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OBJECTIVES: To investigate the effect of Chinese medicine He's Yangchao recipe on premature ovarian insufficiency (POI) and its relationship with mitochondrial function of ovarian granulose cells in an animal model. METHODS: Thirty-six female C57BL/6J mice were randomly divided into blank control group, model group, low-, medium- and high-dose He's Yangchao recipe treatment group and coenzyme Q10 (Q10) treatment group (positive control). The POI model was induced by a single intraperitoneal injection of cyclophosphamide (90 mg/kg). The animals were sacrificed after 21 days. Primary granulose cells were obtained from POI mice and treated with He's Yangchao recipe, ERß inhibitor PHTPP, and He's Yangchao recipe+PHTPP in vitro for 24 h, respectively. Ovarian histopathological changes were observed by hematoxylin-eosin (HE) staining, ATP levels were detected by luciferase assay, mtDNA copy numbers were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), mitochondrial structure changes were observed by transmission electron microscopy, protein and mRNA expression levels of estrogen receptor ß (ERß), peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), mitochondrial transcription factor A (TFAM), and superoxide dismutase 2 (SOD2) were detected by Western blotting and qRT-PCR. RESULTS: The ovarian tissue in model group exhibited few secondary and tertiary follicles, whereas the He's Yangchao recipe groups and Q10 group had abundant secondary and tertiary follicles. Compared with the blank control group, ATP and mtDNA levels in model group decreased (P<0.01), mitochondrial crista disappeared or abnormal vacuolated structure increased; the protein and mRNA levels of ERß, PGC1α, TFAM, and SOD2 decreased (all P<0.01). ATP production increased in granulose cells of high-dose He's Yangchao recipe group and Q10 group; mtDNA copy numbers increased (P<0.05 or P<0.01); abnormal mitochondrial structure was reduced; the protein and mRNA expressions of ERß, PGC1α, TFAM, and SOD2 increased (P<0.05 or P<0.01). Compared with the PHTPP intervention group, the proportion of normal mitochondrial structure in the granulose cells of He's Yangchao recipe + PHTPP group was higher; ATP content increased (P<0.05 or P<0.01); mtDNA copy numbers increased (P<0.05 or P<0.01); the protein and mRNA expression of ERß, PGC1α, TFAM and SOD2 increased (P<0.05 or P<0.01). CONCLUSIONS: He's Yangchao recipe can regulate mitochondrial biogenesis through ERß/PGC1α/TFAM pathway to improve ovarian function in POI mice.

Disrupted mitochondrial transcription factor A expression promotes mitochondrial dysfunction and enhances ocular surface inflammation by activating the absent in melanoma 2 inflammasome.

PURPOSE: Severe dry eye disease causes ocular surface damage, which is highly associated with mitochondrial dysfunction. Mitochondrial transcription factor A (TFAM) is essential for packaging mitochondrial DNA (mtDNA) and is crucial for maintaining mitochondrial function. Herein, we aimed to explore the effect of a decreased TFAM expression on ocular surface damage. METHODS: Female C57BL/6 mice were induced ocular surface injury by topical administrating benzalkonium chloride (BAC). Immortalized human corneal epithelial cells (HCECs) were stimulated by tert-butyl hydroperoxide (t-BHP) to create oxidative stress damage. HCECs with TFAM knockdown were established. RNA sequencing was employed to analyze the whole-genome expression. Mitochondrial changes were measured by transmission electron microscopy, Seahorse metabolic flux analysis, mitochondrial membrane potential, and mtDNA copy number. TFAM expression and inflammatory cytokines were determined using RT-qPCR, immunohistochemistry, immunofluorescence, and immunoblotting. RESULTS: In both the corneas of BAC-treated mice and t-BHP-induced HCECs, we observed impaired TFAM expression, accompanied by mitochondrial structure and function defects. TFAM downregulation in HCECs suppressed mitochondrial respiratory capacity, reduced mtDNA content, induced mtDNA leakage into the cytoplasm, and led to inflammation. RNA sequencing revealed the absent in melanoma 2 (AIM2) inflammasome was activated in the corneas of BAC-treated mice. The AIM2 inflammasome activation was confirmed in TFAM knockdown HCECs. TFAM knockdown in t-BHP-stimulated HCECs aggravated mitochondrial dysfunction and the AIM2 inflammasome activation, thereby further triggering the secretion of inflammatory factors such as interleukin (IL) -1ß and IL-18. CONCLUSIONS: TFAM reduction impaired mitochondrial function, activated AIM2 inflammasome and promoted ocular surface inflammation, revealing an underlying molecular mechanism for ocular surface disorders.

