scholarly journals Manganese (II) chloride leads to dopaminergic neurotoxicity by promoting mitophagy through BNIP3-mediated oxidative stress in SH-SY5Y cells

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Yanning Huang ◽  
Qiaolin Wen ◽  
Jinfeng Huang ◽  
Man Luo ◽  
Yousheng Xiao ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Clinical Manifestations ◽  
Receptor Protein ◽  
Ros Generation ◽  
Mitochondrial Morphology ◽  
Human Neuroblastoma ◽  
Protein Levels ◽  
Mitochondrial Membrane Potential Loss

Abstract Background Manganese overexposure can induce neurotoxicity, lead to manganism and result in clinical manifestations similar to those of parkinsonism. However, the underlying molecular mechanism is still unclear. This study demonstrated that MnCl2 induces mitophagy and leads to neurotoxicity by promoting BNIP3-mediated reactive oxygen species (ROS) generation. Methods Human neuroblastoma SH-SY5Y cells were used throughout our experiments. Cell viability was detected by cell proliferation/toxicity test kits. Mitochondrial membrane potential was measured by flow cytometry. ROS generation was detected using a microplate reader. Protein levels were evaluated by Western blot. Transmission electron microscopy was used to evaluate mitochondrial morphology. Co-immunoprecipitation was used to verify the interaction between BNIP3 and LC3. Results MnCl2 led to loss of mitochondrial membrane potential and apoptosis of SH-SY5Y cells by enhancing expression of BNIP3 and conversion of LC3-I to LC3-II. Moreover, MnCl2 reduced expression of the mitochondrial marker protein TOMM20 and promoted interaction between BNIP3 and LC3. The results also indicated that a decrease in BNIP3 expression reduced the mitochondrial membrane potential loss, attenuated apoptosis and reduced mitochondrial autophagosome formation in SH-SY5Y cells after MnCl2 treatment. Finally, we found that manganese-induced ROS generation could be reversed by the antioxidant N-acetyl cysteine (NAC) or silencing BNIP3 expression. Conclusions BNIP3 mediates MnCl2-induced mitophagy and neurotoxicity in dopaminergic SH-SY5Y cells through ROS. Thus, BNIP3 contributes to manganese-induced neurotoxicity by functioning as a mitophagy receptor protein.

scholarly journals Neuroprotective Effects of Hesperidin, a Plant Flavanone, on Rotenone-Induced Oxidative Stress and Apoptosis in a Cellular Model for Parkinson’s Disease

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Kuppusamy Tamilselvam ◽  
Nady Braidy ◽  
Thamilarasan Manivasagam ◽  
Musthafa Mohamed Essa ◽  
Nagarajan Rajendra Prasad ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Neuroprotective Effect ◽  
Neuroblastoma Cell ◽  
Ros Generation ◽  
Enzymatic Antioxidants ◽  
Neuroprotective Effects ◽  
Human Neuroblastoma ◽  
Cyt C

Rotenone a widely used pesticide that inhibits mitochondrial complex I has been used to investigate the pathobiology of PD bothin vitroandin vivo. Studies have shown that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species (ROS), leading to neuronal apoptosis. The current study was carried out to investigate the neuroprotective effects of hesperidin, a citrus fruit flavanol, against rotenone-induced apoptosis in human neuroblastoma SK-N-SH cells. We assessed cell death, mitochondrial membrane potential, ROS generation, ATP levels, thiobarbituric acid reactive substances, reduced glutathione (GSH) levels, and the activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) using well established assays. Apoptosis was determined in normal, rotenone, and hesperidin treated cells, by measuring the protein expression of cytochrome c (cyt c), caspases 3 and 9, Bax, and Bcl-2 using the standard western blotting technique. The apoptosis in rotenone-induced SK-N-SH cells was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, the depletion of GSH, enhanced activities of enzymatic antioxidants, upregulation of Bax, cyt c, and caspases 3 and 9, and downregulation of Bcl-2, which were attenuated in the presence of hesperidin. Our data suggests that hesperidin exerts its neuroprotective effect against rotenone due to its antioxidant, maintenance of mitochondrial function, and antiapoptotic properties in a neuroblastoma cell line.

scholarly journals Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

1996 ◽  
Vol 44 (12) ◽  
pp. 1363-1372 ◽  
Author(s):  
M Poot ◽  
Y Z Zhang ◽  
J A Krämer ◽  
K S Wells ◽  
L J Jones ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Epifluorescence Microscopy ◽  
Mitochondrial Morphology ◽  
Microtubule Network ◽  
Multiple Features ◽  
Permeabilized Cells ◽  
Dye Staining ◽  
And Function

