing loss, seizures, and intellectual disability. Treatment focusing on symptomatic management, and the clinical prognosis remains poor. Here, we describe a novel case of TFAM mutation presenting with progressive neonatal cholestasis, hypoglycemia and abnormal amino acid profiling. The patient progressed to liver failure at 6 months of age but did not exhibit neurological involvement. No morphologic abnormalities were observed in muscle biopsy, while mtDNA copy number was reduced in comparison to age- and tissue-matched controls. After liver transplantation, liver biochemistries and blood amino acid profiling normalized three weeks later. Moreover, the boy was doing well post-transplant without any clinical concerns, and his development and neurological examination remain normal 33 months after liver transplantation. Our report suggests that liver transplantation appears to have a favorable profile in such patients.

Persistent Epstein-Barr Virus Infection of Epithelial Cells Leads to APOBEC3C Expression and Induces Mitochondrial DNA Mutations.

Upon infection with the virus, cells increase the expression of cytidine deaminase APOBEC3 family genes. This leads to the accumulation of C-to-T mutations in the replicating viral genome and suppresses viral propagation. In contrast, herpesviruses, including Epstein-Barr virus (EBV), express genes that counteract APOBEC3 during lytic infection. However, because viral resistance factors are not expressed during EBV latent infection, it is unknown how APOBEC3 functions during latent infection. We observed that in gastric epithelial cells persistently infected with EBV, the expression of APOBEC3 family genes increased, C-to-T mutations in the D-loop genome of mitochondrial DNA (mtDNA) increased, and mtDNA copy number decreased. By introducing and expressing individual APOBEC3 family genes, APOBEC3C was particularly expressed in the cytoplasm, increasing C-to-T mutations in mtDNA and decreasing mtDNA copy number. Furthermore, we confirmed that APOBEC3C co-localized with mitochondria in EBV-infected cells. Expression of the EBV latent gene LMP2A increased APOBEC3C expression. Conversely, APOBEC3C expression was reduced in LMP2A-deficient EBV-infected cells compared to wild-type EBV-infected cells. These results indicate that persistent infection of EBV in gastric epithelial cells reduces the number of mitochondria through mtDNA mutations induced by APOBEC3C expression.

The relationship between mitochondrial DNA haplotype and its copy number on body weight and morphological traits of Wuliangshan black-bone chickens.

Mitochondria play a pivotal role as carriers of genetic information through their circular DNA molecules. The rapid evolution of the D-loop region in mitochondria makes it an ideal molecular marker for exploring genetic differentiation among individuals within species and populations with close kinship. However, the influence of mtDNA D-loop region haplotypes and mtDNA copy numbers on phenotypic traits, particularly production traits in chickens, remains poorly understood. In this comprehensive study, we conducted D-loop region amplification and sequencing in the blood mitochondria of 232 female Wuliangshan black-bone chickens. Our investigation identified a total of 38 haplotypes, with a focus on 10 haplotypes that included more than five individuals. We meticulously analyzed the correlations between these haplotypes and a range of traits, encompassing body weight, tibial length, tibial circumference, body oblique length, chest width, and chest depth. The results unveiled significant disparities in specific tested traits across different haplotypes, indicating a tangible association between mtDNA haplotypes and traits in chickens. These findings underscore the potential impact of mitochondrial DNA variations on energy metabolism, ultimately leading to divergent chicken phenotypes. Furthermore, our examination revealed positive correlations between mtDNA copy numbers and tested traits for select haplotypes, while other haplotypes exhibited non-uniform relationships between traits and mtDNA copy numbers. In addition, phylogenetic analysis disclosed the involvement of two subspecies of red jungle chicken in the origin of Wuliangshan black-bone chickens. Consequently, our research contributes novel insights into mitochondrial genomic selection, augments comprehension of the roles played by haplotypes and mtDNA copy numbers in chicken population genetics and phylogenetic analysis, and furnishes fundamental data crucial for the preservation and provenance determination of black-bone chickens.

