Mitochondrial Loss Research Articles

Attenuation of tacrolimus induced oxidative stress, mitochondrial damage, and cell cycle arrest by Boerhavia diffusa root fraction in mdck cell lines.

Objective(s):The protective effect of ethyl acetate fraction (EAF) of Boerhavia diffusa roots against Tacrolimus (TAC) induced nephrotoxicity was studied using MDCK cell lines.Materials and Methods:Ethanolic root extract of B. diffusa was fractionated using the liquid-liquid partition method. The cytotoxic effect of TAC and protective effect of EAF co-treatment were studied in MDCK cell lines by measuring ROS, LPO, and NO levels; collagen accumulation, effect on mitochondrial membrane integrity and cell cycle analysis were studied. The active component in EAF was quantified by HPLC analysis.Results:TAC induced toxicity, leading to apoptosis and necrosis, was significantly reduced (P<0.001) in EAF co-treatment, with reversal of cell cycle arrest and reduced cell population at sub G0/G1 phase. Further, ROS (P<0.05), LPO and NO (P<0.001), were significantly reduced with EAF co-treatment compared with TAC individually treated cells. TAC induced mitochondrial membrane integrity loss was found to be significantly reduced in co-treated cells, as measured by rhodamine123 (P<0.05) and translocation of cytochrome c (P<0.001) from nucleus to cytoplasm, and caspase 3 release (P<0.001). The same was confirmed through annexin-FITC and PI staining (P<0.05) with reduced apoptotic and necrotic death in co-treated population. Interestingly, EAF co-treatment decreased collagen accumulation (P<0.001) with significant increase in the cell survival of tubular epithelial cells. HPLC analysis showed the presence of Quercetin (87.5 mg/g) in EAF, which may be responsible for the nephroprotective role. Conclusion:Thus, these results provide sound evidence that EAF may be an effective adjuvant therapy to prevent nephrotoxicity induced by TAC.

Ruthenium(II) and Platinum(II) Complexes with Biologically Active Aminoflavone Ligands Exhibit In Vitro Anticancer Activity.

Continuing our studies on the mechanisms underlying the cytotoxicity of potential drugs, we have described several aspects of the in vitro anticancer activity of ruthenium(II) and platinum(II) complexes with bioactive, synthetic aminoflavone ligands. We examined the mechanism of proapoptotic activity of cis-dichlorobis(3-imino-2-methoxyflavanone)ruthenium(II), cis-dichlorobis(3-imino-2-ethoxyflavanone)ruthenium(II), and trans-dichlorobis(3-aminoflavone)platinum(II). Cisplatin was used as a reference compound. The cytotoxicity was investigated by MTT assay. The mechanism of proapoptotic activity of the tested compounds was investigated by evaluation of caspase-8 activity, cytometric analysis of annexin-V positive cells, and mitochondrial potential loss measurement. The results showed that ruthenium compounds break partially or completely the cisplatin resistance by activating the caspase 8-dependent apoptosis pathway and loss of mitochondrial membrane potential. Platinum compounds also have a cytostatic effect, but their action requires more exposure time. Potential mechanisms underlying drug resistance in the two pairs of cancer cell lines were investigated: total glutathione content, P-glycoprotein activity, and differences in the activity of DNA repair induced by nucleotide excision. Results showed that cisplatin-resistant cells have elevated glutathione levels relative to sensitive cells. Moreover, they indicated the mechanisms enabling cells to avoid apoptosis caused by DNA damage. Pg-P activity has no effect on the development of cisplatin resistance in the cell lines described.

Protective Effect of Polysaccharides Extracted from Cudrania tricuspidata Fruit against Cisplatin-Induced Cytotoxicity in Macrophages and a Mouse Model.

