Normal Mitochondrial Structure Research Articles

Mammalian Diaphanous-related formin 1 (mDia1) inducing diabetic cardiomyopathy by regulating metabolic reprogramming

Abstract Background Mammalian Diaphanous-related formin 1 (mDia1) is one of the formin family proteins and acts as a rho-effector molecule which is involved in actin organization. Previous studies showed that mDia1 aggravated compensatory cardiac hypertrophy in response to pressure overload. However, the role of mDia1 in diabetic cardiomyopathy and its mechanisms remain elusive. Objective To clarify the mechanisms of mDia1 in DCM through mediating metabolic reprogramming. Methods 5-week-old of mDia1 ko mice were used to induce DCM by feeding high-fat diet (HFD) for 16 weeks. The blood glucose and body weight of the mice were measured regularly. Echocardiography was used to evaluate cardiac function. HE and WGA staining were used to evaluate hypertrophy of cardiomyocytes. Transmission electron microscopy and mitotracker staining were used to observe the morphology of mitochondrial in heart. Proteomics and metabolomics were used to explore the metabolites in hearts of mice. Primary neonatal cardiomyocytes were isolated and stimulated with 35 mM glucose and 250 μM palmitate in vitro. Adenovirus expressing shRNA of mDia1 was used to knockdown mDia1 in cardiomyocytes. Mitochondria were isolated from primary cardiomyocytes to verify the translocation of mDia1. Co-Immunoprecipitation was used to confirm the interaction between mDia1 with succinate dehydrogenase subunits A (SDHA). Results mDia1 had no effects to blood glucose and body weight after 16-week HFD feeding. Loss of mDia1 ameliorated diastolic dysfunction in DCM mice with shorter isovolumic relaxation time (IVRT) and increased E/A ratio in left ventricular. In addition, cardiac hypertrophy was significantly improved in mDia1 ko mice. mDia1 deficiency tended to maintain the normal mitochondrial structure. Results of proteomics showed that mDia1 was involved in mitochondrial function, ATP synthesis and metabolic pathways. Metabolomics showed that HFD induced accumulation of α-ketoglutaric acid in wild type mice compared with mDia1 ko mice. However, mDia1 ko significantly increased fumaric acid but did not affect succinyl-CoA and succinic acid compared with wild type mice. Furthermore，we found that mDia1 translocated into mitochondrial with high glucose and palmitate stimuli. Results of CoIP showed mDia1 interacted with SDHA both in cardiomyocytes and HEK293 cells. Conclusions mDia1 deteriorated DCM through translocating into mitochondrial and interacting with SDHA, a process dependent on metabolic pathways.Figure of Mechanism

Mechanism of Mitochondrial Damage in Cardiac Dysfunction Induced by Sepsis

Sepsis is a syndrome with a high fatality rate, which is the dysfunction of multiple organs in the whole body caused by the host’s inflammation response caused by infection. The cardiac insufficiency caused by sepsis is called Septic-Induced Myocardial Dysfunction (SIMD), which is mainly characterized by bilateral systolic and diastolic dysfunction of the heart, which is one of the most serious complications of sepsis. Normal mitochondrial structure and function are essential for maintaining mitochondrial homeostasis. Mitochondrial homeostasis plays a central role in the pathophysiology of SIMD, and its structural changes, oxidative stress, inflammation, endoplasmic reticulum stress and autophagy are directly or indirectly related to the occurrence and development of SIMD, which has a promising future as a targeted therapy for SIMD. Therefore, this paper focuses on the molecular mechanism and signaling pathway of mitochondrial homeostasis in the pathogenesis of SIMD, and provides implications for the search for therapeutic targets and biomarkers of SIMD. In addition, many traditional Chinese medicine components play a protective role in maintaining mitochondrial homeostasis in sepsis, and have the potential to be developed as novel therapeutics for SIMD.

