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Keywords:

  • nuclear factor-κB;
  • oxidative stress;
  • phosphatidylinositol 3-kinase;
  • akt pathway;
  • signal transduction;
  • stroke

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

1. Reactive oxygen species and oxidative state are slowly gaining acceptance in having a physiological relevance rather than just being the culprits in pathophysiological processes. The control of the redox environment of the cell provides for additional regulation in relation to signal transduction pathways. Conversely, aberrant regulation of oxidative state manifesting as oxidative stress can predispose a cell to adverse outcome.

2. The phosphatidylinositol 3-kinase/akt pathway is one such pathway that is partially regulated via oxidative state and, in an oxidative stress paradigm such as ischaemic–reperfusion injury, may be inactivated, which can lead to exacerbation of cell death.

3. Activation of nuclear factor (NF)-κB has been associated with oxidative stress. The role of NF-κB in neuronal cell death is widely debated, with major studies highlighting both a pro- and anti-apoptotic role for NF-κB, with the outcome being region, stimulus, dose and duration specific.

4. Oxidative state plays a key role in the regulation and control of numerous signal transduction pathways in the cell. Elucidating the mechanisms behind oxidative stress-mediated neuronal cell death is important in identifying potential putative targets for the treatment of diseases such as stroke.


List of abbreviations
FKHRL-1

Forkhead rank ligand-1

Gpx1

Glutathione peroxidase-1

H2O2

Hydrogen peroxide

IKK

IκB-kinase

IL

Interleukin

MCA

Mid-cerebral artery

Mn-SOD

Manganese-superoxide dismutase

NF-κB

Nuclear factor-κB

NIK

NF-κB-inducing kinase

NO

Nitric oxide

NOS

Nitric oxide synthase

OH

Hydroxy radical

PDGF

Platelet-derived growth factor

PI3-K

Phosphatidylinositol 3-kinase

ROS

Reactive oxygen species

SOD1

Superoxide dismutase 1

O2

Superoxide

TGF-β

Transforming growth factor-β

TNF-α

Tumour necrosis factor-α

TRAF

TNF-α receptor-associated factor

INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

Reactive oxygen species (ROS) have recently been hypothesized to play a role in the coordinated mechanism of cellular signalling.1 They have been found to stimulate a number of signal transduction pathways that are important in maintaining cellular homeostasis in the neuron. The coordination and regulation of ROS are controlled by the cell's endogenous anti-oxidants. It is when this regulation is impaired or is unable to cope with the level of ROS present that oxidative stress ensues. High levels of oxidative stress can subsequently impact on particular signal transduction systems, leading to a predisposition to disease of both an acute and chronic nature. The contribution of oxidative stress can lead to a vicious cycle because it impinges upon mitochondrial dysfunction, excitotoxicity, lipid peroxidation and inflammation (Fig. 1). The present review proposes to focus on two of these signalling systems, the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the transcription factor nuclear factor (NF)-κB, and how oxidative stress influences their responsiveness to the acute neural injury of stroke.

image

Figure 1. Mechanisms of neuronal apoptosis in stroke. Following an ischaemic insult, there is a reduction in the oxygen and glucose supply to the area with a loss of energy. The subsequent depolarization of membranes leads to increased intracellular calcium levels and the release of glutamate. Glutamate receptors are activated and, consequently, a further increase in calcium levels occurs. This can have many damaging effects on the cell, including proteolysis and lipolysis. Although re-oxygenation has been associated with recovery, it does result in the generation of free radicals. Together with increased nitric oxide synthase (NOS) activity and mitochondrial damage, this can lead to caspase activation, the release of inflammatory mediators and oxidative damage to the cell, ultimately leading to its death. DAG, diacylglycerol; PKC, protein kinase C. (Modified from Mattson et al.3)

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ISCHAEMIC STROKE

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

Stroke is characterized by a blockage of blood flow to the brain. In humans, there are basically two types of stroke: (i) that induced by a total loss of blood flow to the brain, such as during a cardiac arrest; or (ii) cerebral ischaemia arising from a focal loss of blood flow to the brain due to an artery blockage.2 Experimental models of stroke have been developed in animals in an attempt to mimic the events of human cerebral ischaemia. The focal model involves the transient or permanent occlusion of the mid-cerebral artery (MCA) to be used as a model of cerebral ischaemia. A global model has also been developed to mimic human cardiac arrest and involves the bilateral occlusion of the carotid and vertebral arteries.2 Following a stroke, there is a disruption in glucose and oxygen supply to the neurons, resulting in necrotic cell death and the formation of an infarct. Surrounding the infarct is an area termed the penumbra, which is a rim of mild to moderately ischaemic tissue lying between tissue that is normally perfused and the area of non-viable tissue. Brain cells within the penumbral zone are supplied with blood by collateral arteries and, therefore, may remain viable for an extended period of time if blood flow returns.3 In focal ischaemia, there is a return of blood flow and, although this is correlated with recovery, there are damaging effects during this period. Although the core of the ischaemic injury is not recoverable, the penumbra remains a target for the development of therapeutic strategies.

