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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 and Drp1 are interdependent. Human neuroblastoma SH-SY5Y cells were transfected with control siRNA, PKCδ siRNA, or Drp1 siRNA. After 48 h, cells were treated with Ang II (1 μM) for 30 min. (A) Total cell lysates were analyzed by Western blot to confirm knockdown of PKCδ (top) and Drp1 (middle panel). GAPDH was used as an internal loading control. (B) The levels of PKCδ and Drp1 in the mitochondrial fractions were analyzed by Western blot at the indicated groups. (C) Histograms depicting the amount of Drp1 (left) and PKCδ (right) associated with the mitochondria of SH-SY5Y cells. The data are expressed as mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. Ang II-treated group.
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Figure 6: PKCδ and Drp1 are interdependent. Human neuroblastoma SH-SY5Y cells were transfected with control siRNA, PKCδ siRNA, or Drp1 siRNA. After 48 h, cells were treated with Ang II (1 μM) for 30 min. (A) Total cell lysates were analyzed by Western blot to confirm knockdown of PKCδ (top) and Drp1 (middle panel). GAPDH was used as an internal loading control. (B) The levels of PKCδ and Drp1 in the mitochondrial fractions were analyzed by Western blot at the indicated groups. (C) Histograms depicting the amount of Drp1 (left) and PKCδ (right) associated with the mitochondria of SH-SY5Y cells. The data are expressed as mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. Ang II-treated group.

Mentions: To directly determine whether the translocations of PKCδ and Drp1 to the mitochondria are dependent on each other, we reduced the cellular levels of PKCδ or Drp1 by transfecting human neuronal SH-SY5Y cells with siRNA for PKCδ or for Drp1, respectively (Figure 6A). Translocation of Drp1 to the mitochondria after 30 min of Ang II treatment was abolished when PKCδ levels were reduced by PKCδ siRNA, as compared with control siRNA–transfected cells (Figure 6, B and C). Conversely, PKCδ translocation to the mitochondria was completely abolished in cells expressing Drp1 siRNA when exposed to the same treatment (Figure 6, B and C). These data show that Drp1 and PKCδ are both required for the translocation of either protein to the mitochondria, further supporting our findings that PKCδ and Drp1 move to the mitochondria as a preformed complex.FIGURE 6:


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 and Drp1 are interdependent. Human neuroblastoma SH-SY5Y cells were transfected with control siRNA, PKCδ siRNA, or Drp1 siRNA. After 48 h, cells were treated with Ang II (1 μM) for 30 min. (A) Total cell lysates were analyzed by Western blot to confirm knockdown of PKCδ (top) and Drp1 (middle panel). GAPDH was used as an internal loading control. (B) The levels of PKCδ and Drp1 in the mitochondrial fractions were analyzed by Western blot at the indicated groups. (C) Histograms depicting the amount of Drp1 (left) and PKCδ (right) associated with the mitochondria of SH-SY5Y cells. The data are expressed as mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. Ang II-treated group.
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Figure 6: PKCδ and Drp1 are interdependent. Human neuroblastoma SH-SY5Y cells were transfected with control siRNA, PKCδ siRNA, or Drp1 siRNA. After 48 h, cells were treated with Ang II (1 μM) for 30 min. (A) Total cell lysates were analyzed by Western blot to confirm knockdown of PKCδ (top) and Drp1 (middle panel). GAPDH was used as an internal loading control. (B) The levels of PKCδ and Drp1 in the mitochondrial fractions were analyzed by Western blot at the indicated groups. (C) Histograms depicting the amount of Drp1 (left) and PKCδ (right) associated with the mitochondria of SH-SY5Y cells. The data are expressed as mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. Ang II-treated group.
Mentions: To directly determine whether the translocations of PKCδ and Drp1 to the mitochondria are dependent on each other, we reduced the cellular levels of PKCδ or Drp1 by transfecting human neuronal SH-SY5Y cells with siRNA for PKCδ or for Drp1, respectively (Figure 6A). Translocation of Drp1 to the mitochondria after 30 min of Ang II treatment was abolished when PKCδ levels were reduced by PKCδ siRNA, as compared with control siRNA–transfected cells (Figure 6, B and C). Conversely, PKCδ translocation to the mitochondria was completely abolished in cells expressing Drp1 siRNA when exposed to the same treatment (Figure 6, B and C). These data show that Drp1 and PKCδ are both required for the translocation of either protein to the mitochondria, further supporting our findings that PKCδ and Drp1 move to the mitochondria as a preformed complex.FIGURE 6:

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