Michio Tamatani

Emerging data indicate that tumor necrosis factor (TNF) exerts a neuroprotective effect in response to brain injury. Here we examined the mechanism of TNF in preventing neuronal death in primary hippocampal neurons. TNF protected neurons against hypoxia- or nitric oxide-induced injury, with an increase in the anti-apoptotic proteins Bcl-2 and Bcl-x as determined by Western blot and reverse transcriptase-polymerase chain reaction analysis. Treatment of neurons with an antisense oligonucleotide to bcl-2 mRNA or that to bcl-x mRNA blocked the up-regulation of Bcl-2 or Bcl-x expression, respectively, and partially inhibited the neuroprotective effect induced by TNF. Moreover, adenovirus-mediated overexpression of Bcl-2 significantly inhibited hypoxia- or nitric oxide-induced neuronal death. To examine the possible involvement of a transcription factor, NFκB, in the regulation of Bcl-2 and Bcl-x expression in TNF-treated neurons, an adenoviral vector capable of expressing a mutated form of IκB was used to infect neurons prior to TNF treatment. Expression of the mutant NFκB completely inhibited NFκB DNA binding activity and inhibited both TNF-induced up-regulation of Bcl-2 and Bcl-x expression and neuroprotective effect. These findings indicate that induction of Bcl-2 and Bcl-x expression through NFκB activation is involved in the neuroprotective action of TNF against hypoxia- or nitric oxide-induced injury. Emerging data indicate that tumor necrosis factor (TNF) exerts a neuroprotective effect in response to brain injury. Here we examined the mechanism of TNF in preventing neuronal death in primary hippocampal neurons. TNF protected neurons against hypoxia- or nitric oxide-induced injury, with an increase in the anti-apoptotic proteins Bcl-2 and Bcl-x as determined by Western blot and reverse transcriptase-polymerase chain reaction analysis. Treatment of neurons with an antisense oligonucleotide to bcl-2 mRNA or that to bcl-x mRNA blocked the up-regulation of Bcl-2 or Bcl-x expression, respectively, and partially inhibited the neuroprotective effect induced by TNF. Moreover, adenovirus-mediated overexpression of Bcl-2 significantly inhibited hypoxia- or nitric oxide-induced neuronal death. To examine the possible involvement of a transcription factor, NFκB, in the regulation of Bcl-2 and Bcl-x expression in TNF-treated neurons, an adenoviral vector capable of expressing a mutated form of IκB was used to infect neurons prior to TNF treatment. Expression of the mutant NFκB completely inhibited NFκB DNA binding activity and inhibited both TNF-induced up-regulation of Bcl-2 and Bcl-x expression and neuroprotective effect. These findings indicate that induction of Bcl-2 and Bcl-x expression through NFκB activation is involved in the neuroprotective action of TNF against hypoxia- or nitric oxide-induced injury. Acute and chronic neurodegenerative disorders are characterized by increased levels of a variety of inflammatory cytokines. Among them, the role of tumor necrosis factor (TNF) 1The abbreviations used are:TNF, tumor necrosis factor; Ad, adenovirus; HA, hemagglutinin; LDH, lactate dehydrogenase; m.o.i., multiplicity of infection; NO, nitric oxide; NFκB, nuclear factor κB; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; SNP, sodium nitroprusside; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2; ANOVA, analysis of variance; ODN, oligodeoxynucleotides.1The abbreviations used are:TNF, tumor necrosis factor; Ad, adenovirus; HA, hemagglutinin; LDH, lactate dehydrogenase; m.o.i., multiplicity of infection; NO, nitric oxide; NFκB, nuclear factor κB; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; SNP, sodium nitroprusside; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2; ANOVA, analysis of variance; ODN, oligodeoxynucleotides. (1Cheng B. Christakos S. Mattson M.P. Neuron. 1994; 12: 139-153Abstract Full Text PDF PubMed Scopus (597) Google Scholar) in injury responses has been indicated (1Cheng B. Christakos S. Mattson M.P. Neuron. 