Until recently, ROS were seen only as harmful by-products of oxidative phosphorylation and reperfusion injury; however, small concentrations of ROS can produce changes in cellular redox state that can, in turn, affect the activity, protein–protein and DNA–protein interactions of enzymes and transcription factors. This is highlighted by the discovery that ligand stimulation via platelet-derived growth factor (PDGF) in vascular endothelial cells required the generation of H2O2 to elicit signalling.4 It has been proposed that ROS may act as growth factors by direct oxidation of critical protein groups, resulting in the activation of growth factors.5 Given that ROS are now known to modulate the signalling environment, it is not unreasonable to assume that chronic levels of oxidative stress will impact the cellular environment and may predispose the cell to certain pathophysiological stimuli and phenomena related to preconditioning.

A major pathway by which ROS generation occurs is through the reduction of molecular oxygen to H2O. The reduction of the dioxygen molecule generates the superoxide anion radical (O2) and hydrogen peroxide (H2O2). In the presence of transition metals, such as Fe2+, H2O2 can undergo the Fenton reaction, which leads to increased levels of the hydroxyl radical (OH). The hydroxyl radical is highly reactive and is thought to be the radical most responsible for oxidative damage. Defence against free radicals is provided by a number of anti-oxidant enzymes, including glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD). Superoxide dismutase converts O2 to H2O2, whereas GPX and CAT convert H2O2 to H2O. These enzymes, coupled with other anti-oxidants, such as ascorbic acid and α-tocopherol, serve to reduce levels of ROS. When a shift occurs, either by increasing free radical formation or decreased anti-oxidant defences, oxidative stress results, leading to lipid, protein and DNA damage. Eventually, this leads to cell death via either apoptotic or necrotic pathways.

The brain is particularly sensitive to oxidative stress owing to the high generation of ROS, with free radical damage arising due to the high lipid content of the brains, an increased oxygen consumption rate and chemical reactions involving dopamine oxidation and glutamate.3 Furthermore, neurons have been shown to have very low amounts of CAT and, therefore, primarily rely on GPX to eliminate H2O2.6 Although the direct initial insult to neurons may not be ROS, the resulting events mediated by increased free radical generation can cause secondary damage that is far greater and is pathologically relevant to the various neurodegenerative disorders.

ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

There is mounting evidence through the generation of transgenic and knockout mutant mice that suggests that anti-oxidants play a protective role in stroke (Table 1). Homozygous SOD1 transgenic mice (fivefold increase in SOD1 activity) demonstrated an infarct volume that was decreased by 35% compared with control mice in a permanent focal ischaemia model.7 However, in contrast, Chan et al.8 suggested SOD1 overexpression to be ineffective in affording protection following permanent cerebral ischaemia. Following transient MCA occlusion, a reduction in neuronal cell death, ranging from 25 to 50%, has been reported in SOD1-overexpressing mice.9–12 This reduction was linked to decreased DNA damage by blocking the early release of cytochrome c from the mitochondria.13,14 Accordingly, mice lacking SOD1 demonstrated an increase in both infarct size and oedema following transient focal cerebral ischaemia.15 However, SOD1-knockout mice do not show any differences in either cerebral or oedema volumes, with no alteration in mitochondrial release of cytochrome c, in a model of permanent focal cerebral ischaemia.16 The disparities between transient and permanent ischaemia models suggests SOD1 protection may be important in the reperfusion event, where it plays a role in scavenging cytosolic ROS produced.

Table 1.  Transgenic and knockout mutant mice used to study the role of oxidants in ischaemic brain injury
GeneEnzyme/proteinTransgene/knockoutStudyEffectReference
  1. FCI, model of focal cerebral ischaemia; +, neuronal protection; NP, no protection; –, exacerbates damage; SOD, superoxide dismutase; NOS, nitric oxide synthase; Gpx, glutathione peroxidase.

  2. Modified from Chan et al.8

SOD-1CuZn-SODHuman transgeneFCI+Kinouchi et al.7
SOD-1CuZn-SODHuman transgeneTransient FCI+Yang et al.9
SOD-1CuZn-SODHuman transgeneTransient global ischaemia+Murakami et al.12
SOD-1CuZn-SODHuman transgenePermanent FCINPChan et al.10
sod-1CuZn-SODHomozygous (–/–)Transient FCIKondo et al.15
sod-2Mn-SODHeterozygousTransient FCIMikawa et al.11
GPX-1Gpx1TransgenicTransient FCI+Ishibashi et al.76
GPX-1Gpx1HomozygousTransient FCICrack et al.20
nNOSNeuronal NOSHomozygous (–/–)Permanent FCI+Huang et al.77
eNOSEndothelial NOSHomozygous (–/–)Permanent FCIHuang et al.78
p53p53 proteinHomozygous (–/–)Permanent FCI+Crumrine et al.79
Bcl-2Bcl-2 proteinHuman transgene: neuronal specificPermanent FCI+Martinou et al.80

Significantly, the Bad pathway has been implicated in the elevated neuronal cell death after transient focal cerebral ischaemia–reperfusion, with this response attenuated in SOD1 transgenic mice.17 Furthermore, induction of caspase-8 expression has been reported following transient focal cerebral ischaemia–reperfusion, but not in a permanent model.14 Caspase-8 induction was reduced in mice overexpressing SOD1 compared with wild-type animals, highlighting the importance of SOD1 in protecting against neuronal apoptosis.

