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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)...

It is well established that the peroxyl radicals formed during the thermal decomposition of 2,2'-azobis(amidinopropane), ABAP, in oxygenated water can cleave double-stranded DNA, from which fact it has been concluded that peroxyl radicals, as a general class, can induce DNA strand scission. However, the ABAP-derived radicals are positively charged, and DNA is a negatively charged polyanion. Moreover, the relatively small and, therefore, free to diffuse peroxyl radicals likely to be formed in vivo will generally be negatively charged or neutral. Plasmid supercoiled DNA [pBR 322, 4361 base pairs (bp)] was reacted with known, equal fluxes of two positively charged peroxyl radicals, a negatively charged peroxyl radical, and a neutral peroxyl radical. The two positively charged peroxyl radicals degraded >/=80% of the supercoiled pBR 322 at a flux of 4 radicals/bp, but the negatively charged and neutral peroxyl radicals had no significant effect even at a flux as high as 24 radicals/bp. The same lack of effect on the DNA was also observed with high fluxes of superoxide/hydroperoxyl radicals. Similar results were obtained with another supercoiled DNA, pUC 19, except that pUC 19 is somewhat more sensitive to strand scission by positively charged peroxyl radicals than pBR 322. We conclude that most of the peroxyl radicals likely to be formed in vivo have little or no ability to induce DNA strand scission and that the potential role of electrostatics in radical/DNA reactions should always be considered.

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Summary:The time course of appearance of cells with DNA damage was studied in rats following transient se vere forebrain ischemia. This DNA damage could be de tected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day I in the stri atum and later in the CAl region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered ap optotic type of internucleosomal cleavage, which per sisted without smearing for up to 7 days of reperfusion. In

gp65 and gp55 are glycoprotein components of CNS synapses that are recognised by a single monoclonal antibody, SMgp65. This antibody has now been used to investigate the molecular properties of these two glycoproteins and the structural relationship between them. Both gp65 and gp55 occur in most brain regions as doublets of apparent molecular masses of 63 and 67 kDa, and 52 and 57 kDa, respectively. Striatal samples, however, are enriched in a novel gp65 isoform of 69 kDa. Removal of oligosaccharide residues from gp65 and gp55 with trifluoromethanesulphonic acid shows that gp65 and gp55 are composed of single polypeptide chains of 40 and 28 kDa, respectively. Removal of sialic acid residues with neuraminidase lowers the apparent molecular mass of both glycoproteins by 5-6 kDa. Triton X-114 phase partitioning and alkaline extraction of synaptic membranes indicate that both gp65 and gp55 are integral membrane glycoproteins. Treatment of synaptic membranes with phosphatidylinositol-specific phospholipase C does not solubilise either glycoprotein. One-dimensional peptide and epitope maps obtained by digestion of gp65 and gp55 with endoproteinase lys C or subtilisin are consistent with a close structural relationship between the two molecules. Tryptic digestion of samples enriched in gp65 and/or gp55 results in the formation of a novel immunoreactive 53-kDa species that is resistant to further trypsin degradation except in the presence of 0.1% (wt/vol) sodium dodecyl sulphate. Trypsin treatment of cultures of forebrain neurones in situ lowers the apparent molecular mass of gp65 to 53 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)