1994; 12: 139-153Abstract Full Text PDF PubMed Scopus (597) Google Scholar, 2Bruce A.J. Boling W. Kindy M.S. Peschon J. Kraemer P.J. Carpenter M.K. Holtsberg F.W. Mattson M.P. Nat. Med. 1996; 2: 788-794Crossref PubMed Scopus (840) Google Scholar). Cell culture studies have shown that TNF can protect cultured neurons against glucose deprivation-induced injury and excitatory amino acid toxicity, by stabilizing intracellular calcium concentration (1Cheng B. Christakos S. Mattson M.P. Neuron. 1994; 12: 139-153Abstract Full Text PDF PubMed Scopus (597) Google Scholar). Moreover, experiments using mice deficient in TNF receptors demonstrated that endogenous TNF protects neurons against ischemic and excitotoxic insults, with induction of an antioxidative pathway (2Bruce A.J. Boling W. Kindy M.S. Peschon J. Kraemer P.J. Carpenter M.K. Holtsberg F.W. Mattson M.P. Nat. Med. 1996; 2: 788-794Crossref PubMed Scopus (840) Google Scholar). However, the exact mechanisms by which TNF protects neurons are largely unknown.Nuclear factor κB (NFκB) is a ubiquitous transcription factor that is activated by a variety of cytokines including TNF (3Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2008) Google Scholar, 4Baldwin A.S. Annu. Rev. Immunol. 1994; 14: 649-681Crossref Scopus (5540) Google Scholar) and is thought to be a key regulator of genes involved in inflammation, response to infection, and stress. Conventional NFκB is a heterodimer that consists of p50 and p65 subunits. The activity of NFκB is strictly regulated by one of the IκB inhibitory proteins, such as IκBα or IκBβ, which forms a complex with NFκB and keeps NFκB in the cytoplasm (3Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2008) Google Scholar, 4Baldwin A.S. Annu. Rev. Immunol. 1994; 14: 649-681Crossref Scopus (5540) Google Scholar). When cells receive signals that activate NFκB, IκBs are phosphorylated and degraded through a ubiquitin/proteasome pathway (5Baeurle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). Coincident with IκB degradation, activated NFκB translocates to the nucleus and participates in the transactivation of a variety of genes, including genes for inflammatory cytokines, chemokines, and cell adhesion molecules (5Baeurle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar).Recently, dominant negative mutations of IκBα that are not phosphorylated and therefore not proteolyzed have been reported (6Brockmann J.A. Scherr D.C. McKinsy T.A. Hall S.M. Qi X. Le W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar, 7Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (929) Google Scholar). One such protein is a mutant IκBα that contains serine-to-alanine mutations at amino acids 32 and 36, which inhibit signal-induced phosphorylation and subsequent proteasome-mediated degradation of IκBα. This form of mutant IκB has been used to demonstrate that inhibition of NFκB induces apoptosis through a variety of cancer agents (8Wang A.S. 1996; PubMed Scopus Google Scholar, T. 1996; PubMed Scopus Google are that to key molecules in the induction or the of One such pathway is regulated by the expression of the and Bcl-2 is an intracellular protein that to and the nuclear D. J. PubMed Scopus Google Scholar, S. S. S. Google Scholar) and has been shown to cell by apoptosis in a variety of cell S. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar, D. Cell. Full Text PDF PubMed Scopus Google Scholar). The bcl-x to cell death. Bcl-x are a and form or a form the T. X. G. Cell. Full Text PDF PubMed Scopus Google Scholar, W. J. Immunol. 1994; Google Scholar). The form cell the form as a dominant bcl-2 and bcl-x apoptosis T. X. G. Cell. Full Text PDF PubMed Scopus Google Scholar, W. J. Immunol. 1994; Google Scholar). forms a with Bcl-2 or and the death activity of anti-apoptotic proteins T. S. S. U. S. 1994; Scholar, 1994; PubMed Scopus Google Scholar). the expression of Bcl-2 J. U. S. 1994; PubMed Scopus Google Scholar) and neuronal death D. S. J. Neuron. 