The H2O2-detoxifying enzyme Gpx1 has also been shown to play a significant role in providing neuroprotection against cell death induced by oxidative stress in cerebral ischaemia–reperfusion injury. Mice overexpressing Gpx1 demonstrate a significant reduction in neuronal cell death in a focal model of cerebral ischaemia–reperfusion, with a 48% decrease in infarct volume.18 There was also decreased astrocytic and microglial activation with reduced inflammatory cell infiltration, suggesting a role for Gpx1 in brain inflammatory processes. The decreased inflammatory response seen in the transgenic mice may contribute to the reduced susceptibility of neurons in these animals to cell death following cerebral ischaemia–reperfusion. This is supported by the increased inflammation reported in mice lacking Gpx1 in a cold-induced head trauma model.19 The increase in infarct size in the Gpx1–/– mice following cerebral ischaemia–reperfusion was accompanied by an earlier activation of caspase-3 expression and increased apoptosis in the knockout mice.20

It was not surprising that the SOD1 transgenic mice showed neuroprotection following cerebral ischaemic–reperfusion injury in light of the superoxide-scavenging properties of this anti-oxidant. However, it can be presumed that such an increase in SOD1 activity would lead to the generation of elevated H2O2 levels. The increased neuronal cell death in the Gpx1–/– mice suggested that H2O2 is a significant inducer of oxidative damage. Crack et al.21 addressed the relationship between SOD1 and Gpx1 following transient MCA occlusion though the generation of a SOD1 transgenic/Gpx1 knockout mouse. Interestingly, upon crossing the SOD1 transgenic to a Gpx1–/– mouse, a significant amount of protection previously seen in the overexpressing animal was repressed. This study supported that notion that much of the neuroprotective effects of SOD1 rely on the downstream detoxifying effects of Gpx1 to remove the H2O2 generated. However, SOD1 still plays an important role in providing protection against oxidative stress, because the SOD1 transgenic/Gpx1 knockout mice demonstrated reduced infarct volumes compared with the Gpx1–/– mice.21 This neuroprotection is possibly through a reduction in levels of peroxynitrite generated through a reaction between superoxide and nitric oxide (NO).

SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

The study of signal transduction mechanisms has been exhaustive over the past 50 years; however, the recent surge of interest in many signalling pathways and their links to human disease has led to an increased understanding in the area. This progress has been aided, in part, by developments in structural and genetic analysis, improvements in techniques (such as the generation of genetically modified mice) and also through the use of pharmacological modulators of signalling. In particular, the complex signalling network involving numerous survival/death pathways in the neuronal cell death of stroke and neurodegenerative diseases has been studied extensively. Importantly, as depicted in Fig. 2, a number of these pathways have been reported to be redox mediated. Generally, there are two proposed mechanisms of action concerning oxidative stress-mediated signalling, either: (i) altering the intracellular redox state of a cell to activate specific pathways; or (ii) by direct oxidative modification of proteins.22

image

Figure 2. Putative pathways mediated by oxidative stress. Reactive oxygen species (ROS) activate a number of cell signalling pathways within a cell. Solid lines represent characterized pathways, whereas dashed lines represent hypothesized pathways. There are those that are proposed to be involved in promoting cell death, such as the p38, c-Jun N-terminal kinase (JNK) and p53 pathways; other cascades, such as those involving extracellular signal-regulated kinase (ERK) 1/2 and Akt, have demonstrated a protective role against oxidative stress-induced cell. Heat shock factor-1 (HSF1) is involved in cellular survival, whereas while ataxia telangiectasia mutant (ATM) and the janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway is involved in cellular death. The nature of nuclear factor (NF)-κB signalling, whether it is pro- or anti-apoptotic has been widely debated. PLC, phospholipase C; PKC, protein kinase C; PI3-K, phosphatidylinositol 3-kinase. (Modified from Martindale and Holbrook.23)

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The signalling cascades involved in cell survival are complex and, although studied widely, the mechanisms behind their activation and modulation by factors including ROS are not well understood. Although the activation of c-Jun N-terminal kinase (JNK) and p38 by ROS has generally been implicated in promoting cell death, the extracellular signal-regulated kinase (ERK) 1/2 pathway has been shown to be protective against cell death.23 The PI3-K/Akt pathway is also a key player in preventing apoptosis and, indeed, activation of Akt and its downstream effectors have been shown to be necessary for the survival of a number of cell types, including neurons.24 Interestingly, Akt has been implicated in the activation of the transcription factor NF-κB.25,26 Significantly, both Akt and NF-κB are activated following cerebral ischaemia–reperfusion injury.27–30