Targeting Sumoylation Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment

Acute myeloid leukemia (AML) is a hematopoietic cancer characterized by aberrant immature myeloid progenitor blasts in bone marrow and peripheral blood. Venetoclax (VEN), a BCL-2 inhibitor, is FDA-approved for AML treatment with hypomethylating agents (HMA). Despite the improvement of VEN and HMA, not all patients respond, and most patients develop resistance. This presents an urgent need for new therapies to overcome VEN resistance and enhance VEN efficacy. We propose SUMOylation inhibition as a novel therapy to address this need. SUMOylation regulates protein function by covalently attaching Small Ubiquitin-like MOdifier (SUMO) proteins to target proteins. SUMOylation plays an important role in tumor progression and immune response. Our study aims to evaluate the effects of SUMOylation inhibition on anti-AML activity of VEN and the mechanism. We firstly generated VEN-resistant cell lines Molm13-VR and MV411-VR by culturing Molm13 and MV4-11 cells in increasing doses of VEN. SUMO E1 (SAE2) and global SUMOylation levels were remarkably upregulated in VEN-resistant cell lines, suggesting SUMOylation plays a role in VEN resistance. We evaluated the antileukemic effects of TAK-981 (subasumstat), a novel and specific SUMO E1 inhibitor, alone and in combination with VEN, using AML cell lines and primary blasts isolated from refractory/relapsed (R/R) AML patients. Different AML cell lines and blast samples showed various sensitivities to VEN (IC 50 10nM ~ 5,000nM), but significant synergism was observed with TAK-981 and VEN in cell viability assay. TAK-981 synergized with VEN at inducing AML cell apoptosis determined by annexin-V staining with flow cytometry as well as increased levels of cleaved-PARP and cleaved caspase-3 by western blotting. The mechanism of action of VEN is dependent on BCL-2 mediated mitochondrial function and metabolism. We assessed mitochondrial membrane potential using TMRM probe. TAK-981 reduced mitochondrial membrane potential in both VEN-sensitive and resistant AML lines, further in combination with VEN. Because mitochondrial membrane potential is maintained by normal mitochondrial structure, we performed electron microscopy to determine mitochondrial structure. VEN had limited effect on mitochondria in resistant cells. However, TAK-981 induced abnormal ultrastructure mitochondria with fewer cristae, worsened by TAK-981+VEN combination. Since TAK-981 treatment caused mitochondrial structure defects, we determine the mitochondrial function using Seahorse XF assay. VEN decreased oxygen consumption rate (OCR), indicating inhibition of oxidative phosphorylation (OXPHOS), further suppressed by TAK-981. Metabolomic profiling by mass spectrometry indicated TAK-981+VEN treated AML cells displayed striking decreases in TCA cycle intermediates and reduced electron transport chain (ETC) activity. Pathway analysis of the metabolomics data revealed significant enrichment in amino acids metabolism. Notably, RNA-sequencing and GSEA analysis showed consistent findings that TAK-981+VEN showed suppressed signaling pathways in mitochondrial function, glycolysis and amino-acid metabolisms, indicating TAK-981 targeted mitochondrial metabolism to enhance VEN activity. We then determined whether TAK-981 enhanced antileukemic effect of VEN ex vivo and in vivo. TAK-981 synergized with VEN at reducing colony formation capacity in colony formation assay using leukemia stem cells (LSCs) isolated from relapse AML blasts. More importantly, TAK-981 showed remarkable therapeutic effect in patient-derived xenograft (PDX) mouse model engrafted with blasts isolated from R/R AML patients, and significantly synergized with VEN at extending mice survival in vivo. In conclusion, TAK-981 enhances the antileukemic activity of VEN by targeting mitochondria function and metabolism, offering a potent therapy for refractory/relapsed AML.

Evolution of cisplatin resistance through coordinated metabolic reprogramming of the cellular reductive state.

Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring. To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics. Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function. Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways.

Efficient gene transfection to lung cancer cells via Folate-PEI-Sorbitol gene transporter.

