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Aberrant mitochondrial fission in neurons induced by protein kinase C{delta} under oxidative stress conditions in vivo.

Qi X, Disatnik MH, Shen N, Sobel RA, Mochly-Rosen D - Mol. Biol. Cell (2010)

Bottom Line: Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration.However, the triggers for this mitochondrial dysregulation are not known.Further, we found that Drp1 Ser 579 phosphorylation by PKCδ is associated with Drp1 translocation to the mitochondria under oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration. However, the triggers for this mitochondrial dysregulation are not known. Here we show excessive mitochondrial fission and mitochondrial structural disarray in brains of hypertensive rats with hypertension-induced brain injury (encephalopathy). We found that activation of protein kinase Cδ (PKCδ) induced aberrant mitochondrial fragmentation and impaired mitochondrial function in cultured SH-SY5Y neuronal cells and in this rat model of hypertension-induced encephalopathy. Immunoprecipitation studies indicate that PKCδ binds Drp1, a major mitochondrial fission protein, and phosphorylates Drp1 at Ser 579, thus increasing mitochondrial fragmentation. Further, we found that Drp1 Ser 579 phosphorylation by PKCδ is associated with Drp1 translocation to the mitochondria under oxidative stress. Importantly, inhibition of PKCδ, using a selective PKCδ peptide inhibitor (δV1-1), reduced mitochondrial fission and fragmentation and conferred neuronal protection in vivo and in culture. Our study suggests that PKCδ activation dysregulates the mitochondrial fission machinery and induces aberrant mitochondrial fission, thus contributing to neurological pathology.

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PKCδ phosphorylated Drp1 at Ser 579. (A) The mitochondrial and cytosolic fractions were isolated from the cortex of hypertensive rats and cultured neurons treated with H2O2 (200 μM, 2 h). Then 200 μg of proteins was subjected to immunoprecipitation (IP) with anti-Drp1 antibody, and the immunoprecipitates were analyzed by immunoblotting (IB) with a mixture of serine and threonine antibodies. The input is 10 μg protein. (B) In vitro phosphorylation assays were carried out with recombinant protein GST-PKCδ and His-Drp1 in the presence or absence of the PKC activators phosphatidylserine and diacylglycerol (PS/DG). Shown are representative data from three independent experiments. (C) Mass spectroscopy analysis was carried out after in vitro phosphorylation using recombinant proteins GST-PKCδ and GST-Drp1 in the presence or absence of PKC activators. The site phosphorylated by PKCδ in Drp1 was identified and highlighted in purple among species (see mass spectrometry analysis in Supplementary Figure 4). (D) Modeling of predicted human Drp1 structure (isoform 3). Blue: GTPase domain; green: middle domain and variable domain; red: GTPase effector domain (GED). The phosphorylation site (Ser 579) of Drp1 by PKCδ is highlighted in pink.
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Figure 3: PKCδ phosphorylated Drp1 at Ser 579. (A) The mitochondrial and cytosolic fractions were isolated from the cortex of hypertensive rats and cultured neurons treated with H2O2 (200 μM, 2 h). Then 200 μg of proteins was subjected to immunoprecipitation (IP) with anti-Drp1 antibody, and the immunoprecipitates were analyzed by immunoblotting (IB) with a mixture of serine and threonine antibodies. The input is 10 μg protein. (B) In vitro phosphorylation assays were carried out with recombinant protein GST-PKCδ and His-Drp1 in the presence or absence of the PKC activators phosphatidylserine and diacylglycerol (PS/DG). Shown are representative data from three independent experiments. (C) Mass spectroscopy analysis was carried out after in vitro phosphorylation using recombinant proteins GST-PKCδ and GST-Drp1 in the presence or absence of PKC activators. The site phosphorylated by PKCδ in Drp1 was identified and highlighted in purple among species (see mass spectrometry analysis in Supplementary Figure 4). (D) Modeling of predicted human Drp1 structure (isoform 3). Blue: GTPase domain; green: middle domain and variable domain; red: GTPase effector domain (GED). The phosphorylation site (Ser 579) of Drp1 by PKCδ is highlighted in pink.

Mentions: Further, we found that inhibition of PKCδ by δV1-1 treatment blocked phosphorylation of mitochondria-associated Drp1 on serine/threonine residues in hypertensive rat brains and in vitro–cultured SH-SY5Y cells (Figure 3A, left panel). However, we did not find increased phosphorylation of Drp1 in the cytosolic fractions in response to HTNE (Figure 3A, right panel). Next, an in vitro kinase assay confirmed that recombinant PKCδ can directly phosphorylate recombinant Drp1 in the presence of PKC activators (Figure 3B).FIGURE 3:


Aberrant mitochondrial fission in neurons induced by protein kinase C{delta} under oxidative stress conditions in vivo.

