Methods for assessing mitochondrial quality control mechanisms and cellular consequences in cell culture

Redmann, Matthew; Benavides, Gloria A.; Wani, Willayat Yousuf; Berryhill, Taylor F.; Ouyang, Xiaosen; Johnson, Michelle S.; Ravi, Saranya; Mitra, Kasturi; Barnes, Stephen; Darley‐Usmar, Victor; Zhang, Jianhua

doi:10.1016/j.redox.2018.04.005

Cited by 38 publications

(39 citation statements)

References 48 publications

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“…Indeed, our previous study in FRDA fibroblasts found that low FXN levels led to increased mtDNA damage and decreased mtDNA repair capacity (Bhalla et al, 2016). To determine whether the integrity of mtDNA was compromised in cells expressing only Fxn G127V , we analyzed mtDNA isolated from WT and MUT MEFs using a quantitative PCR assay based on the principle that DNA lesions can slow down or block the progression of DNA polymerase (Bhalla et al, 2016;Ponti et al, 1991;Redmann et al, 2018). This type of assay detects various forms of DNA damage, from single-or double-strand DNA breaks and gaps to specific chemical lesions (Lehle et al, 2014).…”

Section: Mitochondrial Integrity In Fxn G127v Mut Mefs Decreases Overmentioning

confidence: 99%

Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia

Fil

Chacko

Conley

et al. 2020

Disease Models &Amp; Mechanisms

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Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeat sequences in intron 1 of FXN, whereas a fraction of patients are compound heterozygotes, with a missense or nonsense mutation in one FXN allele and expanded GAAs in the other. A prevalent missense mutation among FRDA patients changes a glycine at position 130 to valine (G130V). Herein, we report generation of the first mouse model harboring an Fxn point mutation. Changing the evolutionarily conserved glycine 127 in mouse Fxn to valine results in a failure-to-thrive phenotype in homozygous animals and a substantially reduced number of offspring. Like G130V in FRDA, the G127V mutation results in a dramatic decrease of Fxn protein without affecting transcript synthesis or splicing. FxnG127V mouse embryonic fibroblasts exhibit significantly reduced proliferation and increased cell senescence. These defects are evident in early passage cells and are exacerbated at later passages. Furthermore, increased frequency of mitochondrial DNA lesions and fragmentation are accompanied by marked amplification of mitochondrial DNA in FxnG127V cells. Bioenergetics analyses demonstrate higher sensitivity and reduced cellular respiration of FxnG127V cells upon alteration of fatty acid availability. Importantly, substitution of FxnWT with FxnG127V is compatible with life, and cellular proliferation defects can be rescued by mitigation of oxidative stress via hypoxia or induction of the NRF2 pathway. We propose FxnG127V cells as a simple and robust model for testing therapeutic approaches for FRDA.

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Section: Mitochondrial Integrity In Fxn G127v Mut Mefs Decreases Overmentioning

confidence: 99%

Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia

Fil

Chacko

Conley

et al. 2020

Disease Models &Amp; Mechanisms

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“…As research progresses, hepatic fatty acid–induced mitochondrial dysfunction has also been proved to play an important role in the development of hepatic insulin resistance (Wang et al, 2017b; Wang et al, 2018; Wang et al, 2019b). Recently, it has been widely recognized that mitochondrial autophagy (mitophagy), a catabolic process, can selectively remove damaged mitochondria by autophagolysosomes to maintain mitochondrial function and energy metabolism (Redmann et al, 2018; Li et al, 2018a). As a mitochondrial quality control mechanism, mitophagy can target and degrade damaged mitochondria to suppress damaged mitochondria-derived reactive oxygen species (ROS), which can dramatically impair healthy mitochondria, leading to mitochondrial dysfunction.…”

Section: Introductionmentioning

confidence: 99%

Mitophagy in Hepatic Insulin Resistance: Therapeutic Potential and Concerns

Nie

Huang

et al. 2019

Front. Pharmacol.

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Metabolic syndrome, characterized by central obesity, hypertension, and hyperlipidemia, increases the morbidity and mortality of cardiovascular disease, type 2 diabetes, nonalcoholic fatty liver disease, and other metabolic diseases. It is well known that insulin resistance, especially hepatic insulin resistance, is a risk factor for metabolic syndrome. Current research has shown that hepatic fatty acid accumulation can cause hepatic insulin resistance through increased gluconeogenesis, lipogenesis, chronic inflammation, oxidative stress and endoplasmic reticulum stress, and impaired insulin signal pathway. Mitochondria are the major sites of fatty acid β-oxidation, which is the major degradation mechanism of fatty acids. Mitochondrial dysfunction has been shown to be involved in the development of hepatic fatty acid–induced hepatic insulin resistance. Mitochondrial autophagy (mitophagy), a catabolic process, selectively degrades damaged mitochondria to reverse mitochondrial dysfunction and preserve mitochondrial dynamics and function. Therefore, mitophagy can promote mitochondrial fatty acid oxidation to inhibit hepatic fatty acid accumulation and improve hepatic insulin resistance. Here, we review advances in our understanding of the relationship between mitophagy and hepatic insulin resistance. Additionally, we also highlight the potential value of mitophagy in the treatment of hepatic insulin resistance and metabolic syndrome.

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“…Many diseases, such as aging, cancer, cardiovascular disease, diabetes, and neurodegenerative diseases, are associated with mitochondrial dysfunction [7][8][9][10][11][12]; in brief, mitochondrial function is closely related to health and disease development. Mitochondrial quality control (MQC) is one of the critical processes maintaining mitochondrial health, and autophagy is an indispensable cellular process that greatly contributes to MQC [13,14].…”

Section: Introductionmentioning

confidence: 99%

Sulfuretin protects hepatic cells through regulation of ROS levels and autophagic flux

Xiao

et al. 2018

Acta Pharmacol Sin

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Palmitate (PA) exposure induces stress conditions featuring ROS accumulation and upregulation of p62 expression, resulting in autophagic flux blockage and cell apoptosis. Sulfuretin (Sul) is a natural product isolated from Rhus verniciflua Stokes; the cytoprotective effect of Sul on human hepatic L02 cells and mouse primary hepatocytes under PA-induced stress conditions was investigated in this study. Sul induced mitophagy by activation of p-TBK1 and LC3 and produced a concomitant decline in p62 expression. Autophagosome formation and mitophagy were assessed by the sensitive dual fluorescence reporter mCherry-EGFP-LC3B, and mitochondrial fragmentation was analyzed using MitoTracker Deep Red FM. A preliminary structure-activity relationship (SAR) for Sul was also investigated, and the phenolic hydroxyl group was found to be pivotal for maintaining the cytoprotective bioactivity of Sul. Furthermore, experiments using flow cytometry and western blots revealed that Sul reversed the cytotoxic effect stimulated by the autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ), and its cytoprotective effect was almost eliminated when the autophagy-related 5 (Atg5) gene was knocked down. These studies suggest that, in addition to its antioxidative effects, Sul stimulates mitophagy and restores impaired autophagic flux, thus protecting hepatic cells from apoptosis, and that Sul has potential future medical applications for hepatoprotection.

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Methods for assessing mitochondrial quality control mechanisms and cellular consequences in cell culture

Cited by 38 publications

References 48 publications

Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia

Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia

Mitophagy in Hepatic Insulin Resistance: Therapeutic Potential and Concerns

Sulfuretin protects hepatic cells through regulation of ROS levels and autophagic flux

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