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Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Sanderson TH, Mahapatra G, Pecina P, Ji Q, Yu K, Sinkler C, Varughese A, Kumar R, Bukowski MJ, Tousignant RN, Salomon AR, Lee I, Hüttemann M - PLoS ONE (2013)

Bottom Line: Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration.This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion.These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America ; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

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Insulin prevents neuronal cell death in the CA1 hippocampus following brain ischemia.Cresyl violet stained sections (top row) show CA1 hippocampus densely populated with pyramidal neurons in sham-operated controls (Sham), and triple-label immunofluorescence (bottom row) shows these cells to be NeuN-positive (red). After 8 min of global brain ischemia followed by 14 days of reperfusion (I/R, n = 5) there is a 90% loss of CA1 neurons and an increase in Iba-1-positive microglia and GFAP-positive astrocytes (green and magenta, respectively). Animals exposed to 8 min of ischemia followed by 14 days of reperfusion with a single bolus of insulin given at the onset of reperfusion (I/R + Ins, n = 4) demonstrate a 49% increase in NeuN-positive neurons (p < 0.05) while Iba-1 and GFAP-positive cells remain unchanged.
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pone-0078627-g002: Insulin prevents neuronal cell death in the CA1 hippocampus following brain ischemia.Cresyl violet stained sections (top row) show CA1 hippocampus densely populated with pyramidal neurons in sham-operated controls (Sham), and triple-label immunofluorescence (bottom row) shows these cells to be NeuN-positive (red). After 8 min of global brain ischemia followed by 14 days of reperfusion (I/R, n = 5) there is a 90% loss of CA1 neurons and an increase in Iba-1-positive microglia and GFAP-positive astrocytes (green and magenta, respectively). Animals exposed to 8 min of ischemia followed by 14 days of reperfusion with a single bolus of insulin given at the onset of reperfusion (I/R + Ins, n = 4) demonstrate a 49% increase in NeuN-positive neurons (p < 0.05) while Iba-1 and GFAP-positive cells remain unchanged.

Mentions: We first investigated the effect of insulin treatment on release of Cytc from the mitochondria into the cytosol and subsequent cell death in a rat model of global brain ischemia/reperfusion injury. Cytc release was detected by immunoblotting rat hippocampal homogenates fractionated into cytosolic and mitochondrial fractions with an anti-Cytc antibody. Antibodies against cytochrome c oxidase subunit IV (CcOIV) and β-actin were used as loading controls and to determine the purity of mitochondrial and cytosolic fractions. We previously demonstrated that Cytc release following global brain ischemia reaches a maximum level by 24 hours of reperfusion [4]. Therefore, we investigated whether insulin could prevent the peak Cytc release at this late reperfusion interval, i.e., 24 hours of reperfusion, in order to determine if Cytc release can be prevented and not just delayed. Western blot of cytosolic fractions showed a significant 4-fold increase in cytosolic Cytc in the untreated controls (Fig. 1). This was associated with a trend towards a reduction in the overall mitochondrial pool of Cytc, although this effect failed to reach statistical significance (p = 0.08). A single bolus of 20 U/Kg insulin administered at the onset of reperfusion prevented increased cytosolic Cytc levels, maintaining cytosolic Cytc levels similar to sham-operated controls (p = 0.98, Fig. 1; Sham vs. T24). To confirm if this effect on Cytc release was associated with neuroprotection we used cresyl violet and triple-label immunofluorescence against a neuron marker (NeuN-Red), an astrocyte marker (GFAP-Magenta), and a microglial marker (Iba-1-Green). Sham-operated control animals exhibit a CA1 hippocampus densely populated with pyramidal neurons positive for NeuN, with few astrocytes and microglia (Fig. 2). Eight-minutes of ischemia followed by 14 days of reperfusion results in extensive loss of CA1 pyramidal neurons and an increase in GFAP-positive astrocytes and microglia/macrophages positive for Iba-1. The neuronal loss is partially prevented (49%) by insulin administration; however, gliosis remains unchanged.


Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Sanderson TH, Mahapatra G, Pecina P, Ji Q, Yu K, Sinkler C, Varughese A, Kumar R, Bukowski MJ, Tousignant RN, Salomon AR, Lee I, Hüttemann M - PLoS ONE (2013)

Insulin prevents neuronal cell death in the CA1 hippocampus following brain ischemia.Cresyl violet stained sections (top row) show CA1 hippocampus densely populated with pyramidal neurons in sham-operated controls (Sham), and triple-label immunofluorescence (bottom row) shows these cells to be NeuN-positive (red). After 8 min of global brain ischemia followed by 14 days of reperfusion (I/R, n = 5) there is a 90% loss of CA1 neurons and an increase in Iba-1-positive microglia and GFAP-positive astrocytes (green and magenta, respectively). Animals exposed to 8 min of ischemia followed by 14 days of reperfusion with a single bolus of insulin given at the onset of reperfusion (I/R + Ins, n = 4) demonstrate a 49% increase in NeuN-positive neurons (p < 0.05) while Iba-1 and GFAP-positive cells remain unchanged.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818486&req=5

pone-0078627-g002: Insulin prevents neuronal cell death in the CA1 hippocampus following brain ischemia.Cresyl violet stained sections (top row) show CA1 hippocampus densely populated with pyramidal neurons in sham-operated controls (Sham), and triple-label immunofluorescence (bottom row) shows these cells to be NeuN-positive (red). After 8 min of global brain ischemia followed by 14 days of reperfusion (I/R, n = 5) there is a 90% loss of CA1 neurons and an increase in Iba-1-positive microglia and GFAP-positive astrocytes (green and magenta, respectively). Animals exposed to 8 min of ischemia followed by 14 days of reperfusion with a single bolus of insulin given at the onset of reperfusion (I/R + Ins, n = 4) demonstrate a 49% increase in NeuN-positive neurons (p < 0.05) while Iba-1 and GFAP-positive cells remain unchanged.
Mentions: We first investigated the effect of insulin treatment on release of Cytc from the mitochondria into the cytosol and subsequent cell death in a rat model of global brain ischemia/reperfusion injury. Cytc release was detected by immunoblotting rat hippocampal homogenates fractionated into cytosolic and mitochondrial fractions with an anti-Cytc antibody. Antibodies against cytochrome c oxidase subunit IV (CcOIV) and β-actin were used as loading controls and to determine the purity of mitochondrial and cytosolic fractions. We previously demonstrated that Cytc release following global brain ischemia reaches a maximum level by 24 hours of reperfusion [4]. Therefore, we investigated whether insulin could prevent the peak Cytc release at this late reperfusion interval, i.e., 24 hours of reperfusion, in order to determine if Cytc release can be prevented and not just delayed. Western blot of cytosolic fractions showed a significant 4-fold increase in cytosolic Cytc in the untreated controls (Fig. 1). This was associated with a trend towards a reduction in the overall mitochondrial pool of Cytc, although this effect failed to reach statistical significance (p = 0.08). A single bolus of 20 U/Kg insulin administered at the onset of reperfusion prevented increased cytosolic Cytc levels, maintaining cytosolic Cytc levels similar to sham-operated controls (p = 0.98, Fig. 1; Sham vs. T24). To confirm if this effect on Cytc release was associated with neuroprotection we used cresyl violet and triple-label immunofluorescence against a neuron marker (NeuN-Red), an astrocyte marker (GFAP-Magenta), and a microglial marker (Iba-1-Green). Sham-operated control animals exhibit a CA1 hippocampus densely populated with pyramidal neurons positive for NeuN, with few astrocytes and microglia (Fig. 2). Eight-minutes of ischemia followed by 14 days of reperfusion results in extensive loss of CA1 pyramidal neurons and an increase in GFAP-positive astrocytes and microglia/macrophages positive for Iba-1. The neuronal loss is partially prevented (49%) by insulin administration; however, gliosis remains unchanged.

Bottom Line: Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration.This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion.These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America ; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Show MeSH
Related in: MedlinePlus