1994; Full Text PDF PubMed Scopus Google and expression of Bcl-2 and has been in neurons that are to ischemic J. K. J. PubMed Scopus Google Scholar) brain injury. the expression is increased in neurons in the ischemic brain S. G. S. PubMed Scopus Google Scholar). However, the mechanism by which expression of is is we increased expression of Bcl-2 and Bcl-x primary neurons with TNF. inhibition of NFκB activation by overexpression of the mutant form of IκB the up-regulation of Bcl-2 and Bcl-x expression and the neuroprotective effect induced by studies have demonstrated that overexpression of Bcl-2 in neurons can or cell death induced by a variety of PubMed Scopus Google Scholar, S. Cell. Full Text PDF PubMed Scopus Google Scholar) and in J. U. S. 1994; PubMed Scopus Google Scholar, D. S. J. Neuron. 1994; Full Text PDF PubMed Scopus Google Scholar). of protects neurons apoptosis induced by factor P.A. 1995; Scopus Google Scholar). we levels of Bcl-2 expression in hypoxia- and nitric oxide-induced neuronal apoptosis S. G. Cell PubMed Scopus Google Scholar, S. Mol. PubMed Scopus Google Scholar). Bcl-2 and Bcl-x are induced in neurons to S. G. S. PubMed Scopus Google Scholar) and J. K. J. PubMed Scopus Google Scholar). is that the endogenous levels of Bcl-2 and Bcl-x neuronal data demonstrated that TNF protects neurons against hypoxia- and nitric oxide-induced and Bcl-2 and Bcl-x of neurons with Bcl-2 or Bcl-x antisense which up-regulation of Bcl-2 or Bcl-x expression, respectively, significantly the neuroprotective effect induced by that Bcl-2 and Bcl-x induction is involved in the neuroprotective action of of both the antisense ODN, which up-regulation of Bcl-2 and Bcl-x expression, to not inhibit TNF-induced studies that cell is by Bcl-2 or T. S. S. U. S. 1994; Scholar, 1994; PubMed Scopus Google Scholar). the of to Bcl-2 and Bcl-x to be for cell TNF-induced expression of Bcl-2 and Bcl-x the of expression was by TNF. Moreover, the that overexpression of Bcl-2 protects neurons anti-apoptotic protein as a factor for neuronal against hypoxia- or nitric oxide-induced injury. of TNF has been in an ischemic brain injury T. J. PubMed Scopus Google in which the is in the expression of Bcl-2 and Bcl-x has been in neurons that are to ischemic brain injury J. K. J. PubMed Scopus Google Scholar). be that TNF by brain cells Bcl-2 and Bcl-x expression in neurons and protects ischemic cells including neurons receptors for TNF. receptors have been characterized and TNF receptor and TNF receptor the which not in cells J.A. J. W. EMBO J. 1994; PubMed Scopus Google induced Bcl-2 and Bcl-x expression and protected hippocampal neurons against hypoxia- and is to a pathway a that induces of which in activation of the transcription factor NFκB Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). demonstrated that induced NFκB activity in cultured hippocampal neurons as determined by gel in with a by Mattson M.P. W. K. J. PubMed Scopus Google Scholar). experiments using that inhibition of NFκB activation completely the neuroprotective effect and inhibited the induction of Bcl-2 and Bcl-x expression in TNF-treated These indicate that the neuroprotective effect and up-regulation of Bcl-2 and Bcl-x expression induced by TNF are by activation of NFκB and apoptosis was by studies that inhibition of NFκB, by the IκB (8Wang A.S. 1996; PubMed Scopus Google Scholar, T. 1996; PubMed Scopus Google Scholar) or in cells Baltimore D. 1996; PubMed Scopus Google in increased to has been shown that NFκB activation induces apoptosis in cells K. K. T. 1994; PubMed Scopus Google Scholar, J. D. B. P.J. Cell. Full Text PDF PubMed Scopus Google Scholar) including cells and is not at NFκB activation is involved in apoptosis in cells and is for preventing apoptosis in neuronal has been reported that activation of NFκB protected hippocampal neurons against using κB DNA M.P. W. K. J. PubMed Scopus Google and that inhibition of NFκB in cells by to