BCL2/adenovirus E1B 19 kDa Protein-interacting Protein 3 Research Articles

Dysfunctional mitochondria during accelerated senescence and their impact on Alzheimer’s disease neuropathology

AbstractBackgroundAlthough aging is the leading risk factor for Alzheimer’s disease (AD), the exact mechanisms through which aging becomes pathogenic and triggers neurodegeneration have not yet been identified. Recent evidence suggests that AD is tightly correlated with the appearance of senescent cells and that mitochondrial dysfunction is a pathological feature occurring during senescence and AD. Additionally, studies have suggested that mitochondrial dysfunction and altered mitophagy might lead to Aβ accumulation. Our goal was to explore if a reduced removal of dysfunctional mitochondria during aging is a key contributor to amyloid pathology and neurodegeneration in AD.MethodSince telomere shortening is a known trigger of cellular senescence, a telomerase‐deficient mouse model was used: Terc−/−. The brain proteome of Terc−/− mice was evaluated to validate classical and novel markers of senescence and investigate the alteration of key cellular pathways. Primary cultures and brain tissue from Terc−/− mice were evaluated for mitochondrial function/content and mitophagy alterations. Then, we explored the relationship between amyloid pathology and mitochondrial dysfunction by crossing the Terc−/− mice with an AD‐related amyloid mouse model (5xFAD) and exploring mitophagy alterations and amyloid‐β (Aβ) accumulation in disease‐vulnerable regions.ResultProteomic analysis revealed changes in proteins related to autophagy and mitochondrial functions during accelerated senescence. The mitophagy modulator BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) was found downregulated in Terc−/− mice. We evidenced the presence of mitochondrial dysfunction and accumulation in senescent neurons. Additionally, we observed that senescence induces intraneuronal Aβ accumulation in specific brain regions (e.g., the subiculum) of an AD mouse model and we suggest that mitochondrial dysfunction and altered mitophagy might lead to Aβ accumulation.ConclusionOur results indicate that dysfunctional mitochondria accumulate during pathological aging due to a mitophagy impairment, which might contribute to AD neuropathology.

SGLT2 inhibitor empagliflozin alleviates cardiac remodeling and contractile anomalies in a FUNDC1-dependent manner in experimental Parkinson's disease.

Recent evidence shows a close link between Parkinson's disease (PD) and cardiac dysfunction with limited treatment options. Mitophagy plays a crucial role in the control of mitochondrial quantity, metabolic reprogramming and cell differentiation. Mutation of the mitophagy protein Parkin is directly associated with the onset of PD. Parkin-independent receptor-mediated mitophagy is also documented such as BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and FUN14 domain containing 1 (FUNDC1) for receptor-mediated mitophagy. In this study we investigated cardiac function and mitophagy including FUNDC1 in PD patients and mouse models, and evaluated the therapeutic potential of a SGLT2 inhibitor empagliflozin. MPTP-induced PD model was established. PD patients and MPTP mice not only displayed pronounced motor defects, but also low plasma FUNDC1 levels, as well as cardiac ultrastructural and geometric anomalies (cardiac atrophy, interstitial fibrosis), functional anomalies (reduced E/A ratio, fractional shortening, ejection fraction, cardiomyocyte contraction) and mitochondrial injury (ultrastructural damage, UCP2, PGC1α, elevated mitochondrial Ca2+ uptake proteins MCU and VDAC1, and mitochondrial apoptotic protein calpain), dampened autophagy, FUNDC1 mitophagy and apoptosis. By Gene set enrichment analysis (GSEA), we found overtly altered glucose transmembrane transport in the midbrains of MPTP-treated mice. Intriguingly, administration of SGLT2 inhibitor empagliflozin (10 mg/kg, i.p., twice per week for 2 weeks) in MPTP-treated mice significantly ameliorated myocardial anomalies (with exception of VDAC1), but did not reconcile the motor defects or plasma FUNDC1. FUNDC1 global knockout (FUNDC1-/- mice) did not elicit any phenotype on cardiac geometry or function in the absence or presence of MPTP insult, but it nullified empagliflozin-caused cardioprotection against MPTP-induced cardiac anomalies including remodeling (atrophy and fibrosis), contractile dysfunction, Ca2+ homeostasis, mitochondrial (including MCU, mitochondrial Ca2+ overload, calpain, PARP1) and apoptotic anomalies. In neonatal and adult cardiomyocytes, treatment with PD neurotoxin preformed fibrils of α-synuclein (PFF) caused cytochrome c release and cardiomyocyte mechanical defects. These effects were mitigated by empagliflozin (10 μM) or MCU inhibitor Ru360 (10 μM). MCU activator kaempferol (10 μM) or calpain activator dibucaine (500 μM) nullified the empagliflozin-induced beneficial effects. These results suggest that empagliflozin protects against PD-induced cardiac anomalies, likely through FUNDC1-mediated regulation of mitochondrial integrity.

