Mitochondrial DNA-encoded Gene Expression Research Articles

Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function.

Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3-targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r2 > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3, which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.

STEM-21. INHIBITING ADENINE SYNTHESIS ATTENUATES GLIOBLASTOMA CELL STEMNESS AND TEMOZOLOMIDE RESISTANCE

Abstract Glioblastoma (GBM) is a lethal brain cancer that exhibits high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work has shown that homozygous MTAP deletion, a frequent genetic alteration in GBM, promotes the stemness of GBM cells. Here, exploiting the MTAP loss-conferred deficiency in adenine salvage, we demonstrated that subtle levels of adenine blockade via treatment with Alanosine, an inhibitor of de novo adenine synthesis, attenuate the stemness of MTAP-null GBM cells. Transcriptomic profiling performed on Alanosine-treated GBM cells revealed a reduction of mitochondrial DNA-encoded gene expression. Furthermore, Seahorse XF analysis showed that Alanosine treatment led to a reduction in mitochondrial respiration and eliminated GBM cells’ spare respiratory capacity, an important metabolic measure of cell fitness that is representative of their ability to respond to oxidative stress. Importantly, long term adenine shortage via treatment with low doses of Alanosine resulted in similarly compromised mitochondria functionality and attenuated GBM cells’ stemness. In further supporting this adenine blockade – compromised mitochondria – reduced stemness cascade, treatment with a mitochondrial respiration inhibitor attenuated the stemness of GBM cells, suggesting the importance of mitochondrial function in maintaining GBM stemness. Finally, in agreement with the diminished stemness and compromised mitochondrial function, we showed that Alanosine sensitized GBM cells to temozolomide (TMZ) in both in vitro cultures and in an orthotopic GBM model. Collectively, these results identify critical roles of adenine supply in maintaining stemness and mitochondrial function in GBM cells, suggest a targeted method of abrogating stemness and chemoresistance in GBMs, and support targeting adenine synthesis as a complementary approach for treating the half of GBMs harboring MTAP deletions.

The Genetic Evidence of Burn-Induced Cardiac Mitochondrial Metabolism Dysfunction.

Burn-induced cardiac dysfunction is thought to involve mitochondrial dysfunction, although the mechanisms responsible are unclear. In this study, we used our established model of in vivo burn injury to understand the genetic evidence of burn-induced mitochondrial confusion dysfunction by describing cardiac mitochondrial metabolism-related gene expression after burn. Cardiac tissue was collected at 24 hours after burn injury. An O2K respirometer system was utilized to measure the cardiac mitochondrial function. Oxidative phosphorylation complex activities were determined using enzyme activity assays. RT Profiler PCR array was used to identify the differential regulation of genes involved in mitochondrial biogenesis and metabolism. The quantitative qPCR and Western blotting were applied to validate the differentially expressed genes. Burn-induced cardiac mitochondrial dysfunction was supported by the finding of decreased state 3 respiration, decreased mitochondrial electron transport chain activity in complex I, III, IV, and V, and decreased mitochondrial DNA-encoded gene expression as well as decreased levels of the corresponding proteins after burn injury. Eighty-four mitochondrial metabolism-related gene profiles were measured. The mitochondrial gene profile showed that 29 genes related to mitochondrial energy and metabolism was differentially expressed. Of these 29 genes, 16 were more than 2-fold upregulated and 13 were more than 2-fold downregulated. All genes were validated using qPCR and partial genes were correlated with their protein levels. This study provides preliminary evidence that a large percentage of mitochondrial metabolism-related genes in cardiomyocytes were significantly affected by burn injury.

Heat exposure impairs porcine oocyte quality with suppressed actin expression in cumulus cells and disrupted F-actin formation in transzonal projections