Qi X, Disatnik MH, Shen N, Sobel RA, Mochly-Rosen D - Mol. Biol. Cell (2010)

PKCδ phosphorylated Drp1 at Ser 579. (A) The mitochondrial and cytosolic fractions were isolated from the cortex of hypertensive rats and cultured neurons treated with H2O2 (200 μM, 2 h). Then 200 μg of proteins was subjected to immunoprecipitation (IP) with anti-Drp1 antibody, and the immunoprecipitates were analyzed by immunoblotting (IB) with a mixture of serine and threonine antibodies. The input is 10 μg protein. (B) In vitro phosphorylation assays were carried out with recombinant protein GST-PKCδ and His-Drp1 in the presence or absence of the PKC activators phosphatidylserine and diacylglycerol (PS/DG). Shown are representative data from three independent experiments. (C) Mass spectroscopy analysis was carried out after in vitro phosphorylation using recombinant proteins GST-PKCδ and GST-Drp1 in the presence or absence of PKC activators. The site phosphorylated by PKCδ in Drp1 was identified and highlighted in purple among species (see mass spectrometry analysis in Supplementary Figure 4). (D) Modeling of predicted human Drp1 structure (isoform 3). Blue: GTPase domain; green: middle domain and variable domain; red: GTPase effector domain (GED). The phosphorylation site (Ser 579) of Drp1 by PKCδ is highlighted in pink.
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Figure 3: PKCδ phosphorylated Drp1 at Ser 579. (A) The mitochondrial and cytosolic fractions were isolated from the cortex of hypertensive rats and cultured neurons treated with H2O2 (200 μM, 2 h). Then 200 μg of proteins was subjected to immunoprecipitation (IP) with anti-Drp1 antibody, and the immunoprecipitates were analyzed by immunoblotting (IB) with a mixture of serine and threonine antibodies. The input is 10 μg protein. (B) In vitro phosphorylation assays were carried out with recombinant protein GST-PKCδ and His-Drp1 in the presence or absence of the PKC activators phosphatidylserine and diacylglycerol (PS/DG). Shown are representative data from three independent experiments. (C) Mass spectroscopy analysis was carried out after in vitro phosphorylation using recombinant proteins GST-PKCδ and GST-Drp1 in the presence or absence of PKC activators. The site phosphorylated by PKCδ in Drp1 was identified and highlighted in purple among species (see mass spectrometry analysis in Supplementary Figure 4). (D) Modeling of predicted human Drp1 structure (isoform 3). Blue: GTPase domain; green: middle domain and variable domain; red: GTPase effector domain (GED). The phosphorylation site (Ser 579) of Drp1 by PKCδ is highlighted in pink.
Mentions: Further, we found that inhibition of PKCδ by δV1-1 treatment blocked phosphorylation of mitochondria-associated Drp1 on serine/threonine residues in hypertensive rat brains and in vitro–cultured SH-SY5Y cells (Figure 3A, left panel). However, we did not find increased phosphorylation of Drp1 in the cytosolic fractions in response to HTNE (Figure 3A, right panel). Next, an in vitro kinase assay confirmed that recombinant PKCδ can directly phosphorylate recombinant Drp1 in the presence of PKC activators (Figure 3B).FIGURE 3:

Bottom Line: Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration.However, the triggers for this mitochondrial dysregulation are not known.Further, we found that Drp1 Ser 579 phosphorylation by PKCδ is associated with Drp1 translocation to the mitochondria under oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration. However, the triggers for this mitochondrial dysregulation are not known. Here we show excessive mitochondrial fission and mitochondrial structural disarray in brains of hypertensive rats with hypertension-induced brain injury (encephalopathy). We found that activation of protein kinase Cδ (PKCδ) induced aberrant mitochondrial fragmentation and impaired mitochondrial function in cultured SH-SY5Y neuronal cells and in this rat model of hypertension-induced encephalopathy. Immunoprecipitation studies indicate that PKCδ binds Drp1, a major mitochondrial fission protein, and phosphorylates Drp1 at Ser 579, thus increasing mitochondrial fragmentation. Further, we found that Drp1 Ser 579 phosphorylation by PKCδ is associated with Drp1 translocation to the mitochondria under oxidative stress. Importantly, inhibition of PKCδ, using a selective PKCδ peptide inhibitor (δV1-1), reduced mitochondrial fission and fragmentation and conferred neuronal protection in vivo and in culture. Our study suggests that PKCδ activation dysregulates the mitochondrial fission machinery and induces aberrant mitochondrial fission, thus contributing to neurological pathology.

Show MeSH
Related in: MedlinePlus