VEGFR2 blockade inhibits glioblastoma cell proliferation by enhancing mitochondrial biogenesis.

BACKGROUND: Glioblastoma is an aggressive brain tumor linked to significant angiogenesis and poor prognosis. Anti-angiogenic therapies with vascular endothelial growth factor receptor 2 (VEGFR2) inhibition have been investigated as an alternative glioblastoma treatment. However, little is known about the effect of VEGFR2 blockade on glioblastoma cells per se. METHODS: VEGFR2 expression data in glioma patients were retrieved from the public database TCGA. VEGFR2 intervention was implemented by using its selective inhibitor Ki8751 or shRNA. Mitochondrial biogenesis of glioblastoma cells was assessed by immunofluorescence imaging, mass spectrometry, and western blot analysis. RESULTS: VEGFR2 expression was higher in glioma patients with higher malignancy (grade III and IV). VEGFR2 inhibition hampered glioblastoma cell proliferation and induced cell apoptosis. Mass spectrometry and immunofluorescence imaging showed that the anti-glioblastoma effects of VEGFR2 blockade involved mitochondrial biogenesis, as evidenced by the increases of mitochondrial protein expression, mitochondria mass, mitochondrial oxidative phosphorylation (OXPHOS), and reactive oxygen species (ROS) production, all of which play important roles in tumor cell apoptosis, growth inhibition, cell cycle arrest and cell senescence. Furthermore, VEGFR2 inhibition exaggerated mitochondrial biogenesis by decreased phosphorylation of AKT and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which mobilized PGC1α into the nucleus, increased mitochondrial transcription factor A (TFAM) expression, and subsequently enhanced mitochondrial biogenesis. CONCLUSIONS: VEGFR2 blockade inhibits glioblastoma progression via AKT-PGC1α-TFAM-mitochondria biogenesis signaling cascade, suggesting that VEGFR2 intervention might bring additive therapeutic values to anti-glioblastoma therapy.

Mitochondrial-dependent oxidative phosphorylation is key for postnatal metabolic adaptation of alveolar macrophages in the lung.

Alveolar macrophages (AMs) seed in lung during embryogenesis and become mature in perinatal period. Establishment of acclimatization to environmental challenges is important, whereas the detailed mechanisms that drive metabolic adaptation of AMs remains to be elucidated. Here, we showed that energy metabolism of AMs was transformed from glycolysis prenatally to oxidative phosphorylation (OXPHOS) postnatally accompanied by up-regulated expression of mitochondrial transcription factor A (TFAM). TFAM deficiency disturbed mitochondrial stability and decreased OXPHOS, which finally impaired AM maintenance and function, but not AM embryonic development. Mechanistically, Tfam-deletion resulted in impaired mitochondrial respiration and decreased ATP production, which triggered endoplasmic reticulum (ER) stress to cause B cell lymphoma 2 ovarian killer (BOK) accumulation and abnormal distribution of intracellular Ca2+, eventually led to induce AM apoptotic death. Thus, our data illustrated mitochondrial-dependent OXPHOS played a key role in orchestrating AM postnatal metabolic adaptation.

Therapeutic effects of genistein in experimentally induced ulcerative colitis in rats via affecting mitochondrial biogenesis.

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the mucosa of the colon, resulting in severe inflammation and ulcers. Genistein is a polyphenolic isoflavone present in several vegetables, such as soybeans and fava beans. Therefore, we conducted the following study to determine the therapeutic effects of genistein on UC in rats by influencing antioxidant activity and mitochondrial biogenesis and the subsequent effects on the apoptotic pathway. UC was induced in rats by single intracolonic administration of 2 ml of 4% acetic acid. Then, UC rats were treated with 25-mg/kg genistein. Colon samples were obtained to assess the gene and protein expression of nuclear factor erythroid 2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1), peroxisome proliferator-activated receptor-gamma coactivator (PGC-1), mitochondrial transcription factor A (TFAM), B-cell lymphoma 2 (BCL2), BCL2-associated X (BAX), caspase-3, caspase-8, and caspase-9. In addition, colon sections were stained with hematoxylin/eosin to investigate the cell structure. The microimages of UC rats revealed inflammatory cell infiltration, hemorrhage, and the destruction of intestinal glands, and these effects were improved by treatment with genistein. Finally, treatment with genistein significantly increased the expression of PGC-1, TFAM, Nrf2, HO-1, and BCL2 and reduced the expression of BAX, caspase-3, caspase-8, and caspase-9. In conclusion, genistein exerted therapeutic effects against UC in rats. This therapeutic activity involved enhancing antioxidant activity and increasing mitochondrial biogenesis, which reduced cell apoptosis.

