*12.2.3. Protein kinase C*

is accompanied by decreases in affinity of PKA for cAMP and PKA activity [94]. In contrast, the peri-ischemic and less-injured areas exhibit increased PKA-mediated phosphorylation compared to the ischemic core, which suggests that active PKA is associated with the survival of neuronal tissue [94]. Derangement of cAMP-dependent signal transduction is associated with ischemic neuronal damage and activation of the PKA is important for neuronal survival in acute cerebral ischemia [94]. Phosphorylation of Bad at S155 by mitochondria-anchored PKA leads to cytosolic sequestration of Bad and blocks its mitochondrial translocation, the release of cytochrome c, and neuronal apoptosis [77]. Also, PKA-mediated phosphorylation of Drp1 at S637 blocks its translocation to mitochondria and their fission and promotes mitochondrial fusion. In hippocampal neurons, expression of a constitutively active catalytic subunit of PKA targeted to the mitochondrion promotes the fusion of mitochondria into networks [95]. Finally, PKA suppresses autophagy and mitochondrial degradation in neurons by phosphorylating and inhibiting the microtubule-associated protein 1A/1B-light chain 3 (LC3)

Akt can localize to the mitochondrial matrix and the inner and outer membranes of cardiac, neuronal, and kidney cells [97, 98]. Protein levels of mitochondrial Akt are regulated by a variety of extracellular signals and stresses, which induce rapid translocation of active (phosphorylated) Akt to the mitochondria [97, 98]. Mitochondrial localization of Akt is associated with cardioprotection against ischemic injury and renoprotective actions against chemical toxicity [97–100]. The cardioprotective effect of mitochondrial Akt is attributed to its action on mitoK(ATP) channels and to decreasing apoptosis [99]. Akt phosphorylates pro-apoptotic Bad on S136 and Bax on S184, and prevents MPT in neuronal cells [101]. Phosphorylated Bad associates with 14-3-3 proteins in the cytosol and cannot form complexes with mitochondrial Bcl-2/Bcl-XL to induce permeabilization of the mitochondrial outer membrane and MPT [101]. Bax phosphorylation by Akt promotes dimerization of Bax with Bcl-XL or Mcl-1 proteins, which sequesters Bax away from mitochondria and prevents their permeabilization and apoptosis [101]. Akt prevents the MPT in cardiomyocytes by phosphorylating hexokinase II, stabilizing it in the outer mitochondrial membrane, and promoting its binding to VDAC [102]. Inhibition of Akt or targeted disruption of the association of hexokinase II with mitochondria

We have identified mitochondria as a subcellular target of protective actions of Akt against necrosis in injured RPTC [98]. Mitochondrial levels of active Akt decrease in injured RPTC and this is associated with mitochondrial dysfunction [98, 100]. Selective activation of Akt increases the levels of Akt in mitochondria, improves state 3 respiration, activities of com-

tial (ΔΨm), increases ATP production, and reduces ATP deficits [100]. Selective inhibition of Akt exacerbates mitochondrial dysfunction, energy deficits, and necrosis in injured RPTC [100]. These results are consistent with a report that Akt phosphorylates the β-subunit of ATP synthase and improves its activity and increases ATP production to prevent energy deficits in injured cells [97]. Also, active Akt increases activity of PDC and oxidative metabolism of carbohydrates by inhibiting phosphorylation of PDC by GSK-3β [103]. Thus, signals from cell


and increases neurite outgrowth [94, 96].

*12.2.2. Protein kinase B (PKB/Akt)*

192 Mitochondrial Diseases

abolishes cardioprotection [102].