Lung cancer is known to be one of the fatal diseases in the world and is experiencing treatment difficulties. Many treatments have been discovered and implemented, but death rate of patients with lung cancer continues to remain high. Current treatments for cancer such as chemotherapy, immunotherapy, and radiotherapy have shown considerable results, yet they are accompanied by side effects. One effective method for reducing the cytotoxicity of these treatments is via the use of a nanoparticle-mediated siRNA delivery strategy with selective silencing effects and non-viral vectors. In this study, a folate (FA) moiety ligand-conjugated poly(sorbitol-co-PEI)-based gene transporter was designed by combining low-molecular weight polyethyleneimine (LMW PEI) and D-sorbitol with FA to form FPS. Since folate receptors are commonly overexpressed in various cancer cells, folate-conjugated nanoparticles may be more effectively delivered to selective cancer cells. Additionally, siOPA1 was used to induce apoptosis through mitochondrial fusion. The OPA1 protein stability level is important for maintaining normal mitochondrial cristae structure and function, conserving the inner membrane structure, and protecting cells from apoptosis. Consequently, when FPS/siOPA1 was used for lung cancer in-vitro and in-vivo, it improved cell viability and cellular uptake.

A pair of transporters controls mitochondrial Zn2+ levels to maintain mitochondrial homeostasis

Zn2+ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn2+ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn2+. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn2+ exporter. Loss of SLC-30A9 leads to mitochondrial Zn2+ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn2+ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn2+ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn2+ pool from which mitochondrial Zn2+ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn2+ levels for normal mitochondrial structure and functions.

Circulating Cell-Free Mitochondrial DNA in Cerebrospinal Fluid as a Biomarker for Mitochondrial Diseases.

Mitochondrial diseases (MD) are genetic metabolic disorders that impair normal mitochondrial structure or function. The aim of this study was to investigate the status of circulating cell-free mitochondrial DNA (ccfmtDNA) in cerebrospinal fluid (CSF), together with other biomarkers (growth differentiation factor-15 [GDF-15], alanine, and lactate), in a cohort of 25 patients with a molecular diagnosis of MD. Measurement of ccfmtDNA was performed by using droplet digital PCR. The mean copy number of ccfmtDNA was approximately 6 times higher in the MD cohort compared to the control group; patients with mitochondrial deletion and depletion syndromes (MDD) had the higher levels. We also detected the presence of both wild-type mtDNA and mtDNA deletions in CSF samples of patients with single deletions. Patients with MDD with single deletions had significantly higher concentrations of GDF-15 in CSF than controls, whereas patients with point mutations in mitochondrial DNA presented no statistically significant differences. Additionally, we found a significant positive correlation between ccfmtDNA levels and GDF-15 concentrations (r = 0.59, P = 0.016). CSF ccfmtDNA levels are significantly higher in patients with MD in comparison to controls and, thus, they can be used as a novel biomarker for MD research. Our results could also be valuable to support the clinical outcome assessment of MD patients.

OTUD1 Activates Caspase-Independent and Caspase-Dependent Apoptosis by Promoting AIF Nuclear Translocation and MCL1 Degradation.

Apoptosis‐inducing factor (AIF) plays a dual role in regulating cell survival and apoptosis, acting as a prosurvival factor in mitochondria via its NADH oxidoreductase activity and activating the caspase‐independent apoptotic pathway (i.e., parthanatos) after nuclear translocation. However, whether one factor conjunctively controls the separated functions of AIF is not clear. Here, it is shown that OTU deubiquitinase 1 (OTUD1) acts as a link between the two functions of AIF via deubiquitination events. Deubiquitination of AIF at K244 disrupts the normal mitochondrial structure and compromises oxidative phosphorylation, and deubiquitination of AIF at K255 enhances its DNA‐binding ability to promote parthanatos. Moreover, OTUD1 stabilizes DDB1 and CUL4 associated factor 10 (DCAF10) and recruits the cullin 4A (CUL4A)‐damage specific DNA binding protein 1 (DDB1) complex to promote myeloid cell leukemia sequence 1 (MCL1) degradation, thereby activating caspase‐dependent apoptotic signaling. Collectively, these results reveal the central role of OTUD1 in activating both caspase‐independent and caspase‐dependent apoptotic signaling and propose decreased OTUD1 expression as a key event promoting chemoresistance in esophageal squamous cell carcinoma.