Impaired mitophagy induces antimicrobial responses in macrophages infected with Mycobacterium tuberculosis

BackgroundMitophagy, mitochondrial selective autophagy, plays a pivotal role in the maintenance of cellular homeostasis in response to cellular stress. However, the role of mitophagy in macrophages during infection has not been elucidated. To determine whether mitophagy regulates intracellular pathogen survival, macrophages were infected with Mycobacterium tuberculosis (Mtb), an intracellular bacterium.ResultsWe showed that Mtb-infected macrophages induced mitophagy through BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) activation. In contrast, BNIP3-deficient macrophages failed to induce mitophagy, resulting in reduced mitochondrial membrane potential in response to Mtb infection. Moreover, the accumulation of damaged mitochondria due to BNIP3 deficiency generated higher levels of mitochondrial reactive oxygen species (mROS) compared to the control, suppressing the intracellular survival of Mtb. We observed that siBNIP3 suppressed intracellular Mtb in mice lungs.ConclusionWe found that BNIP3 plays a critical role in the regulation of mitophagy during Mtb infection. The inhibition of mitophagy suppresses Mtb growth in macrophages through increased mROS production. Therefore, BNIP3 might be a novel therapeutic target for tuberculosis treatment.

A crosstalk between phosphorylation and ubiquitination of BNIP3 regulates mitophagy under hypoxia

BNIP3 (BCL2/adenovirus e1B 19 kDa protein interacting protein 3) is a mitochondrial outer membrane protein that is sensitive to hypoxia and mediates mitophagy, a process important for mitochondrial quality control and to maintain energetic and redox homeostasis under hypoxia. It has been reported that up-regulation of BNIP3, which acts as mitophagy receptor, promotes mitophagy. In our recent study, we found that the post-translational modification of BNIP3 is crucial to induce mitophagy, and that a crosstalk between phosphorylation/dephosphorylation and ubiquitination acts as a switch to control BNIP3-mediated mitophagy under hypoxia. We demonstrated that the phosphorylation of BNIP3 at S60 and T66 by MAPK8/9 (mitogen-activated protein kinase 8/9) under hypoxia blocks the degradation of BNIP3 via the ubiquitin-proteasome pathway and enhances its interaction with MAP1LC3 (microtubule associated protein 1 light chain 3), thereby promoting mitophagy. In contrast, dephosphorylation of BNIP3 by members of the PP1/2A (protein phosphatase PP1 and PP2A) phosphatase subfamily under hypoxia accelerates degradation of BNIP3 via the ubiquitin-proteasome pathway, thereby suppressing mitophagy. Altogether, these findings provide knowledge necessary to devise intervention strategies for hypoxia-related diseases and/or hypoxia-related developmental processes.Abbreviations: BCL2: BCL2 apoptosis regulator; BCL2L1: BCL2 like 1; BECN1: beclin 1, autophagy related; BH3: BCL2 homology 3; BNIP3: BCL2/adenovirus e1B 19 kDa protein interacting protein 3; LIR: MAP1LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; MAPK8: mitogen-activated protein kinase 8; MAPK9: mitogen-activated protein kinase 9; PEST: rich in amino acids P, E, S, T, and D; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; PEST: rich in amino acids P, E, S, T, and D

Relationship between Mitochondrial Quality Control Markers, Lower Extremity Tissue Composition, and Physical Performance in Physically Inactive Older Adults.

Altered mitochondrial quality and function in muscle may be involved in age-related physical function decline. The role played by the autophagy-lysosome system, a major component of mitochondrial quality control (MQC), is incompletely understood. This study was undertaken to obtain initial indications on the relationship between autophagy, mitophagy, and lysosomal markers in muscle and measures of physical performance and lower extremity tissue composition in young and older adults. Twenty-three participants were enrolled, nine young (mean age: 24.3 ± 4.3 years) and 14 older adults (mean age: 77.9 ± 6.3 years). Lower extremity tissue composition was quantified volumetrically by magnetic resonance imaging and a tissue composition index was calculated as the ratio between muscle and intermuscular adipose tissue volume. Physical performance in older participants was assessed via the Short Physical Performance Battery (SPPB). Protein levels of the autophagy marker p62, the mitophagy mediator BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), the lysosomal markers transcription factor EB, vacuolar-type ATPase, and lysosomal-associated membrane protein 1 were measured by Western immunoblotting in vastus lateralis muscle biopsies. Older adults had smaller muscle volume and lower tissue composition index than young participants. The protein content of p62 and BNIP3 was higher in older adults. A negative correlation was detected between p62 and BNIP3 and the tissue composition index. p62 and BNIP3 were also related to the performance on the 5-time sit-to-stand test of the SPPB. Our results suggest that an altered expression of markers of the autophagy/mitophagy-lysosomal system is related to deterioration of lower extremity tissue composition and muscle dysfunction. Additional studies are needed to clarify the role of defective MQC in human muscle aging and identify novel biological targets for drug development.