Unveiling the mystery: Investigating the debate surrounding mitochondrial DNA copy number and Sjögren syndrome using Mendelian randomization analysis.

Numerous studies have investigated the relationship between mitochondrial DNA (mtDNA) copy number and Sjögren syndrome (SS). However, the conclusions remain inconclusive, with conflicting findings. The genome-wide association study summary statistics for mtDNA copy number were obtained from 2 sources: a cohort of 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium and the UK Biobank, and a dataset of 395,718 UK Biobank participants. Additionally, we obtained 2 sets of genome-wide association study summary statistics for SS through datasets from FinnGen and the UK Biobank, involving a total of 809,836 participants. Furthermore, we conducted a two-sample bidirectional Mendelian randomization analysis, primarily utilizing the inverse variance weighted method, complemented by 4 other validation methods, to explore the association between mtDNA copy number and SS. Following our comprehensive investigation, no discernible causal relationship was identified between mtDNA copy number and SS in either the training or validation cohorts (inverse variance weighted, P > .05). Similarly, the reverse Mendelian randomization analysis yielded negative results (inverse variance weighted, P > .05). Furthermore, all analyses indicated an absence of horizontal pleiotropy or heterogeneity. Our analysis revealed no causal relationship between mtDNA copy number and SS.

КОРРЕКЦИЯ МИТОХОНДРИАЛЬНОЙ ДИСФУНКЦИИ ПРИ КОМБИНИРОВАННОМ ПРИМЕНЕНИИ ПРЕПАРАТОВ МЕТРОГИЛ И АИКАР В ЭКСПЕРИМЕНТАЛЬНОЙ МОДЕЛИ ПАРОДОНТИТА

Background: It is known that mitochondrial dysfunction is involved in the pathogenesis and progression of periodontitis, affecting oxidative stress and regulating inflammatory reactions. Therefore, in recent years, research on mitochondria-directed therapeutic approach to periodontal tissue diseases has been rapidly growing. AIM: Evaluate the effect of the drugs Metrogyl and Aicar on structural and functional disorders in the mitochondria of periodontal tissues in an experimental model of periodontitis in laboratory rats. Materials and methods: Male white Wistar rats weighing 221 ± 7.5 g at the age of 4 months were used in this work. The animals were divided by simple randomization into four groups, 10 animals in each: Group 1 – intact (control group); Group 2 – rats with modeled periodontitis; Group 3 – rats with modeled periodontitis and the use of Metrogyl; Group 4 – rats with modeled periodontitis and combined use of Metrogyl and Aicar. Experimental periodontitis (EP) in rats was modeled by the ligature method by suturing a polyfilament non-absorbable thread into the gum in the area of the lower incisors. The following molecular genetic and biochemical parameters were used: damage to nuclear DNA (nDNA) and mitochondrial DNA (mtDNA), mtDNA copy number and the level of heteroplasmy, as well as the levels of malondialdehyde (MDA) and reduced glutathione (GSH). Results: The data from this study showed that on the 14th day after ligation, animals in the periodontal tissue have an increased level of damage and mtDNA heteroplasmy, compared to the control group. The same animals showed a decrease in the GSH level, while the MDA level was increased compared to the control animals. In other groups of rats with EP, the modulation of these damages was assessed using Metrogyl, as well as the combined use of Metrogyl and Aicar. It was shown that for all the parameters studied, the combined use of Metrogyl and Aicar led to a more effective reduction in mitochondrial disorders, compared to the group of rats that received only Metrogyl. Conclusions: The combined use of Metrogyl and Aicar drugs had a more effective effect on reducing mitochondrial dysfunction in periodontal tissue in rats with EP, in contrast to the group in which only Metrogyl was used. Thus, the use of traditional drugs in combination with mitochondria-targeted compounds that reduce oxidative stress may serve as a new therapeutic approach to various periodontal tissue diseases.

PDE4B abrogation extenuates angiotensin II-induced endothelial dysfunction related to hypertension through up-regulation of AMPK/Sirt1/Nrf2/ARE signaling.