Although cisplatin is one of most effective chemotherapeutic drugs that is widely used to treat various types of cancer, it can cause undesirable damage in immune cells and normal tissue because of its strong cytotoxicity and non-selectivity. This study was conducted to investigate the cytoprotective effects of Cudrania tricuspidata fruit-derived polysaccharides (CTPS) against cisplatin-induced cytotoxicity in macrophages, lung cancer cell lines, and a mouse model, and to explore the possibility of application of CTPS as a supplement for anticancer therapy. Both cisplatin alone and cisplatin with CTPS induced a significant cytotoxicity in A549 and H460 lung cancer cells, whereas cytotoxicity was suppressed by CTPS in cisplatin-treated RAW264.7 cells. CTPS significantly attenuated the apoptotic and necrotic population, as well as cell penetration in cisplatin-treated RAW264.7 cells, which ultimately inhibited the upregulation of Bcl-2-associated X protein (Bax), cytosolic cytochrome c, poly (adenosine diphosphateribose) polymerase (PARP) cleavage, and caspases-3, -8, and -9, and the downregulation of B cell lymphoma-2 (Bcl-2). The CTPS-induced cytoprotective action was mediated with a reduction in reactive oxygen species production and mitochondrial transmembrane potential loss in cisplatin-treated RAW264.7 cells. In agreement with the results obtained above, CTPS induced the attenuation of cell damage in cisplatin-treated bone marrow-derived macrophages (primary cells). In in vivo studies, CTPS significantly inhibited metastatic colonies and bodyweight loss as well as immunotoxicity in splenic T cells compared to the cisplatin-treated group in lung metastasis-induced mice. Furthermore, CTPS decreased the level of CRE and BUN in serum. In summation, these results suggest that CTPS-induced cytoprotective action may play a role in alleviating the side effects induced by chemotherapeutic drugs.

Frequency characteristics of horizontal semicircular canals damage and the ultrastructure analysis of crista ampullaris in patients with Meniere's disease

Objective: To investigate the frequency characteristics and the pathological characteristics of the horizontal crista ampullaris in patients with Meniere's disease,and to analyse its structural basis. Methods: Between March, 2019 and November, 2019, seventy-two patients diagnosed as Meniere's disease (27 males and 45 females, aged from 13 to 74 years, with a course of disease ranging from 4 months to 32 years)in Shandong Provincial ENT Hospital were included.Caloric test, sinusoidal harmonic acceleration test (SHA), video-head impulse test (v-HIT), Gadolinium-enhanced inner-ear 3D-FLAIR MRI and pure tone audiometry were conducted in the patients. The function of the horizontal semicircular canal in these patients were analysed as well as its relationship with the degree of endolymphatic hydrops,clinical stage and duration. Light microscopy and transmission electron microscopy were used to observe the ultrastructure of horizontal semicircular canal crista ampullaris from six patients with refractory Meniere's disease who underwent labyrinthectomy. The number of type Ⅰ and type Ⅱ vestibular hair cells, the common pathophysiological changes of horizontal semicircular canal crista ampullaris were investigated in these patients. Statistical analysis was performed using SPSS 19.0. Results: With the increase of detection frequency, the abnormal rate decreased gradually. The abnormal rate of caloric test was 69.4% (50/72), SHA 51.4% (37/72), V-HIT 36.1% (26/72), comparation of the positive rate among the three tests showed statistically significant differences(P<0.05).Neither caloric test nor SHA had correlation with the degree of hydrops(P>0.05), but v-HIT(r=0.434,P<0.01).There was correlation with clinical stage to SHA and v-HIT(r=0.338,0.462,P<0.01), except caloric test(P>0.05).No significant relation was found with caloric test, SHA, v-HIT and course of disease(P>0.05).Morphological observation found abnormal monolayer epithelialization of the horizontal semicircular canal crista ampullaris significantly decreased number of type Ⅱ hair cells compared with type Ⅰhair cells. Hair cells showed perinuclear vacuolization, cytoplasmic vacuoles, mitochondrial electron density increasement and loss of stereocilia. Conclusions: The horizontal semicircular canal damage in the patients with Meniere's disease has a frequency-dependent characteristic, mainly occurres in low frequency area. With progress of the disease, the high frequency area of ampulla will be impaired gradually, and it is related to the degree of endolymphatic hydrops and hearing level. Hair cell injury would be observed,the frequency characteristics may be more associated with the disorder of type Ⅱ hair cells.

Thymoquinone protects against cardiac mitochondrial DNA loss, oxidative stress, inflammation and apoptosis in isoproterenol-induced myocardial infarction in rats

