Nuclear translocation of X-linked inhibitor of apoptosis (XIAP) determines cell fate after hypoxia ischemia in neonatal brain.

The inhibitors of apoptosis (IAPs) are emerging as key proteins in the control of cell death. In this study, we evaluated the expression and subcellular distribution of the antiapoptotic protein X-linked IAP (XIAP), and its interactions with the XIAP-associated factor 1 (XAF1) in neonatal rat brain following hypoxia-ischemia (HI). HI triggered the mitochondrial release of cytochrome c, Smac/DIABLO, and caspase 3 activation. Confocal microscopy detected XIAP-specific immunofluorescence in the cytoplasm under normal condition, which exhibited a diffuse distribution at 6 h post-HI and by 12 h the majority of XIAP was redistributed into the nucleus. XIAP nuclear translocation was confirmed by subcellular fractionations and by expressing FLAG-tagged XIAP in primary cortical neurons. Over-expression of XIAP significantly reduced, whereas XIAP gene silencing further enhanced cell death, demonstrating a specific requirement of cytoplasmic XIAP for cell survival. An elevated level of cytosolic XIAP was also evident under the conditions of neuroprotection by fibroblast growth factor-1. XAF1 expression was increased temporally and there was increased nuclear co-localization with XIAP in hypoxic-ischemic cells. XIAP co-immunoprecipitated > 9-fold XAF1 protein concurrent with decreased association with caspases 9 and 3. This is evidenced by the enhanced caspase 3 activity and neuronal death. Our findings implicate XIAP nuclear translocation in neuronal death and point to a novel mechanism in the regulation of hypoxic-ischemic brain injury.

Neuronal pentraxin 1: a novel mediator of hypoxic-ischemic injury in neonatal brain.

Neonatal hypoxic-ischemic brain injury is a major cause of neurological disability and mortality. Its therapy will likely require a greater understanding of the discrete neurotoxic molecular mechanism(s) triggered by hypoxia-ischemia (HI). Here, we investigated the role of neuronal pentraxin 1 (NP1), a member of a newly recognized subfamily of "long pentraxins," in the HI injury cascade. Neonatal brains developed marked infarcts in the ipsilateral cerebral hemisphere at 24 hr and showed significant loss of ipsilateral striatal, cortical, and hippocampal volumes at 7 d after HI compared with the contralateral hemisphere and sham controls. Immunofluorescence analyses revealed elevated neuronal expression of NP1 in the ipsilateral cerebral cortex from 6 hr to 7 d and in the hippocampal CA1 and CA3 regions from 24 hr to 7 d after HI. These same brain areas developed infarcts and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells within 24-48 hr of HI. In primary cortical neurons, NP1 protein was induced >2.5-fold (p < 0.001) after their exposure to hypoxia that caused approximately 30-40% neuronal death. Transfecting cortical neurons with antisense oligodeoxyribonucleotides directed against NP1 mRNA (NP1AS) significantly inhibited (p < 0.01) hypoxia-induced NP1 protein induction and neuronal death (p < 0.001), demonstrating a specific requirement of NP1 in hypoxic neuronal injury. NP1 protein colocalized and coimmunoprecipitated with the fast excitatory AMPA glutamate receptor subunit (GluR1) in primary cortical neurons, and hypoxia induced a time-dependent increase in NP1-GluR1 interactions. NPIAS also protected against AMPA-induced neuronal death (p < 0.05), implicating a role for NP1 in the excitotoxic cascade. Our results show that NP1 induction mediates hypoxic-ischemic injury probably by interacting with and modulating GluR1 and potentially other excitatory glutamate receptors.

Neuronal pentraxin 1 induction in hypoxic-ischemic neuronal death is regulated via a glycogen synthase kinase-3α/ß dependent mechanism.

Intracellular signaling pathways that regulate the production of lethal proteins in central neurons are not fully characterized. Previously, we reported induction of a novel neuronal protein neuronal pentraxin 1 (NP1) in neonatal brain injury following hypoxia-ischemia (HI); however, how NP1 is induced in hypoxic-ischemic neuronal death remains elusive. Here, we have elucidated the intracellular signaling regulation of NP1 induction in neuronal death. Primary cortical neurons showed a hypoxic-ischemia time-dependent increase in cell death and that NP1 induction preceded the actual neuronal death. NP1 gene silencing by NP1-specific siRNA significantly reduced neuronal death. The specificity of NP1 induction in neuronal death was further confirmed by using NP1 (-/-) null primary cortical neurons. Declines in phospho-Akt (i.e. deactivation) were observed concurrent with decreased phosphorylation of its downstream substrate GSK-3α/ß (at Ser21/Ser9) (i.e. activation) and increased GSK-3α and GSK-3ß kinase activities, which occurred prior to NP1 induction. Expression of a dominant-negative inhibitor of Akt (Akt-kd) blocked phosphorylation of GSK-3α/ß and subsequently enhanced NP1 induction. Whereas, overexpression of constitutively activated Akt (Akt-myr) or wild-type Akt (wtAkt) increased GSK-α/ß phosphorylation and attenuated NP1 induction. Transfection of neurons with GSK-3α siRNA completely blocked NP1 induction and cell death. Similarly, overexpression of the GSK-3ß inhibitor Frat1 or the kinase mutant GSK-3ßKM, but not the wild-type GSK-3ßWT, blocked NP1 induction and rescued neurons from death. Our findings clearly implicate both GSK-3α- and GSK-3ß-dependent mechanism of NP1 induction and point to a novel mechanism in the regulation of hypoxic-ischemic neuronal death.

