Isolation of functionally intact pig retina mitochondria

Schild, Lorenz; Westphal, Sabine; Scheithauer, S.; Holfeld, M.; Augustin, Wolfgang; Sabel, Bernhard A.

doi:10.1111/j.1600-0420.1996.tb00707.x

Cited by 7 publications

(2 citation statements)

References 14 publications

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“…We used a procedure adapted from that for adrenal cortical mitochondria separation [41] to isolate mitochondria rich-fraction from retinal tissue [42] . Briefly, each individual experiment used three pairs of mice retinas from the same experimental group.…”

Section: Methodsmentioning

confidence: 99%

Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue

et al. 2019

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Dysfunction in mitochondrial oxidative phosphorylation (OXPHOS) underlies a wide spectrum of human ailments known as mitochondrial diseases. Deficiencies in complex I of the electron transport chain (ETC) contribute to 30–40% of all cases of mitochondrial diseases, and leads to eye disease including optic nerve atrophy and retinal degeneration. The mechanisms responsible for organ damage in mitochondrial defects may include energy deficit, oxidative stress, and an increase in the NADH/NAD+ redox ratio due to decreased NAD+ regeneration. Currently, there is no effective treatment to alleviate human disease induced by complex I defect.Photoreceptor cells have the highest energy demand and dependence on OXPHOS for survival, and the lowest reserve capacity indicating that they are sensitive to OXPHOS defects. We investigated the effect of mitochondrial OXPHOS deficiency on retinal photoreceptors in a model of mitochondrial complex I defect (apoptosis inducing factor, AIF-deficient mice, Harlequin mice), and tested the protective effect of a mitochondrial redox compound (methylene blue, MB) on mitochondrial and photoreceptor integrity. MB prevented the reduction in the retinal thickness and protein markers for photoreceptor outer segments, Muller and ganglion cells, and altered mitochondrial integrity and function induced by AIF deficiency. In rotenone-induced complex I deficient 661 W cells (an immortalized mouse photoreceptor cell line) MB decreased the NADH/NAD+ ratio and oxidative stress without correcting the energy deficit, and improved cell survival. MB deactivated the mitochondrial stress response pathways, the unfolding protein response and mitophagy. In conclusion, preserving mitochondrial structure and function alleviates retinal photoreceptor degeneration in mitochondrial complex I defect.

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Section: Methodsmentioning

confidence: 99%

Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue

et al. 2019

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“…In order to examine the subcellular localization of PGRMC1 in retinal membranes, tissues were fractionated to yield microsomal/plasma membrane, crude and density gradient purified mitochondria and synaptosomes (Method A, Schild et al, 1996). Since recent studies (Prokisch et al, 2004; Zischka et al, 2003) have demonstrated high purity of organelles separated by zone free flow electrophoresis (BD Biosciences), we further purified retinal mitochondria from the density gradient by ZE‐FFE according to (Zischka et al, 2003).…”

Section: Methodsmentioning

confidence: 99%

Membrane‐initiated effects of progesterone on calcium dependent signaling and activation of VEGF gene expression in retinal glial cells

Lange¹,

Stampfl

Hauck³

et al. 2007

Glia

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Neurosteroids, such as progesterone, influence central nervous system development and function by regulating a broad spectrum of physiological processes. Here, we investigated membrane-initiated actions of progesterone in the retina and identified the membrane-associated progesterone receptor component 1 (PGRMC1). We found PGRMC1 expressed mainly in retinal Muller glia (RMG) and retinal pigment epithelium, and localized uniquely to microsomal and plasma membrane fractions. In RMG, membrane-impermeable progesterone conjugate induced calcium influx and subsequent phosphatidylinositol 3-kinase-mediated phosphorylation of PKC and ERK-1/2. Induction by progesterone also led to PKC-dependent activation of VEGF gene expression and protein synthesis, suggesting a contribution of membrane-initiated hormone effects to VEGF induced neovascularization within retina.

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Mitochondrial polarization in rat hippocampal astrocytes is resistant to cytosolic Ca²⁺ loads

Kahlert

Schild

Reiser

2001

J of Neuroscience Research

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The influence of physiological Ca(2+)-inducing stimuli and agents mimicking ischemic conditions on mitochondrial potential was studied in postnatal (P1) hippocampal astrocytes. Cytosolic Ca(2+) loads with characteristic kinetics of rise and duration, detected by Fura-2, were provoked by extracellular Ca(2+) influx, release from InsP(3)-sensitive intracellular stores, or inhibition of the reloading of endoplasmic reticulum Ca(2+) stores. Inhibitors of mitochondrial respiration caused only moderate release of Ca(2+) from intracellular stores, inducing a rise of less than 60 nM. The maximal Ca(2+) rise was found with InsP(3)-mediated responses (500 nM; via ATP) or with ionophore (4-Br-A23187)-mediated Ca(2+) influx from extracellular medium (770 nM). Remarkably, all these agents causing significant rise of cytosolic Ca(2+), only minimally depolarized the mitochondria. Membrane potential of mitochondria was monitored by Rh123 or TMRE. Depolarization was only found with very high cytosolic Ca(2+) levels (above 60 microM; measured by fura FF). These were achieved with external Ca(2+) influx by ionophore in combination with inhibition of glycolysis. Thus, mitochondria in the astrocytes are obviously not sensitive to moderate cytosolic Ca(2+) loads, irrespective of the source of Ca(2+). Furthermore, isolated rat brain mitochondria display a low sensitivity of respiratory activity to Ca(2+), which is consistent with the data obtained with the astrocytes in vitro. The capacity of isolated mitochondria to build up a potential was gradually reduced at low micromolar Ca(2+) and totally compromised only at Ca(2+) concentrations in the 100 microM range.

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Isolation of functionally intact pig retina mitochondria

Cited by 7 publications

References 14 publications

Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue

Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue

Membrane‐initiated effects of progesterone on calcium dependent signaling and activation of VEGF gene expression in retinal glial cells

Mitochondrial polarization in rat hippocampal astrocytes is resistant to cytosolic Ca²⁺ loads

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Isolation of functionally intact pig retina mitochondria

Cited by 7 publications

References 14 publications

Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue

Apoptosis inducing factor deficiency causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue

Membrane‐initiated effects of progesterone on calcium dependent signaling and activation of VEGF gene expression in retinal glial cells

Mitochondrial polarization in rat hippocampal astrocytes is resistant to cytosolic Ca2+ loads

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Mitochondrial polarization in rat hippocampal astrocytes is resistant to cytosolic Ca²⁺ loads