THE PI3-K/AKT PATHWAY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

The PI3-K/Akt pathway is activated by various growth factors in a number of cells ranging from fibroblasts to neurons.31 Furthermore, it is well established that the anti-apoptotic effects of these growth factors can be attributed, in part, to the activation of this pathway, first reported in PC12 cells by Yao and Cooper.24

Activation of Akt is necessary for cell survival, with this demonstrated by a number of studies. Apoptosis induced by growth factor withdrawal, ultraviolet (UV) irradiation, DNA damage and transforming growth factor (TGF)-β treatment in a variety of cell types is reduced through the transfection of constitutively active Akt. In contrast, the introduction of dominant negative Akt or inactive Akt constructs blocks the survival of cells in the presence of growth factors, highlighting the significance of this pathway in preventing cell death.32

ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

Akt has been identified as having a diverse role. It is involved in regulating the cell cycle (through its involvement with cyclin D and p21), insulin signalling and is a key player in protecting against cell death.33 Numerous studies have identified that transfection of a variety of cell types, with constitutively active Akt alleles blocking apoptosis induced by growth factor withdrawal, UV irradiation, DNA damage and TGF-β treatment. Furthermore, growth factor-mediated survival requires Akt. Transfection of dominant negative or inactive Akt constructs blocks the survival of cells in response to growth factors.31 Akt primarily prevents apoptosis through its direct interaction with the pro-apoptotic molecule Bad,34 thus preventing it from homodimerizing (and also from forming heterodimers with the antiapoptotic Bcl-2). Consequently, cytochrome c release from the mitochondria cannot occur and subsequent caspase activation is prevented (Fig. 3).

image

Figure 3. Role of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in cell survival. This cell survival pathway is initiated by growth factor and H2O2 treatment through growth factor receptors. Phosphatidylinositol 3-kinase is involved in the phosphorylation (and activation) of the downstream molecule Akt; with the pro-apoptotic molecule Bad phosphorylated by Akt, it is sequestered in the cytoplasm through its interaction with 14-3-3. This proves to be important in that Bax homodimers and heterodimers (with the pro-apoptotic Bcl-XL) cannot form and, thus, cytochrome c release from the mitochondria is prevented. Consequently, it cannot form a complex with apoptotic protease activating factor (Apaf)-1 and procaspase-9 and subsequent cleavage of downstream caspases, such as caspase-3, does not occur. FKHRL-1, forkhead rank ligand-1.

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Akt may also interact with other molecules to promote cell survival. Akt phosphorylates and inactivates the transcription factor forkhead rank ligand-1 (FKHR-L1) and, therefore, has been implicated in modulating Bcl-2 family members, such as the anti-apoptotic Mcl-1 and pro-apoptotic Bim, through the upregulation and downregulation of expression, respectively.35,36 Furthermore, direct phosphorylation of caspase-9 at Ser196 by Akt has also been demonstrated, although the mechanism underlying the inactivation of caspase-9 by Akt is unknown.31 Evidence also suggests that there is a relationship between Akt and p53, possibly through the p53 regulator Mdm2. Mdm2 contains two potential Akt phosphorylation sites, as does apoptotic protease activating factor-1 (APAF-1); therefore, these proteins may be counter-regulated by Akt.31 Furthermore, the recent discoveries of Diablo/Smac (inhibitors of the inhibitor of apoptosis proteins (IAP)) may play a role, with these possibly being regulated by Akt at the transcriptional or post-transcriptional level.37

ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

The requirement of cell survival pathways to protect against the increased neuronal cell damage following cerebral ischaemia is well established. In models of cerebral ischaemia, a temporal increase in Akt phosphorylation has been reported.38 Noshita et al.29 have further studied the pattern of Akt phosphorylation following ischaemia and have similarly reported an increase in immunoreactivity 4 h post-reperfusion, with a decrease by 24 h. However, at the core of the infarct area (the caudate putamen), Akt phosphorylation was decreased. This was attributed to an inability of the cells within this area to activate the PI3-K/Akt pathway owing to too much cellular damage. This was in contrast with the cortex, where damage was not as extensive. Cells that were identified as being positive for phosphorylated Akt colocalized with neurons (through NeuN staining); however, they were not terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labeling (TUNEL) positive and did not display features of apoptosis, which suggests a neuroprotective mechanism. The importance of the PI3-K/Akt pathway in preventing cellular damage following ischaemia–reperfusion was highlighted by an increase in TUNEL-positive cells and also DNA fragmentation upon PI3-K inhibition.29