Contamination of Aflatoxins Induces Severe Hepatotoxicity Through Multiple Mechanisms

Aflatoxins (AFs) are commonly contaminating mycotoxins in foods and medicinal materials. Since they were first discovered to cause “turkey X” disease in the United Kingdom in the early 1960s, the extreme toxicity of AFs in the human liver received serious attention. The liver is the major target organ where AFs are metabolized and converted into extremely toxic forms to engender hepatotoxicity. AFs influence mitochondrial respiratory function and destroy normal mitochondrial structure. AFs initiate damage to mitochondria and subsequent oxidative stress. AFs block cellular survival pathways, such as autophagy that eliminates impaired cellular structures and the antioxidant system that copes with oxidative stress, which may underlie their high toxicities. AFs induce cell death via intrinsic and extrinsic apoptosis pathways and influence the cell cycle and growth via microribonucleic acids (miRNAs). Furthermore, AFs induce the hepatic local inflammatory microenvironment to exacerbate hepatotoxicity via upregulation of NF-κB signaling pathway and inflammasome assembly in the presence of Kupffer cells (liver innate immunocytes). This review addresses the mechanisms of AFs-induced hepatotoxicity from various aspects and provides background knowledge to better understand AFs-related hepatoxic diseases.

ATPIF1 maintains normal mitochondrial structure which is impaired by CCM3 deficiency in endothelial cells

BackgroundNumerous signaling pathways have been demonstrated experimentally to affect the pathogenesis of cerebral cavernous malformations (CCM), a disease that can be caused by CCM3 deficiency. However, the understanding of the CCM progression is still limited. The objective of the present work was to elucidate the role of CCM3 by RNA-seq screening of CCM3 knockout mice.ResultsWe found that ATPIF1 was decreased in siCCM3-treated Human Umbilical Vein Endothelial Cells (HUVECs), and the overexpression of ATPIF1 attenuated the changes in cell proliferation, adhesion and migration caused by siCCM3. The probable mechanism involved the conserved ATP concentration in mitochondria and the elongated morphology of the organelles. By using the CRISPR-cas9 system, we generated CCM3-KO Endothelial Progenitor Cells (EPCs) and found that the knockout of CCM3 destroyed the morphology of mitochondria, impaired the mitochondrial membrane potential and increased mitophagy. Overexpression of ATPIF1 contributed to the maintenance of normal structure of mitochondria, inhibiting activation of mitophagy and other signaling proteins (e.g., KLF4 and Tie2). The expression of KLF4 returned to normal in CCM3-KO EPCs after 2 days of re-overexpression of CCM3, but not other signaling proteins.ConclusionATPIF1 maintains the normal structure of mitochondria, inhibiting the activation of mitophagy and other signaling pathway in endothelial cells. Loss of CCM3 leads to the destruction of mitochondria and activation of signaling pathways, which can be regulated by KLF4.

SUN-572 Estrogen Synergistically Interacts with Optic Atrophy Protein 1 to Promote Thrombosis