BNIP3 phosphorylation by JNK1/2 promotes mitophagy via enhancing its stability under hypoxia

Mitophagy is an important metabolic mechanism that modulates mitochondrial quality and quantity by selectively removing damaged or unwanted mitochondria. BNIP3 (BCL2/adenovirus e1B 19 kDa protein interacting protein 3), a mitochondrial outer membrane protein, is a mitophagy receptor that mediates mitophagy under various stresses, particularly hypoxia, since BNIP3 is a hypoxia-responsive protein. However, the underlying mechanisms that regulate BNIP3 and thus mediate mitophagy under hypoxic conditions remain elusive. Here, we demonstrate that in hypoxia JNK1/2 (c-Jun N-terminal kinase 1/2) phosphorylates BNIP3 at Ser 60/Thr 66, which hampers proteasomal degradation of BNIP3 and drives mitophagy by facilitating the direct binding of BNIP3 to LC3 (microtubule-associated protein 1 light chain 3), while PP1/2A (protein phosphatase 1/2A) represses mitophagy by dephosphorylating BNIP3 and triggering its proteasomal degradation. These findings reveal the intrinsic mechanisms cells use to regulate mitophagy via the JNK1/2-BNIP3 pathway in response to hypoxia. Thus, the JNK1/2-BNIP3 signaling pathway strongly links mitophagy to hypoxia and may be a promising therapeutic target for hypoxia-related diseases.

Branched-chain amino acid supplementation suppresses the detraining-induced reduction of mitochondrial content in mouse skeletal muscle.

Exercise training enhances oxidative capacity whereas detraining reduces mitochondrial content in skeletal muscle. The strategy to suppress the detraining-induced reduction of mitochondrial content has not been fully elucidated. As previous studies reported that branched-chain amino acid (BCAA) ingestion increased mitochondrial content in skeletal muscle, we evaluated whether BCAA supplementation could suppress the detraining-induced reduction of mitochondrial content. Six-week-old male Institute of Cancer Research (ICR) mice were randomly divided into four groups as follows: control (Con), endurance training (Tr), detraining (DeTr), and detraining with BCAA supplementation (DeTr + BCAA). Mice in Tr, DeTr, and DeTr + BCAA performed treadmill running exercises [20-30 m/min, 60 min, 5 times/week, 4 weeks]. Then, mice in DeTr and DeTr + BCAA were administered with water or BCAA [0.6mg/g of body weight, twice daily] for 2 weeks of detraining. In whole skeletal muscle, mitochondrial enzyme activities and protein content were decreased after 2 weeks of detraining, but the reduction was suppressed by BCAA supplementation. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein content, a master regulator of mitochondrial biogenesis, was decreased by detraining irrespective of BCAA ingestion. Regarding mitochondrial degradation, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a mitophagy-related protein, was significantly higher in the Tr group than in the DeTr + BCAA group, but not different from in the DeTr group. With respect to mitochondrial quality, BCAA ingestion did not affect oxygen consumption rate (OCR) and reactive oxygen species (ROS) production in isolated mitochondria. Our findings suggest that BCAA ingestion suppresses the detraining-induced reduction of mitochondrial content partly through inhibiting mitophagy.

