Hepatitis C Virus Non-Structural Protein 5A (NS5A) Disrupts Mitochondrial Dynamics and Induces Mitophagy

Mitophagy is a selective form of autophagy, targeting damaged mitochondria for lysosomal degradation. Although HCV infection has been shown to induce mitophagy, the precise underlying mechanism and the effector protein responsible remain unclear. Herein, we demonstrated that the HCV non-structural protein 5A (NS5A) plays a key role in regulating cellular mitophagy. Specifically, the expression of HCV NS5A in the hepatoma cells triggered hallmarks of mitophagy including mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin translocation to the mitochondria. Furthermore, mitophagy induction through the expression of NS5A led to an increase in autophagic flux as demonstrated by an accumulation of LC3II in the presence of bafilomycin and a time-dependent decrease in p62 protein level. Intriguingly, the expression of NS5A concomitantly enhanced reactive oxygen species (ROS) production, and treatment with an antioxidant attenuated the NS5A-induced mitophagy event. These phenomena are similarly recapitulated in the NS5A-expressing HCV subgenomic replicon cells. Finally, we demonstrated that expression of HCV core, which has been documented to inhibit mitophagy, blocked the mitophagy induction both in cells harboring HCV replicating subgenomes or expressing NS5A alone. Our results, therefore, identified a new role for NS5A as an important regulator of HCV-induced mitophagy and have implications to broadening our understanding of the HCV-mitophagy interplay.

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Diminished YAP1 affects mitochondrial dynamics in IDH1 mutant glioma

Journal of Cell Science ◽

10.1242/jcs.259188 ◽

2021 ◽

Author(s):

Shruti Patrick ◽

Pruthvi Gowda ◽

Kirti Lathoria ◽

Vaishali Suri ◽

Ellora Sen

Keyword(s):

Mitochondrial Dynamics ◽

Redox Homeostasis ◽

Luciferase Reporter ◽

Ros Generation ◽

Mitochondrial Fragmentation ◽

Isocitrate Dehydrogenase 1 ◽

Idh1 R132h ◽

Important Regulator ◽

Reporter Assays ◽

Induced Apoptosis

Mutation in isocitrate dehydrogenase 1 (IDH1) gene, leading to the production of oncometabolite D-2-hydroxyglutarate (2-HG) from α-ketoglutarate, is associated with better prognosis in glioma. As Yes-associated protein 1 (YAP1) is an important regulator of tumor progression, its role in glioma expressing IDH1 R132H mutation was investigated. Diminished nuclear YAP1 in IDH1 mutant patient gliomas and cell lines was accompanied by decreased TFAM levels. Luciferase reporter assays and chromatin immunoprecipitation indicated the functionality of TEAD2 site on TFAM promoter in mediating its YAP1-dependent expression. YAP1-dependent mitochondrial fragmentation and ROS generation was accompanied by decreased TERT levels and increased mitochondrial TERT localization in IDH1 R132H cells. Treatment with Bosutinib that prevents extranuclear TERT shuttle, further elevated ROS in IDH1 R132H cells and triggered apoptosis. Importantly, Bosutinib elevated ROS levels and induced apoptosis in IDH1 WT cells upon concurrent depletion of YAP1. These findings highlight the involvement of YAP1 in coupling mitochondrial dysfunction with TERT mitochondrial shuttle to constitute an essential non-canonical function of YAP1 in regulating redox homeostasis.

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Abstract 18238: Cardiomyocyte-specific Bag3+ Mutation P209L Induces Mitochondrial Fragmentation, Increased Apoptosis, and Activates p38 Signaling in vivo

Circulation ◽

10.1161/circ.132.suppl_3.18238 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Megan T Quintana ◽

Samuel C Eaton ◽

Cecelia C Yates ◽

Shinichi Takayama ◽

Monte S Willis

Keyword(s):