Expression of mitochondrial transcription factor A in granulosa cells: implications for oocyte maturation and in vitro fertilization outcomes.

OBJECTIVE: In vitro fertilization-embryo transfer (IVF-ET) is a widely used treatment for infertility, with oocyte maturation and quality having a significant impact on oocyte fertilization, embryo development, and fetal growth. Mitochondrial transcription factor A (TFAM) is essential for maintaining the mitochondrial oxidative respiratory chain and supplying energy for oocyte development, fertilization, and embryonic development. In this study, we aimed to examine TFAM expression in women undergoing IVF-ET and assess its impact on the IVF outcomes. METHODS: We recruited 85 women who underwent IVF-ET treatment for infertility. On the date of egg collection, granulosa cells were extracted from the clear follicular fluid of the first mature egg using ultrasound-guided needle aspiration. The collected granulosa cells served three purposes: (1) detecting TFAM gene expression in granulosa cells via immunocytochemistry, (2) determining TFAM mRNA expression using reverse transcription-PCR (RT-PCR), and (3) measuring TFAM protein expression through western blotting. RESULT: Based on the results, we found that TFAM was localized and expressed in the cytoplasm of granulosa cells, whereas no expression was detected in the nucleus. Granulosa cells exhibited a linear correlation between TFAM mRNA and TFAM protein expression. The study participants were divided into three groups using the ternary method based on relative TFAM mRNA expression thresholds of 33% and 76%: the low-expression group (n = 30), the moderate-expression group (n = 27), and the high-expression group (n = 28). When compared to the other two groups, the moderate expression group exhibited a significantly higher egg utilization rate, 2 pronucleus rate, fertilization rate, and clinical pregnancy rate (P < 0.05). CONCLUSION: TFAM was detected in the cytoplasm of human ovarian granulosa cells. Women with moderate TFAM expression demonstrate enhanced outcomes in IVF.

The Protective Mechanism of TFAM on Mitochondrial DNA and its Role in Neurodegenerative Diseases.

Mitochondrial transcription factor A (TFAM) is a mitochondrial protein encoded by nuclear genes and transported from the cytoplasm to the mitochondria. TFAM is essential for the maintenance, expression, and delivery of mitochondrial DNA (mtDNA) and can regulate the replication and transcription of mtDNA. TFAM is associated with the formation of mtDNA nucleomimetic structures, mtDNA repair, and mtDNA stability. However, the mechanism by which TFAM protects mtDNA is still being studied. This review provides a summary of the protective mechanism of TFAM on mtDNA including the discrete regulatory effects of TFAM acetylation and phosphorylation on mtDNA, the regulation of Ca2+ levels by TFAM to activate transcription in mitochondria, and the increased binding of TFAM to mtDNA damage hot spots. This review also discusses the association between TFAM and some neurodegenerative diseases.

Kaempferia galanga L. extract and its main component, ethyl p-methoxycinnamate, inhibit the proliferation of Ehrlich ascites tumor cells by suppressing TFAM expression.