F1

plexes I and III and F0

PKC plays a key role in mediating I/R injury in the brain, heart, and kidneys; however, elucidation of this role is complicated due to the presence of 11 distinct PKC isozymes, which have unique tissue and cellular localizations and often play quite opposite roles in ischemic injury even within the same organ. Alterations in PKC activity or subcellular localization during ischemia occur in the brain, heart, liver, and kidney [25, 104–106]. Major isozymes of PKC (α, β, γ, δ, ε) translocate to mitochondria in response to ischemia and/or reperfusion and oxidative stress, and have been implicated in regulating mitochondrial functions in ischemic and postischemic tissues [104]. The classical PKC isozymes (α, β, γ) are dependent on Ca2+ and 1,2-diacylglycerol, and ischemia increases the levels of both activators through the stimulation of phospholipase C. PKC-α activation has been implicated in ischemia-induced heart failure and cerebral barrier breakdown after ischemic stroke [107, 108]. PKC-α inhibition or deletion protects the heart from decompensation and cardiomyopathy and attenuates cerebral barrier breakdown after ischemia [107, 108]. In contrast, PKC-α translocation to mitochondria is protective against mitochondrial dysfunction, ATP deficits, and cell death caused by ischemia, hypoxia, and oxidative stress in RPTC [109]. We have shown that ATP synthase (F<sup>0</sup> F1 -ATPase) is a target of PKC-α, which associates with the α-, β-, and γ-subunits of the F<sup>1</sup> domain of ATP synthase [52]. Injury or inactive PKC-α disrupt, whereas active PKC-α promotes this association and increases the levels of α-, β-, and γ-subunits in injured RPTC [52]. Active PKC-α promotes phosphorylation of the γ-subunit on S146 and F<sup>0</sup> F1 -ATPase activity after injury [52]. PKC-α also reduces apoptosis by phosphorylating mitochondrial Bcl-2 on S70 [77, 110]. Thus, activation of PKC-α exerts differential effects in different types of cells and tissues.

PKC-γ is expressed exclusively in neurons of the brain and spinal cord and is activated rapidly during ischemia and inhibited during reperfusion [111–114]. PKC-γ plays a detrimental role in the early stages of ischemia mediating events leading to cell death. PKC-γ knockouts show smaller infarct areas after cerebral ischemia [112]. Also, PKC-γ may directly (or through the activation of a Src tyrosine kinase) phosphorylate NMDA receptors, stimulate their function, and increase the concentration of intracellular Ca2+, which results in mitochondrial dysfunction, ATP deficits, increased ROS formation, and neuronal death [112]. However, after reperfusion, PKC-γ may mediate protection against cell death [112]. Thus, the same PKC isozyme may play opposing roles at different stages of ischemic injury.

PKC-δ is rapidly activated by signaling initiated by reperfusion. PKC-δ plays a detrimental role in ischemic stroke injury and mediates oxidative stress, cell death, and inflammation associated with reperfusion [111, 112]. Selective inhibition or deletion of PKC-δ reduces infarct size and ischemic brain injury caused by middle cerebral artery occlusion, specifically, reperfusion-induced death of parenchymal cells [115, 116]. PKC-δ inhibition activates PKB/Akt, inhibits translocation of Bad to mitochondria, and decreases apoptosis [112]. PKC-δ mediates neutrophil infiltration, which is responsible for the detrimental effects of PKC-δ during reperfusion [112]. Similarly, reperfusion after cardiac ischemia activates PKC-δ, which translocates to mitochondria and reduces state 3 respiration, TCA cycle, and ATP production, increases generation of mitochondrial ROS, and induces release of cytochrome c and cell death [84, 113, 115, 116]. Selective inhibition of PKC-δ translocation to mitochondria blocks these changes and protects the heart from ischemic injury [117]. PKC-δ is also activated by oxidative stress in the kidney, exacerbates RPTC and kidney injury by activating Bax, inducing cytochrome c release and apoptosis, and blocking autophagy. PKC-δ inhibition protects kidneys from injury by upregulating autophagy [118].

ERK1/2 associates with the outer mitochondrial membrane and protects against MPTP opening and mitochondrial depolarization [127]. These events are thought to contribute to the cardioprotective effects of ERK1/2 activation against I/R injury. In contrast, ERK1/2 activation is detrimental to mitochondria and cell viability in RPTC [128]. We have shown that ERK1/2 activation and translocation to mitochondria in response to oxidant injury mediates mitochondrial dysfunction and cell death in RPTC [128]. ERK1/2 activation mediates decreases in state 3 respiration, activities of aconitase of the TCA cycle and complex I of the electron transport chain, and ATP production in injured RPTC [128]. ERK1/2 inhibition restores respiration, complex I activity, ΔΨm and ATP production, and decreases RPTC death [128]. Recently, it was shown that ERK1/2 downregulates mitochondrial function through the EGFR/ERK1/2/FOXO3a/1/ PGC-1α pathway by phosphorylating the upstream regulators of PGC-1α and decreasing mitochondrial biogenesis [129]. Similarly, ERK1/2 activation mediates inflammatory changes, infiltration by neutrophils, apoptosis, and severe injury after ischemia in the lung [130].