Mitochondrial Dynamics ◽

Systolic Function ◽

Cardiac Complications ◽

Autophagic Flux ◽

Mitochondrial Fragmentation ◽

Downstream Signaling ◽

Transmission Electron ◽

Confocal Immunofluorescence ◽

Amyloid Oligomers

Introduction: There are 6 members of the Bcl2-related anthanogene (BAG) protein family, which act as co-chaperones expressed at high levels in skeletal and cardiac muscle. BAG3 is involved in chaperone-assisted selective autophagy (CASA). Mutations in the Z-disc protein BAG3 have recently been found to cause myofibrillar myopathy (MFM) with cardiac complications (dilated cardiomyopathy). The missense mutation Pro209Leu (P209L) is one of ten BAG3 mutations identified in human disease to date. Hypothesis: Cardiac BAG3 P209L expression inhibits CASA, evidenced by less autophagic flux of misfolded proteins. Methods: Cardiac specific transgenic mice with alphaMHC-driven human Bag3P209L were created and analyzed functionally over 12 months. Histological analysis of apoptosis (TUNEL), pre-amyloid oligomers (PAO), and fibroblasts were performed in parallel with transmission electron microscopy (TEM) and molecular analysis of autophagy and mitochondrial dynamics. Results: Starting at 8 months, Bag3 P209L Tg+ hearts had significantly depressed systolic function. By 12 months, significant deficits in both diastolic and systolic function were identified, along with increased number of fragmented mitochondria and significant alterations in genes regulating mitochondrial fusion (Opa1) and Fission (Drp1). Immunofluorescence analysis revealed no differences in apoptosis (TUNEL) or autophagic flux (LC3II). But increased levels of cardiomyocyte pre-amyloid oligomers (PAO) were identified by confocal immunofluorescence. As PAO proteotoxic intermediates found in neurodegenerative diseases reportedly activate p38 MAPK, we assayed BAG3 P209L Tg+ hearts and found a significant ~50% increase in phospho-p38/p38. Conclusion: As increased activated p38 activity has negative inotropic and restrictive diastolic effects in vivo, we conclude that inhibiting p38 activity in BAG3 P209L patients may attenuate and/or delay the onset of heart failure. Studies are underway testing this hypothesis using specific inhibitors of P38 and downstream signaling pathways in vivo.

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Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

Cell Death and Disease ◽

10.1038/s41419-021-03752-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Anthony R. Anzell ◽

Garrett M. Fogo ◽

Zoya Gurm ◽

Sarita Raghunayakula ◽

Joseph M. Wider ◽

...

Keyword(s):

Cortical Neurons ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Mitochondrial Fission ◽

Conditional Knockout ◽

Mitochondrial Morphology ◽

Primary Neurons ◽

Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

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A combination of CMC and α-MSH inhibited ROS activated NLRP3 inflammasome in hyperosmolarity stressed HCECs and scopolamine-induced dry eye rats

Scientific Reports ◽

10.1038/s41598-020-80849-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ying Lv ◽

Chenchen Chu ◽

Ke Liu ◽

Yusha Ru ◽

Yan Zhang ◽

...

Keyword(s):

Dry Eye ◽

Nlrp3 Inflammasome ◽

Ocular Surface ◽

Combined Treatment ◽

Underlying Mechanism ◽

Oxygen Species ◽

Corneal Epithelial ◽

Melanocyte Stimulating Hormone ◽

Human Corneal Epithelial Cells ◽

Conjunctival Goblet Cells

AbstractAn important mechanism involved in dry eye (DE) is the association between tear hyperosmolarity and inflammation severity. Inflammation in DE might be mediated by the NLRP3 inflammasome, which activated by exposure to reactive oxygen species (ROS). A combination of carboxymethylcellulose (CMC) and α-melanocyte stimulating hormone (α-MSH) may influence DE through this mechanism, thus avoiding defects of signal drug. In this study, we assessed whether treatment comprising CMC combined with α-MSH could ameliorate ocular surface function; we found that it promoted tear secretion, reduced the density of fluorescein sodium staining, enhanced the number of conjunctival goblet cells, and reduced the number of corneal apoptotic cells. Investigation of the underlying mechanism suggested that the synergistic effect of combined treatment alleviated DE inflammation through reduction of ROS level and inhibition of the NLRP3 inflammasome in human corneal epithelial cells. These findings indicate that combined CMC + α-MSH treatment could ameliorate lesions and restore ocular surface function in patients with DE through reduction of ROS level and inhibition of NLRP3 signalling.