BNIP3 regulates cardiac energy metabolism by interaction with F1FO ATP-synthase

Abstract Background The oxidative phosphorylation (OXPHOS) that takes place in the mitochondria produces chemical energy in form of ATP, the main energy source of the heart. Proper mitochondrial function determines the contractility of the heart. Changes in myocardial ATP levels are often associated with cardiovascular disease. However, mitochondrial dysfunction remains an unmet therapeutic challenge. The F1FO ATP-synthase protein complex catalyses the last step of OXPHOS, synthesising ATP and determining the respiratory function of cardiac mitochondria. An increased ATP pool during cardiovascular challenges, regulated by F1FO ATP-synthase, could therefore significantly improve the clinical outcome of affected patients. In neurons, Bcl2-familiy members like BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) have been suggested to be involved in the regulation of metabolic efficiency through interaction with the mitochondrial F1FO ATP synthase. A similar metabolic regulation in the cardiac context remains unclear. Furthermore, BNIP3 has a significant impact on the loss of cardiomyocytes under various pathological conditions and is thus a potential mediator of energy metabolism in cardiomyocytes. Methods and results With native gel-electrophoresis, cardiac BNIP3 of C57BL/6J mice was first identified in higher oligomeric complexes in similar molecular weight range as mitochondrial F1FO ATP-synthase. Using 60Å-precise proximity-ligation assays to determine possible binding partners, F1FO ATP-synthase was uncovered for the first time as an interacting protein of BNIP3. This interaction was confirmed by co-immunoprecipitation of BNIP3 and F1FO ATP-synthase and by spatial localization using electron microscopy. Peptide microarray studies elucidate the f subunit of F1FO ATP-synthase as the structural interaction site. Functional analysis using extracellular flux analyser technology revealed significantly elevated mitochondrial respiratory activity in Bnip3 knock out mice and human cardiomyocytes during BNIP3 inhibition. Increased ATP levels in C57BL/6J mice after acute inhibition of BNIP3 confirmed the observed regulatory effect of BNIP3 on F1FO ATP-synthase activity. Finally, depletion of BNIP3 in mice improves stress resistance with an augmented chronotropic capacity during dobutamine-induced cardiac stress. Conclusion Depletion of BNIP3 suggests a regulatory role of BNIP3 in cardiac mitochondrial energy homeostasis via interaction with F1F0 ATP-synthase in oligomeric complexes. Since acute inhibition of BNIP3 activity allows positive modulation of cardiac performance by elevating the available ATP pool, this may serve as a beneficial treatment for patients with cardiovascular disease in the future. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German research foundation (DFG)

IGF-1 Signaling Regulates Mitochondrial Remodeling during Myogenic Differentiation.

Skeletal muscle is essential for locomotion, metabolism, and protein homeostasis in the body. Mitochondria have been considered as a key target to regulate metabolic switch during myo-genesis. The insulin-like growth factor 1 (IGF-1) signaling through the AKT/mammalian target of rapamycin (mTOR) pathway has a well-documented role in promoting muscle growth and regeneration, but whether it is involved in mitochondrial behavior and function remains un-examined. In this study, we investigated the effect of IGF-1 signaling on mitochondrial remodeling during myogenic differentiation. The results demonstrated that IGF-1 signaling stimulated mitochondrial biogenesis by increasing mitochondrial DNA copy number and expression of genes such as Cox7a1, Tfb1m, and Ppargc1a. Moreover, the level of mitophagy in differentiating myoblasts elevated significantly with IGF-1 treatment, which contributed to mitochondrial turnover. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) were identified as two key mediators of IGF-1-induced mitochondrial biogenesis and mitophagy, respectively. In addition, IGF-1 supplementation could alleviate impaired myoblast differentiation caused by mitophagy deficiency, as evidenced by increased fusion index and myosin heavy chain expression. These findings provide new insights into the role of IGF-1 signaling and suggest that IGF-1 signaling can serve as a target for the research and development of drugs and nutrients that support muscle growth and regeneration.

ROS-mediated BNIP3-dependent mitophagy promotes coelomocyte survival in Apostichopus japonicus in response to Vibrio splendidus infection.

Organisms produce high levels of reactive oxygen species (ROS) to kill pathogens or act as signaling molecules to induce immune responses; however, excessive ROS can result in cell death. To maintain ROS balance and cell survival, mitophagy selectively eliminates damaged mitochondria via mitophagy receptors in vertebrates. In marine invertebrates, however, mitophagy and its functions remain largely unknown. In the current study, Vibrio splendidus infection damaged mitochondrial morphology in coelomocytes and reduced mitochondrial membrane potential (ΔΨm) and mitophagosome formation. The colocalization of mitochondria and lysosomes further confirmed that lipopolysaccharide (LPS) treatment increased mitophagy flux. To explore the regulatory mechanism of mitophagy, we cloned Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a common mitophagy receptor, from sea cucumber Apostichopus japonicus (AjBNIP3) and confirmed that AjBNIP3 was significantly induced and accumulated in mitochondria after V. splendidus infection and LPS exposure. At the mitochondrial membrane, AjBNIP3 interacts with microtubule-associated protein 1 light chain 3 (LC3) on phagophore membranes to mediate mitophagy. After AjBNIP3 interference, mitophagy flux decreased significantly. Furthermore, AjBNIP3-mediated mitophagy was activated by ROS following the addition of exogenous hydrogen peroxide (H2O2), ROS scavengers, and ROS inhibitors. Finally, inhibition of BNIP3-mediated mitophagy by AjBNIP3 small interfering RNA (siRNA) or high concentrations of lactate increased apoptosis and decreased coelomocyte survival. These findings highlight the essential role of AjBNIP3 in damaged mitochondrial degradation during mitophagy. This mitophagy activity is required for coelomocyte survival in A. japonicus against V. splendidus infection.