Thrombosis is a major concern in: premenopausal females on oral contraceptives, menopausal women undergoing hormone replacement therapy, post-menopausal women and transgender individuals receiving estrogen supplementation. The mechanisms linking estrogen exposure with increased thrombosis risk is incompletely understood. Analysis of platelet transcripts in the Framingham cohort identified Optic atrophy 1 (OPA1) expression as being highly predictive of female sex and correlating with increased risk of diabetes and cardiovascular disease. OPA1 regulates mitochondrial fusion, electron transport chain (ETC), complex assembly, cristae morphology and apoptosis. Thus, to determine the functional relationship between platelet OPA1 expression and platelet function in relation to sex steroids we generated mice with platelet specific deletion of OPA-1 protein (pOPA1KO). Male pOPA1KO exhibited structurally and functionally compromised mitochondria with a 50% reduction in mitochondrial DNA and respiration. Male pOPA1KO mice exhibited a prothrombotic phenotype they had: increased agonist-induced activation, shortened time to stable occlusion of the carotid artery as assessed in vivo by (rose Bengal) photochemical injury (~25 min knockout vs ~ 35 min control), and were more prone to develop a thrombus (14/15 knockouts vs. 4/8 controls) following permanent ligation of the inferior vena cava. In contrast, female pOPA1KO mice had normal mitochondrial structure, function and DNA. Both agonist-induced platelet activation and thrombus formation was unchanged in pOPA1KO females. Paradoxically, pOPA-1 KO female mice had increased time to stable occlusion of the carotid artery as assessed by photochemical injury (~75 min Knockout vs ~35 min control). Notably, when platelets from pOPA-1 KO or control males were transferred into females following depletion of endogenous platelets, the reconstituted male platelets acquired the phenotype of female pOPA-1 KO mice. Thus, the time to stable occlusion of the carotid artery following photochemical injury was increased. Similarly, reconstitution of male mice with female pOPA1KO platelets were no longer prothrombotic. Gonadectomized pOPA1KO males had an increased time to stable occlusion and gonadectomized female pOPA1KO no longer exhibited increased time to stable carotid artery occlusion. Eugonadal pOPA1KO male mice treated with estrogen exhibited the pOPA1KO female thrombotic phenotype, with increased time to stable occlusion relative to control males. Additionally, OPA-1 levels were positively correlated with increased platelet aggregation and increased estrogen levels in third trimester pregnant human females. Together, these findings reveal a synergistic interaction between platelet OPA1 levels and estrogen to promote thrombosis.

PTEN inhibitor VO-OHpic attenuates GC-associated endothelial progenitor cell dysfunction and osteonecrosis of the femoral head via activating Nrf2 signaling and inhibiting mitochondrial apoptosis pathway

BackgroundGlucocorticoid (GC)-associated osteonecrosis of the femoral head (ONFH) is the most common in non-traumatic ONFH. Despite a strong relationship between GC and ONFH, the detailed mechanisms have remained elusive. Recent studies have shown that GC could directly injure the blood vessels and reduce blood supply in the femoral head. Endothelial progenitor cells (EPCs), which were inhibited quantitatively and functionally during ONFH, play an important role in maintaining the normal structure and function of vascular endothelium. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that promotes cell apoptosis, and its expression was found to be elevated in GC-associated ONFH patients. However, whether direct inhibition of PTEN attenuates GC-associated apoptosis and dysfunction of the EPCs remains largely unknown.MethodsWe investigated the effect of, VO-OHpic, a potent inhibitor of PTEN, in attenuating GC-associated apoptosis and dysfunction of EPCs and the molecular mechanism. SD rats were used to study the effect of VO-OHpic on angiogenesis and osteonecrosis in vivo.ResultsThe results revealed that methylprednisolone (MPS) obviously inhibit angiogenesis of EPCs by inducing apoptosis, destroying the normal mitochondrial structure, and disrupting function of mitochondria. VO-OHpic treatment is able to reverse the harmful effects by inhibiting the mitochondrial apoptosis pathway and activating the NF-E2-related factor 2 (Nrf2) signaling. Si-Nrf2 transfection significantly reduced the protective effects of VO-OHpic on EPCs. Our in vivo studies also showed that intraperitoneal injection of VO-OHpic obviously attenuates the osteonecrosis of the femoral head induced by MPS and potently increases the blood supply in the femoral head.ConclusionTaken together, the data suggests that inhibition of PTEN with VO-OHpic attenuates apoptosis and promotes angiogenesis of EPCs in vitro via activating Nrf2 signaling pathway and inhibiting the mitochondrial apoptosis pathway. Moreover, VO-OHpic also mitigates GC-associated ONFH and potentiates angiogenesis in the femoral head.