IntroductionMyocardial infarction (MI) is an ischemic life-threatening disease with exaggerated oxidative stress state that vigorously damages the cardiomyocyte membrane and subcellular structures, including the vital mitochondrial DNA (mtDNA). The mtDNA is responsible for the proper functionality of the mitochondria, which are abundant in cardiomyocytes due to their dynamic nature and energy production requirements. Furthermore, oxidative stress triggers an inflammatory cascade and eventual apoptosis, which exacerbates cardiac injuries and dysfunction. AimThe present study used an isoproterenol (ISP)-induced MI rat model to investigate the role of the main active constituent of Nigella Sativa seeds, thymoquinone (TQ), in preserving the cardiac mtDNA content and ameliorating oxidative stress, inflammation, and apoptosis. MethodsRats in the (TQ + ISP) group were pre-treated with TQ (20 mg/kg/day) for 21 days before the MI induction using ISP (85 mg/kg/day). In addition, negative control and ISP groups were included in the study for comparison. A histopathological examination was performed and serum cardiac parameters (cTnI and LDH) were assessed. In addition, mtDNA content, oxidative stress parameters (MDA, GSH, SOD, GPx, and CAT), inflammatory mediators (IL-6, IL-1β, and TNF-α), and apoptosis markers (BAX, Bcl2, and caspase-3) were detected. ResultsThe results showed that pre- and co-treatment with TQ in the (TQ + ISP) group reversed the histoarchitecture changes, caused a significant decrease in serum cardiac markers, oxidative stress markers, inflammatory cytokines, the apoptosis process, and preserved the cardiac mtDNA content. ConclusionTQ is a cardioprotective agent with an extended effect on preserving the cardiac mtDNA content, in addition to its powerful antioxidant, anti-inflammatory, and anti-apoptotic action.

Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.

Mitochondrial localization and moderated activity are key to murine erythroid enucleation

Mammalian red blood cells (RBCs), which primarily contain hemoglobin, exemplify an elaborate maturation process, with the terminal steps of RBC generation involving extensive cellular remodeling. This encompasses alterations of cellular content through distinct stages of erythroblast maturation that result in the expulsion of the nucleus (enucleation) followed by the loss of mitochondria and all other organelles and a transition to anaerobic glycolysis. Whether there is any link between erythroid removal of the nucleus and the function of any other organelle, including mitochondria, remains unknown. Here we demonstrate that mitochondria are key to nuclear clearance. Using live and confocal microscopy and high-throughput single-cell imaging, we show that before nuclear polarization, mitochondria progressively move toward one side of maturing erythroblasts and aggregate near the nucleus as it extrudes from the cell, a prerequisite for enucleation to proceed. Although we found active mitochondrial respiration is required for nuclear expulsion, levels of mitochondrial activity identify distinct functional subpopulations, because terminally maturing erythroblasts with low relative to high mitochondrial membrane potential are at a later stage of maturation, contain greatly condensed nuclei with reduced open chromatin–associated acetylation histone marks, and exhibit higher enucleation rates. Lastly, to our surprise, we found that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation, primarily through pyruvate but independent of in situ glycolysis. These findings demonstrate the critical but unanticipated functions of mitochondria during the erythroblast enucleation process. They are also relevant to the in vitro production of RBCs as well as to disorders of the erythroid lineage.

Hyoscyamus albus nortropane alkaloids reduce hyperglycemia and hyperinsulinemia induced in HepG2 cells through the regulation of SIRT1/NF-kB/JNK pathway

BackgroundChronic superphysiological glucose and insulin concentrations are known to trigger several tissue and organ failures, including insulin resistance, oxidative stress and chronic low-grade inflammation. Hence, the screening for molecules that may counteract such conditions is essential in current existing therapeutic strategies, thereby the use of medicinal plant derivatives represents a promising axis in this regard.MethodsIn this study, the effect of a selected traditional medicinal plant, Hyoscyamus albus from which, calystegines have been isolated, was investigated in an experimental model of hyperinsulinemia and hyperglycemia induced on HepG2 cells. The mRNA and protein expression levels of different insulin signaling, gluconeogenic and inflammatory pathway- related molecules were examined. Additionally, cell viability and apoptosis, oxidative stress extent and mitochondrial dysfunctions were assayed using flow cytometric and qRT-PCR techniques.ResultsTreatment of IR HepG2 cells with calystegines strongly protected the injured cells from apoptosis, oxidative stress and mitochondrial integrity loss. Interestingly, nortropane alkaloids efficiently regulated the impaired glucose metabolism in IR HepG2 cells, through the stimulation of glucose uptake and the modulation of SIRT1/Foxo1/G6PC/mTOR pathway, which is governing the hepatic gluconeogenesis. Furthermore, the alkaloidal extract restored the defective insulin signaling pathway, mainly by promoting the expression of Insr at the mRNA and protein levels. What is more, treated cells exhibited significant mitigated inflammatory response, as evidenced by the modulation and the regulation of the NF- κB/JNK/TLR4 axis and the downstream proinflammatory cytokines recruitment.ConclusionOverall, the present investigation demonstrates that calystegines from Hyoscyamus albus provide cytoprotection to the HepG2 cells against insulin/glucose induced insulin resistance and apoptosis due to the regulation of SIRT1/Foxo1/G6PC/mTOR and NF-κB/JNK/TLR4 signaling pathways.1oVDx9MMsFkfL9sCGFfm2tVideo

Impact of Fatty Acid-Binding Proteins in α-Synuclein-Induced Mitochondrial Injury in Synucleinopathy.