BackgroundTranszonal projections (TZPs) constitute a structural basis for the communication between the oocyte and its surrounding cumulus cells (CCs), which play critical roles in promoting the oocyte maturation. Previously we found that heat stress (HS) causes loss of TZPs in porcine cumulus-oocyte complexes (COCs) with decreased density of filamentous actin (F-actin). However, the time-course responses of F-actin and its monomeric actins (β-actin and γ-actin) during the in vitro maturation of oocytes remain unclear.ResultsIn this study, excised porcine ovaries were exposed to HS at 41.5 °C for 1 h before COCs were isolated and matured in vitro for 44 h. HS significantly reduced oocyte quality, characterized by impaired cumulus expansion, delayed meiotic resumption and lower survival rate and polar body extrusion rate, as well as decreased expression of mitochondrial DNA-encoded genes and elevated mitochondrial reactive oxygen species concentration. Expression of β-actin and γ-actin in CCs increased gradually with oocytes maturation, which was significantly reduced in HS group, especially at 24 h and/or 44 h of in vitro maturation. By contrast, the number of TZPs and the fluorescence intensity of F-actin in zona pellucida decreased gradually during oocytes maturation, which were significantly reduced by HS at 24 h of in vitro maturation. Moreover, colocalization analyses revealed both β-actin and γ-actin contribute to the F-actin formation in porcine TZPs, and the colocalization of F-actin with GJ protein connexin 45 was significantly reduced in heat-exposed COCs.ConclusionsThe results indicate that the suppression of actin expressions in CCs, which may lead to the F-actin unstabilization in TZPs, will subsequently contribute to the compromised quality of oocytes under HS.

Expression of Mitochondrial-Encoded Genes in Blood Differentiate Acute Renal Allograft Rejection.

Despite potent immunosuppression, clinical and biopsy confirmed acute renal allograft rejection (AR) still occurs in 10–15% of recipients, ~30% of patients demonstrate subclinical rejection on biopsy, and ~50% of them can show molecular inflammation, all which increase the risk of chronic dysfunction and worsened allograft outcomes. Mitochondria represent intracellular endogenous triggers of inflammation, which can regulate immune cell differentiation, and expansion and cause antigen-independent graft injury, potentially enhancing the development of acute rejection. In the present study, we investigated the role of mitochondrial DNA encoded gene expression in biopsy matched peripheral blood (PB) samples from kidney transplant recipients. Quantitative PCR was performed in 155 PB samples from 115 unique pediatric (<21 years) and adult (>21 years) renal allograft recipients at the point of AR (n = 61) and absence of rejection (n = 94) for the expression of 11 mitochondrial DNA encoded genes. We observed increased expression of all genes in adult recipients compared to pediatric recipients; separate analyses in both cohorts demonstrated increased expression during rejection, which also differentiated borderline rejection and showed an increasing pattern in serially collected samples (0–3 months prior to and post rejection). Our results provide new insights on the role of mitochondria during rejection and potentially indicate mitochondria as targets for novel immunosuppression.

Increased expression of mitochondrial DNA-encoded genes in human renal mesangial cells in response to high glucose-induced reactive oxygen species.

Reactive oxygen species (ROS)-mediated disruption of mitochondrial respiratory function has been implicated in the complications of diabetes. The present study examined changes in the gene expression of mitochondrial DNA (mtDNA)-encoded subunits of electron transport chain complexes in response to high glucose-induced ROS overproduction in an in vitro model of diabetic nephropathy using human renal mesangial cells. Mitochondrial ROS generation was assessed by confocal microscopy and flow cytometry in the cells following culture in 5 and 25 mM glucose. The mRNA expression levels of nicotinamide adenine dinucleotide dehydrogenase 2 (ND2) of complex I, cytochrome b (CYTB) of complex III, cytochrome c oxidase (COI) of complex IV and ATPase 6 of complex V were analyzed by reverse transcription-quantitative polymerase chain reaction. The protein expression levels of ND2, CYTB, COI and ATPase 6 were analyzed by western blotting. A significant increase in mitochondrial ROS production was observed in the cells cultured in 25 mM glucose, compared with cells cultured in 5 mM glucose (P<0.05). The mRNA expression of ND2, CYTB, CO1 and ATPase 6 was significantly increased following culture in 25 mM glucose, compared with the cells cultured in 5 mM glucose (P<0.05). This increase in mRNA expression was accompanied by significant increases in protein expression following incubation in 25 mM glucose (P<0.05). The increase in mtDNA-encoded gene expression in the electron transport subunits following exposure to high glucose-induced ROS may be a compensatory response mechanism for the decline in mitochondrial function, which may be important in the development of diabetic nephropathy through enhanced ROS generation.

Lipopolysaccharide significantly influences the hepatic triglyceride metabolism in growing pigs.