κB DNA or induced apoptosis that not be by factor G. J. PubMed Scopus Google Scholar). These with indicate that the NFκB is involved in of apoptosis in neuronal of the of NFκB activation in the regulation of expression of proteins that anti-apoptotic be demonstrated that up-regulation of Bcl-2 and Bcl-x expression induced by TNF is completely inhibited by IκB that Bcl-2 and Bcl-x are the of The bcl-x genes have complex and the have not been the of NFκB has been demonstrated in the of bcl-2 J.A. PubMed Scopus Google Scholar). the regulation of Bcl-2 and Bcl-x expression that we have to be by NFκB in neurons is to an in the bcl-2 to bcl-2 and expression of genes be induced by TNF through NFκB has been reported that the expression of is induced by which is by κB in hippocampal neurons M.P. W. K. J. PubMed Scopus Google Scholar). Moreover, in the the neuroprotective effect of TNF was blocked by IκB by antisense against bcl-2 in both of which TNF-induced up-regulation of Bcl-2 and Bcl-x expression was completely This that proteins that a effect be induced by NFκB activation in TNF-treated findings demonstrated that induction of Bcl-2 and Bcl-x expression through NFκB activation is involved in the neuroprotective effect of TNF. that inhibition of NFκB activation by expression of a mutant form of IκB the neuroprotective effect of TNF inhibition of TNF-induced up-regulation of Bcl-2 and Bcl-x expression by the antisense that genes for anti-apoptotic activity are induced by NFκB of the genes to the regulation of neuronal and to for and chronic neurodegenerative Acute and chronic neurodegenerative disorders are characterized by increased levels of a variety of inflammatory cytokines. Among them, the role of tumor necrosis factor (TNF) 1The abbreviations used are:TNF, tumor necrosis factor; Ad, adenovirus; HA, hemagglutinin; LDH, lactate dehydrogenase; m.o.i., multiplicity of infection; NO, nitric oxide; NFκB, nuclear factor κB; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; SNP, sodium nitroprusside; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2; ANOVA, analysis of variance; ODN, oligodeoxynucleotides.1The abbreviations used are:TNF, tumor necrosis factor; Ad, adenovirus; HA, hemagglutinin; LDH, lactate dehydrogenase; m.o.i., multiplicity of infection; NO, nitric oxide; NFκB, nuclear factor κB; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; SNP, sodium nitroprusside; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2; ANOVA, analysis of variance; ODN, oligodeoxynucleotides. (1Cheng B. Christakos S. Mattson M.P. Neuron. 1994; 12: 139-153Abstract Full Text PDF PubMed Scopus (597) Google Scholar) in injury responses has been indicated (1Cheng B. Christakos S. Mattson M.P. Neuron. 1994; 12: 139-153Abstract Full Text PDF PubMed Scopus (597) Google Scholar, 2Bruce A.J. Boling W. Kindy M.S. Peschon J. Kraemer P.J. Carpenter M.K. Holtsberg F.W. Mattson M.P. Nat. Med. 1996; 2: 788-794Crossref PubMed Scopus (840) Google Scholar). Cell culture studies have shown that TNF can protect cultured neurons against glucose deprivation-induced injury and excitatory amino acid toxicity, by stabilizing intracellular calcium concentration (1Cheng B. Christakos S. Mattson M.P. Neuron. 1994; 12: 139-153Abstract Full Text PDF PubMed Scopus (597) Google Scholar). Moreover, experiments using mice deficient in TNF receptors demonstrated that endogenous TNF protects neurons against ischemic and excitotoxic insults, with induction of an antioxidative pathway (2Bruce A.J. Boling W. Kindy M.S. Peschon J. Kraemer P.J. Carpenter M.K. Holtsberg F.W. Mattson M.P. Nat. Med. 1996; 2: 788-794Crossref PubMed Scopus (840) Google Scholar). However, the exact mechanisms by which TNF protects neurons are largely factor κB (NFκB) is a ubiquitous transcription factor that is activated by a variety of cytokines including TNF (3Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2008) Google Scholar, 4Baldwin A.S. Annu. Rev. Immunol. 