Synucleinopathies are diverse diseases with motor and cognitive dysfunction due to progressive neuronal loss or demyelination, due to oligodendrocyte loss in the brain. While the etiology of neurodegenerative disorders (NDDs) is likely multifactorial, mitochondrial injury is one of the most vital factors in neuronal loss and oligodendrocyte dysfunction, especially in Parkinson’s disease, dementia with Lewy body, multiple system atrophy, and Krabbe disease. In recent years, the abnormal accumulation of highly neurotoxic α-synuclein in the mitochondrial membrane, which leads to mitochondrial dysfunction, was well studied. Furthermore, fatty acid-binding proteins (FABPs), which are members of a superfamily and are essential in fatty acid trafficking, were reported to trigger α-synuclein oligomerization in neurons and glial cells and to target the mitochondrial outer membrane, thereby causing mitochondrial loss. Here, we provide an updated overview of recent findings on FABP and α-synuclein interactions and mitochondrial injury in NDDs.

Metabolic Compensation Associated with Progressive Mitochondrial Dysfunction

The decline in mitochondrial function has been associated and correlated with aging and various neurogenerative diseases. The mitochondria lie at a critical junction of several metabolic pathways, and metabolic reactions are highly compartmentalized across distinct tissues. As such, progressive mitochondrial defects will have diverse metabolic and phenotypic consequences that could reflect physiological changes that occur with aging. Here, we comprehensively characterized metabolic alterations in PolgD257A mitochondrial DNA mutator mice throughout the aging process. Plasma alanine increased dramatically with progressive loss of mitochondria, with lactate and other organic acids accumulating to a lesser extent. These changes are reflective of an increased glycolytic flux and rapid glucose turnover, and a reduced ability to oxidize keto acids, as demonstrated by [13C]glucose tracing. Amino acid changes were also suggestive of dysfunctional nitrogen metabolism, which was confirmed by quantifying [15N]ammonium incorporation into urea and amino acids. Additionally, progressive mitochondrial dysfunction led to alterations in the lipidome including sphingolipids. We observed the accumulation of 1-deoxysphingolipids, which are synthesized by serine palmitoyltransferase (SPT) using alanine. Consistent with this metabolic rewiring, PolgD257A mice exhibit accelerated thermal hypoalgesia. These results highlight the distinct changes that occur in carbon and nitrogen metabolism upon mitochondrial loss and highlight key metabolic mechanisms which can drive aging associated neuropathy.