Kaempferia galanga L. shows anti-cancer effects; however, the underling mechanism remains unclear. In this study, we explored the underlying mechanism of the anti-cancer effects of Kaempferia galanga L. Kaempferia galanga L. rhizome extracts (KGEs) suppressed Ehrlich ascites tumor cell (EATC) proliferation by inhibiting S-phase progression. The main component of KGE is ethyl p-methoxycinnamate (EMC), which exhibits the same anti-proliferative effect as KGE. Furthermore, EMC induced the downregulation of cyclin D1 and upregulation of p21. EMC also decreased the expression of mitochondrial transcription factor A (TFAM) but did not significantly change mitochondrial DNA copy number and membrane potential. Phosphorylation at Ser62 of c-Myc, a transcription factor of TFAM, was decreased by EMC treatment, which might be due to the suppression of H-ras expression. These results indicate that EMC is the active compound responsible for the anti-cancer effect of KGE and suppresses EATC proliferation by regulating the protein expression of cyclin D1 and p21; TFAM may also regulate the expression of these genes. In addition, we investigated the anticancer effects of KGE and EMC in vivo using EATC bearing mice. The volume of ascites fluid was significantly increased by intraperitoneal administration of EATC. However, the increase in the volume of ascites fluid was suppressed by oral administration of EMC and KGE. This study provides novel insights into the association between the anti-cancer effects of natural compounds and TFAM, indicating that TFAM might be a potential therapeutic target.

35 Years of TFAM Research: Old Protein, New Puzzles.

Transcription Factor A Mitochondrial (TFAM), through its contributions to mtDNA maintenance and expression, is essential for cellular bioenergetics and, therefore, for the very survival of cells. Thirty-five years of research on TFAM structure and function generated a considerable body of experimental evidence, some of which remains to be fully reconciled. Recent advancements allowed an unprecedented glimpse into the structure of TFAM complexed with promoter DNA and TFAM within the open promoter complexes. These novel insights, however, raise new questions about the function of this remarkable protein. In our review, we compile the available literature on TFAM structure and function and provide some critical analysis of the available data.

Mitochondrial replication, transcription, and function in obstructive sleep apnea.

We assessed mitochondrial replication, transcription, and function in the upper airways of obstructive sleep apnea (OSA) patients and the effects of uvulopalatopharyngoplasty. Twenty subjects with mild and 40 with moderate to severe OSA requiring uvulopalatopharyngoplasty were included. Mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in uvula specimens were assessed by immunohistochemical staining, and their mRNA and protein expression was examined using reverse-transcription polymerase chain reaction and western blotting, respectively. The mitochondrial to nuclear DNA (Mt/N) ratio in the blood, exhaled breath condensate (EBC), and uvula was measured using quantitative reverse-transcription polymerase chain reaction. TFAM and PGC-1α protein concentrations in the plasma and EBC were determined using enzyme-linked immunosorbent assay. All tested parameters were higher in the OSA group than in the control. Three months later, 21 uvulopalatopharyngoplasty-responsive patients with OSA showed decreased TFAM and PGC-1α concentrations and EBC Mt/N ratio while these remained high in 19 uvulopalatopharyngoplasty-unresponsive patients. The OSA group showed severe inflammation, increased mitochondrial replication and transcription-related signaling, and mitochondrial dysfunction in the uvula. Successful OSA treatment using uvulopalatopharyngoplasty restored the TFAM and PGC-1α levels and EBC Mt/N ratio.

TFAM-Mediated mitochondrial transfer of MSCs improved the permeability barrier in sepsis-associated acute lung injury.

Vascular endothelial cell barrier disruption is a hallmark of sepsis-induced acute lung injury (ALI). Mesenchymal stem cells (MSCs)-based therapy has been regarded as a promising treatment for repairing injured lungs, and mitochondrial transfer was shown to be important for the therapeutic effects of MSCs. Here we investigated the ability of MSCs to modulate endothelial barrier integrity through mitochondrial transfer in sepsis-induced ALI. We found that mitochondrial transfer from MSCs to LPS-induced PMVECs through forming tunneling nanotubes (TNTs). Due to the inhibition of TNTs (using LAT-A), MSCs-mediated reparation on PMVECs functions, including cell apoptosis, MMP, ATP generation, TEER level and monolayer permeability of FITC-dextran were greatly inhibited. In addition, silencing of mitochondrial transcription factor A (TFAM) in MSCs could also partly inhibit the TNTs formation and aggravate the LPS-induced mitochondrial dysfunction and permeability barrier in PMVECs. Furthermore, the LPS-induced pulmonary edema and higher pulmonary vascular permeability were alleviated by MSCs while that of lung tissue bounced back after MSCs were pre-incubated by LAT-A and or down-regulation of TFAM. Therefore, we firstly revealed that regulation of TFAM expression in MSCs played a critical role to improve the permeability barrier of PMVECs by TNTs mediating mitochondrial transfer in sepsis-associated ALI. This study provided a new therapeutic strategy for the treatment of sepsis-induced ALI.