In a recent study, Shibata et al.30 reported an increase in Akt phosphorylation by western blot analysis as early as 1 h after permanent occlusion; however, there was no detectable increase in phosphorylated Akt thereafter. The phospho-Akt-positive cells were confirmed by colocalization with NeuN to be neurons. Immunoreactivity was localized to the cytoplasm of neurons in the peripheral part of the MCA region, whereas at the central part of this area, staining was more nuclear and perinuclear cytoplasmic. As reported by Noshita et al.,29 immunoreactivity of activated caspase-3 was absent where Akt phosphorylation was present, whereas caspase-3-positive cells were present in the central part of the ischaemic region where little phospho-Akt staining was detected. Akt kinase activity was increased after 1 h ischaemia in both nuclear and cytoplasmic fractions.30 It has been proposed that nuclear translocation of phosphorylated Akt to the nucleus is critical in providing neuroprotection through its phosphorylation and inactivation of the forkhead family transcription factors.39 This family of proteins resides predominantly in the nucleus, where they promote the transcription of pro-apoptotic genes, such as Bim and Fas-L. Upon its phosphorylation by Akt, FKHRL1 is exported from the nucleus to the cytoplasm, where it is sequestered by 14-3-3 proteins.40

The neuroprotective actions of the PI3-K/Akt pathway in cerebral ischaemia have been widely reported. It appears that activation of Akt is extremely important in protecting against neuronal cell death. Confusingly, Akt has been implicated in the activation of the NF-κB, whose role in neuronal cell death is less well understood and, indeed, controversial.

ROLE OF NF-κB IN NEURONAL CELL DEATH

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

Nuclear factor-κB has been shown to be involved in a variety of processes in the central nervous system, including neural development and plasticity, neuronal apoptosis and a variety of neuropathological conditions (Alzheimer's disease, Parkinson's disease) and brain injury (cerebral ischaemia and seizures). Although NF-κB has been shown to play an important role in neuronal cell death, a full understanding of the mechanisms of its activation and how this affects neurons has not been established. The implication of NF-κB in a number of different cellular responses arises from the wide variety of genes responsive to NF-κB. These include pro-inflammatory cytokines, adhesion molecules, chemokines, growth factors and anti-oxidant enzymes.41 Much of our knowledge of NF-κB has relied on evidence from electrophoretic mobility shift assays (EMSA). However, increased DNA binding activity does not always correlate with increased transactivation and, therefore, interpreting results from these earlier studies and associating them with a functional outcome has been difficult.

OXIDATIVE STRESS AND NF-κB

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

Oxidative stress has been associated with the activation of NF-κB. This stems from three basic findings. First, many agents that activate NF-κB are involved in the formation of ROS or are oxidants themselves. Second, NF-κB can be activated by H2O2 or hydroperoxides in a number of cell types. Third, activation of NF-κB is inhibited by a variety of anti-oxidants.42

It is well established that NF-κB is a key signalling molecule in the apoptotic process; however, there are two differing viewpoints on its role. Although a number of studies suggest that the transcription factor protects against cell death, NF-κB has also been implicated in promoting apoptosis.26 To elucidate its role, many strategies have been used to block NF-κB transcription. Many of the studies have either relied on using genetically modified mice or, alternatively, have used NF-κB inhibitors or κB-decoy DNA in vitro.

Studies of p65–/– mice have demonstrated the importance of NF-κB in cell survival, with a fetal lethal phenotype due to massive apoptosis of the liver. Furthermore, p65–/– fibroblasts and macrophages have been shown to be more sensitive to tumour necrosis factor (TNF)-α-induced cytotoxicity.43 These studies implicate p65 in playing a protective role against cell death. Studies with IκB-kinase (IKK) α- and IKKβ-knockout mice have also provided some insight into the role of NF-κB in apoptosis.43 The IKKα–/– mice show developmental abnormalities associated with decreased proliferation; however, there is normal activation of NF-κB in response to various cytokines. In contrast, IKKβ–/– mice show a perturbation in cytokine-induced NF-κB activation (highlighting that it is IKKβ and not IKKα that is necessary for IKK activation in response to inflammatory stimuli) and also show a similar phenotype to the p65–/– mouse in that they are embryonic lethal at embryonic day (E) 12–13 owing to massive apoptosis of liver cells.44 This phenotype can be overcome by mating the IKKβ-deficient mice to TNF-R1–/– mice.45 Double mutants (IKKα–/– mice mated to IKKβ–/– mice) were fetal lethal at E12, which was associated with defects in neurulation in the form of increased apoptosis of the neuroepithelium.46 Wang et al.47 suggested that activated NF-κB was critical to a cell's survival, with increased levels of apoptosis induced by a variety of stimuli, including DNA-damaging agents, seen when NF-κB was inactivated. Together, these studies of knockout mice suggest that NF-κB activation is involved in preventing apoptosis.

However, evidence from studies of the p50–/– mice are at odds with this and suggest that NF-κB may not be protective against cell death. Unlike the p65–/– mice, a lack of p50 activity does not result in fetal lethality. Furthermore, the p50–/– mice demonstrate an increased susceptibility to cerebral ischaemia.27 However, the role of p50 in cell survival is found to be more complex through in vitro studies identifying an increased susceptibility of p50–/– neurons to excitotoxic insult.48 Therefore, although knockout mice of the NF-κB subunits and other members of the activation cascade implicate the NF-κB pathway in cell survival, these studies have also provided conflicting results concerning its exact role. This is especially apparent when comparing these findings with those of in vitro studies.

