Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily ref lects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism.

Neuroglobin (Ngb), a protein related to myoglobin and hemoglobin but expressed predominantly in the brain, is induced by neuronal hypoxia and cerebral ischemia and protects against hypoxic or ischemic neuronal injury. We engineered transgenic mice that overexpress murine Ngb under the control of a chicken ␤-actin promoter, resulting in enhanced Ngb expression in multiple cell types and multiple tissues, including brain and heart. In Ngb-overexpressing transgenic mice compared with wild-type littermates, the volume of cerebral infarcts after occlusion of the middle cerebral artery was reduced by Ϸ30%, and the volume of myocardial infarcts produced by occlusion of the left anterior descending coronary artery was reduced by Ϸ25%. Ngb overexpression was associated with enhanced expression of endothelial nitric oxide synthase in vascular endothelial cells. These findings extend prior evidence for cytoprotection by Ngb and suggest both direct (parenchymatous) and indirect (vasomotor) protective mechanisms.endothelial nitric oxide synthase ͉ myocardial infarction ͉ stroke

B and T lymphocytes undergoing apoptosis in response to anti-immunoglobulin M antibodies and dexamethasone, respectively, were found to have increased amounts of messenger RNA for the inositol 1,4,5-trisphosphate receptor (IP3R) and increased amounts of IP3R protein. Immunohistochemical analysis revealed that the augmented receptor population was localized to the plasma membrane. Type 3 IP3R (IP3R3) was selectively increased during apoptosis, with no enhancement of type 1 IP3R (IP3R1). Expression of IP3R3 antisense constructs in S49 T cells blocked dexamethasone-induced apoptosis, whereas IP3R3 sense, IP3R1 sense, or IP3R1 antisense control constructs did not block cell death. Thus, the increases in IP3R3 may be causally related to apoptosis.

T cell activation is dependent on both a primary signal delivered through the T cell receptor and a secondary costimulatory signal mediated by coreceptors. Although controversial, costimulation is thought to act through the specific redistribution and clustering of membrane and intracellular kinase-rich lipid raft microdomains at the contact site between T cells and antigen-presenting cells. This site has been termed the immunological synapse. Endogenous mediators of raft clustering in lymphocytes have not been identified, although they are essential for T cell activation. We now demonstrate that agrin, an aggregating protein crucial for formation of the neuromuscular junction, is also expressed in lymphocytes and is important in reorganization of membrane lipid microdomains and setting the threshold for T cell signaling. Our data show that agrin induces the aggregation of signaling proteins and the creation of signaling domains in both immune and nervous systems through a common lipid raft pathway.

Cerebral hypoxia and ischemia trigger endogenous protective mechanisms that can prevent or limit brain damage. Understanding these mechanisms may lead to new therapeutic strategies for stroke and related disorders. Neuroglobin (Ngb), a recently discovered protein that is distantly related to hemoglobin and myoglobin, is expressed predominantly in brain neurons, and appears to modulate hypoxic-ischemic brain injury. Evidence includes the observations that neuronal hypoxia and cerebral ischemia induce Ngb expression, that enhancing Ngb expression reduces-and knocking down Ngb expression increases-hypoxic neuronal injury in vitro and ischemic cerebral injury in vivo, and that Ngb-overexpressing transgenic mice are resistant to cerebral infarction. However, the mechanisms that underlie hypoxic induction of and neuroprotection by Ngb are still unclear.