BackgroundIn the practical commercial pig farms, inflammation is a perennial problem, yet most of studies on inflammation are focused on immune response. Actually, inflammation can induce body metabolism disorder which will finally influence animals’ growth. In this study, we investigated the effect of acute inflammation on the triglyceride (TG) metabolism in the liver of growing pigs and the possible underlying mechanisms.MethodsTwelve male growing pigs were randomly divided into two groups, a control group (received saline) and a LPS group (intramuscular injected with 15 μg/kg LPS). Six hours after LPS injection, the pigs were euthanized and sampled. Biochemical indexes, inflammation factors, lipid metabolism related parameters and mitochondrial function were evaluated. The relationship between glucocorticoid receptor (GR) and the key enzymes of de novo lipogenesis were also investigated by chromatin immunoprecipitation assay (ChIP).ResultsLPS induced a serious inflammation in the liver of growing pigs proved by liver morphologic changes, the up-regulated plasma cortisol, tumor necrosis factor-α (TNF-α) content and gene expression of inflammation related genes in liver. For de novo lipogenesis, LPS significantly decreased the gene expression of fatty acid synthase (FAS), Acetyl-CoA carboxylase-1 (ACC-1) and Stearoyl-CoA desaturase-1 (SCD-1), and the protein expression of ACC-1 and SCD-1. For lipolysis, only the gene expression of adipose triglyceride lipase (ATGL) was decreased. LPS did nothing to the gene expression of hormone-sensitive lipase (HSL) and the lipolytic enzymes activities. For β-oxidation, LPS significantly increased the protein expression of CPT-1α, but the gene expression of mitochondrial DNA-encoded genes and the activities of mitochondrial complex IV and V demonstrated no obviously changes. Furthermore, ChIP results showed that LPS significantly decreased the level of GR binding to ACC-1 promoter.ConclusionLPS infection has a profound impact on hepatic TG metabolism. This impact is mainly demonstrated by the significantly deceased de novo lipogenesis, and GR may involve in its regulation.

Results of a Multi-Center Study Identify Mitochondria as Tissue and Blood Damage Associated Molecular Patterns in the Development of Renal Allograft Injury

Purpose: Investigating peripheral mitochondrial DNA encoded gene expression and graft tissue anti-mitochondrial protein antibody levels in kidney transplant recipients as novel (donor-specific) allograft damage and/or tolerance associated molecular patterns. Methods: A discovery set of 19 whole blood samples (PaxGene) from Stanford renal transplant recipients included 6 cases of acute rejection (AR), 8 without rejection (no-AR), and 5 tolerant cases (TOL), defined as absence of any immunosuppression and stable function for one year minimum. An independent validation set from Pittsburgh University included 29 whole blood samples from 9 renal AR and 20 no-AR. Expression of mt-DNA encoded genes was analyzed by high throughput QPCR (Fluidigm, Biomark). Negative cross analysis was performed using publicly available gene expression data from 130 patients with autoimmune diseases and 45 healthy controls (HC). In a third set of kidney transplant recipients (11 AR; 11 no-AR) antibody levels against mt-proteins were investigated by ProtoArray (HumanProteinMicorarray v5.0, Invitrogen, Carlsbad, CA). Results: The discovery set revealed upregulation of 11 mt-DNA encoded genes in patients with AR compared to non-AR and TOL (p< 0.05, T-test) independent from time post-transplantation. The average gene expression in TOL patients was lower compared to all other patients. Upregulation of 3 of the 11 genes was validated in the Pittsburgh cohort of AR and no-AR patients, of these, 2 belonged to a gene family known for high mutation rates and currently used in DNA-barcode analyses to distinguish closely related species. Cross-analyses of mt-gene expression in auto-immune diseases showed no differences between diseased patients compared to HC. Subsequent ProtoArray analysis identified 145 post-transplant renal allograft immunogenic mt-proteins (average signal intensity >500). Of these, 4 were encoded by the same gene family upregulated in AR patient whole blood from both discovery and validation set. Conclusions: mt-proteins present novel antigens in transplantation, and together with peripheral mt-DNA encoded genes, may serve as biomarkers for patient monitoring of allograft injury.