Protein Abundance of NOR‐1 is Diminished During Aging and Skeletal Muscle Disuse

INTRODUCTION Skeletal muscle mass and function are crucial for metabolic health and quality of life. Mitochondrial defects and loss of proteostasis driven by inactivity and aging predispose skeletal muscle to apoptosis and greater degradative signaling. Nuclear orphan receptor NOR-1 has been shown to be responsive to exercise and promotes an oxidative phenotype. NOR-1 is also an important regulator of glucose uptake, fatty acid oxidation, and mitochondrial biogenesis. A gap in our knowledge is that we do not know if NOR-1 is altered by aging or muscle disuse. PURPOSE Exercise is beneficial for skeletal muscle and overall health. However, exercise participation is low among the elderly, which represent a clinically relevant population prone to inactivity and bed rest. Disuse due to prolonged immobility can result in significant atrophy and metabolic impairments to the immobilized limb. Identifying genes that are dysregulated during aging or disuse is crucial for discovering therapeutic targets that might be modulated to delay or mitigate detriments to skeletal muscle mass and function. Although NOR-1 is responsive to exercise and is associated with improved metabolic and mitochondrial function, the role of NOR-1 in disuse and aging-related muscle atrophy has yet to be investigated. Therefore, the purpose of this study was to determine if aging and limb immobilization impact skeletal muscle protein content of NOR-1. We hypothesized that both disuse and aging reduce skeletal muscle protein abundance of NOR-1 in mice. METHODS Aged (33 months) and adult (6-10 months) C57/BL6 mice were used in this study. To investigate the effect of age, the tibialis anterior (TA) of aged and adult control mice were harvested and analyzed for skeletal muscle protein content of NOR-1. In a separate set of experiments, adult mice were subjected to 14 days of unilateral cast immobilization to induce disuse associated atrophy in the hindlimb plantarflexors. After immobilization, the gastrocnemius and soleus muscles of both the immobilized and contralateral limb were harvested, weighed, and analyzed for NOR-1 protein content. Skeletal muscle abundance of NOR-1 protein was measured by western blot. Paired t-test were used to determine differences between immobilized and contralateral limbs. To compare aged and adult animals, data were analyzed via unpaired t-test. RESULTS Protein abundance of NOR-1 was reduced (72%; p<0.05) in aged mice when compared to adult control mice. Additionally, hindlimb immobilization in adult mice resulted in marked (35% and 18%; p<0.05) decrease in the wet weight of soleus and gastrocnemius muscles respectively and reduced NOR-1 protein abundance (51% and 42%; p<0.05) in soleus and gastrocnemius muscles respectively, as compared to the contralateral control muscles. Taken together, these data suggest muscle protein content of NOR-1 is reduced during disuse and aging.

Mitocytosis, a migrasome-mediated mitochondrial quality-control process

Damaged mitochondria need to be cleared to maintain the quality of the mitochondrial pool. Here, we report mitocytosis, a migrasome-mediated mitochondrial quality-control process. We found that, upon exposure to mild mitochondrial stresses, damaged mitochondria are transported into migrasomes and subsequently disposed of from migrating cells. Mechanistically, mitocytosis requires positioning of damaged mitochondria at the cell periphery, which occurs because damaged mitochondria avoid binding to inward motor proteins. Functionally, mitocytosis plays an important role in maintaining mitochondrial quality. Enhanced mitocytosis protects cells from mitochondrial stressor-induced loss of mitochondrial membrane potential (MMP) and mitochondrial respiration; conversely, blocking mitocytosis causes loss of MMP and mitochondrial respiration under normal conditions. Physiologically, we demonstrate that mitocytosis is required for maintaining MMP and viability in neutrophils invivo. We propose that mitocytosis is an important mitochondrial quality-control process in migrating cells, which couples mitochondrial homeostasis with cell migration.

Mechanism of hepatocyte mitochondrial NDUFA13 deficiency-induced liver fibrogenesis: the role of abnormal hepatic stellate cell activation

To investigate the role of hepatocyte mitochondrial NDUFA13 loss in the liver fibrogenesis in mice and explore the possible mechanisms. We used liver-specific NDUFA13 heterozygous knockout mouse models (NDUFA13fl/-; Alb-Cre) established previously by intercrossing NDUFA13fl/fl and Alb-Cre mice, with their littermate control NDUFA13fl/fl mice as the control (n=8). The mice were euthanized at the age of 4 weeks and 2 years, and the liver tissues were collected for HE and Masson staining to observe the pathological changes and fibrosis phenotypes. Western blotting was performed to detect the expression of NDUFA13 protein in the liver tissues, and the infiltration of F4/80+ macrophages and the expressions of TGF-β1, TNF-α and IL-1β were analyzed by immunofluorescence assay. The expression levels of α-SMA, matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteases 1 (TIMP-1), collagen-Ⅰ and collagen-Ⅲ were assayed by immunohistochemistry. HE and Masson staining showed obvious inflammatory infiltration but no significant fibrosis in the liver tissues of 4-week-old NDUFA13fl/- mice, but severe liver damage with massive fibrosis was observed in 2-year-old NDUFA13fl/- mice. NDUFA13 expression in 2-year-old NDUFA13fl/- mice markedly decreased compared with that in the control NDUFA13fl/fl mice as shown by Western blotting (P < 0.05). Immunohistochemistry showed obvious infiltration of F4/80+ macrophages in the liver tissue with a large amount of TGF-β1 production (P < 0.05) and TNF-α and IL-1β secretions in NDUFA13fl/- mice (P < 0.05). NDUFA13 knockout obviously promoted α-SMA expression (P < 0.05) and collagen-Ⅰ and collagen-Ⅲ deposition (P < 0.05) while significantly decreased MMP-9 and increased TIMP-1 expression in the liver (P < 0.05). Hepatocytes-specific NDUFA13 deficiency can trigger spontaneous and chronic liver fibrosis phenotypes in mice probably in association with abnormal activation of hepatic stellate cells induced by macrophages and inflammatory factors.