Transgenic expression of human FGF-1 protects against hypoxic-ischemic injury in perinatal brain by intervening at caspase-XIAP signaling cascades.

Perinatal hypoxia-ischemia (HI) is a major cause of neurological disability and mortality in infant and children. In the present study, we explored the neuroprotective efficacy of FGF-1 in a rat model of perinatal HI. Carotid ligation combined with hypoxia caused marked infarctions in the ipsilateral cerebral hemisphere with significant loss of ipsilateral striatal, cortical and hippocampal volumes. Morphological analyses revealed both apoptotic and necrotic form of neuronal death determined by Nissl histology, dark-field microscopy and TUNEL staining. HI induced a marked increase in activated caspase-9, caspase-3 and PARP cleavage at 12 h to 7 days after HI in brain areas displaying TUNEL (+) cells. In addition, expression of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) was decreased under similar conditions of HI. Expression of human FGF-1 in brain significantly reduced the extent of both apoptotic and necrotic injury caused by HI. FGF-1 attenuated the HI-induced increase in activated caspase-3, caspase-9 and cleaved PARP protein levels and markedly blocked the HI-induced decrease in XIAP expression under the conditions at which FGF-1 showed significant neuroprotection. These findings demonstrate that FGF-1 prevents the onset of both apoptotic and necrotic death in neurons otherwise "destined to die" following hypoxic-ischemic injury by intervening at the level of caspase-signaling cascades and by restoring prosurvival protein XIAP expression in central neurons.

Vascular endothelial growth factor mediates vasogenic edema in acute lead encephalopathy.

Brain injury from inorganic Pb(2+) is considered the most important environmental childhood health hazard worldwide. The microvasculature of the developing brain is uniquely susceptible to high level Pb(2+) toxicity (ie, Pb(2+) encephalopathy) characterized by cerebellar hemorrhage, increased blood-brain barrier permeability, and vasogenic edema. However, the specific molecular mediators of Pb(2+) encephalopathy have been elusive. We found that Pb(2+) induces vascular endothelial growth factor/vascular permeability factor (VEGF) in cultured astrocytes (J Biol Chem, 2000;275:27874-27882). The study presented here asks if VEGF dysregulation contributes mechanistically to Pb(2+) encephalopathy. Neonatal rats exposed to 4% Pb-carbonate develop the histopathological features of Pb(2+) encephalopathy seen in children. Cerebellar VEGF expression increased approximately twofold (p < 0.01) concurrent with the development of cerebellar microvascular hemorrhage, enhanced vascular permeability to serum albumin, and vasogenic cerebellar edema (p < 0.01). No change in VEGF expression occurred in cerebral cortex that does not develop these histopathological complications of acute Pb(2+) intoxication. Pb(2+) exposure increased phosphorylation of cerebellar Flk-1 VEGF receptors and the Flk-1 inhibitor CEP-3967 completely blocked cerebellar edema formation without affecting microhemorrhage formation or blood-brain barrier permeability. This establishes that Pb(2+)-induced vasogenic edema formation develops via a Flk-1-dependent mechanism and suggests that the vascular permeability caused by Pb(2+) is Flk-1 independent.

Neuroprotection by scatter factor/hepatocyte growth factor and FGF-1 in cerebellar granule neurons is phosphatidylinositol 3-kinase/akt-dependent and MAPK/CREB-independent.

Neuroprotective actions of scatter factor/hepatocyte growth factor (SF/HGF) have not been described. We examined the effects of SF/HGF in comparison to acidic fibroblast growth factor-1 (FGF-1) on N-methyl-D-aspartate (NMDA) and quinolinic acid (QUIN)-induced excitotoxicity in primary cerebellar granule neurons. Exposure to NMDA or QUIN for 24 h resulted in concentration-dependent cell death (p < 0.001) that was completely attenuated (p < 0.001) by pre-treatment of cells with SF/HGF (50 ng/mL) or FGF-1 (40 ng/mL). SF/ HGF and FGF-1 activated both Akt and MAP-kinase > threefold (p < 0.001). Neither SF/HGF nor FGF-1 activated cyclic AMP-response element binding protein (CREB), a downstream target of MAP-kinase, whereas brain-derived neurotrophic factor (BDNF) activated both MAP-kinase and CREB in granule neurons. Neuroprotection against NMDA or QUIN by SF/HGF and FGF-1 was negated by the addition of LY294002 (10 microM) or wortmannin (100 microM), two distinct inhibitors of phosphatidylinositol 3-kinase (P13-K), but not by the MAP-kinase kinase (MEK) inhibitor PD98059 (33 microm). Likewise, expression of a dominant-negative mutant of Akt (Akt-kd) completely prevented the neuroprotective actions of SF/HGF and FGF-1. Overexpression of a constitutively activated Akt (Akt-myr) or wild-type Akt (wtAkt) attenuated excitotoxic cell death. These data show that both SF/HGF and FGF-1 protect cerebellar granule neurons against excitotoxicity with similar potency in a P13-K/Akt-dependent and MAP-kinase/CREB-independent manner.