Astragaloside IV attenuates podocyte apoptosis through ameliorating mitochondrial dysfunction by up-regulated Nrf2-ARE/TFAM signaling in diabetic kidney disease.

Defective antioxidant system as well as mitochondrial dysfunction contributes to the pathogenesis and progression of diabetic kidney disease (DKD). Nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling is the central defensive mechanism against oxidative stress and therefore pharmacological activation of Nrf2 is a promising therapeutic strategy. In this study, using molecular docking we found that Astragaloside IV (AS-IV), an active ingredient from traditional formula of Huangqi decoction (HQD), exerted a higher potential to promote Nrf2 escape from Keap1-Nrf2 interaction via competitively bind to amino acid sites in Keap1. When podocyte exposed to high glucose (HG) stimulation, mitochondrial morphological alterations and podocyte apoptosis were presented and accompanied by Nrf2 and mitochondrial transcription factor A (TFAM) downregulation. Mechanistically, HG promoted a decrease in mitochondria-specific electron transport chain (ETC) complexes, ATP synthesis and mtDNA content as well as increased ROS production. Conversely, all these mitochondrial defects were dramatically alleviated by AS-IV, but suppression of Nrf2 with inhibitor or siRNA and TFAM siRNA simultaneously alleviated the AS-IV efficacy. Moreover, experimental diabetic mice exhibited significant renal injury as well as mitochondrial disorder, corresponding with the decreased expression of Nrf2 and TFAM. On the contrary, AS-IV reversed the abnormality and the Nrf2 and TFAM expression were also restored. Taken together, the present findings demonstrate the improvement of AS-IV on mitochondrial function, thereby resistance to oxidative stress-induced diabetic kidney injury and podocyte apoptosis, and the process is closely associated with activation of Nrf2-ARE/TFAM signaling.

How to Quantify DNA Compaction by TFAM with Acoustic Force Spectroscopy and Total Internal Reflection Fluorescence Microscopy.

Mitochondrial transcription factor A (TFAM) plays a key role in the organization and compaction of the mitochondrial genome. However, there are only a few simple and accessible methods available to observe and quantify TFAM-dependent DNA compaction. Acoustic Force Spectroscopy (AFS) is a straightforward single-molecule force spectroscopy technique. It allows one to track many individual protein-DNA complexes in parallel and to quantify their mechanical properties. Total internal reflection fluorescence (TIRF) microscopy is a high-throughput single-molecule technique that permits the real-time visualization of the dynamics of TFAM on DNA, parameters inaccessible with classical biochemistry tools. Here we describe, in detail, how to set up, perform, and analyze AFS and TIRF measurements to study DNA compaction by TFAM.

Assessing TFAM Binding to Human Mitochondrial DNA.

Mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA)-binding protein that plays a crucial dual role in the initiation of mitochondrial transcription initiation and mtDNA maintenance. Because TFAM directly interacts with mtDNA, assessing its DNA-binding property can provide useful information. This chapter describes two in vitro assay methods, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay with recombinant TFAM proteins, which both require simple agarose gel electrophoresis. These are used to investigate the effects of mutations, truncation, and posttranslational modifications on this key mtDNA regulatory protein.

Effects of the anti-inflammatory drug celecoxib on cell death signaling in human colon cancer.

The anti-inflammatory drug celecoxib, the only inhibitor of cyclooxygenase-2 (COX-2) with anticancer activity, is used to treat rheumatoid arthritis and can cause endoplasmic reticulum (ER) stress by inhibiting sarco/ER Ca2 +-ATPase activity in cancer cells. This study aimed to investigate the correlation between celecoxib-induced ER stress and the effects of celecoxib against cell death signaling. Treatment of human colon cancer HCT116 cells with celecoxib reduced their viability and resulted in a loss of mitochondrial membrane potential ([Formula: see text]). Additionally, celecoxib treatment reduced the expression of genes involved in mitochondrial biogenesis and metabolism such as mitochondrial transcription factor A (TFAM) and uncoupling protein 2 (UCP2). Furthermore, celecoxib reduced transmembrane protein 117 (TMEM117), and RNAi-mediated knockdown of TMEM117 reduced TFAM and UCP2 expressions. These results suggest that celecoxib treatment results in the loss of [Formula: see text] by reducing TMEM117 expression and provide insights for the development of novel drugs through TMEM117 expression.