Nuclear factor-κB activation in cultured neurons has been shown to be protective against both excitotoxic and metabolic insults, such and glutamate exposure, glucose deprivation, low K+, hypoxia, β-amyloid toxicity and oxidative stress.49 Treatment of cultured cortical neurons with NF-κB inhibitors, such as the synthetic peptide SN50, have also suggested a protective role. Inhibition of NF-κB resulted in a marked increase in the mitochondrial release of cytochrome c, activation of both caspase-3 and -9, poly (ADP-ribose) polymerase (PARP) cleavage and features of apoptosis, such as nuclear fragmentation, chromatin condensation and membrane blebbing. Furthermore, treatment with these inhibitors also repressed transcription of the anti-apoptotic Bcl-2, A1 and Bcl-xL.50

It has been proposed that glial activation by NF-κB can be damaging to neurons.28In vitro studies have demonstrated that cytokines mediate the activation of microglia, with the subsequent damage caused by oxyradical production prevented by inhibitors of NF-κB. Furthermore, activation of NF-κB in astrocytes has been reported to increase both NO synthase (NOS) and NO production, which may be detrimental to cells owing to the possible formation of peroxynitrites.51

Coculture studies of astrocytes and neurons have provided significant evidence that the relationship between the two cell types is important in the activation of NF-κB. Although cultured neurons constitutively express activated NF-κB, Kaltschmidt and Kaltschmidt52 found that coculturing neurons with astrocytes repressed this activation of NF-κB. The authors proposed a number of models behind this repression. First, it was possible to attribute the repression to diffusible factors, such as growth factors, that are produced by glial cells. Second, glial glutamate transporters may decrease glutamate from neurons to levels that are not sufficient for NF-κB activation. Third, astrocytes may be involved in controlling redox status that otherwise involves the activation of NF-κB by reactive oxygen intermediates (ROI), such as those generated by glutamate. Therefore, the complex relationship between astrocytes and neurons in vivo may sometimes be lost when studying neuronal responses in a culture system. This may account for the conflicting findings between in vitro systems and in vivo studies.

It has been proposed that the discrepancies between the various studies on the role of NF-κB in cell death may be as a result of the dual role of this transcription factor in signal transduction. Induction of NF-κB by mild cellular stresses to low levels may be protective; however, greater activation of NF-κB by a larger insult may promote cell death.53 In support of this theory, Clemens et al.54 suggest that the transient activation of NF-κB may promote the induction of factors that are protective (such as Mn-SOD), whereas persistent activation of NF-κB results in cell death. Significantly, the neuroprotective effects of TNF-α and ceramide when neurons were exposed to oxidative and metabolic insults were overcome in the presence of κB decoy DNA, with the level of protection corresponding with levels of Mn-SOD.54 Such a notion is of interest when studying the activation of NF-κB by oxidative stress. As discussed previously, initial activation of NF-κB may result in elevated gene transcription of anti-oxidants, such as Mn-SOD and Gpx1, to provide a negative feedback loop. However, a persistent insult would overcome this increased anti-oxidant defence, thus leading to cell death. This may be relevant in studying the role of NF-κB in cerebral ischaemia.

ROLE OF NF-κB IN CEREBRAL ISCHAEMIA

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

Nuclear factor-κB is expressed widely throughout the central nervous system, seen in all cell types, with constitutive expression in neurons.55 Although NF-κB activation induced by injury or other mechanisms may protect a cell from apoptosis, it may lead to indirect apoptosis in other cells through the production of cytotoxic agents (e.g. NOS). Indeed, microglia have been shown to produce ROS and excitotoxins when activated that can subsequently be damaging to neurons.51

The first reports correlating NF-κB activation with cerebral ischaemia were identified in glial cells of post-mortem human brains, primarily in the penumbra.56 Elevated NF-κB activation has also been demonstrated in various mouse models of stroke, with initial reports suggesting that this was promoting cell death. In a transient global ischaemia model, western blot analysis identified that p50 NF-κB was increased by 6 h, with a further increase from 12 to 48 h. There was, however, a decrease noted by 96 h after ischaemia.57 Both p50 and p65 immunoreactivity identified a similar time-course of staining in the nuclei of neurons in the CA1 and other regions of the hippocampus.57 The NF-κB immunoreactivity correlated with neuronal cell death (at 72 h), suggesting NF-κB is involved in promoting cell death in cerebral ischaemia. Furthermore, increased mRNA levels of the pro-apoptotic Bcl-Xs were identified in areas of NF-κB activation. Studies of focal models of cerebral ischaemia have also reported extensive NF-κB activation. In a 2 h model, Stevenson et al.58 identified immunoreactivity in the nuclei of both cortical and striatal neurons on the ischaemic side of the brain at 2, 6 and 12 h after reperfusion. It has been suggested that activation of NF-κB is even more long lasting, such as 1 week after ischaemia (in neurons) and even 1 year later (in microglia and macrophages), which was reported in a rat model of brain trauma.59 Nuclear factor-κB activation in microglia suggests its involvement in the complex inflammatory processes occurring after ischaemia.