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Metformin Collaborates With PINK1/Mfn2 Overexpression Prevented Isoproterenol-Induced Cardiomyocyte Injury By Improving Mitochondrial Function

2021 ◽  
Author(s):  
Zhuang Ma ◽  
Zuheng Liu ◽  
Yuting Xue ◽  
Hao Zhang ◽  
Wenjun Xiong ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Energy Metabolism ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Mitochondrial Morphology ◽  
Dependent Manner ◽  
Combination Strategy ◽  
Mitochondrial Energy Metabolism ◽  
Atp Production

Abstract Background: Both mitochondrial quality control and energy metabolism are critical in maintaining the physiological function of cardiomyocytes. Previous studies indicated that PGC-1α is a transcription co-activator in promoting mitochondrial energy metabolism which would be beneficial for cardiomyocytes. However, PGC-1α overexpression in heart tissues could also result in the development of cardiomyopathy. This discrepancy in vivo and in vitro might be due to neglecting the elimination of damaged mitochondrial. Thus, an integration strategy of mitochondrial biogenesis and mitophagy might be beneficial.Methods: We studied the function of PINK1 in mitophagy in isoproterenol (Iso)-induced cardiomyocyte injury. Adenovirus was used to provoke an overexpression of the PINK1/Mfn2 protein. Mitochondrial morphology was examined via electron microscopy and confocal microscopy. Cardiomyocytes injury were measured by mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and apoptosis. Metformin was used to increase mitochondrial biogenesis, the level of which was detected via immunoblotting. Additionally, mitochondrial respiratory function was measured by ATP production and oxygen consumption rate (OCR). Results: Cardiomyocytes treated with Iso had high levels of PINK1 and low levels of Mfn2 in a time-dependent manner. PINK1 overexpression promoted mitophagy, alleviated Iso-induced reduction in MMP, reduced ROS production and the apoptotic rate. In addition to increasing mitophagy, metformin could promote mitochondrial biogenensis and the overexpression of Mfn2 induce mitochondrial fusion. Moreover, metformin treatment and PINK1/Mfn2 overexpression reduced the mitochondrial dysfunction by inhibiting the generation of ROS, and leading to an increase in both ATP production and mitochondrial membrane potential in Iso-induced cardiomyocytes injury. Conclusion: Our findings indicate that a combination strategy may help ameliorate myocardial injury through mitophagy and mitochondrial biogenesis.

Potential Crosstalk Of Ca2+-ROS Dependent Mechanism Involved In Kasumi cells’ Apoptosis Induced By Lentivirus-Mediated HO-1 siRNA

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5558-5558
Author(s):  
Jishi Wang ◽  
Wei Sixi ◽  
Wang Yating ◽  
Chai Qixiang
Keyword(s):  
Membrane Potential ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Cell Apoptosis ◽  
Mitochondrial Membrane ◽  
The Other ◽  
Ros Generation ◽  
Dependent Manner ◽  
Realtime Pcr ◽  
Protein Expressions