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Syntaxin-17-Dependent Mitochondrial Dynamics is Essential for Protection Against Oxidative-Stress-Induced Apoptosis

Antioxidants ◽

10.3390/antiox8110522 ◽

2019 ◽

Vol 8 (11) ◽

pp. 522 ◽

Cited By ~ 4

Author(s):

Wang ◽

Xiao ◽

Huang ◽

Liu

Keyword(s):

Oxidative Stress ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Autophagic Flux ◽

Wild Type ◽

Dual Roles ◽

Defective Mutant ◽

U2os Cells ◽

Induced Apoptosis

In this study, cell death induced by the oxidant tert-butylhydroperoxide (tBH) was observed in U2OS cells; this phenotype was rescued by Syntaxin 17 (STX17) knockout (KO) but the mechanism is unknown. STX17 plays dual roles in autophagosome–lysosome fusion and mitochondrial fission. However, the contribution of the two functions of STX17 to apoptosis has not been extensively studied. Here, we sought to dissect the dual roles of STX17 in oxidative-stress-induced apoptosis by taking advantage of STX17 knockout cells and an autophagosome–lysosome fusion defective mutant of STX17. We generated STX17 knockout U2OS cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and the STX17 knockout cells were reconstituted with wild-type STX17 and its autophagosome–lysosome fusion defective mutant. Autophagy was assessed by autophagic flux assay, Monomer red fluorescent protein (mRFP)–GFP–LC3 assay and protease protection assay. Golgi, endoplasmic reticulum (ER)/ER–Golgi intermediate compartment (ERGIC) and mitochondrial dynamics were examined by staining the different indicator proteins. Apoptosis was evaluated by caspase cleavage assay. The general reactive oxygen species (ROS) were detected by flow cytometry. In STX17 complete knockout cells, sealed autophagosomes were efficiently formed but their fusion with lysosomes was less defective. The fusion defect was rescued by wild-type STX17 but not the autophagosome–lysosome fusion defective mutant. No obvious defects in Golgi, ERGIC or ER dynamics were observed. Mitochondria were significantly elongated, supporting a role of STX17 in mitochondria fission and the elongation caused by STX17 KO was reversed by the autophagosome–lysosome fusion defective mutant. The clearance of protein aggregation was compromised, correlating with the autophagy defect but not with mitochondrial dynamics. This study revealed a mixed role of STX17 in autophagy, mitochondrial dynamics and oxidative stress response. STX17 knockout cells were highly resistant to oxidative stress, largely due to the function of STX17 in mitochondrial fission rather than autophagy.

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S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux

Cells ◽

10.3390/cells10040929 ◽

2021 ◽

Vol 10 (4) ◽

pp. 929

Author(s):

Aleksandra Hać ◽

Karolina Pierzynowska ◽

Anna Herman-Antosiewicz

Keyword(s):

Degradation Process ◽

Underlying Mechanism ◽

Stress Conditions ◽

Autophagic Flux ◽

Ratiometric Fluorescence ◽

Mouse Embryonic Fibroblasts ◽

Double Knockout ◽

Embryonic Fibroblasts ◽

Microtubule Network ◽

Tubulin Acetylation

Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.

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Clock knockdown attenuated reactive oxygen species-mediated senescence of chondrocytes through restoring autophagic flux

Life Sciences ◽

10.1016/j.lfs.2021.119036 ◽

2021 ◽

Vol 269 ◽

pp. 119036

Author(s):

Junlong Zhong ◽

Bin Wang ◽

Biao Wu ◽

Jie Shang ◽

Ning Jiang ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Autophagic Flux ◽

Oxygen Species

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Mfn2 Overexpression Attenuates MPTP Neurotoxicity In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms22020601 ◽

2021 ◽

Vol 22 (2) ◽

pp. 601

Author(s):

Fanpeng Zhao ◽

Quillan Austria ◽

Wenzhang Wang ◽

Xiongwei Zhu

Keyword(s):

Mitochondrial Dysfunction ◽

Mitochondrial Dynamics ◽

Significant Loss ◽

Critical Event ◽

Mitochondrial Fragmentation ◽

Wild Type Littermate ◽

Littermate Control ◽

Mptp Administration

Mitochondrial dysfunction represents a critical event in the pathogenesis of Parkinson’s disease (PD). Increasing evidence demonstrates that disturbed mitochondrial dynamics and quality control play an important role in mitochondrial dysfunction in PD. Our previous study demonstrated that MPP+ induces mitochondrial fragmentation in vitro. In this study, we aimed to assess whether blocking MPTP-induced mitochondrial fragmentation by overexpressing Mfn2 affords neuroprotection in vivo. We found that the significant loss of dopaminergic neurons in the substantia nigra (SN) induced by MPTP treatment, as seen in wild-type littermate control mice, was almost completely blocked in mice overexpressing Mfn2 (hMfn2 mice). The dramatic reduction in dopamine neuronal fibers and dopamine levels in the striatum caused by MPTP administration was also partially inhibited in hMfn2 mice. MPTP-induced oxidative stress and inflammatory response in the SN and striatum were significantly alleviated in hMfn2 mice. The impairment of motor function caused by MPTP was also blocked in hMfn2 mice. Overall, our work demonstrates that restoration of mitochondrial dynamics by Mfn2 overexpression protects against neuronal toxicity in an MPTP-based PD mouse model, which supports the modulation of mitochondrial dynamics as a potential therapeutic target for PD treatment.

