Spinal cord injury is a devastating condition, with much of the clinical disability resulting from disruption of white matter tracts. Recent reports suggest a component of glutamate excitotoxicity in spinal cord injury. In this study, the role of glutamate and mechanism of release of this excitotoxin were investigated in rat dorsal column slices subjected to 60 min of anoxia or 15 sec of mechanical compression at a force of 2 gm in vitro. The broad-spectrum glutamate antagonist kynurenic acid (1 mm) and the selective AMPA antagonist GYKI52466 (30 microm) were protective against anoxia (compound action potential amplitude recovered to 56 vs 27% without drug). GYKI52466 was also effective against trauma (65 vs 35%). Inhibition of Na(+)-dependent glutamate transport with dihydrokainate or l-trans-pyrrolidine-2,4-dicarboxylic acid (1 mm each) protected against anoxia (65-75 vs 25%) and trauma (70 vs 35%). The depletion of cytosolic glutamate in axon cylinders and oligodendrocytes by anoxia was completely prevented by glutamate transport inhibition. Immunohistochemistry revealed that a large component of injury occurred in the myelin sheath and was prevented by AMPA receptor blockade or glutamate transport inhibitors. We conclude that release of glutamate by reversal of Na(+)-dependent glutamate transport with subsequent activation of AMPA receptors is an important mechanism in spinal cord white matter anoxic and traumatic injury.

Excitatory synaptic transmission in the central nervous system is mediated primarily by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. The myriad intracellular responses arising from the activation of the NMDA and AMPA receptors have previously been attributed to the flow of Ca2+ and/or Na+ through these ion channels. Here we report that the binding of the agonist AMPA to its receptor can generate intracellular signals that are independent of Ca2+ and Na+ in rat cortical neurons. In the absence of intracellular Ca2+ and Na+, AMPA, but not NMDA, brought about changes in a guanine-nucleotide-binding protein (Galpha[il]) that inhibited pertussis toxin-mediated ADP-ribosylation of the protein in an in vitro assay. This effect was observed in intact neurons treated with AMPA as well as in isolated membranes exposed to AMPA, and was also found in MIN6 cells, which express functional AMPA receptors but have no metabotropic glutamate receptors. AMPA also inhibited forskolin-stimulated activity of adenylate cyclase in neurons, demonstrating that Gi proteins were activated. Moreover, both Gbetagamma blockage and co-precipitation experiments demonstrated that the modulation of the Gi protein arose from the association of Galpha(il) with the glutamate receptor-1 (GluR1) subunit. These results suggest that, as well as acting as an ion channel, the AMPA receptor can exhibit metabotropic activity.

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Abstract:The transcription factor E2F1 is known to mediate apoptosis in isolated quiescent and postmitotic cardiac myocytes, and its absence decreases the size of brain infarction following cerebral ischemia. To demonstrate directly that E2F1 modulates neuronal apoptosis, we used cultured cortical neurons to show a temporal association of the transcription and expression of E2F1 in neurons with increased neuronal apoptosis. Cortical neurons lacking E2F1 expression (derived from E2F1 Ϫ/Ϫ mice) were resistant to staurosporine-induced apoptosis as evidenced by the significantly lower caspase 3-like activity and a lesser number of cells with apoptotic morphology in comparison with cortical cultures derived from wild-type mice. Furthermore, overexpressing E2F1 alone using replication-deficient recombinant adenovirus was sufficient to cause neuronal cell death by apoptosis, as evidenced by the appearance of hallmarks of apoptosis, such as the threefold increase in caspase 3-like activity and increased laddered DNA fragmentation, in situ endlabeled DNA fragmentation, and numbers of neuronal cells with punctate nuclei. Taken together, we conclude that E2F1 plays a key role in modulating neuronal apoptosis. Key Words: Cortical neurons-Apoptosis-E2F1-Caspase -DNA fragmentation-Replication-deficient adenovirus. J. Neurochem. 75, 91-100 (2000).Neuronal apoptosis has been implicated in the pathogenesis of several neurodegenerative disorders, such as that occurring during delayed neuronal cell death after ischemia, Huntington's disease, Parkinson's disease, and Alzheimer's disease (Thompson, 1995;Rudin and Thompson, 1997). Although the exact molecular pathways underlying neuronal apoptosis after ischemia or other insults are complex and not fully understood (MacManus and Linnik, 1997;D'Mello, 1998;Lee et al., 1999), we hypothesized that several known cell death elements may play critical roles. One of these elements is the transcription factor E2F1.E2F1, a member of a family of six related growth regulatory transcription factors, was first recognized to promote G1 to S-phase transition by trans-activation of genes involved in DNA synthesis, e.g., dihydrofolate reductase and DNA polymerase ␣, and cell cycle control, e.g., cyclin E and cyclin A (reviewed by Dyson, 1998;Nevins, 1998). In cycling cells, the activity of E2F1 is regulated by the retinoblastoma gene product pRb. Hypophosphorylated pRb forms a complex with E2F1 and represses transcription, perhaps by inhibition of histone deacetylase activities (Brehm et al., 1998;Luo et al., 1998;Magnaghi-Jaulin et al., 1998). During G1/S transition, pRb becomes highly phosphorylated by cell cycledependent kinases, such as Cdk4/6 (Taya, 1997), and releases E2F1. An inappropriate increase in content of free E2F1 has been described as a key regulator of cell death by apoptosis in cycling cells, quiescent cells (Johnson et al., 1993;Qin et al., 1994;Kowalik et al., 1995;DeGregori et al., 1997), and postmitotic myocardium and cardiac myocytes (Kirshenbaum et al., 1996;Agah et al., 1997)...

Recent studies have demonstrated a critical role for the oocyte in proliferation and differentiation of granulosa cells and expansion of the cumulus oophorus in vitro. The purpose of this study was to determine if steroid production by cumulus granulosa cells was also modulated by oocytes. Mouse oocyte-cumulus cell complexes (intact) and complexes from which the oocytes were removed microsurgically (oocytectomized; OOX) were cultured for 24 h in the presence or absence of follicle-stimulating hormone (FSH; 150 ng/ml), testosterone (T; 5 x 10(-7) M) or both. Oocytectomy had no effect on the ability of cumulus cells to produce progesterone or estradiol in control cultures or in response to T. However, OOX complexes produced 17- and 36-fold more progesterone than intact complexes when cultured in the presence of FSH or FSH+T, respectively. Oocyte-conditioned medium (maximum 1 oocyte/2 microliters) had no effect on progesterone production by intact cumulus complexes, but reduced the progesterone production by OOX complexes by 75%. This inhibition was directly proportional to the number of oocytes used to condition the medium. Oocytectomy caused a slight decrease (29%) in estradiol production by complexes in the presence of FSH and T; however, OOX complexes in oocyte-conditioned medium produced almost twice as much estradiol as complexes in unconditioned medium. These results indicate that mouse oocytes secrete a factor(s) that inhibits progesterone and stimulates estradiol production by cumulus granulosa cells.