1994; 14: 649-681Crossref Scopus (5540) Google Scholar) and is thought to be a key regulator of genes involved in inflammation, response to infection, and stress. Conventional NFκB is a heterodimer that consists of p50 and p65 subunits. The activity of NFκB is strictly regulated by one of the IκB inhibitory proteins, such as IκBα or IκBβ, which forms a complex with NFκB and keeps NFκB in the cytoplasm (3Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2008) Google Scholar, 4Baldwin A.S. Annu. Rev. Immunol. 1994; 14: 649-681Crossref Scopus (5540) Google Scholar). When cells receive signals that activate NFκB, IκBs are phosphorylated and degraded through a ubiquitin/proteasome pathway (5Baeurle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). Coincident with IκB degradation, activated NFκB translocates to the nucleus and participates in the transactivation of a variety of genes, including genes for inflammatory cytokines, chemokines, and cell adhesion molecules (5Baeurle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). dominant negative mutations of IκBα that are not phosphorylated and therefore not proteolyzed have been reported (6Brockmann J.A. Scherr D.C. McKinsy T.A. Hall S.M. Qi X. Le W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar, 7Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (929) Google Scholar). One such protein is a mutant IκBα that contains serine-to-alanine mutations at amino acids 32 and 36, which inhibit signal-induced phosphorylation and subsequent proteasome-mediated degradation of IκBα. This form of mutant IκB has been used to demonstrate that inhibition of NFκB induces apoptosis through a variety of cancer agents (8Wang A.S. 1996; PubMed Scopus Google Scholar, T. 1996; PubMed Scopus Google Scholar). are that to key molecules in the induction or the of One such pathway is regulated by the expression of the and Bcl-2 is an intracellular protein that to and the nuclear D. J. PubMed Scopus Google Scholar, S. S. S. Google Scholar) and has been shown to cell by apoptosis in a variety of cell S. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar, D. Cell. Full Text PDF PubMed Scopus Google Scholar). The bcl-x to cell death. Bcl-x are a and form or a form the T. X. G. Cell. Full Text PDF PubMed Scopus Google Scholar, W. J. Immunol. 1994; Google Scholar). The form cell the form as a dominant bcl-2 and bcl-x apoptosis T. X. G. Cell. Full Text PDF PubMed Scopus Google Scholar, W. J. Immunol. 1994; Google Scholar). forms a with Bcl-2 or and the death activity of anti-apoptotic proteins T. S. S. U. S. 1994; Scholar, 1994; PubMed Scopus Google Scholar). the expression of Bcl-2 J. U. S. 1994; PubMed Scopus Google Scholar) and neuronal death D. S. J. Neuron. 1994; Full Text PDF PubMed Scopus Google and expression of Bcl-2 and has been in neurons that are to ischemic J. K. J. PubMed Scopus Google Scholar) brain injury. the expression is increased in neurons in the ischemic brain S. G. S. PubMed Scopus Google Scholar). However, the mechanism by which expression of is is we increased expression of Bcl-2 and Bcl-x primary neurons with TNF. inhibition of NFκB activation by overexpression of the mutant form of IκB the up-regulation of Bcl-2 and Bcl-x expression and the neuroprotective effect induced by TNF. studies have demonstrated that overexpression of Bcl-2 in neurons can or cell death induced by a variety of PubMed Scopus Google Scholar, S. Cell. Full Text PDF PubMed Scopus Google Scholar) and in J. U. S. 1994; PubMed Scopus Google Scholar, D. S. J. Neuron. 