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Abstract 14412: Activation of Autophagic Flux Maintains Mitochondrial Homeostasis During Cardiac Ischemia/reperfusion Injury

Circulation ◽

10.1161/circ.142.suppl_3.14412 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Jing Yang ◽

Geoffrey W CHO ◽

Lihao He ◽

Yuxin Chu ◽

Jin He ◽

...

Keyword(s):

Infarct Size ◽

Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Systolic Function ◽

Ischemia Reperfusion ◽

Border Zone ◽

Autophagic Flux ◽

Hdac Inhibition ◽

Infarct Border Zone ◽

Infarct Border

Background and Hypothesis: Reperfusion injury accounts for ~50% of myocardial infarct size, and clinically efficacious therapies are lacking. Histone deacetylase (HDAC) inhibition enhances cardiomyocyte autophagic activity, mitochondria biogenesis, and blunts ischemia/reperfusion (I/R) injury when given at the time of reperfusion. However, as HDAC inhibition has pleiotropic effects, we will test whether augmentation of autophagic flux using a specific autophagy-inducing peptide, Tat-Beclin (TB), is cardioprotective. Methods: 8-12-week-old, wild-type, C57BL6 mice were randomized into three groups: vehicle control, Tat-Scrambled (TS) peptide, or Tat-Beclin (TB) peptide. Each group was subjected to I/R surgery (45min ischemia, 24h reperfusion). Infarct size, systolic function, and mitochondrial dynamics were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were used to test for cardiomyocyte specificity. Conditional cardiomyocyte ATG7 knockout (ATG7 KO) mice and ATG7 knockdown by siRNA in NRVMs were used to evaluate the role of autophagy. Results: TB treatment at reperfusion reduced infarct size by 20.1±6.3% (n=23, p<0.02) and improved systolic function. Increased autophagic flux and reduced reactive oxygen species (ROS) were observed in the infarct border zone. The cardioprotective effects of TB were abolished in ATG7 KO mice. TB increased mtDNA content in the border zone significantly. In NRVMs subjected to I/R, TB reduced cell death by 41±6% (n=12, p<0.001), decreased ROS, and increased mtDNA content significantly by ~50%. Moreover, TB promoted expression of PGC1α (a major driver of mitochondrial biogenesis) both in the infarct border zone and NRVMs subjected to I/R by ~40%, and increased levels of mitochondrial dynamics gene transcripts Drp1, Fis1, and MFN1 / 2. Conversely, ATG7 knockdown in NRVMs and cardiac ATG7 KO abolished the beneficial effects of TB on mitochondria DNA content. Conclusions: Autophagic flux is an essential process to mitigate myocardial reperfusion injury acting, at least in part, by inducing PGC1α-mediated mitochondrial biogenesis. Augmentation of autophagic flux may emerge as a viable clinical therapy to reduce reperfusion injury in myocardial infarction.

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The Na/K-ATPase Signaling: From Specific Ligands to General Reactive Oxygen Species

International Journal of Molecular Sciences ◽

10.3390/ijms19092600 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2600 ◽

Cited By ~ 11

Author(s):

Rebecca Pratt ◽

Cameron Brickman ◽

Cameron Cottrill ◽

Joseph Shapiro ◽

Jiang Liu

Keyword(s):

Reactive Oxygen Species ◽

Positive Feedback ◽

Reactive Oxygen ◽

Underlying Mechanism ◽

Short Review ◽

Oxygen Species ◽

Signaling Function ◽

Amplification Loop

The signaling function of the Na/K-ATPase has been established for 20 years and is widely accepted in the field, with many excellent reports and reviews not cited here. Even though there is debate about the underlying mechanism, the signaling function is unquestioned. This short review looks back at the evolution of Na/K-ATPase signaling, from stimulation by cardiotonic steroids (also known as digitalis-like substances) as specific ligands to stimulation by reactive oxygen species (ROS) in general. The interplay of cardiotonic steroids and ROS in Na/K-ATPase signaling forms a positive-feedback oxidant amplification loop that has been implicated in some pathophysiological conditions.

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