Mitophagy Protects the Retina Against Anti-Vascular Endothelial Growth Factor Therapy-Driven Hypoxia via Hypoxia-Inducible Factor-1α Signaling.

Anti-VEGF drugs are first-line treatments for retinal neovascular diseases, but these anti-angiogenic agents may also aggravate retinal damage by inducing hypoxia. Mitophagy can protect against hypoxia by maintaining mitochondrial quality, thereby sustaining metabolic homeostasis and reducing reactive oxygen species (ROS) generation. Here we report that the anti-VEGF agent bevacizumab upregulated the hypoxic cell marker HIF-1α in photoreceptors, Müller cells, and vascular endothelial cells of oxygen-induced retinopathy (OIR) model mice, as well as in hypoxic cultured 661W photoreceptors, MIO-MI Müller cells, and human vascular endothelial cells. Bevacizumab also increased expression of mitophagy-related proteins, and mitophagosome formation both in vivo and in vitro, but did not influence cellular ROS production or apoptosis rate. The HIF-1α inhibitor LW6 blocked mitophagy, augmented ROS production, and triggered apoptosis. Induction of HIF-1α and mitophagy were associated with upregulation of BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3) and FUN14 domain containing 1 (FUNDC1), and overexpression of these proteins in culture reversed the effects of HIF-1α inhibition. These findings suggest that bevacizumab does induce retinal hypoxia, but that concomitant activation of the HIF-1α-BNIP3/FUNDC1 signaling pathway also induces mitophagy, which can mitigate the deleterious effects by reducing oxidative stress secondary. Promoting HIF-1α-BNIP3/FUNDC1-mediated mitophagy may enhance the safety of anti-VEGF therapy for retinal neovascular diseases and indicate new explanation and possible new target of the anti-VEGF therapy with suboptimal effect.

Hypoxia Inducible Factor (HIF)‐1α is a Transcriptional Mediator for Thiamine Insufficiency Induced Neurotoxicity

Age-dependent decline in vitamin B1 (thiamine) levels has a devastating impact on the brain parenchyma. The neurotoxicity promoted by chronic thiamine insufficiency (TI) is a well-established comorbidity of Alzheimer's Disease (AD), the most prevalent age-related metabolic neurodegenerative disease. Inadequate levels of blood thiamine and derivates highly correlate with senile dementia prognosis as predictive peripheral AD biomarker. Impaired activity of thiamine-dependent enzymes due to TI, dramatically decreased cerebral oxidative metabolism promoting oxidative stress, mitochondrial damage, inflammation, and eventually neuronal loss. Furthermore, age-related decline in TI potentiates the AD neuropathology triggering the amyloid plaque formation and hyperphosphorylation of tau. We previously showed that TI stabilizes Hypoxia Inducible Factor -1α (HIF-1α), the main transcription factor involved in hypoxic stress, leading to HIF-1α induced pro-apoptotic and pro-inflammatory response. Among the transcriptionally activated HIF-1α target genes, TI triggered the expression of the pro-apoptotic protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 (BNIP-3). Induction of BNIP-3 leads to its mitochondria migration where eventually triggers cell death via loss of membrane potential and increase in reactive oxygen species. To date, despite the well-established correlation between TI and neuropathogenesis, the molecular mechanism underlying the TI mediated neurotoxicity is still unknown. Therefore, we hypothesize that HIF-1α may be a critical upstream mediator between thiamine insufficiency and neurotoxicity. In this study, hippocampal murine cells (HT22) treated in TI conditions exhibited a significant increase in protein and mRNA levels of HIF-1α. Increase in BNIP-3 and other HIF-1α target genes (LDHA, VEGF, BACE-1, MCP-1) implicated in pro-inflammatory and pro-apoptotic response was also detected. Treatment with 50 μM and 100 μM of the thiamine antagonist Pyrithiamine (PT) up to 5 days significantly decrease HT22 viability to 30% and 20%, respectively. HIF-1α silencing via shRNA attenuated the neurotoxic response improving viability up to 60% with the same PT doses. In order to better characterize the HIF-1α response, BNIP-3 was also silenced via shRNA. In congruency with HIF-1α shRNA result, we found a 2-fold decrease in PT-induced toxicity in BNIP-3 shRNA transduced cells. Localization of BNIP-3 in mitochondria and loss of mitochondrial potential after PT treatment further highlighted the involvement of the HIF-1α - BNIP-3 response in TI induced neuronal death. Overall, these findings suggest a central role for HIF-1α as upstream regulator in TI mediated neurotoxicity.