Fucoidan induces ROS-dependent epigenetic modulation in cervical cancer HeLa cell

Fucoidan is a sulfated polysaccharide obtained from marine algae and known for various pharmacological activities. In this study, we investigated the effect of Fucoidan on cell viability, redox balance, cytoskeletal component F-actin, HDAC inhibition, autophagy, and senescence phenomenon in human cervical cancer HeLa cell line in comparison to positive control suberoylanilide hydroxamic acid by flow cytometry, fluorescence microscopy, and western blotting. Our observations revealed that Fucoidan exposure induces cytotoxicity in HeLa cells via ROS and mitochondrial superoxide generation and loss of ATP. Colorimetrical studies suggested that Fucoidan impairs the function of HDAC expression. Fucoidan treatment also contributes to the change in the granularity of cells, senescence-associated heterochromatin foci formation that leads to senescence in HeLa cells. Moreover, we visualize that Fucoidan exhibits autophagosomes formation with monodansylcadaverine, and flow cytometry analysis by acridine orange further substantiates that Fucoidan triggers autophagy in HeLa cells. Additionally, the changes in the expression of proteins p21, p16, BECN1, and HDAC1 were seen as markers of senescence, autophagy, and HDAC inhibition by FACS and immunoblotting. Molecular docking study validates Fucoidan–HDAC1 association in corroboration with the experimental data. Collectively, these mechanistic studies demonstrated that Fucoidan could be a therapeutic molecule for targeting HDACs in cervical cancer.

Mitochondrial Dysfunction: Cause or Consequence of Vascular Calcification?

Mitochondria are crucial bioenergetics powerhouses and biosynthetic hubs within cells, which can generate and sequester toxic reactive oxygen species (ROS) in response to oxidative stress. Oxidative stress-stimulated ROS production results in ATP depletion and the opening of mitochondrial permeability transition pores, leading to mitochondria dysfunction and cellular apoptosis. Mitochondrial loss of function is also a key driver in the acquisition of a senescence-associated secretory phenotype that drives senescent cells into a pro-inflammatory state. Maintaining mitochondrial homeostasis is crucial for retaining the contractile phenotype of the vascular smooth muscle cells (VSMCs), the most prominent cells of the vasculature. Loss of this contractile phenotype is associated with the loss of mitochondrial function and a metabolic shift to glycolysis. Emerging evidence suggests that mitochondrial dysfunction may play a direct role in vascular calcification and the underlying pathologies including (1) impairment of mitochondrial function by mineral dysregulation i.e., calcium and phosphate overload in patients with end-stage renal disease and (2) presence of increased ROS in patients with calcific aortic valve disease, atherosclerosis, type-II diabetes and chronic kidney disease. In this review, we discuss the cause and consequence of mitochondrial dysfunction in vascular calcification and underlying pathologies; the role of autophagy and mitophagy pathways in preventing mitochondrial dysfunction during vascular calcification and finally we discuss mitochondrial ROS, DRP1, and HIF-1 as potential novel markers and therapeutic targets for maintaining mitochondrial homeostasis in vascular calcification.

An HSP90 Cochaperone Ids2 Maintains the Stability of Mitochondrial DNA and ATP Synthase

A previous study demonstrates crosstalk among three hallmarks of aging: deregulated nutrient sensing, loss of proteostasis, and mitochondrial dysfunction. Nutrient overconsumption impairs the folding activity of the HSP90-Ids2 complex, but it remains unclear through what mechanisms proteostasis controls mitochondrial functions. Here we uncover Ids2 as an essential cochaperone for maintaining mitochondrial DNA and functions. A screen of the databases of Hsc82 physical interactors, mitochondrial components, and mutants with respiratory defect identified Atp3, a subunit of the complex V ATP synthase, as a client of Ids2. Deletion of IDS2 destabilizes Atp3, and an a-helix at the middle region of Ids2 recruits Atp3 to the folding system. Shortage of Ids2 or Atp3 leads to the loss of mitochondrial DNA. The intermembrane space protease Yme1 is critical for Atp3's quality control. Moreover, Ids2 is highly induced when cells carry out oxidative respiration. In summary, we identify the first folding client of Ids2 and explain why the deficiency of Ids2 triggers mitochondrial DNA loss and mitochondrial dysfunction.