Abstract Aim Using lentivirus-mediated HO-1 siRNA (lenti-siHO-1-GFP) to silence the HO-1 gene in Kasumi cells so as to explore the role and mechanism of HO-1 on cell apoptosis. Methods To infect Kasumi cells with lenti-siHO-1-GFP and check the infection efficiency by using fluorescence microscopy and flow cytometry (FCM). Experimental group was divided into three groups: untreated Kasumi (K), infected Kasumi by empty vector (lenti-GFP-K) and infected Kasumi by lentivirus-mediated HO-1 siRNA (lenti-siHO-1-K). The HO-1 expression of each group was detected by realtime PCR. Fluo3-AM method was used to detect the intracellular Ca2+ accumulation. DCFH-DA was used for the measurement of intracellular ROS. The change of mitochondrial membrane potential was evaluated by JC-1 stainning by using FCM. After being treated with various concentrations of daunorubicin for 24, 48, and 72 h respectively, cell viability was determined by MTT assay. Cell apoptosis was determined by FCM following with cells dual-stained with Annexin-V-FITC and propidium iodide (PI). The mRNA of HO-1 and apoptosis-related genes were analyzed by realtime PCR and, the expressions of their corresponding protein were determined by western blot. Additionally, After treating with 10mM Ca2+chelator BAPTA-AM and 0.5mM NAC for 12h, Ca2+ accumulation, ROS generation, the expression of HO-1 and apoptosis-related genes were detected respectively. Result presented in mean±sd manner. Results After lenti-siHO-1-GFP infection for 48h, we could observe the fluorescence clear, the fluorescent intensity was 95.87% after 72 hours. The HO-1 silencing efficiency of lenti-siHO-1-K was 77.00%. MTT result showed that daunorubicin exerted moderate inhibitory effects on cell proliferation in a dose and time dependent manner. With the same treating conditions, the cell viability of lenti-siHO-1-K group was significantly lower than the other two groups(e.g 49.20±1.30% survival in lenti-siHO-1-K group, 72.40±1.90% in K group and 74.10±2.10% in lenti-GFP-K group after being treated by 5ug/ml DNR,respectively, p=0.014), while the apoptosis rate was higher than the other two groups(e.g 75.77±3.41% in lenti-siHO-1-K group, 23.72±2.03% in K group and 26.10±1.95% in lenti-GFP-K group after being treated by 5ug/ml DNR,respectively, p=0.011). Compared with other two groups, the lenti-siHO-1-K group showed a downregulation in the mRNA and protein expression of HO-1. The mRNA and protein expressions of cyto-C, caspase3, caspase8, caspase9 and caspase12 in lenti-siHO-1-K group were upregulated after exposure to 5ug/ml daunorubicin for 24 hours. Compared with K and lenti-GFP-K groups, Ca2+ accumulation in lenti-siHO-1-K group was increased significantly(e.g 40.35±2.10% in lenti-siHO-1-K group, 17.30±1.81% in K group and 14.15±1.75% in lenti-GFP-K group,respectively, p=0.041). The ROS generation was higher than the other two groups(e.g 47.65±2.05% in lenti-siHO-1-K group, 21.30±1.94% in K group and19.90±2.01% in lenti-GFP-K group,respectively, p=0.037). The ratio of Green/Red fluorescence intensity increased significantly in lenti-siHO-1-K group(e.g 0.704±0.06 in lenti-siHO-1-K group, 0.57±0.09 in K group and 0.527±0.05 in lenti-GFP-K group, respectively, p=0.042). After exposure to 10mM BAPTA-AM and 0.1mM NAC alone or combined with, both the intracellular Ca2+accumulation and the ROS level in lenti-siHO-1-K group reduced(17.59±1.01% of Ca2+acumulation and 19.78±1.3% of ROS production after BAPTA-AM treatment alone, 23.42±1.97% of Ca2+and 15.47±1.14% of ROS after being treated by NAC alone, 16.52±1.23% of Ca2+and 14.37±1.21% of ROS after treatment by both agent) , while the mRNA and protein expressions of cyto-C, caspase3, caspase8, caspase9 and caspase12, decreased significantly. Conclusion HO-1 gene silencing played a role in pro-apoptosis in Kasumi cells. The mechanism may be related to the endoplasmic reticulum stress and abnormal accumulation of intracellular Ca2+, ROS generation, descending of the mitochondrial membrane potential and release cyto-C, then further activated the caspases cascade and promoted apoptosis. However, it tended to be initiated by crosstalk in Ca2+-ROS pathway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Short Isoform of DNAJB6 Protects against 1-Methyl-4-phenylpridinium Ion-Induced Apoptosis in LN18 Cells via Inhibiting Both ROS Formation and Mitochondrial Membrane Potential Loss

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Yeon-Mi Hong ◽  
Yohan Hong ◽  
Yeong-Gon Choi ◽  
Sujung Yeo ◽  
Soo Hee Jin ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Molecular Mechanisms ◽  
Mitochondrial Pathway ◽  
Caspase 9 ◽  
Ros Formation ◽  
Mitochondrial Membrane Potential Loss ◽  
Induced Apoptosis ◽  
Shock Proteins

In a previous study, we found that the short isoform of DNAJB6 (DNAJB6(S)) had been decreased in the striatum of a mouse model of Parkinson’s disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). DNAJB6, one of the heat shock proteins, has been implicated in the pathogenesis of PD. In this study, we explored the cytoprotective effect of DNAJB6(S) against 1-methyl-4-phenylpyridinium ion- (MPP+-) induced apoptosis and the underlying molecular mechanisms in cultured LN18 cells from astrocytic tumors. We observed that MPP+ significantly reduced the cell viability and induced apoptosis in LN18 glioblastoma cells. DNAJB6(S) protected LN18 cells against MPP+-induced apoptosis not only by suppressing Bax cleavage but also by inhibiting a series of apoptotic events including loss of mitochondrial membrane potential, increase in intracellular reactive oxygen species, and activation of caspase-9. These observations suggest that the cytoprotective effects of DNAJB6(S) may be mediated, at least in part, by the mitochondrial pathway of apoptosis.

scholarly journals Rhomboid Protease PARL Mediates the Mitochondrial Membrane Potential Loss-induced Cleavage of PGAM5