Results of a Multi-Center Study Identify Mitochondria as Tissue and Blood Damage Associated Molecular Patterns in the Development of Renal Allograft Injury

Purpose: Investigating peripheral mitochondrial DNA encoded gene expression and graft tissue anti-mitochondrial protein antibody levels in kidney transplant recipients as novel (donor-specific) allograft damage and/or tolerance associated molecular patterns. Methods: A discovery set of 19 whole blood samples (PaxGene) from Stanford renal transplant recipients included 6 cases of acute rejection (AR), 8 without rejection (no-AR), and 5 tolerant cases (TOL), defined as absence of any immunosuppression and stable function for one year minimum. An independent validation set from Pittsburgh University included 29 whole blood samples from 9 renal AR and 20 no-AR. Expression of mt-DNA encoded genes was analyzed by high throughput QPCR (Fluidigm, Biomark). Negative cross analysis was performed using publicly available gene expression data from 130 patients with autoimmune diseases and 45 healthy controls (HC). In a third set of kidney transplant recipients (11 AR; 11 no-AR) antibody levels against mt-proteins were investigated by ProtoArray (HumanProteinMicorarray v5.0, Invitrogen, Carlsbad, CA). Results: The discovery set revealed upregulation of 11 mt-DNA encoded genes in patients with AR compared to non-AR and TOL (p< 0.05, T-test) independent from time post-transplantation. The average gene expression in TOL patients was lower compared to all other patients. Upregulation of 3 of the 11 genes was validated in the Pittsburgh cohort of AR and no-AR patients, of these, 2 belonged to a gene family known for high mutation rates and currently used in DNA-barcode analyses to distinguish closely related species. Cross-analyses of mt-gene expression in auto-immune diseases showed no differences between diseased patients compared to HC. Subsequent ProtoArray analysis identified 145 post-transplant renal allograft immunogenic mt-proteins (average signal intensity >500). Of these, 4 were encoded by the same gene family upregulated in AR patient whole blood from both discovery and validation set. Conclusions: mt-proteins present novel antigens in transplantation, and together with peripheral mt-DNA encoded genes, may serve as biomarkers for patient monitoring of allograft injury.

Abnormal expression of the genes involved in cytokine networks and mitochondrial function in systemic juvenile idiopathic arthritis identified by DNA microarray analysis

Objectives:Systemic juvenile idiopathic arthritis (sJIA) is a rheumatic disease in childhood characterised by systemic symptoms and a relatively poor prognosis. Peripheral leukocytes are thought to play a pathological role in...

Homologs of Mitochondrial Transcription Factor B, Sparsely Distributed Within the Eukaryotic Radiation, Are Likely Derived from the Dimethyladenosine Methyltransferase of the Mitochondrial Endosymbiont

Mitochondrial transcription factor B (mtTFB), an essential component in regulating the expression of mitochondrial DNA-encoded genes in both yeast and humans, is a dimethyladenosine methyltransferase (DMT) that has acquired a secondary role in mitochondrial transcription. So far, mtTFB has only been well studied in Opisthokonta (metazoan animals and fungi). Here we investigate the phylogenetic distribution of mtTFB homologs throughout the domain Eucarya, documenting the first examples of this protein outside of the opisthokonts. Surprisingly, we identified putative mtTFB homologs only in amoebozoan protists and trypanosomatids. Phylogenetic analysis together with conservation of intron positions in amoebozoan and human genes supports the grouping of the putative mtTFB homologs as a distinct clade. Phylogenetic analysis further demonstrates that the mtTFB is most likely derived from the DMT of the mitochondrial endosymbiont.

Fetal and Early Postnatal Protein Malnutrition Cause Long-Term Changes in Rat Liver and Muscle Mitochondria

Epidemiologic data suggest a strong association between low birth weight and increased risk of metabolic syndrome in adult life. However, the underlying mechanisms are largely unknown. To test the hypothesis that mitochondrial changes may serve as a link between poor nutrition in early life and insulin resistance in later life, we investigated the effect of protein malnutrition during gestation and lactation on mitochondria of the liver and skeletal muscle. Female offspring of Sprague-Dawley rats fed a low protein diet (casein, 80 g/kg) were randomly divided into two groups and weaned onto either the low protein diet or a control diet (casein, 180 g/kg). As a control group, offspring of rats fed the control diet were weaned onto the control diet. The rats in each group were randomly divided into four groups that were killed at 5, 10, 15 and 20 wk of age. Both mitochondrial DNA content and the expression of mitochondrial DNA-encoded genes in liver and muscle were measured. Mitochondrial transcription factor A and antioxidant enzyme activities were also determined. The mitochondrial DNA content of the liver and skeletal muscle were reduced in fetal and early postnatal malnourished rats even when proper nutrition was supplied after weaning. These changes were accompanied by a decrease in mitochondrial DNA-encoded gene expression; however, they were not dependent on mitochondrial transcriptional factor A. Our findings indicate that poor nutrition in early life causes long-lasting changes in mitochondria that may contribute to the development of insulin resistance in later life.