Fis1 deficiencies differentially affect mitochondrial quality in skeletal muscle

Mitochondrial dynamics and mitophagy are important aspects of mitochondrial quality control, and are linked to neurodegenerative diseases and muscular diseases. Fis1, a protein on the mitochondrial outer membrane, is thought to mediate mitochondrial fission. However, Fis1 null worms and mammalian cells only display mild fission defects but show aberrant mitophagy. To assess Fis1 function in vivo, we generated conditional knock-out Fis1 mice to allow for specific Fis1 deletion in adult skeletal muscle. In the absence of Fis1 in Type I muscle, mitochondrial hyperfusion, respiratory chain deficiency, and increased mitophagy were found. Moreover, abnormal mitophagy was aggravated by endurance exhaustive exercise stress (EEE), suggesting that Fis1 is involved in maintaining normal mitophagy in mitochondria-rich Type I muscle during exercise. Additionally, Fis1 loss induced delayed onset muscle ultrastructure change (DOMUC) in Type I muscle and strong inflammation in response to acute exhaustive exercise (EE). Thus, we identify a role for Fis1 in maintaining normal mitochondrial structure and function at rest and under exercise stress.

In silico molecular modeling and docking studies on the Leishmania mitochondrial iron transporter-1 (LMIT1)

Leishmania mitochondrial iron transporter-1 (LMIT1) is a transmembrane protein required for normal mitochondrial structure and function. This protein is essential for the replication, survival, virulence, and the differentiation of promastigotes into infective amastigotes. Regarding the important role of LMIT1 in the iron-regulated process in Leishmania parasites, it can be considered as a promising target for structure-based drug design against leishmaniasis. In the present study, the three-dimensional (3D) structure of LMIT1 was determined by various in silico modeling approaches. The quality of the built models was evaluated using the different servers. The best model, which was predicted by Robetta, was selected for following analyses. Thereafter, the obtained model was successfully refined and used for determination of its probable inhibitors. Virtual screening of an approved compound library was employed to find the probable LMIT1 inhibitors. Ultimately, six molecules were found to inhibit the iron interaction with the LMIT1 in proper orientation and binding energy. The inhibitor candidates are shown to interact with functional residues of LMIT1. The achieved compounds could pave the way toward the development of new drugs against leishmaniasis in future studies.

Molecular mechanisms mediating the adaptive regulation of human colonic thiamine pyrophosphate (TPP) uptake process

Thiamin(vitamin B1) is indispensable for normal cellular metabolism due to itsinvolvement in critical metabolic pathways (e.g., energy metabolism, maintenanceof normal cellular redox state, normal mitochondrial structure and function). Human(mammals) cannot synthesize thiamin, and thus, must obtain the vitamin fromexogenous sources, i. e., diet and gut microbiota. A substantial amount of themicrobiota‐generated thiamin exists in the form of TPP. We have previously shown the existence of a specific and high‐affinity carrier‐mediated uptake process for TPP in human colonocytes (Am J Physiol Gastrointest Liver Physiol. 2012; 303: G389‐95), and have recently determined the molecular identity of the system involved (product of the SLC44A4 gene) (J Biol Chem. 2014; 289: 4405‐16). We have also shown that the colonic TPP uptake process is adaptively regulated by the extracellular substrate level, but nothing is known about the molecular mechanism(s) that mediates this regulation. We addressed this issue in the current investigation using the human colonepithelial NCM460 cells as a model. Maintaining these cells in the presence of high levels of TPP (1 mM) for 2 and 9 days was found to lead to a significant(p ≤ 0.01) reduction in the rate of TPP uptake compared to uptake by cells maintained in the absence of added TPP. While no change in the level of expression of the SLC44A4 mRNA was found in cells maintained for 2 days in the presence of high TPP level [suggesting possible involvement of post translation mechanism(s)], a significant (p ≤ 0.01) decrease in level of SLC44A4 mRNA was found in cells maintained for 9 days. We also observed a decrease in activity of the SLC44A4 promoter in cells maintained for 9 days in high TPP level. The latter was associated with clear histone modifications at the SLC44A4 promoter that involve significant decrease in histone H3K4‐trimethylation and H3K9‐acetylation (euchromatin/activating markers)and a significant increase in histone H3K9‐trimethylation and H3K27‐trimethylation (heterochromatin/repressive markers). While maintaining the cells for 9 days in the presence of high TPP level did not affect the level of expression of the two transcription factors, ELF3 and CREB‐1, that drive the activity of the SLC44A4promoter, their binding to the SLC44A4promoter was decreased as shown by Chip‐qPCR analysis. These studies demonstrate, for the first time, that multiple mechanisms are involved in the adaptive regulation of colonic TPP uptake by extracellular substrate level. At early stages, this regulation involves post‐translational mechanism(s), while long‐term adaptive regulation involves transcriptional and epigenetic mechanisms.Support or Funding InformationSupported by grants from the DVA and the NIH (DK56061 and DK58057).