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JNK and p38 MAPK are activated and localize in cardiac mitochondria after ischemia or oxidant exposure and mediate mitochondria-initiated apoptosis [131]. p38 MAPK inhibition attenuates the loss of ΔΨm, mitochondrial swelling, and ultrastructural changes, reduces cardiomyocyte apoptosis and infarct size, and improves left ventricular function after ischemia [131, 132]. Inhibition of p38 MAPK also decreases phosphorylation of p53 and Bax expression and reduces cytochrome c release from mitochondria and the levels of active caspase 3 [132].

GSK3β activated by cardiac I/R docks to mitochondria, phosphorylates VDAC1, and leads to phosphorylation of ANT and cyclophilin D, and MPTP opening [55, 133]. This suggests that GSK3β plays a role in mitochondria-mediated apoptosis in cardiac tissue. GSK3β also regulates mitochondrial oxidative metabolism by phosphorylating and inhibiting PDC [65]. Activation enhances whereas inhibition of GSK3β activates mTOR pathway, inhibits mTORdependent autophagy, and reduces myocardial I/R injury [134]. Inhibition of GSK-3β also

The mitochondrial serine-threonine protein kinase, PTEN (phosphatase and tensin homolog on chromosome 10)-induced kinase 1 (PINK1) localizes to both mitochondrial membranes and regulates mitochondrial function and dynamics [136]. PINK1 phosphorylates mitofusin MFN2, a mitochondrial receptor for Parkin (a protein related to Parkinson's disease) [136]. PINK1 regulates mitochondrial dynamics in ischemic stroke and prevents damage to neurons by reducing mitochondrial translocation of Drp1 and fission, and preventing the collapse of ΔΨm and ATP synthesis [137, 138]. Deletion of PINK1 causes defects in the turnover of proteins of the electron transport chain, impairs mitochondrial respiration and activity of complex I in cardiomyocytes, induces MPTP opening, decreases production of ATP, exacerbates oxidative stress, and increases the size of ischemic myocardial infarct, which suggests increased susceptibility to I/R injury in PINK1-deficient hearts [139]. Overexpressing PINK1

These data suggests that active p38 MAPK mediates apoptosis.

attenuates brain infarct volume after cerebral I/R-induced injury [135].

*12.2.5. Glycogen synthase kinase*

*12.2.6. PTEN-induced kinase 1*

PKC-ε, another novel isozyme, is activated and translocates to mitochondria during I/R. These events have been implicated in the cardio- and neuro-protection against ischemic injury and in the reduction of myocardial infarct [119, 120]. PKC-ε activation is a pivotal signaling event in the cardioprotective mechanisms of ischemic preconditioning, and it is thought that this protection is mediated through mitochondrial and transport mechanisms [120]. Conversely, inhibition of PKC-ε eliminates the cardioprotection [120]. Several substrates of PKC-ε are present in cardiac mitochondria. Active PKC-ε induces opening of the mitoK(ATP) channels, maintains ATP production, and reduces ROS production [120]. Specifically, PKC-ε regulates interactions between conexin43 and the mitoK(ATP) subunit, which leads to mitoK(ATP) opening when ATP levels decrease, resulting in cardioprotection [121]. Active PKC-ε also increases phosphorylation of subunit IV and the activity of complex IV [122]. Thus, the PKC-ε-mediated resistance to cardiac ischemia may also be due to increased activity of the electron transport chain and a greater ΔΨm for ATP synthesis. Also, PKC-ε phosphorylates VDAC1 on T51, which modifies its gating and interaction with proteins of the MPT pore to inhibit its opening and maintain ΔΨm and ATP synthesis [60]. These data support protective and prosurvival actions of mitochondrial PKC-ε in cardiac I/R. In contrast, translocation of the active PKC-ε to mitochondria in RPTC results in mitochondrial dysfunction, decreases in ATP levels, mitochondrial fission, and RPTC death [25, 72]. Active PKC-ε produces the classical hallmarks of mitochondrial dysfunction in RPTC: decreases in state 3 respiration and activity of complex I, increases in ΔΨm, ROS production, and mitochondrial fission [25, 72]. Inhibition of PKC-ε protects against mitochondrial dysfunction induced by hypoxia and oxidative stress [72]. Deletion of PKC-ε *in vivo* ameliorates I/R-induced decreases in respiration and activities of complexes I, III, and IV, and reduces oxidant production and morphological damage in ischemic kidneys [25]. Deletion of PKC-ε reduces the inflammatory response and apoptosis, and promotes renal function and survival after I/R-induced acute kidney injury in mice [25, 123]. Thus, in contrast to the cardio- and neuroprotective effects of PKC-ε, activation of this PKC isozyme in the renal cortex is detrimental to mitochondria, cell viability, and kidney functions [25, 123].