1994; Full Text PDF PubMed Scopus Google Scholar). of protects neurons apoptosis induced by factor P.A. 1995; Scopus Google Scholar). we levels of Bcl-2 expression in hypoxia- and nitric oxide-induced neuronal apoptosis S. G. Cell PubMed Scopus Google Scholar, S. Mol. PubMed Scopus Google Scholar). Bcl-2 and Bcl-x are induced in neurons to S. G. S. PubMed Scopus Google Scholar) and J. K. J. PubMed Scopus Google Scholar). is that the endogenous levels of Bcl-2 and Bcl-x neuronal data demonstrated that TNF protects neurons against hypoxia- and nitric oxide-induced and Bcl-2 and Bcl-x of neurons with Bcl-2 or Bcl-x antisense which up-regulation of Bcl-2 or Bcl-x expression, respectively, significantly the neuroprotective effect induced by that Bcl-2 and Bcl-x induction is involved in the neuroprotective action of of both the antisense ODN, which up-regulation of Bcl-2 and Bcl-x expression, to not inhibit TNF-induced studies that cell is by Bcl-2 or T. S. S. U. S. 1994; Scholar, 1994; PubMed Scopus Google Scholar). the of to Bcl-2 and Bcl-x to be for cell TNF-induced expression of Bcl-2 and Bcl-x the of expression was by TNF. Moreover, the that overexpression of Bcl-2 protects neurons anti-apoptotic protein as a factor for neuronal against hypoxia- or nitric oxide-induced injury. of TNF has been in an ischemic brain injury T. J. PubMed Scopus Google in which the is in the expression of Bcl-2 and Bcl-x has been in neurons that are to ischemic brain injury J. K. J. PubMed Scopus Google Scholar). be that TNF by brain cells Bcl-2 and Bcl-x expression in neurons and protects ischemic cells including neurons receptors for TNF. receptors have been characterized and TNF receptor and TNF receptor the which not in cells J.A. J. W. EMBO J. 1994; PubMed Scopus Google induced Bcl-2 and Bcl-x expression and protected hippocampal neurons against hypoxia- and is to a pathway a that induces of which in activation of the transcription factor NFκB Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). demonstrated that induced NFκB activity in cultured hippocampal neurons as determined by gel in with a by Mattson M.P. W. K. J. PubMed Scopus Google Scholar). experiments using that inhibition of NFκB activation completely the neuroprotective effect and inhibited the induction of Bcl-2 and Bcl-x expression in TNF-treated These indicate that the neuroprotective effect and up-regulation of Bcl-2 and Bcl-x expression induced by TNF are by activation of NFκB and apoptosis was by studies that inhibition of NFκB, by the IκB (8Wang A.S. 1996; PubMed Scopus Google Scholar, T. 1996; PubMed Scopus Google Scholar) or in cells Baltimore D. 1996; PubMed Scopus Google in increased to has been shown that NFκB activation induces apoptosis in cells K. K. T. 1994; PubMed Scopus Google Scholar, J. D. B. P.J. Cell. Full Text PDF PubMed Scopus Google Scholar) including cells and is not at NFκB activation is involved in apoptosis in cells and is for preventing apoptosis in neuronal has been reported that activation of NFκB protected hippocampal neurons against using κB DNA M.P. W. K. J. PubMed Scopus Google and that inhibition of NFκB in cells by to κB DNA or induced apoptosis that not be by factor G. J. PubMed Scopus Google Scholar). These with indicate that the NFκB is involved in of apoptosis in neuronal of the of NFκB activation in the regulation of expression of proteins that anti-apoptotic be demonstrated that up-regulation of Bcl-2 and Bcl-x expression induced by TNF is completely inhibited by IκB that Bcl-2 and Bcl-x are the of The bcl-x genes have complex and the have not been the of NFκB has been demonstrated in the of bcl-2 J.A. PubMed Scopus Google Scholar). the regulation of Bcl-2 and Bcl-x expression that we have to be by NFκB in neurons is to an in the bcl-2 to bcl-2 and expression of genes be induced by TNF through NFκB has been reported that the expression of is induced by which is by κB in hippocampal neurons M.P. W. K. J. PubMed Scopus Google Scholar). Moreover, in the the neuroprotective effect of TNF was blocked by IκB by antisense against bcl-2 in both of which TNF-induced up-regulation of Bcl-2 and Bcl-x expression was completely This that proteins that a effect be induced by NFκB activation in TNF-treated findings demonstrated that induction of Bcl-2 and Bcl-x expression through NFκB activation is involved in the neuroprotective effect of TNF. that inhibition of NFκB activation by expression of a mutant form of IκB the neuroprotective effect of TNF inhibition of TNF-induced up-regulation of Bcl-2 and Bcl-x expression by the antisense that genes for anti-apoptotic activity are induced by NFκB of the genes to the regulation of neuronal and to for and chronic neurodegenerative studies have demonstrated that overexpression of Bcl-2 in neurons can or cell death induced by a variety of PubMed Scopus Google Scholar, S. Cell. Full Text PDF PubMed Scopus Google Scholar) and in J. U. S. 1994; PubMed Scopus Google Scholar, D. S. J. Neuron. 