The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure

The protein optic atrophy 1 (OPA1) is a dynamin-related protein associated with the inner mitochondrial membrane and functions in mitochondrial inner membrane fusion and cristae maintenance. Inner membrane-anchored long OPA1 (L-OPA1) undergoes proteolytic cleavage resulting in short OPA1 (S-OPA1). It is often thought that S-OPA1 is a functionally insignificant proteolytic product of L-OPA1 because the accumulation of S-OPA1 due to L-OPA1 cleavage is observed in mitochondrial fragmentation and dysfunction. However, cells contain a mixture of both L- and S-OPA1 in normal conditions, suggesting the functional significance of maintaining both OPA1 forms, but the differential roles of L- and S-OPA1 in mitochondrial fusion and energetics are ill-defined. Here, we examined mitochondrial fusion and energetic activities in cells possessing L-OPA1 alone, S-OPA1 alone, or both L- and S-OPA1. Using a mitochondrial fusion assay, we established that L-OPA1 confers fusion competence, whereas S-OPA1 does not. Remarkably, we found that S-OPA1 alone without L-OPA1 can maintain oxidative phosphorylation function as judged by growth in oxidative phosphorylation-requiring media, respiration measurements, and levels of the respiratory complexes. Most strikingly, S-OPA1 alone maintained normal mitochondrial cristae structure, which has been commonly assumed to be the function of OPA1 oligomers containing both L- and S-OPA1. Furthermore, we found that the GTPase activity of OPA1 is critical for maintaining cristae tightness and thus energetic competency. Our results demonstrate that, contrary to conventional notion, S-OPA1 is fully competent for maintaining mitochondrial energetics and cristae structure.

Protection of mitochondria prevents high-fat diet–induced glomerulopathy and proximal tubular injury

Obesity is a major risk factor for the development of chronic kidney disease, even independent of its association with hypertension, diabetes, and dyslipidemia. The primary pathologic finding of obesity-related kidney disease is glomerulopathy, with glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis. Proposed mechanisms leading to renal pathology include abnormal lipid metabolism, lipotoxicity, inhibition of AMP kinase, and endoplasmic reticulum stress. Here we report dramatic changes in mitochondrial structure in glomerular endothelial cells, podocytes, and proximal tubular epithelial cells after 28 weeks of a high-fat diet in C57BL/6 mice. Treatment with SS-31, a tetrapeptide that targets cardiolipin and protects mitochondrial cristae structure, during high-fat diet preserved normal mitochondrial structure in all kidney cells, restored renal AMP kinase activity, and prevented intracellular lipid accumulation, endoplasmic reticulum stress, and apoptosis. SS-31 had no effect on weight gain, insulin resistance or hyperglycemia. However, SS-31 prevented loss of glomerular endothelial cells and podocytes, mesangial expansion, glomerulosclerosis, macrophage infiltration, and upregulation of proinflammatory (TNF-α, MCP-1, NF-κB) and profibrotic (TGF-β) cytokines. Thus, mitochondria protection can overcome lipotoxicity in the kidney and represent a novel upstream target for therapeutic development.

Interaction of MDM33 with mitochondrial inner membrane homeostasis pathways in yeast.