#### *12.2.4. Mitogen-activated protein kinases (MAPK)*

ERK1/2 localize to the mitochondria of cardiac, brain, and renal epithelial cells. Brain and cardiac ischemia activates ERK1/2, which form signaling modules with PKC-ε and translocate to mitochondria [124–126]. ERK1/2–PKC-ε modules play a role in the phosphorylation and inactivation of Bad, which blocks the intrinsic pathway of apoptosis [126]. Furthermore, active ERK1/2 associates with the outer mitochondrial membrane and protects against MPTP opening and mitochondrial depolarization [127]. These events are thought to contribute to the cardioprotective effects of ERK1/2 activation against I/R injury. In contrast, ERK1/2 activation is detrimental to mitochondria and cell viability in RPTC [128]. We have shown that ERK1/2 activation and translocation to mitochondria in response to oxidant injury mediates mitochondrial dysfunction and cell death in RPTC [128]. ERK1/2 activation mediates decreases in state 3 respiration, activities of aconitase of the TCA cycle and complex I of the electron transport chain, and ATP production in injured RPTC [128]. ERK1/2 inhibition restores respiration, complex I activity, ΔΨm and ATP production, and decreases RPTC death [128]. Recently, it was shown that ERK1/2 downregulates mitochondrial function through the EGFR/ERK1/2/FOXO3a/1/ PGC-1α pathway by phosphorylating the upstream regulators of PGC-1α and decreasing mitochondrial biogenesis [129]. Similarly, ERK1/2 activation mediates inflammatory changes, infiltration by neutrophils, apoptosis, and severe injury after ischemia in the lung [130].

JNK and p38 MAPK are activated and localize in cardiac mitochondria after ischemia or oxidant exposure and mediate mitochondria-initiated apoptosis [131]. p38 MAPK inhibition attenuates the loss of ΔΨm, mitochondrial swelling, and ultrastructural changes, reduces cardiomyocyte apoptosis and infarct size, and improves left ventricular function after ischemia [131, 132]. Inhibition of p38 MAPK also decreases phosphorylation of p53 and Bax expression and reduces cytochrome c release from mitochondria and the levels of active caspase 3 [132]. These data suggests that active p38 MAPK mediates apoptosis.

#### *12.2.5. Glycogen synthase kinase*

mediates neutrophil infiltration, which is responsible for the detrimental effects of PKC-δ during reperfusion [112]. Similarly, reperfusion after cardiac ischemia activates PKC-δ, which translocates to mitochondria and reduces state 3 respiration, TCA cycle, and ATP production, increases generation of mitochondrial ROS, and induces release of cytochrome c and cell death [84, 113, 115, 116]. Selective inhibition of PKC-δ translocation to mitochondria blocks these changes and protects the heart from ischemic injury [117]. PKC-δ is also activated by oxidative stress in the kidney, exacerbates RPTC and kidney injury by activating Bax, inducing cytochrome c release and apoptosis, and blocking autophagy. PKC-δ inhibition protects

