Biochemical and Biophysical Methods for Studying Mitochondrial Iron Metabolism

Holmes-Hampton, Gregory P.; Tong, Wing-Hang; Rouault, Tracey A.

doi:10.1016/b978-0-12-801415-8.00015-1

Cited by 13 publications

(10 citation statements)

References 145 publications

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“…Lower maximal respiration was observed in the D2 ON, starting at 6 months and extending to 10 m. Since maximal respiration is primarily determined by substrate supply and oxidation, lower maximal respiration corroborates the potentially compromised substrate delivery expected in the D2 ON that has lower levels of GLUT1 and MCT2 [3], restricting substrate getting across the plasma or mitochondrial membrane [29]. The number of mitochondria and/or the cristae density can also limit maximal respiration.…”

Section: Discussionmentioning

confidence: 81%

“…The extracellular acidification rate (ECAR) represents the release of protons into the extracellular milieu as a byproduct of glycolysis (from lactate production) but also the protons from substrate oxidation that are released with the export of CO 2 that becomes hydrated to H 2 CO 3 and dissociates to HCO 3 − + H + [29]. Acidification from respiration compared to glycolysis varies widely by cell type [30]; however, as shown below in the fluorocitrate experiments, roughly half of the ECAR in D2G ON can be attributed to glycolytic activity.…”

Section: Resultsmentioning

confidence: 99%

“…MCT2 functions to bring monocarboxylates like lactate, pyruvate, and ketone bodies into the axon. Lack of substrate availability is one contributor to decreased ATP production [29]. Another source of decreased ATP production is likely the diminished activity of electron transport chain complexes I, III, and IV that were observed as early at 5 months of age in the D2 ON [31].…”

Section: Discussionmentioning

confidence: 99%

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Higher Reliance on Glycolysis Limits Glycolytic Responsiveness in Degenerating Glaucomatous Optic Nerve

et al. 2019

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Metabolic dysfunction accompanies neurodegenerative disease and aging. An important step for therapeutic development is a more sophisticated understanding of the source of metabolic dysfunction, as well as to distinguish disease-associated changes from aging effects. We examined mitochondrial function in ex vivo aging and glaucomatous optic nerve using a novel approach, the Seahorse Analyzer. Optic nerves (ON) from the DBA/2J mouse model of glaucoma and the DBA/2- Gpnmb + control strain were isolated, and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), the discharge of protons from lactate release or byproducts of substrate oxidation, were measured. The glial-specific aconitase inhibitor fluorocitrate was used to limit the contribution of glial mitochondria to OCR and ECAR. We observed significant decreases in maximal respiration, ATP production, and spare capacity with aging. In the presence of fluorocitrate, OCR was higher, with more ATP produced, in glaucoma compared to aged ON. However, glaucoma ON showed lower maximal respiration. In the presence of fluorocitrate and challenged with ATPase inhibition, glaucoma ON was incapable of further upregulation of glycolysis to compensate for the loss of oxidative phosphorylation. Inclusion of 2-deoxyglucose as a substrate during ATPase inhibition indicated a significantly higher proportion of ECAR was derived from TCA cycle substrate oxidation than glycolysis in glaucoma ON. These data indicate that glaucoma axons have limited ability to respond to increased energy demand given their lower maximal respiration and inability to upregulate glycolysis when challenged. The higher ATP output from axonal mitochondria in glaucoma optic nerve compensates for this lack of resiliency but is ultimately inadequate for continued function. Electronic supplementary material The online version of this article (10.1007/s12035-019-1576-4) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Higher Reliance on Glycolysis Limits Glycolytic Responsiveness in Degenerating Glaucomatous Optic Nerve

et al. 2019

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“…In order to obtain sufficient amounts of protein, a tagged version of a candidate protein will need to be overexpressed in bacteria that are transformed with the ISC (bacterial ironsulfur cluster) operon to optimize insertion of Fe-S clusters 11 . The protein could then be immunoprecipitated under anaerobic conditions to protect the presumed Fe-S cluster and analyzed using inductively coupled plasma mass spectrometry (ICP-MS), a type of mass spectrometry that is capable of detecting iron and sulfur at concentrations as low as 50 parts per billion 15,25 . ICP-MS requires much smaller amounts of sample protein for analysis than the other major spectrometry techniques.…”

Section: Workflow For Fe-s Protein Discoverymentioning

confidence: 99%

Iron-sulfur proteins hiding in plain sight

Rouault

2015

Nat Chem Biol

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“…To understand the genesis of mitochondrial iron overload, it is necessary to employ various techniques to quantify iron in mitochondria. A range of these techniques is elegantly summarized by Holmes-Hampton et al (93) and includes colorimetric determination, atomic absorption spectroscopy, inductively coupled plasma-optical and plasma-mass emission spectroscopy, electron microscopy (in combination with iron histochemical staining such as Perls's and Turnbull's, X-ray microscopy, and electron energy loss spectroscopy), synchrotron X-ray fluorescence microscopy imaging, confocal Raman microscopy, ultraviolet (UV)-vis spectroscopy, electron paramagnetic resonance, X-ray absorption spectroscopy, and Mössbauer spectroscopy. Several of these methods can be used to measure iron in isolated mitochondria from cells and tissues with varying degrees of sensitivity.…”

Section: A Word Of Caution: Methodology To Measure Mitochondrial Ironmentioning

confidence: 99%

Mitochondrial Iron in Human Health and Disease

Ward

Cloonan

2019

Annu. Rev. Physiol.

131

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Mitochondria are an iconic distinguishing feature of eukaryotic cells. Mitochondria encompass an active organellar network that fuses, divides, and directs a myriad of vital biological functions, including energy metabolism, cell death regulation, and innate immune signaling in different tissues. Another crucial and often underappreciated function of these dynamic organelles is their central role in the metabolism of the most abundant and biologically versatile transition metals in mammalian cells, iron. In recent years, cellular and animal models of mitochondrial iron dysfunction have provided vital information in identifying new proteins that have elucidated the pathways involved in mitochondrial homeostasis and iron metabolism. Specific signatures of mitochondrial iron dysregulation that are associated with disease pathogenesis and/or progression are becoming increasingly important. Understanding the molecular mechanisms regulating mitochondrial iron pathways will help better define the role of this important metal in mitochondrial function and in human health and disease.

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Biochemical and Biophysical Methods for Studying Mitochondrial Iron Metabolism

Cited by 13 publications

References 145 publications

Higher Reliance on Glycolysis Limits Glycolytic Responsiveness in Degenerating Glaucomatous Optic Nerve

Higher Reliance on Glycolysis Limits Glycolytic Responsiveness in Degenerating Glaucomatous Optic Nerve

Iron-sulfur proteins hiding in plain sight

Mitochondrial Iron in Human Health and Disease

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