Endothelial dysfunction is commonly perceived as a precursor in the process of hypertension, a severe cardiovascular disorder. Phosphodiesterase 4B (PDE4B) inactivation has been proposed to exert cardioprotective effects and prevent pulmonary hypertension. However, the role of PDE4B in endothelial dysfunction in hypertension remains inexplicit, which will be investigated in the present work. In angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs), RT-qPCR and Western blotting were used to analyze PDE4B expression. CCK-8 method was used to detect cell viability. Flow cytometry assay and Caspase 3 assay kit were used to detect cellular apoptotic level. Wound healing and tube formation assays were respectively used to detect cell migration and angiogenesis. Western blotting and corresponding assay kits were respectively used to analyze the expressions and contents of endothelial dysfunction markers. JC-1 assay, RT-qPCR and relevant assay kit were respectively used to detect mitochondrial membrane potential (ΔΨm), quantify mitochondrial DNA (mtDNA) copy number and mitochondrial permeability transition pore (mPTP) opening. Besides, Western blotting was used to analyze the expressions of endoplasmic reticulum stress (ERS) and AMP-activated protein kinase (AMPK)/sirtuin 1 (Sirt1)/nuclear factor-erythroid 2 related factor 2 (Nrf2)/antioxidant response element (ARE) signaling-associated proteins. PDE4B expression was increased in Ang II- induced HUVECs. PDE4B knockdown promoted the viability, migration, angiogenesis while inhibiting the apoptosis, endothelial dysfunction, ERS and mitochondrial damage in Ang II-induced HUVECs. Additionally, PDE4B silence activated AMPK/Sirt1/Nrf2/ARE pathway and AMPK inhibitor Compound C (CC) partially reversed the effects of PDE4B down-regulation on Ang II-induced HUVECs. Conclusively, PDE4B inhibition might protect against Ang II-induced endothelial dysfunction in HUVECs via up-regulating AMPK/Sirt1/Nrf2/ARE pathway, which might be mediated by the suppression of ERS and mitochondrial damage.

Analysis of mitochondrial DNA replisome in autism spectrum disorder: Exploring the role of replisome genes.

Autism spectrum disorder (ASD) is a neurodevelopmental condition often associated with mitochondrial dysfunction, including increased mitochondrial DNA (mtDNA) copy number and impaired energy production. This study investigates the role of the mitochondrial replisome-specifically, the genes TFAM, TWNK, POLG, and TOP1MT-in mtDNA replication and its potential contribution to ASD pathophysiology. We analyzed samples from the oral mucosa of children with ASD and typically developing (TD) controls, assessing mtDNA copy number, gene expression, and protein levels. Our findings revealed a significant increase in mtDNA copy number in the oral mucosa of ASD children, along with partially deleted mtDNA molecules. However, there were no significant changes in the expression of TFAM, TWNK, POLG, or MT-TL1 genes between ASD and TD samples. Additionally, TFAM protein levels, including monomeric, dimeric, and trimeric forms, did not differ significantly. We also observed increased oxidative stress and inflammatory markers in the oral mucosa of ASD children, suggesting that mitochondrial alterations may be linked to inflammation and oxidative damage in ASD. To further investigate the functional impact of TFAM, we overexpressed it in human HEK293 cells and cortical neurons (CN1.4). TFAM overexpression led to increased mtDNA copy number, cell proliferation, and ATP production in HEK293 cells, but did not significantly alter mitochondrial gene expression, protein oxidation, or mtDNA integrity. In CN1.4 neurons, TFAM overexpression increased mitochondrial membrane potential and length, indicating potential changes in mitochondrial dynamics. Overall, our study suggests that while mtDNA alterations are present in ASD, they are not directly driven by changes in mitochondrial replisome gene expression. These findings highlight the complexity of mitochondrial dysfunction in ASD and suggest the need for further investigation into the underlying molecular mechanisms.