NF-κB-Activated miR-574 Promotes Multiple Malignant and Metastatic Phenotypes by Targeting BNIP3 in Thyroid Carcinoma.

Thyroid cancer is the most common endocrine malignancy, and miR-574 is significantly upregulated in thyroid cancer. However, the role and underlying mechanism of miR-574 in thyroid cancer development are poorly understood. In this study, we showed that NF-κB/p65 signaling pathway was activated and miR-574 was upregulated in thyroid cancer cells. p65 directly bound to the promoter of miR-574 and activated miR-574 transcription. Functionally, miR-574 inhibited apoptosis, promoted proliferation and migration of thyroid cancer cells, and stimulated thyroid cancer-induced tube formation of endothelial cells. On the molecular level, miR-574 inhibited the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) by binding to 3'-UTR of BNIP3. miR-574 also downregulated the expression of apoptosis-inducing factor (AIF), while elevated the levels of MMP2, MMP9, and VEGFA. In vivo, miR-574 promoted xenograft growth, which was associated with reduced apoptosis and enhanced angiogenesis. NF-κB/miR-574 signaling presents multiple oncogenic activities on thyroid cancer development by directly regulating the BNIP3/AIF pathway. Therefore, targeting NF-κB/miR-574 signaling may reduce the aggressiveness of thyroid cancer. IMPLICATIONS: miR-574, directly regulated by NF-κB/p65, promotes tumorigenesis of thyroid cancer via inhibiting BNIP3/AIF pathway.

Iron deficiency-induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria-containing vesicles.

Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes.

Methylene Blue Protects Against Sevoflurane-Induced Cognitive Dysfunction by Suppressing Drp1 deSUMOylation in Aged Mice.

Exposure to sevoflurane and other inhalational anesthetics can induce cognitive impairment in elderly patients. Studies have indicated that methylene blue (MB) has beneficial effects on multiple neurodegenerative diseases and the mechanism involves mitochondrial function preservation. However, whether MB can attenuate the cognitive decline induced by sevoflurane in aged mice requires further investigation. Forty-five 18-month-old C57/BL mice were used to establish a model of sevoflurane-induced cognitive impairment in which the mice were exposed to 3% sevoflurane for 2h. Mice in the MB group were intraperitoneally injected with MB at a dose of 5mg/kg before sevoflurane inhalation. The Morris water maze test was used to evaluate the learning and memory performances. We also examined changes in mitochondrial morphology as well as the expression and interaction of related proteins in the aged hippocampus. Parkin, BCL2/adenovirus E1B 19kDa protein-interacting protein 3 (BNIP3), mitochondrial dynamin-related protein 1 (Drp1), small ubiquitin-like modifier (SUMO2/3), SUMO-specific proteases 3 (SENP3), and ubiquitin-like conjugating enzyme 9 expression in the mouse hippocampus was detected by western blotting, and SUMO2/3-Drp1 was examined by coimmunoprecipitation. Exposure to sevoflurane increased SENP3 expression and Drp1 deSUMOylation in the aged hippocampus and resulted in cognitive deficiency. MB attenuated sevoflurane-induced memory loss and mitochondrial fragmentation and decreased Drp1 deSUMOylation in the aged hippocampus. This neuroprotective effect provides a mechanistic explanation for how the SUMOylation status of Drp1 acts as a key switch in the cognitive dysfunction induced by sevoflurane.

IGF-1 Signalling Regulates Mitochondria Dynamics and Turnover through a Conserved GSK-3β-Nrf2-BNIP3 Pathway.

The Insulin-like Growth Factor I (IGF-1) signalling pathway is essential for cell growth and facilitates tumourogenic processes. We recently reported that IGF-1 induces a transcriptional programme for mitochondrial biogenesis, while also inducing expression of the mitophagy receptor BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), suggesting that IGF-1 has a key mitochondria-protective role in cancer cells. Here, we investigated this further and delineated the signaling pathway for BNIP3 induction. We established that IGF-1 induced BNIP3 expression through a known AKT serine/threonine kinase 1 (AKT)-mediated inhibitory phosphorylation on Glycogen Synthase Kinase-3β (GSK-3β), leading to activation of Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2/Nrf2) and acting through the downstream transcriptional regulators Nuclear Respiratory Factor-1 (NRF1) and Hypoxia-inducible Factor 1 subunit α (HIF-1α). Suppression of IGF-1 signaling, Nrf2 or BNIP3 caused the accumulation of elongated mitochondria and altered the mitochondrial dynamics. IGF-1R null Mouse Embryonic Fibroblasts (MEFs) were impaired in the BNIP3 expression and in the capacity to mount a cell survival response in response to serum deprivation or mitochondrial stress. IGF-1 signalling enhanced the cellular capacity to induce autophagosomal turnover in response to activation of either general autophagy or mitophagy. Overall, we conclude that IGF-1 mediated a mitochondria-protective signal that was coordinated through the cytoprotective transcription factor Nrf2. This pathway coupled mitochondrial biogenesis with BNIP3 induction, and increased the cellular capacity for autophagosome turnover, whilst enhancing survival under conditions of metabolic or mitochondrial stress.