1994; Full Text PDF PubMed Scopus Google Scholar). of protects neurons apoptosis induced by factor P.A. 1995; Scopus Google Scholar). we levels of Bcl-2 expression in hypoxia- and nitric oxide-induced neuronal apoptosis S. G. Cell PubMed Scopus Google Scholar, S. Mol. PubMed Scopus Google Scholar). Bcl-2 and Bcl-x are induced in neurons to S. G. S. PubMed Scopus Google Scholar) and J. K. J. PubMed Scopus Google Scholar). is that the endogenous levels of Bcl-2 and Bcl-x neuronal data demonstrated that TNF protects neurons against hypoxia- and nitric oxide-induced and Bcl-2 and Bcl-x of neurons with Bcl-2 or Bcl-x antisense which up-regulation of Bcl-2 or Bcl-x expression, respectively, significantly the neuroprotective effect induced by that Bcl-2 and Bcl-x induction is involved in the neuroprotective action of of both the antisense ODN, which up-regulation of Bcl-2 and Bcl-x expression, to not inhibit TNF-induced studies that cell is by Bcl-2 or T. S. S. U. S. 1994; Scholar, 1994; PubMed Scopus Google Scholar). the of to Bcl-2 and Bcl-x to be for cell TNF-induced expression of Bcl-2 and Bcl-x the of expression was by TNF. Moreover, the that overexpression of Bcl-2 protects neurons anti-apoptotic protein as a factor for neuronal against hypoxia- or nitric oxide-induced injury. of TNF has been in an ischemic brain injury T. J. PubMed Scopus Google in which the is in the expression of Bcl-2 and Bcl-x has been in neurons that are to ischemic brain injury J. K. J. PubMed Scopus Google Scholar). be that TNF by brain cells Bcl-2 and Bcl-x expression in neurons and protects ischemic injury. cells including neurons receptors for TNF. receptors have been characterized and TNF receptor and TNF receptor the which not in cells J.A. J. W. EMBO J. 1994; PubMed Scopus Google induced Bcl-2 and Bcl-x expression and protected hippocampal neurons against hypoxia- and is to a pathway a that induces of which in activation of the transcription factor NFκB Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). demonstrated that induced NFκB activity in cultured hippocampal neurons as determined by gel in with a by Mattson M.P. W. K. J. PubMed Scopus Google Scholar). experiments using that inhibition of NFκB activation completely the neuroprotective effect and inhibited the induction of Bcl-2 and Bcl-x expression in TNF-treated These indicate that the neuroprotective effect and up-regulation of Bcl-2 and Bcl-x expression induced by TNF are by activation of The NFκB and apoptosis was by studies that inhibition of NFκB, by the IκB (8Wang A.S. 1996; PubMed Scopus Google Scholar, T. 1996; PubMed Scopus Google Scholar) or in cells Baltimore D. 1996; PubMed Scopus Google in increased to has been shown that NFκB activation induces apoptosis in cells K. K. T. 1994; PubMed Scopus Google Scholar, J. D. B. P.J. Cell. Full Text PDF PubMed Scopus Google Scholar) including cells and is not at NFκB activation is involved in apoptosis in cells and is for preventing apoptosis in neuronal has been reported that activation of NFκB protected hippocampal neurons against using κB DNA M.P. W. K. J. PubMed Scopus Google and that inhibition of NFκB in cells by to κB DNA or induced apoptosis that not be by factor G. J. PubMed Scopus Google Scholar). These with indicate that the NFκB is involved in of apoptosis in neuronal of the of NFκB activation in the regulation of expression of proteins that anti-apoptotic be demonstrated that up-regulation