Maternal Low-Protein Diet Leads to Mitochondrial Dysfunction and Impaired Energy Metabolism in the Skeletal Muscle of Male Rats.

A prenatal low-protein (LP) diet disrupts glucose homeostasis in adult offspring. Skeletal muscles are one of the main sites of glucose clearance, and mitochondria residing in the muscle fibers are central to glucose homeostasis. Our previous studies indicated that impaired mitochondrial health is central to dysregulated glucose metabolism in the gastrocnemius muscle of the LP-programmed female rats. In addition, dysfunctional mitochondria are often an indicator of underlying irregularities in energy metabolism and metabolic inflexibility. Therefore, this study examined the mitochondrial function and metabolic flexibility in the skeletal muscles of prenatal LP-programmed adult male rats. Pregnant Wistar rats were randomly allotted to a control diet (20% protein) or an isocaloric LP diet (6% protein). Standard laboratory rat chow was given to the dams and the pups after delivery and weaning. Gene and protein expressions, mtDNA copy number, and electron microscopy were assessed in gastrocnemius (GS) muscle, and the mitochondrial oxygen consumption rate was determined using isolated flexor digitorum brevis muscle fibers. The genes associated with mitochondrial outer membrane fusion, mitofusin1 and 2 (Mfn1 and Mfn2), fission (Fis1), and biogenesis (Pgc1B, Nrf1, and Esrra) were lower in the LP group. Further, our functional studies showed that the ATP-linked oxygen consumption rate (OCR), maximal, spare respiratory, and non-mitochondrial respiration-associated OCRs were lower in the LP rats. Further, the mRNA and protein expressions of Ndufb8, a key factor involved in the complex-I catalytic activity, were downregulated in the LP group. In addition, the expression of genes linked to mitochondrial pyruvate transport (Mpc1) and metabolism (Pdha1) was lower in the LP group. In contrast, the expression of mitochondrial fatty acid transporters (Cpt1a and Cpt2) was higher in the LP when compared to the control group. However, electron microscopic analysis exhibited no difference in the mitochondrial ultrastructure in the LP muscle compared to the control. Altogether, our results indicate that the LP diet affects the mitochondrial complex-I integrity and dynamics and leads to altered expression of genes associated with substrate oxidation and mitochondrial dysfunction in the skeletal muscle of the male LP offspring.

Decreased mitochondrial transcription factor A and mitochondrial DNA copy number promote cyclin-dependent kinase inhibitor 1A expression and reduce tumorigenic properties of colorectal cancer cells

PurposeColorectal cancer is one of the most common and deadliest cancer types worldwide. In the last years, changes in the mitochondrial DNA (mtDNA) copy number have been described to correlate with the prognostic outcome for colorectal cancer patients by impacting different tumorigenic properties. One key regulator of mtDNA is the mitochondrial transcription factor A (TFAM) that acts as a limiting factor of mtDNA copy number. Here, we investigated the effect of TFAM deficiency on mtDNA and tumorigenic properties in the human colorectal cancer cell line SW480.MethodsTFAM expression was stably downregulated in the colorectal cancer cell line SW480 using the CRISPR-Cas9 approach. To dissect the molecular alterations induced by deletion of TFAM, RNA sequencing and gene set enrichment analysis was performed on TFAM-wild-type and TFAM-deficient SW480 cells. Functional consequences of TFAM downregulation were assessed in cellular assays.ResultsWe showed that TFAM deficiency leads to decreased mtDNA copy number and reduced expression of mtDNA-encoded genes. TFAM-deficient cells also revealed higher activity of senescence-associated β-galactosidase and decreased cell growth parameters. Moreover, RNA sequencing showed that the expression of cyclin dependent kinase inhibitor 1A (CDKN1A/p21) is significantly increased in TFAM-deficient cells.ConclusionOur results suggest that TFAM-induced changes of the mitochondrial genome lead to upregulated CDKN1A/p21 expression in colorectal cancer cells identifying p21 as a new possible linker between mitochondria and nucleus.