MiR-182-5p inhibited proliferation and migration of ovarian cancer cells by targeting BNIP3.

To investigate the potential effect of microRNA-182-5p (miR-182-5p) on the development of ovarian cancer (OC) and the relevant mechanism. The expression levels of miR-182-5p in OC tissues and paracancerous normal tissues were detected. The miR-182-5p expression in OC cells and ovarian epithelial cells was also determined. Through online prediction (TargetScan, miRDB), the potential target of miR-182-5p was screened and further confirmed by the Luciferase reporter gene assay. The effects of the miR-182-5p on human ovarian serous papillary cystadenocarcinoma cell line (SKOV3) cells were determined by in vitro experiments. The low expression of miR-182-5p in OC was confirmed by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay. BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) was identified as a direct target of miR-182-5p. Subsequent experiments showed that the BNIP3 knockdown resulting from the up-regulation of miR-182-5p inhibited cell proliferation, clone formation and migration ability of OC cells. Our research showed the inhibitory function of miR-182-5p in OC by targeting BNIP3, thus providing an experimental basis for the treatment of OC.

Hepatocyte‐Specific Deletion of eNOS Impairs Mitochondrial Function and Exacerbates Hepatic Steatosis

IntroductionWhole body loss of endothelial nitric oxide synthase (eNOS) worsens hepatic mitochondrial function and exacerbates nonalcoholic fatty liver disease (NAFLD) development and progression. However, the precise role of eNOS in hepatocytes in the contribution to NAFLD has not been established. Here, we examine the effects of in vivo hepatocyte‐specific ablation of eNOS on hepatic steatosis and NAFLD susceptibility.MethodsHepatocyte‐specific eNOS knockout (eNOShep−/−) mice were generated by crossing homozygous eNOS floxed (eNOSfl/fl) mice on a C57/BL6J background with albumin‐Cre recombinase transgenic mice. At 10 weeks of age, male eNOSfl/fl and eNOShep−/− mice (n = 12–14) were fed a control diet (10% fat) for 16 weeks.ResultsBody weight, body fat, and food intake did not differ between eNOSfl/fl and eNOShep−/− mice. eNOShep−/− mice developed elevated histological hepatic steatosis and increased fasting serum insulin (~30%) compared to eNOSfl/fl mice (p<0.05), along with trends for elevations in serum liver enzymes AST and ALT (p<0.17). Elevated hepatic steatosis in eNOShep−/− was related to significant reductions (−50%) in hepatic mitochondrial fatty acid oxidation and ADP‐stimulated and maximal uncoupled hepatic mitochondrial respiration (p<0.05). However, hepatic citrate synthase activity, a marker of mitochondrial mass, was significantly elevated in eNOShep−/− mice compared to eNOSfl/fl mice (p<0.05), while protein content of oxidative phosphorylation complexes and peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) did not differ between groups. Furthermore, eNOShep−/− mice presented with significant reductions (p<0.05) in key proteins involved in mitophagy and autophagy including PTEN‐induced kinase 1 (PINK1) and phosphorylated Unc‐51 Like Autophagy Activating Kinase 1 (pUlk1/Ulk1) and tended to have significant reductions in BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) compared to eNOSfl/fl mice.ConclusionHere we demonstrate for the first time that hepatocyte‐specific deletion of eNOS impairs hepatic mitochondrial function, reduces markers of autophagy/mitophagy, and exacerbates hepatic steatosis development. Further studies are necessary to determine the mechanisms by which deletion of eNOS regulates mitochondrial homeostasis.Support or Funding InformationVA Merit Grant I01BX003271‐01 (R.S.R.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of Genes that Comprise the Core Molecular Clock are Altered in the Atrophied Skeletal Muscle by Androgen Deprivation