Membrane homeostasis affects mitochondrial dynamics, morphology, and function. Here we report genetic and proteomic data that reveal multiple interactions of Mdm33, a protein essential for normal mitochondrial structure, with components of phospholipid metabolism and mitochondrial inner membrane homeostasis. We screened for suppressors of MDM33 overexpression-induced growth arrest and isolated binding partners by immunoprecipitation of cross-linked cell extracts. These approaches revealed genetic and proteomic interactions of Mdm33 with prohibitins, Phb1 and Phb2, which are key components of mitochondrial inner membrane homeostasis. Lipid profiling by mass spectrometry of mitochondria isolated from Mdm33-overexpressing cells revealed that high levels of Mdm33 affect the levels of phosphatidylethanolamine and cardiolipin, the two key inner membrane phospholipids. Furthermore, we show that cells lacking Mdm33 show strongly decreased mitochondrial fission activity indicating that Mdm33 is critical for mitochondrial membrane dynamics. Our data suggest that MDM33 functionally interacts with components important for inner membrane homeostasis and thereby supports mitochondrial division.

The integrin-adhesome is required to maintain muscle structure, mitochondrial ATP production, and movement forces in Caenorhabditis elegans.

The integrin-adhesome network, which contains >150 proteins, is mechano-transducing and located at discreet positions along the cell-cell and cell-extracellular matrix interface. A small subset of the integrin-adhesome is known to maintain normal muscle morphology. However, the importance of the entire adhesome for muscle structure and function is unknown. We used RNA interference to knock down 113 putative Caenorhabditis elegans homologs constituting most of the mammalian adhesome and 48 proteins known to localize to attachment sites in C. elegans muscle. In both cases, we found >90% of components were required for normal muscle mitochondrial structure and/or proteostasis vs. empty vector controls. Approximately half of these, mainly proteins that physically interact with each other, were also required for normal sarcomere and/or adhesome structure. Next we confirmed that the dystrophy observed in adhesome mutants associates with impaired maximal mitochondrial ATP production (P < 0.01), as well as reduced probability distribution of muscle movement forces compared with wild-type animals. Our results show that the integrin-adhesome network as a whole is required for maintaining both muscle structure and function and extend the current understanding of the full complexities of the functional adhesome in vivo.—Etheridge, T., Rahman, M., Gaffney, C. J., Shaw, D., Shephard, F., Magudia, J., Solomon, D. E., Milne, T., Blawzdziewicz, J., Constantin-Teodosiu, D., Greenhaff, P. L., Vanapalli, S. A., Szewczyk, N. J. The integrin-adhesome is required to maintain muscle structure, mitochondrial ATP production, and movement forces in Caenorhabditis elegans.

Inhibition of ERK-DLP1 signaling and mitochondrial division alleviates mitochondrial dysfunction in Alzheimer's disease cybrid cell

Mitochondrial dysfunction is an early pathological feature of Alzheimer’s disease (AD). The underlying mechanisms and strategies to repair it remain unclear. Here, we demonstrate for the first time the direct consequences and potential mechanisms of mitochondrial functional defects associated with abnormal mitochondrial dynamics in AD. Using cytoplasmic hybrid (cybrid) neurons with incorporated platelet mitochondria from AD and age-matched non-AD human subjects into mitochondrial DNA (mtDNA)-depleted neuronal cells, we observed that AD cybrid cells had significant changes in morphology and function; such changes associate with altered expression and distribution of dynamin-like protein (DLP1) and mitofusin 2 (Mfn2). Treatment with antioxidant protects against AD mitochondria-induced extracellular signal-regulated kinase (ERK) activation and mitochondrial fission-fusion imbalances. Notably, inhibition of ERK activation not only attenuates aberrant mitochondrial morphology and function but also restores the mitochondrial fission and fusion balance. These effects suggest a role of oxidative stress-mediated ERK signal transduction in modulation of mitochondrial fission and fusion events. Further, blockade of the mitochondrial fission protein DLP1 by a genetic manipulation with a dominant negative DLP1 (DLP1K38A), its expression with siRNA-DLP1, or inhibition of mitochondrial division with mdivi-1 attenuates mitochondrial functional defects observed in AD cybrid cells. Our results provide new insights into mitochondrial dysfunction resulting from changes in the ERK-fission/fusion (DLP1) machinery and signaling pathway. The protective effect of mdivi-1 and inhibition of ERK signaling on maintenance of normal mitochondrial structure and function holds promise as a potential novel therapeutic strategy for AD.