A49 BUTYRATE PRESERVES THE EPITHELIAL MITOCHONDRIAL NETWORK DISRUPTED BY THE CROHN’S DISEASE PATHOBIONT ADHERENT-INVASIVE E. COLI

Abstract Background Mitochondria exist in a dynamic network that undergoes continuous cycles of fission and fusion that is tightly controlled. Mitochondrial dysfunction is implicated in several autoimmune and inflammatory diseases. Adherent-invasive E. coli (AIEC) is a pathogenic strain of bacteria associated with Crohn’s disease (CD), that can evoke pro-inflammatory responses. Data from our lab showed that AIEC (strain LF82) infection in gut epithelial cells (T84 human cell line) caused dramatic mitochondrial network fragmentation and loss of mitochondrial membrane potential. Short chain fatty acids (SCFA) produced by commensal bacteria in the gut have a wide range of benefits including enhancing mitochondrial biogenesis. Aims To determine (1) if sodium butyrate (NaB) treatment can protect against mitochondrial dysfunction induced by AIEC (strain LF82) infection in T84 epithelial cells and then (2) to identify the mechanism by which NaB restores mitochondrial functions. Methods We assessed changes in mitochondrial network morphology through confocal microscopy live cell imaging of mitotracker-stained T84 epithelial cells based on unbiased Hoechst-stained nuclei to select the field of view. The effect of NaB on the proliferation of bacteria and their invasion of T84-epithelia was assessed by growth curve analysis and bacterial internalisation assays. Finally, mRNA and protein expression of peroxisome proliferating activator receptor gamma co-activator 1 alpha (PGC1α), a regulator of mitochondrial biogenesis, were assessed by qPCR and Western Blot. Results We confirmed that AIEC infection (strain LF82, 108cfu, 4h) induces massive mitochondrial fragmentation in T84 cells (2x105). We also found that cotreatment of T84 cells with NaB (10mM) and LF82 showed increased percentage of fused mitochondrial networks compared to LF82-treated cells. This result was also seen in cells treated with mitochondrial uncoupler dinitrophenol (DNP; 0.1 mM, 2h) and NaB. Moreover, the protective effect of NaB was not related to inhibition of proliferation of the bacteria as we demonstrated that LF82 growth and its invasive phenotype was not compromised by NaB (3-20mM; 0-24hr). In agreement with the effect of LF82 on mitochondrial functions, LF82 significantly reduced PGC1α mRNA expression in T84 cells, that was prevented by co-treatment with NaB. Conclusions These data suggest the pathogen disruption of the epithelial mitochondrial network is a component of IBD; thus, identifying mitochondrial fission and fusion pathways as novel therapeutic targets to control enteric inflammation. The data underscore the complex interplay between bacteria and the epithelium, such that commensal organisms may preserve the mitochondrial network in the face of challenge from pathogens that seek to disrupt mitochondrial form and function Funding Agencies CIHR

Autoinducer N-(3-oxododecanoyl)-l-homoserine lactone induces calcium and reactive oxygen species-mediated mitochondrial damage and apoptosis in blood platelets

Acylated homoserine lactones (AHL) such as N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12 HSL) and N-butyryl-l-homoserine lactone (C4 HSL) are the most common autoinducer molecules in Pseudomonas aeruginosa. These AHL molecules not only regulate the expression of virulence factors but also have been shown to interfere with the host cell and modulate its functions. Recently, we reported that 3-oxo-C12 HSL but not C4 HSL causes cytosolic Ca2+ rise and ROS production in platelets. In this study, we examined the potential of AHLs to induce apoptosis in the human blood platelet. Our result showed that 3-oxo-C12 HSL but not C4 HSL causes phosphatidylserine (PS) exposure, mitochondrial dysfunction (mitochondrial transmembrane potential loss, and mitochondrial permeability transition pore (mPTP) formation). Besides, 3-oxo-C12 HSL also inhibited thrombin-induced platelet aggregation and clot retraction. The pretreatment of an intracellular calcium chelator BAPTA-AM or ROS inhibitor (DPI) significantly attenuated the 3-oxo-C12 HSL induced apoptotic characters such as PS exposure and mitochondrial dysfunctions. These data, including our previous findings, confirmed that 3-oxo-C12 HSL induced intracellular Ca2+ mediated ROS production results in the activation and subsequent induction of apoptotic features in platelets. Our results demonstrated that the 3-oxo-C12 HSL modulates the functions of platelets that may cause severe thrombotic complications in P. aeruginosa infected individuals.

