Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Lampl, Thomas; Crum, Jo A.; Davis, Taylor; Milligan, Carol; Moore, Victoria Del Gaizo

doi:10.3791/52076

Cited by 31 publications

(25 citation statements)

References 39 publications

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“…We separated the nucleus and mitochondria using previously described methods 23, 24 Interestingly, approximately 25% of DON was detected in the nucleus, but no DON was detected in mitochondria, suggesting that the mitochondria may be the main target of DON.…”

Section: Resultsmentioning

confidence: 99%

Carrier-Mediated and Energy-Dependent Uptake and Efflux of Deoxynivalenol in Mammalian Cells

Wen

et al. 2017

Sci Rep

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Deoxynivalenol (DON) is one of the most abundant mycotoxins and exerts many adverse effects on humans and animals. To date, the transporting mechanism of DON in mammalian cells remains unclear. In this study, the parallel artificial membrane permeability assay (PAMPA), Transwell models and metabolic inhibitors were used to determine the possible transporting mechanisms of DON in Caco-2, MDCK and HepG2 cells. PAMPA and Transwell models showed reduced passive transport and increased intestinal absorption, indicating a carrier-mediated transporting mechanism. Furthermore, higher unidirectional transport of DON was observed in the basolateral-to-apical direction than in the apical-to-basolateral direction, indicating the existence of efflux proteins. Interestingly, DON was accumulated in the nucleus, and no DON was detected in mitochondria, indicating that the nucleus may be the main target organelle of DON. Moreover, the use of various transporter inhibitors in different cells shows that organic anion transporters, organic cation transporters, and organic anion-transporting polypeptides participate in DON uptake, and P-glycoprotein is the major efflux protein. Importantly, DON uptake is strongly inhibited by metabolic inhibitors and is highly dependent on temperature. In summary, carrier-mediated and energy-dependent uptake and efflux mechanisms for DON in mammalian cells are reported, aiding in improving our understanding of its toxicological mechanisms.

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Section: Resultsmentioning

confidence: 99%

Carrier-Mediated and Energy-Dependent Uptake and Efflux of Deoxynivalenol in Mammalian Cells

Wen

et al. 2017

Sci Rep

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“…Mitochondria enriched fractions were prepared using previously published protocols with slight modifications [ 21 – 23 ]. Briefly, cells were isolated by centrifugation and washed twice with PBS at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Deficiency in Cardiolipin Reduces Doxorubicin-Induced Oxidative Stress and Mitochondrial Damage in Human B-Lymphocytes

Aryal

Rao

2016

PLoS ONE

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Cardiolipin (CL) is an inner mitochondrial membrane phospholipid which plays an important role in mitochondrial function. Perturbation in CL biosynthesis alters mitochondrial bioenergetics causing a severe genetic disorder commonly known as Barth syndrome. Barth syndrome patients are known to have a reduced concentration and altered composition of CL. Cardiolipin is also known to have a high affinity for the chemotherapeutic agent doxorubicin (Dox), resulting in an extensive mitochondrial accumulation of the drug. Our results indicate that B-lymphocytes from healthy individuals are more sensitive to Dox-induced oxidative stress and cellular toxicity compared to the B-lymphocytes from Barth syndrome as indicated by greater cell death and greater level of cleaved caspase-3 following Dox treatment. Barth lymphocytes, when compared to healthy lymphocytes, showed a greater basal level of mitochondrial reactive oxygen species (mito-ROS), yet exhibited a lower level of induced mito-ROS production in response to Dox. Significantly less ATP content and slightly greater OXPHOS protein levels were detected in healthy cells compared to Barth cells after Dox treatment. Consistent with greater mitochondrial ROS, treatment with Dox induced a higher level of lipid peroxidation and protein carbonylation in healthy lymphocytes compared to Barth lymphocytes. The final remodeling of CL during CL synthesis is catalyzed by the tafazzin protein. Knockdown of tafazzin gene in H9c2 cardiomyocytes using siRNA showed decreased oxidant-induced damage, as observed in Barth lymphocytes. Our findings demonstrate that a deficiency in CL might provide a therapeutic advantage in favor of oxidant-induced anticancer activities.

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“…Isolated mitochondria from the mouse midbrain were stained with TMRE as described previously . Briefly, isolated mitochondria (75 μg) were incubated with EB buffer (125 mM KCl, 10 mM Tris–MOPS, 5 mM glutamate, 2.5 mM malate, 1 mM K phosphate and 10 mM EGTA–Tris, pH 7.4) containing 1 μM TMRE for 10 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Knockdown of the mitochondria‐localized protein p13 protects against experimental parkinsonism

et al. 2018

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Mitochondrial dysfunction in the nigrostriatal dopaminergic system is a critical hallmark of Parkinson's disease (PD). Mitochondrial toxins produce cellular and behavioural dysfunctions resembling those in patients with PD Causative gene products for familial PD play important roles in mitochondrial function. Therefore, targeting proteins that regulate mitochondrial integrity could provide convincing strategies for PD therapeutics. We have recently identified a novel 13-kDa protein (p13) that may be involved in mitochondrial oxidative phosphorylation. In the current study, we examine the mitochondrial function of p13 and its involvement in PD pathogenesis using mitochondrial toxin-induced PD models. We show that p13 overexpression induces mitochondrial dysfunction and apoptosis. p13 knockdown attenuates toxin-induced mitochondrial dysfunction and apoptosis in dopaminergic SH-SY5Y cells via the regulation of complex I. Importantly, we generate -deficient mice using the CRISPR/Cas9 system and observe that heterozygous knockout prevents toxin-induced motor deficits and the loss of dopaminergic neurons in the substantia nigra. Taken together, our results suggest that manipulating p13 expression may be a promising avenue for therapeutic intervention in PD.

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Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Cited by 31 publications

References 39 publications

Carrier-Mediated and Energy-Dependent Uptake and Efflux of Deoxynivalenol in Mammalian Cells

Carrier-Mediated and Energy-Dependent Uptake and Efflux of Deoxynivalenol in Mammalian Cells

Deficiency in Cardiolipin Reduces Doxorubicin-Induced Oxidative Stress and Mitochondrial Damage in Human B-Lymphocytes

Knockdown of the mitochondria‐localized protein p13 protects against experimental parkinsonism

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