PKC-ε, another novel isozyme, is activated and translocates to mitochondria during I/R. These events have been implicated in the cardio- and neuro-protection against ischemic injury and in the reduction of myocardial infarct [119, 120]. PKC-ε activation is a pivotal signaling event in the cardioprotective mechanisms of ischemic preconditioning, and it is thought that this protection is mediated through mitochondrial and transport mechanisms [120]. Conversely, inhibition of PKC-ε eliminates the cardioprotection [120]. Several substrates of PKC-ε are present in cardiac mitochondria. Active PKC-ε induces opening of the mitoK(ATP) channels, maintains ATP production, and reduces ROS production [120]. Specifically, PKC-ε regulates interactions between conexin43 and the mitoK(ATP) subunit, which leads to mitoK(ATP) opening when ATP levels decrease, resulting in cardioprotection [121]. Active PKC-ε also increases phosphorylation of subunit IV and the activity of complex IV [122]. Thus, the PKC-ε-mediated resistance to cardiac ischemia may also be due to increased activity of the electron transport chain and a greater ΔΨm for ATP synthesis. Also, PKC-ε phosphorylates VDAC1 on T51, which modifies its gating and interaction with proteins of the MPT pore to inhibit its opening and maintain ΔΨm and ATP synthesis [60]. These data support protective and prosurvival actions of mitochondrial PKC-ε in cardiac I/R. In contrast, translocation of the active PKC-ε to mitochondria in RPTC results in mitochondrial dysfunction, decreases in ATP levels, mitochondrial fission, and RPTC death [25, 72]. Active PKC-ε produces the classical hallmarks of mitochondrial dysfunction in RPTC: decreases in state 3 respiration and activity of complex I, increases in ΔΨm, ROS production, and mitochondrial fission [25, 72]. Inhibition of PKC-ε protects against mitochondrial dysfunction induced by hypoxia and oxidative stress [72]. Deletion of PKC-ε *in vivo* ameliorates I/R-induced decreases in respiration and activities of complexes I, III, and IV, and reduces oxidant production and morphological damage in ischemic kidneys [25]. Deletion of PKC-ε reduces the inflammatory response and apoptosis, and promotes renal function and survival after I/R-induced acute kidney injury in mice [25, 123]. Thus, in contrast to the cardio- and neuroprotective effects of PKC-ε, activation of this PKC isozyme in the renal cortex is detrimental to mitochondria, cell viability, and

ERK1/2 localize to the mitochondria of cardiac, brain, and renal epithelial cells. Brain and cardiac ischemia activates ERK1/2, which form signaling modules with PKC-ε and translocate to mitochondria [124–126]. ERK1/2–PKC-ε modules play a role in the phosphorylation and inactivation of Bad, which blocks the intrinsic pathway of apoptosis [126]. Furthermore, active

kidneys from injury by upregulating autophagy [118].

194 Mitochondrial Diseases

kidney functions [25, 123].

*12.2.4. Mitogen-activated protein kinases (MAPK)*

GSK3β activated by cardiac I/R docks to mitochondria, phosphorylates VDAC1, and leads to phosphorylation of ANT and cyclophilin D, and MPTP opening [55, 133]. This suggests that GSK3β plays a role in mitochondria-mediated apoptosis in cardiac tissue. GSK3β also regulates mitochondrial oxidative metabolism by phosphorylating and inhibiting PDC [65]. Activation enhances whereas inhibition of GSK3β activates mTOR pathway, inhibits mTORdependent autophagy, and reduces myocardial I/R injury [134]. Inhibition of GSK-3β also attenuates brain infarct volume after cerebral I/R-induced injury [135].

#### *12.2.6. PTEN-induced kinase 1*

The mitochondrial serine-threonine protein kinase, PTEN (phosphatase and tensin homolog on chromosome 10)-induced kinase 1 (PINK1) localizes to both mitochondrial membranes and regulates mitochondrial function and dynamics [136]. PINK1 phosphorylates mitofusin MFN2, a mitochondrial receptor for Parkin (a protein related to Parkinson's disease) [136]. PINK1 regulates mitochondrial dynamics in ischemic stroke and prevents damage to neurons by reducing mitochondrial translocation of Drp1 and fission, and preventing the collapse of ΔΨm and ATP synthesis [137, 138]. Deletion of PINK1 causes defects in the turnover of proteins of the electron transport chain, impairs mitochondrial respiration and activity of complex I in cardiomyocytes, induces MPTP opening, decreases production of ATP, exacerbates oxidative stress, and increases the size of ischemic myocardial infarct, which suggests increased susceptibility to I/R injury in PINK1-deficient hearts [139]. Overexpressing PINK1 reduces these changes [139]. Thus, this study suggests mitochondrial PINK1 as a target for cardioprotection against ischemia in the heart.

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