Targeting Mitochondria by SS-31 Ameliorates the Whole Body Energy Status in Cancer- and Chemotherapy-Induced Cachexia.

Simple SummaryCancer cachexia is a debilitating syndrome, caused by both tumor growth and chemotherapy. The skeletal muscle is one of the main tissues affected during cachexia, presenting with altered metabolism and function, leading to progressive tissue wasting. In the current study we aimed at counteracting cachexia by pharmacologically improving metabolic function with the mitochondria-targeted compound SS-31. Experimental cancer cachexia was obtained using C26-bearing mice either receiving chemotherapy (oxaliplatin plus 5-fluorouracil) or not. SS-31 proved effective in rescuing some of the metabolic impairments imposed by both tumor and chemotherapy in the skeletal muscle and the liver, improving systemic energy control. Unfortunately, such effects were no longer present at late disease stages when refractory cachexia ensued. Overall, we provide evidence of potential new treatments targeting mitochondrial function in order to counteract or delay cancer cachexia.Objective: Cachexia is a complex metabolic syndrome frequently occurring in cancer patients and exacerbated by chemotherapy. In skeletal muscle of cancer hosts, reduced oxidative capacity and low intracellular ATP resulting from abnormal mitochondrial function were described. Methods: The present study aimed at evaluating the ability of the mitochondria-targeted compound SS-31 to counteract muscle wasting and altered metabolism in C26-bearing (C26) mice either receiving chemotherapy (OXFU: oxaliplatin plus 5-fluorouracil) or not. Results: Mitochondrial dysfunction in C26-bearing (C26) mice associated with alterations of cardiolipin fatty acid chains. Selectively targeting cardiolipin with SS-31 partially counteracted body wasting and prevented the reduction of glycolytic myofiber area. SS-31 prompted muscle mitochondrial succinate dehydrogenase (SDH) activity and rescued intracellular ATP levels, although it was unable to counteract mitochondrial protein loss. Progressively increased dosing of SS-31 to C26 OXFU mice showed transient (21 days) beneficial effects on body and muscle weight loss before the onset of a refractory end-stage condition (28 days). At day 21, SS-31 prevented mitochondrial loss and abnormal autophagy/mitophagy. Skeletal muscle, liver and plasma metabolomes were analyzed, showing marked energy and protein metabolism alterations in tumor hosts. SS-31 partially modulated skeletal muscle and liver metabolome, likely reflecting an improved systemic energy homeostasis. Conclusions: The results suggest that targeting mitochondrial function may be as important as targeting protein anabolism/catabolism for the prevention of cancer cachexia. With this in mind, prospective multi-modal therapies including SS-31 are warranted.

NME4 Loss-of-Function Alters Mitochondria, Triggers Retrograde Signaling and Leads to Cellular Reprogramming

NME4 (or nucleoside diphosphate kinase D, NDPK-D, NM23-H4) is a mitochondrial protein with multiple key functions in bioenergetics and signaling. The hexameric NME4 is mainly localized in the intermembrane space, bound to the inner membrane. As we showed earlier, NME4 provides GTP to the mitochondrial GTPase OPA1, stimulates respiration, binds to cardiolipin and promotes its inter-membrane transfer to the mitochondrial surface. Here we deleted either its NDP kinase activity or its cardiolipin interaction and expressed these mutants or wild-type protein in HeLa cells that are almost devoid of endogenous NME4. Both mutations independently led to fragmentation of the mitochondrial network and loss of mitochondrial mass, consistent with disturbed GTP-fueling of the pro-fusion GTPase OPA1. NME4 loss-of-function mutants also showed increased ROS generation and a metabolic shift with reduced cellular respiration and increased aerobic glycolysis. The mitochondrial alterations triggered a specific change in cellular protein expression, with down-regulation of several mitochondrial proteins and altered cell signaling. At the cellular level, NME4 loss-of-function manifested in a morphotypic switch, characterized by inhibited intercellular adhesion, and increased migratory and invasive potential. These data suggest that NME4 loss of function leads to primary alterations in mitochondria that trigger retrograde signaling, finally leading to downstream events responsible for pro-invasive reprogramming of the cell.