The mitochondrial Ahi1/GR participates the regulation on mtDNA copy numbers and brain ATP levels and modulates depressive behaviors in mice.

BACKGROUND: Previous studies have shown that depression is often accompanied by an increase in mtDNA copy number and a decrease in ATP levels; however, the exact regulatory mechanisms remain unclear. METHODS: In the present study, Western blot, cell knockdown, immunofluorescence, immunoprecipitation and ChIP-qPCR assays were used to detect changes in the Ahi1/GR-TFAM-mtDNA pathway in the brains of neuronal Abelson helper integration site-1 (Ahi1) KO mice and dexamethasone (Dex)-induced mice to elucidate the pathogenesis of depression. In addition, a rescue experiment was performed to determine the effects of regular exercise on the Ahi1/GR-TFAM-mtDNA-ATP pathway and depression-like behavior in Dex-induced mice and Ahi1 KO mice under stress. RESULTS: In this study, we found that ATP levels decreased and mitochondrial DNA (mtDNA) copy numbers increased in depression-related brain regions in Dex-induced depressive mice and Ahi1 knockout (KO) mice. In addition, Ahi1 and glucocorticoid receptor (GR), two important proteins related to stress and depressive behaviors, were significantly decreased in the mitochondria under stress. Intriguingly, GR can bind to the D-loop control region of mitochondria and regulate mitochondrial replication and transcription. Importantly, regular exercise significantly increased mitochondrial Ahi1/GR levels and ATP levels and thus improved depression-like behaviors in Dex-induced depressive mice but not in Ahi1 KO mice under stress. CONCLUSIONS: In summary, our findings demonstrated that the mitochondrial Ahi1/GR complex and TFAM coordinately regulate mtDNA copy numbers and brain ATP levels by binding to the D-loop region of mtDNA Regular exercise increases the levels of the mitochondrial Ahi1/GR complex and improves depressive behaviors. Video Abstract.

Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A.

The maintenance of human mitochondrial DNA (mtDNA) is critical for proper cellular function as damage to mtDNA, if left unrepaired, can lead to a diverse array of pathologies. Of the pathways identified to participate in DNA repair within the mitochondria, base excision repair (BER) is the most extensively studied. Protein-protein interactions drive the step-by-step coordination required for the successful completion of this pathway and are important for crosstalk with other mitochondrial factors involved in genome maintenance. Human NEIL1 is one of seven DNA glycosylases that initiates BER in both the nuclear and mitochondrial compartments. In the current work, we scrutinized the interaction between NEIL1 and mitochondrial transcription factor A (TFAM), a protein that is essential for various aspects of mtDNA metabolism. We note, for the first time, that both the N- and C- terminal domains of NEIL1 interact with TFAM revealing a unique NEIL1 protein-binding interface. The interaction between the two proteins, as observed biochemically, appears to be transient and is most apparent at concentrations of low salt. The presence of DNA (or RNA) also positively influences the interaction between the two proteins, and molar mass estimates indicate that duplex DNA is required for complex formation at higher salt concentrations. Hydrogen deuterium exchange mass spectrometry data reveal that both proteins exchange less deuterium upon DNA binding, indicative of an interaction, and the addition of NEIL1 to the TFAM-DNA complex alters the interaction landscape. The transcriptional activity of TFAM appears to be independent of NEIL1 expression under normal cellular conditions, however, in the presence of DNA damage, we observe a significant reduction in the mRNA expression of TFAM-transcribed mitochondrial genes in the absence of NEIL1. Overall, our data indicate that the interaction between NEIL1 and TFAM can be modulated by local environment such as salt concentrations, protein availability, the presence of nucleic acids, as well as the presence of DNA damage.

Evaluation of Ectopic Mitochondrial DNA in HeLa Cells.