It has been proposed that the NF-κB activation in microglia promotes neuronal degeneration, whereas NF-κB in neurons may promote their survival.60 The complex induction of gene expression by NF-κB may play an extremely important role and account for the both the cell- and stimulus-specific nature of the response upon activation of NF-κB. As discussed previously, NF-κB can upregulate the expression of a number of anti-oxidant genes, including Mn-SOD and Gpx1. However, Cechetto53 has correlated NF-κB activation with increased mRNA levels of the pro-apoptotic Bcl-Xs in the hippocampus following stroke. Nuclear factor-κB activates many genes that have been associated with the pathology of cerebral ischaemia, including those involved in the inflammatory response, such inducible NOS, interleukin (IL)-1, TNF-α, intercellular adhesion molecule-1, cyclo-oxygenase-2 and IL-6. Indeed, TNF-α or IL-1 production has been reported to be upregulated following ischaemia–reperfusion injury, with this linked to NF-κB activation.61 Therefore, the activation of NF-κB in neuronal and glial cells and the differing responses may influence the outcome of the brain following cerebral ischaemia.

Nuclear factor-κB activation following stroke has been attributed to increased oxidative stress. However, the contribution of NF-κB to the neuronal cell death has been widely debated. The reduced neuronal cell damage displayed by the p50–/– mouse following cerebral ischaemia was pivotal in suggesting NF-κB is cell death promoting in stroke.27 Furthermore, NF-κB activation has been shown to be decreased by the administration of N-acetylcysteine and, although this does provide some neuroprotection, it does not definitely implicate NF-κB in neurodegeneration.62 Other reports suggest that NF-κB activation by oxidants is actually preventing cellular damage following cerebral ischaemia. Tumour necrosis factor-α receptor-knockout mice demonstrate an exacerbation of neuronal cell damage that has been associated with decreased NF-κB activation and reduced Mn-SOD expression.63 Further evidence that NF-κB serves a protective function in neurons comes from studies identifying increased levels of the κB-responsive IAP, neuronal apoptosis inhibitory protein-1 (NAIP), in neurons resistant to ischaemic brain injury. Furthermore, overexpression of NAIP increases the resistance of neurons to ischaemic injury in vivo.64 In contrast, mice overexpressing Mn-SOD have reduced neuronal cell damage following transient focal cerebral ischaemia, with a concurrent downregulation in both NF-κB and c-myc expression.65 However, the majority of these studies are correlative and do not definitely associate the levels of NF-κB activation with stroke outcome.

Despite this limited knowledge, NF-κB has been proposed as being a candidate in preventing the cellular damage seen in stroke. Administration of a proteasome inhibitor was reported to be effective in reducing infarct size following transient MCA occlusion.66 This was attributed to an inhibition of NF-κB activation via decreased IκB degradation. In vitro studies with aspirin and sodium salicylate, both shown to inhibit NF-κB, possibly through inhibiting the phosphorylation and/or degradation of IκB, have resulted in decreased cell death following glutamate exposure.53 Although this suggests that inhibition of NF-κB activation may be a potential therapy for preventing neuronal cell damage following stroke, once again these findings were only correlative. In particular, evidence arising from the use of proteasome inhibitors need to be analysed with caution owing to the non-specificity of such compounds. It cannot be discounted that the beneficial effects of agents, such as aspirin, are not directly through reduced NF-κB activation. Further understanding of the mechanisms of activation of NF-κB and its downstream targets is necessary before implicating NF-κB as a therapeutic target for the treatment of stroke.

ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES

It has been suggested that the prosurvival actions of Akt are through the activation of NF-κB and the subsequent upregulation of anti-apoptotic genes.67 However, the evidence surrounding the link between the PI3-K/Akt and NF-κB pathways is controversial and the caveat must be added that some of the following studies were performed in non-neuronal models. Romashkova and Makarov26 demonstrated PDGF activation of NF-κB in fibroblasts, together with increased Akt phosphorylation. It was concluded from this study that Akt induces NF-κB activation through its association with the IKKs. The phosphorylation of IκB results in its subsequent ubiquitination and degradation, thus allowing the p65–p50 dimer to translocate to the nucleus (Fig. 4). Specifically, it has been reported that TNF-α-induced IκBα phosphorylation is through a direct interaction with the p85 regulatory subunit of PI3-K;68 however, other reports suggest that it is the catalytic p110 subunit of PI3-K that regulates the activation of NF-κB.69 Beraud et al.70 suggest that both PI3-K subunits are involved in NF-κB activation through a tyrosine phosphorylation-mediated pathway. However, other studies have seen very weak activation or no NF-κB activation by PDGF or epidermal growth factor in smooth muscle cells.71 Furthermore, the phosphorylation of Akt by PDGF in embryonic kidney and carcinoma cells, and also Jurkat cells, has been identified to occur in the absence of NF-κB activation.72

image

Figure 4. Nuclear factor (NF)-κB activation: a role for the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway? The PI3-K pathway has been implicated as being involved in the activation of NF-κB either through a direct interaction between PI3-K or Akt with IκB-kinase (IKK) or, alternatively, between Akt and IκBα. Other studies have seen no such relationship between the pathways and propose that NF-κB activation occurs through other signalling pathways, such as that involving tumour necrosis factor (TNF)-α receptor-associated factor (TRAF)2/TRAF6 and NF-κB-inducing kinase (NIK), which results in the phosphorylation of the IKKs upon TNF-α treatment. It is also possible that other pathways involving the mitogen-activated protein kinase activate NF-κB through the IKKs. However, some of these mechanisms are yet to be studied in neuronal models. IL, interleukin; SOD, superoxide dismutase.