Skeletal muscle atrophy increases the risk of morbidity and mortality during various pathological conditions. In males, a decrease in the production and/or bioavailability of androgens (termed hypogonadism) directly contributes to muscle atrophy during various pathological conditions. While it is known that androgens prevent muscle atrophy, the mechanism(s) by which androgens mediate this effect are largely undefined. Our laboratory previously showed that mitochondrial turnover is enhanced in the tibialis anterior (TA) muscle of mice by androgen deprivation induced by castration surgery, and the magnitude of turnover was related to the degree of muscle atrophy. These data suggest that potentially dysfunctional mitochondria contribute to the muscle atrophy observed following androgen deprivation. To gain a better understanding of that factors that might contribute to changes in mitochondrial quality control during androgen deprived conditions, we subjected total RNA from the TA of sham and castrated mice to microarray analysis. This unbiased approach identified significant changes in expression of genes that comprise the core molecular clock. qRT‐PCR confirmed that expression of Brain and Muscle Arntl 1 (Bmal1) was decreased, while expression of Period 1, Period 2, and Period 3 (Per1, 2, & 3) were increased in the TA of castrated mice. When measured across a diurnal cycle, the change in expression of Bmal1, Per1, and Per2 exhibited reduced amplitude under androgen‐deprived conditions. Interestingly, strong relationships were observed between the castration‐mediated changes of core clock components and the measures of mitochondrial turnover. Specifically, Bmal1 expression was directly related to BCL2/adenovirus E1B 19 kDA protein‐interacting protein 3 (BNIP3) protein content (R2 = 0.88), and the expression of core clock components were also directly related to the content of various mitochondrial proteins. Expression of core clock components were also related to the autophagy marker, p62, and the mass of the TA. Ex post facto analysis of the microarray also identified changes in genes regulating polyamine biosynthesis. As polyamines are known to alter core clock function, we determined whether androgen deprivation altered polyamine content. While castration did not alter Spermine content, there was a significant reduction in the content of Spermidine in the TA of castrated mice. Overall, these data suggest that reduced Spermidine concentrations may contribute to alterations in the core molecular clock in the skeletal muscle under androgen‐deprived conditions, which may in turn contribute to reduced mitochondrial quality control and subsequent muscle atrophySupport or Funding InformationNIH‐CA204345 (RAC) and Institute for Successful Longevity (BSG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sex‐Dependent Murine Hepatic Mitochondrial and Mitophagy Responses to High‐Fat Diet and Exercise

Excessive accumulation of hepatic fat (steatosis) is closely associated with obesity and hepatic insulin resistance, a critical factor for the development of type 2 diabetes. Exercise is a therapeutic that mitigates hepatic steatosis putatively through enhanced mitochondrial function and quality, but sex specific differences are unknown. Here we examined if a high fat diet (HFD) elicited sex‐specific reductions in hepatic mitochondrial respiration and mitochondrial recycling (autophagy/mitophagy) pathways and whether exercise (voluntary wheel running [VWR]) restored these impingements. Male and female wild type (WT, C57BL/6J) mice (n=6–7 per sex, per treatment) housed at thermoneutrality (~80°F) were fed low fat diet (LFD) or HFD (45%kcal fat) for 16 weeks with or without access to VWR for the final 8 weeks. Female mice had significantly reduced hepatic triacylglycerol (TAG) compared to males (p<0.0001) and both sexes showed resistance to increases in TAG with HFD, potentially due to thermoneutral housing inducing higher TAG on a control diet. There was a dose‐dependent increase in maximal state 3 (ADP stimulated) respiratory capacity with HFD and HFD+VWR that was potentiated in females. HFD elicited an increase in H2O2 in males that was reduced with exercise while females had reduced mitochondrial H2O2 production in both conditions. Interestingly, neither glutathione (GSH), glutathione disulfide (GSSG) or the GSH:GSSG ratio, markers of oxidative stress, were different between the groups. Males had a consistent reduction in mitochondrial complex I‐V expression with HFD+VWR. Females had reduced expression of complex I with HFD and HFD+VWR but otherwise retained expression of complexes II‐V. Citrate synthase, a marker of hepatic mitochondrial content, was reduced by HFD in both males and females. However, HFD+VWR further reduced citrate synthase in males but restored activity in females to the level of the LFD cohort. Since mitochondrial content is regulated by mitophagy proteins, we examined the mitophagy regulating BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) and Parkin proteins. Males had reduced BNIP3 expression on the LFD that was increased with HFD. HFD elicited no change in females, however, HFD+VWR drastically reduced BNIP3 levels in both sexes. Parkin was unchanged with HFD in both sexes yet had divergent responses with HFD+VWR as males had reduced expression and female expression increased. Overall, these data show sex‐specific hepatic triglyceride, mitochondrial, and mitophagy responses to HFD and exercise and provide cautionary evidence of studying hepatic steatosis in single sex models.Support or Funding InformationFunded by: Veterans Affairs Merit Review; 1I01BX002567‐01 (JPT), KUMC Biomedical Research Training Program (CSM), K‐INBRE grant number P20 GM103418 (CSM), CTSA grant from NCATS awarded to the University of Kansas for Frontiers: University of Kansas Clinical and Translational Science Institute (# TL1TR002368) (CSM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.