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 antiapoptotic 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, NFB, in the regulation of Bcl-2 and Bcl-x expression in TNF-treated neurons, an adenoviral vector capable of expressing a mutated form of IB was used to infect neurons prior to TNF treatment. Expression of the mutant NFB completely inhibited NFB 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 NFB activation is involved in the neuroprotective action of TNF against hypoxia-or nitric oxide-induced injury.

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.

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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.

Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3-like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.