2012 ◽  
Vol 287 (41) ◽  
pp. 34635-34645 ◽  
Author(s):  
Shiori Sekine ◽  
Yusuke Kanamaru ◽  
Masato Koike ◽  
Ayako Nishihara ◽  
Masahiro Okada ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Rhomboid Protease ◽  
Potential Loss ◽  
Mitochondrial Membrane Potential Loss

scholarly journals BKCa channel activation increases cardiac contractile recovery following hypothermic ischemia/reperfusion

2015 ◽  
Vol 309 (4) ◽  
pp. H625-H633 ◽  
Author(s):  
Brenda Cordeiro ◽  
Dmitry Terentyev ◽  
Richard T. Clements
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Left Ventricular ◽  
Bkca Channel ◽  
Ros Generation ◽  
H9c2 Cells ◽  
Bkca Channels ◽  
Channel Activation

Mitochondrial Ca2+-activated large-conductance K+ (BKCa) channels are thought to provide protection during ischemic insults in the heart. Rottlerin (mallotoxin) has been implicated as a potent BKCa activator. The purpose of this study was twofold: 1) to investigate the efficacy of BKCa channel activation as a cardioprotective strategy during ischemic cardioplegic arrest and reperfusion (CP/R) and 2) to assess the specificity of rottlerin for BKCa channels. Wild-type (WT) and BKCa knockout (KO) mice were subjected to an isolated heart model of ischemic CP/R. A mechanism of rottlerin-induced cardioprotection was also investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential and reactive oxygen species (ROS) production. CP/R decreased left ventricular developed pressure, positive and negative first derivatives of left ventricular pressure, and coronary flow (CF) in WT mice. Rottlerin dose dependently increased the recovery of left ventricular function and CF to near baseline levels. BKCa KO hearts treated with or without 500 nM rottlerin were similar to WT CP hearts. H9c2 cells subjected to in vitro CP/R displayed reduced mitochondrial membrane potential and increased ROS generation, both of which were significantly normalized by rottlerin. We conclude that activation of BKCa channels rescues ischemic damage associated with CP/R, likely via effects on improved mitochondrial membrane potential and reduced ROS generation.

scholarly journals EPAC1 Inhibition Protects the Heart from Doxorubicin-Induced Toxicity

2021 ◽  
Author(s):  
Marianne Mazevet ◽  
Maxance Ribeiro ◽  
Anissa Belhadef ◽  
Delphine Dayde ◽  
Anna Llach ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Membrane Potential ◽  
Dilated Cardiomyopathy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Dna Intercalation ◽  
Antitumoral Activity ◽  
Ros Generation

Rationale: The widely used chemotherapeutic agent Doxorubicin (Dox) induces cardiotoxicity leading to dilated cardiomyopathy and heart failure. This cardiotoxicity has been related to ROS generation, DNA intercalation, bioenergetic distress and cell death. However, alternative mechanisms are emerging, focusing on signaling pathways. Objective: We investigated the role of Exchange Protein directly Activated by cAMP (EPAC), key factor in cAMP signaling, in Dox-induced cardiotoxicity. Methods and Results: Dox was administrated in vivo (10 +/- 2 mg/kg, i.v.; with analysis at 2, 6 and 15 weeks post injection) in WT and EPAC1 KO C57BL6 mice. Cardiac function was analyzed by echocardiography and intracellular Ca2+ homeostasis by confocal microscopy in isolated ventricular cardiomyocytes. 15 weeks post-injections, Dox-treated WT mice, developed a dilated cardiomyopathy with decreased ejection fraction, increased telediastolic volume and impaired Ca2+ homeostasis, which were totally prevented in the EPAC1 KO mice. The underlying mechanisms were investigated in neonatal and adult rat cardiac myocytes under Dox treatment (1-10 uM). Flow cytometry, Western blot, BRET sensor assay, and RT-qPCR analysis showed that Dox induced DNA damage and cardiomyocyte cell death with apoptotic features rather than necrosis, including Ca2+-CaMKKβ-dependent opening of the Mitochondrial Permeability Transition Pore, dissipation of the Mitochondrial membrane potential, caspase activation, cell size reduction, and DNA fragmentation. Dox also led to an increase in both cAMP concentration and EPAC1 protein level and activity. The pharmacological inhibition of EPAC1 (CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations including DNA damage, Mitochondrial membrane potential, apoptosis, mitochondrial biogenesis, dynamic, and fission/fusion balance, and respiratory chain activity, suggesting a crucial role of EPAC1 in these processes. Importantly, while preserving cardiomyocyte integrity, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Conclusion: Thus, EPAC1 inhibition could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity.

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