The presence of ectopic DNA in the cytoplasm induces inflammation and cell death. It has been widely reported that leakage of nuclear DNA into the cytoplasm can mainly be sensed by cyclic GMP-AMP synthase (cGAS). We recently reported that mitochondria-derived cytoplasmic double-stranded DNA (dsDNA) that has escaped lysosomal degradation induces significant cytotoxicity in cultured cells and in vivo. Cytoplasmic mitochondrial DNA is assumed to be involved in various diseases and disorders, and more and more papers have been published confirming this. On the other hand, the current method for evaluating mitochondrial DNA in the cytoplasm may not be quantitative. Here, we introduce in detail a method to evaluate ectopic mitochondrial DNA in cells. This method is useful in basic research as well as in the study of aging, Parkinson's disease, Alzheimer's disease, heart failure, autoimmune diseases, cancer, and other conditions.

Salvianolic acid A promotes mitochondrial biogenesis and function via regulating the AMPK/PGC-1α signaling pathway in HUVECs.

Mitochondrial dysregulation is an important pathology that leads to endothelial dysfunction, and the occurrence and development of cardiovascular diseases. Salvianolic acid A (SAA) has been demonstrated to be effective in the treatment of vascular complications of type 2 diabetes mellitus. Limited information has been reported on the effects of SAA on mitochondrial function in endothelial cells. In the present study, the effects of SAA on mitochondrial biogenesis and the related underlying mechanisms were investigated in human umbilical vein endothelial cells (HUVECs). Mitotracker red staining and transmission electron microscopy were used to evaluate the effect of SAA on mitochondrial quality. The effect of SAA treatment on mitochondrial DNA/nuclear DNA ratio of HUVECs was detected by real-time quantitative PCR. Western blot was used to determine the protein expression levels of complex III and Complex IV of mitochondrial oxidative phosphorylation subunit, and ATP production was determined by ATP test kit. Real-time quantitative PCR and Western blot were used to determine the effects of SAA on the expression of peroxisome proliferator-activated receptor γ coactivator (PGC-1α) and its target genes nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) proteins and genes. Finally, in the presence of 5'AMP-activated protein kinase (AMPK) specific inhibitors, the expression of PGC-1α, NRF1 and TFAM proteins and the phosphorylation levels of AMPK and Acetyl CoA Carboxylase (ACC) were detected by Western blot or real-time quantitative PCR. The results showed that SAA treatment significantly promoted mitochondrial biogenesis and enhanced mitochondrial function of HUVECs. SAA significantly increased the expression levels of PGC-1α and its target genes NRF1 and (TFAM), a key regulator of mitochondrial biogenesis in HUVECs. These enhancements were accompanied by significantly increased phosphorylation of AMPK and ACC, and were significantly inhibited by specific AMPK inhibitors. These results suggest that SAA may promote mitochondrial biogenesis in endothelial cells by activating the AMPK-mediated PGC-1α/TFAM signaling pathway. These data provide new insights into the mechanism of action of SAA in treating diabetic vascular complications.

Therapeutic effects of sulforaphane in ulcerative colitis: effect on antioxidant activity, mitochondrial biogenesis and DNA polymerization.

OBJECTIVES: Ulcerative colitis (UC), an inflammatory bowel disease, affects mucosal lining of colon leading to inflammation and ulcers. Sulforaphane is a natural compound obtained from cruciferous vegetables. We aimed to investigate potential therapeutic effects of sulforaphane in experimentally induced UC in rats through affection antioxidant activity, mitochondrial biogenesis and DNA polymerization. METHODS: UC was induced in rats via an intracolonic single administration of 2 ml of 4% acetic acid. UC rats were treated with 15 mg/kg sulforaphane. Samples of colon were used to investigate gene expression and protein levels of peroxisome proliferator-activated receptor-gamma coactivator (PGC-1), mitochondrial transcription factor A (TFAM), mammalian target of rapamycin (mTOR), cyclin D1, nuclear factor erythroid 2-related factor-2 (Nrf2), heme Oxygenase-1 (HO-1) and proliferating cell nuclear antigen (PCNA). RESULTS: UC showed dark distorted Goblet cell nucleus with disarranged mucus granules and no distinct brush border with atypical microvilli. All morphological changes were improved by treating with sulforaphane. Finally, treatment with sulforaphane significantly increased expression of PGC-1, TFAM, Nrf2 and HO-1 associated with reduction in expression of mTOR, cyclin D1 and PCNA. CONCLUSION: Sulforaphane could cure UC in rats. The protective activity can be explained by enhancing antioxidant activity, elevating mitochondrial biogenesis and inhibiting DNA polymerization.