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It has also been reported that cytokines, such as TNF-α, activate NF-κB through an Akt-dependent pathway. Ozes et al.25 reported a link between the PI3-K and NF-κB pathways in 293 cells with reduced TNF-α-induced NF-κB activation following Akt inhibition. The mechanism behind this activation of NF-κB was once again shown to be through the phosphorylation (at Thr23) and subsequent degradation of IκBα, although this seems to be cell type specific. Treatment with IL-1 has been shown to result in PI3-K-dependent NF-κB activation. This was also reported to be through phosphorylation of p65 by the 110 subunit of PI3-K. Surprisingly, however, this NF-κB activation was independent of IκB degradation, NF-κB translocation and also NF-κB DNA-binding activity. Akt has been shown in other studies to increase IκB degradation and mediate NF-κB-induced activation of NF-κB-responsive promoters.73 However, neither the IKKs, IκBs nor NIK contain a consensus sequence for phosphorylation by Akt; therefore, if Akt does play a role in IKK activation, it may be through an indirect mechanism. Kane et al.73 suggest a mitogen-activated protein kinase kinase kinase family member, or alternatively, an unknown pathway may be involved in Akt activation of NF-κB through IKK.

In contrast, other studies have not reported a direct link between NF-κB and Akt following cytokine treatment.74 It is proposed that activation of NF-κB by cytokines such as TNF-α is through a pathway involving the TNF-α receptor-associated factors (TRAF2 and TRAF6), with the subsequent activation of NF-κB-inducing kinase (NIK) and phosphorylation of the IKKs, as depicted in Fig. 4. Furthermore, Beraud et al.70 reported no effect of the PI3-K inhibitor wortmannin on IL-1-induced NF-κB activation. Such conflicting results confirm the disparity between various culture systems involving different cells and also different stimuli. Rauch et al.72 suggest that the involvement of Akt in NF-κB activation may be seen only in cells with high proliferative potential, such as kidney cells, T cells and carcinoma cells, with the activation of Akt being responsible for a prosurvival signal and NF-κB activation promoting proliferation.

Interestingly, it has recently been proposed that Akt is actually a downstream target of NF-κB following TNF-α and lipopolysaccharide treatment.75 Events involved in the activation of NF-κB by these stimulants, such as IκB degradation, nuclear translocation and increased NF-κB DNA binding, occurred before any increases in Akt phosphorylation were detected. Furthermore, Akt phosphorylation was increased by overexpression of p65 or, alternatively, inhibited by IκBα overexpression. The inhibition of Akt phosphorylation by TNF-α in the presence of NF-κB inhibitors suggests that NF-κB is required for Akt activation, contrasting with previous reports, as discussed above, that have suggested Akt is involved in the activation of NF-κB. In contrast, a recent study has suggested that treatment of primary cortical neurons with the NF-κB inhibitors PAR or SN50 resulted in a burst of Akt phosphorylation after a 2 h exposure.50 It was proposed that this elevation in phosphorylated Akt may be an attempt by the cell to prevent apoptosis induced by NF-κB inhibition.

These findings suggest that the relationship between the PI3-K/Akt and NF-κB pathways is both highly cell and stimuli specific. However, the PI3-K/Akt and NF-κB pathways are only two players in a complex signalling cascade of neuronal survival. Many of these pathways have been implicated in the pathogenesis of neurodegenerative disorders, including cerebral ischaemia, with a number of them modulated by oxidative stress. Elucidating the mechanisms behind oxidative stress-mediated neuronal cell death is necessary in identifying potential putative targets for the treatment of stroke.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ISCHAEMIC STROKE
  5. ROLE OF ANTI-OXIDANT ENZYMES IN CEREBRAL ISCHAEMIA
  6. SIGNAL TRANSDUCTION IN NEURONAL CELL DEATH
  7. THE PI3-K/AKT PATHWAY
  8. ROLE OF THE PI3-K/AKT PATHWAY IN NEURONAL CELL DEATH
  9. ACTIVATION OF THE PI3-K/AKT PATHWAY IN CEREBRAL ISCHAEMIA
  10. ROLE OF NF-κB IN NEURONAL CELL DEATH
  11. OXIDATIVE STRESS AND NF-κB
  12. ROLE OF NF-κB IN CEREBRAL ISCHAEMIA
  13. ROLE OF THE PI3-K/AKT PATHWAY IN THE ACTIVATION OF NF-κB
  14. ACKNOWLEDGEMENTS
  15. REFERENCES