Neuroglobin (Ngb), a vertebrate globin expressed primarily in neurons, is induced by and protects against neuronal hypoxia and cerebral ischemia. To investigate the spectrum and mechanism of Ngb's neuroprotective action, we studied the effect of transgenic overexpression of Ngb on NMDA and ␤-amyloid (A␤) toxicity in murine cortical neuron cultures in vitro and on the phenotype of Alzheimer's disease (AD) transgenic (APP Sw,Ind) mice. Compared with cortical neuron cultures from wild-type mice, cultures from Ngb-overexpressing transgenic (Ngb-Tg mice) were resistant to the toxic effects of NMDA and A␤(25-35), as measured by polarization of cell membrane lipid rafts, mitochondrial aggregation, lactate dehydrogenase release, and nuclear fragmentation. In addition, compared with APP Sw,Ind mice, double-transgenic (Ngb-Tg ؋ APP Sw,Ind) mice showed reductions in thioflavin-S-stained extracellular A␤ deposits, decreased levels of A␤(1-40) and A␤(1-42), and improved behavioral performance in a Y-maze test of spontaneous alternations. These findings suggest that the spectrum of Ngb's neuroprotective action extends beyond hypoxic-ischemic insults. Ngb may protect neurons from NMDA and A␤ toxicity by inhibiting the formation of a death-signaling membrane complex, and interventions that increase Ngb expression could have therapeutic application in AD and other neurodegenerative disorders.globin ͉ lipid raft ͉ neurodegeneration ͉ neuroprotection ͉ NMDA N euroglobin (Ngb) is a vertebrate globin that is localized to neurons, binds O 2 and other gaseous messengers, is transcriptionally induced by hypoxia and ischemia, and protects against hypoxic neuronal death and ischemic brain injury (1). However, the mechanism through which Ngb exerts its neuroprotective effect is unclear. We found recently that hypoxia induces polarization of plasma membrane lipid microdomains (rafts) that appear to mediate hypoxic neuronal death because inhibiting their formation enhances survival of hypoxic neurons (A.A.K., unpublished data). Moreover, raft polarization precedes caspase activation and mitochondrial release of cytochrome c, indicating that it is an earlyand, thus, potentially reversible-event in cell-death pathways. Neurons from Ngb-overexpressing transgenic (Ngb-Tg) mice (2, 3) do not undergo membrane polarization in response to hypoxia and are resistant to hypoxic cell death, suggesting that Ngb may protect neurons from hypoxia by interfering with this cell-death signaling complex.In addition to its protective effects against neuronal hypoxia in vitro and cerebral ischemia in vivo, Ngb also reduces oxidative injury from H 2 O 2 or paraquat (4) and ␤-amyloid (A␤)-induced toxicity (5) in cultured neural cell lines. To determine whether inhibition of membrane polarization-dependent neuronal death by Ngb extends to insults other than hypoxia, we investigated the effect of transgenic overexpression of Ngb on the neuronal toxicity of the excitatory amino acid, NMDA, and the Alzheimer's disease (AD)-related peptide, A␤. Compartmentalization by ...

The signal transduction pathways involved in neuronal death are not well understood. Neuroglobin (Ngb), a recently discovered vertebrate globin expressed predominantly in the brain, shows increased expression in neurons in response to oxygen deprivation and protects neurons from ischemic and hypoxic death. The mechanism of this neuroprotection is unclear. We examined the surface distribution of raft membrane microdomains in cortical neuron cultures during hypoxia using the raft marker cholera toxin B (CTx-B) subunit Mechanistically, we demonstrate that hypoxia induces rapid polarization of somal membranes and aggregation of microdomains with the subjacent mitochondrial network. This signaling complex is formed well before neurons commit to die, consistent with an early role in death signal transduction. Ngb-expressing neurons and neurons from Ngboverexpressing transgenic (Ngb-Tg) mice do not undergo microdomain polarization or mitochondrial aggregation in response to, and are resistant to death from hypoxia. We link the protective actions of Ngb to inhibition of Pak1 kinase activity and Rac1-GDI disassociation, and inhibition of actin assembly and death-signaling module polarization.

Immune responses in lymphocytes require cellular accumulation of large amounts of calcium (Ca2+) from extracellular sources. In the T cell tumor line Jurkat, receptors for the Ca(2+)-releasing messenger inositol 1,4,5-trisphosphate (IP3) were localized to the plasma membrane (PM). Capping of the T cell receptor-CD3 complex, which is associated with signal transduction, was accompanied by capping of IP3 receptors. The IP3 receptor on T cells appears to be responsible for the entry of Ca2+ that initiates proliferative responses.