of Bcl-2 and Bcl-x expression induced by TNF is completely inhibited by IκB that Bcl-2 and Bcl-x are the of The bcl-x genes have complex and the have not been the of NFκB has been demonstrated in the of bcl-2 J.A. PubMed Scopus Google Scholar). the regulation of Bcl-2 and Bcl-x expression that we have to be by NFκB in neurons is to an in the bcl-2 to bcl-2 and expression of genes be induced by TNF through NFκB has been reported that the expression of is induced by which is by κB in hippocampal neurons M.P. W. K. J. PubMed Scopus Google Scholar). Moreover, in the the neuroprotective effect of TNF was blocked by IκB by antisense against bcl-2 in both of which TNF-induced up-regulation of Bcl-2 and Bcl-x expression was completely This that proteins that a effect be induced by NFκB activation in TNF-treated neurons. findings demonstrated that induction of Bcl-2 and Bcl-x expression through NFκB activation is involved in the neuroprotective effect of TNF. that inhibition of NFκB activation by expression of a mutant form of IκB the neuroprotective effect of TNF inhibition of TNF-induced up-regulation of Bcl-2 and Bcl-x expression by the antisense that genes for anti-apoptotic activity are induced by NFκB of the genes to the regulation of neuronal and to for and chronic neurodegenerative for and of for and for for of and and for the Bcl-2

ABSTRACT Vascular endothelial growth factor (VEGF) is known as a selective endothelial cell mitogen that promotes angiogenesis and increases blood vessel formation in vivo. Here we report that VEGF has protective effects on primary hippocampal neurons against glutamate toxicity by acting on phosphatidylinositol 3‐kinase (PI3‐K)/Akt pathways and mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) pathways, operating independently of one another. Decrease in the VEGF's neuroprotective effect resulting from inhibition of either pathway alone was significantly enhanced by simultaneous inhibition of both pathways. However, adenovirus‐mediated expression of either the active form of Akt or of MEK significantly inhibited glutamate‐induced neuronal death. Treatment with antisense ODN against Flk‐1, but not against Flt‐1, blocked the effect of VEGF on the activation of Akt and ERK and glutamate‐induced neuronal death. These findings suggest that VEGF has a protective effect on hippocampal neurons against glutamate‐induced toxicity and that this effect is dependent on PI3‐ K/Akt and MEK/ERK signaling pathways mediated primarily through Flk‐1 receptor.

Survival factors suppress apoptosis by activating the serine/threonine kinase Akt. To investigate the molecular mechanism underlying activated Akt's ability to protect neurons from hypoxia or nitric oxide (NO) toxicity, we focused on the apoptosis-related functions of p53 and caspases. We eliminated p53 by employing p53-deficient neurons and increased p53 by infection with recombinant adenovirus capable of transducing p53 expression, and we now show that p53 is implicated in the apoptosis induced by hypoxia or NO treatments of primary cultured hippocampal neurons. Although hypoxia and NO induced p53, treatment with insulin-like growth factor-1 significantly inhibited caspase-3-like activation, neuronal death and transcriptional activity of p53. These insulin-like growth factor-1 effects are prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. Adenovirus-mediated expression of activated-Akt kinase suppressed p53-dependent transcriptional activation of responsive genes such as Bax, suppressed caspase-3-like protease activity and suppressed neuronal cell death with no effect on the cellular accumulation and nuclear translocation of p53. In contrast, overexpression of kinase-defective Akt failed to suppress these same activities. These results suggest a mechanism where Akt kinase activation reduces p53's transcriptional activity that ultimately rescues neurons from hypoxia- or NO-mediated cell death.

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.