Nuclear Factor-kB Contributes to Excitotoxin-Induced Apoptosis in Rat Striatum

Excitotoxin-induced destruction of striatal neurons, proposed as a model of Huntington’s disease, involves a process having the biochemical stigmata of apoptosis. Recent studies suggested that transcription factor nuclear factor (NF)-kB may be involved in excitotoxicity. To further analyze the contribution of NFkB to excitotoxic neuronal death in vivo, changes in binding activities of NFkB and other transcription factors as well as the consequences of inhibiting NFkB nuclear translocation were measured after the infusion of quinolinic acid (120 nmol) into rat striatum. Internucleosomal DNA fragmentation and terminal transferase-mediated dUTP digoxigenin nick end labeling-positive nuclei appeared 12 hr later and intensified over the next 12 hr. NFkB binding activity increased severalfold from 2 to 12 hr, then gradually declined during the next 12 hr. Other transcription factor changes included AP-1, whose binding peaked about 6 hr after quinolinic acid administration, and E2F-1, which was only modestly and transiently elevated. In contrast, quinolinic acid lead to a reduction in OCT-1, beginning after 12 hr, and briefly in SP-1 binding. The NFkB, AP-1, and OCT-1 changes were attenuated both by the N-methyl-D-aspartate receptor antagonist MK-801 and the protein synthesis inhibitor cycloheximide. Moreover, quinolinic acid-induced internucleosomal DNA fragmentation and striatal cell death were significantly reduced by the intrastriatal administration of NFkB SN50, a cell-permeable recombinant peptide that blocks NFkB nuclear translocation. These results illustrate the complex temporal pattern of transcription factor change attending the apoptotic destruction produced in rat striatum by quinolinic acid. They further suggest that NFkB activation contributes to the excitotoxin-induced death of striatal neurons. Neurodegenerative disorders, such as HD, are characterized by the progressive loss of specific central neurons during adult life. Although HD is caused by a polyglutamine expansion in the gene for HD, the exact process by which this abnormality triggers the death of striatal neurons is not yet known. Recent studies of postmortem tissue suggest the degenerative process may involve an apoptotic mechanism (Portera-Cailliau et al., 1995). Glutamate, which can induce oxidative stress in neuronal tissue, has been implicated in the pathogenesis of HD and other neurodegenerative disorders. Indeed, the demise of striatal neurons produced by the glutamate receptor agonist QA as well by kainic acid has been proposed as an animal model of HD (Coyle and Schwarcz, 1976). Recent observations suggest that the QAor kainic acid -induced destruction of striatal cells occurs, at least in part, by an apoptotic mechanism (Ankarcrona et al., 1993; Bonfoco et al., 1995; Filipkowski et al., 1995; Gillardon et al., 1995; Portera-Cailliau et al., 1995; Qin et al., 1996; Simonian et al., 1996). Although the morphological features of the apoptotic process have been well described, just how glutamatergic receptor agonists activate cell death programs at the molecular level remains to be elucidated. Transcription factors, including immediate early genes such as c-jun, E2F-1, OCT-1 and NFkB, have been increasingly implicated in the control of apoptosis (Dragunow and Preston, 1995; Grilli et al., 1996; Ham et al., 1995; Wang and Pittman, 1993). Induction of these regulators of gene expression typically precedes the appearance of internucleosomal DNA fragmentation (Estus et al., 1994). Moreover, antisense, antibody, gene mutation, and pharmacological techniques that selectively inhibit certain transcription factors have been found to block the death of cultured cells, thus suggesting that these factors may be direct contributors to the generation of apoptotic cascades (Estus et al., 1994; Ham et al., 1995; Lin et al., 1995a). NFkB, a member of the Rel transcription factor family, participates in the regulation of a broad array of genes primarily involved in immune and stress defense mechanisms. It has also been linked to the generation of certain cancers and to the control of the cell cycle. In the central nervous system, NFkB is constitutively expressed in both neurons ABBREVIATIONS: HD, Huntington’s disease; QA, quinolinic acid; CHX, cycloheximide; TUNEL, terminal transferase-mediated dUTP-digoxigenin nick end labeling; NMDA, N-methyl-D-aspartate; ANOVA, analysis of variance; IkB, inhibitor kB; NFkB, nuclear factor-kB; AP-1, activator protein 1; CREB, cAMP response element binding protein. 0026-895X/98/010033-10$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:33–42 (1998). 33 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from and glia (Kaltschmidt et al., 1994). A variety of pathogenetic stimuli, including oxidative stress, ischemic insult, and b-amyloid deposition (Kaltschmidt et al., 1997; LegrandPoels et al., 1995; Salminen et al., 1995), can release NFkB from cytosolic sequestration sites where it is bound to a member of the inhibitory protein family, IkB (Beg and Baltimore, 1996; Brown et al., 1993; Liou and Baltimore, 1993). Upon translocated to the nucleus, NFkB acts as a positive regulator of genes favoring either protective or degenerative responses, depending on genetic programs within a particular cell type (Baeuerle, 1991; Baichwal and Baeuerle, 1997; Lipton, 1997). Several NFkB target genes, including P53 and c-Myc, are well established modulators of apoptosis (Wu and Lozano, 1994). Recent in vitro studies have found that stimulation of glutamate receptors strongly activates NFkB (Guerrini et al., 1995; Kaltschmidt et al., 1995). Subsequent reports that NFkB inhibitors such as aspirin and salicylate protect cultured neurons against glutamate-induced neuronal toxicity (Grilli et al., 1996) could thus indicate that NFkB activation contributes to excitotoxic neuronal injury. Unfortunately, interpretation of these results is complicated by the fact that salicylates inhibit other transcription factors and several protein kinases in addition to having many other pharmacologic actions (Frantz and O’Neill, 1995). To more precisely delineate the role of NFkB in excitotoxininduced neuronal apoptosis in vivo, we have examined the temporal pattern of NFkB and other transcription factor alterations in relation to internucleosomal DNA fragmentation after the intrastriatal administration of the potent NMDA receptor agonist QA. We also studied the effect of inhibiting NFkB activity on QA-induced apoptosis using a cell-permeable recombinant peptide (NFkB SN50) to block NFkB nuclear translocation. The results indicate that a complex pattern of transcription factor change precedes the appearance of internucleosomal DNA fragmentation and most noticeably that the activation of NFkB may contribute to the QA-induced apoptosis of striatal neurons. Materials and Methods Animals. Sprague-Dawley rats weighing 300–350 g were purchased from Taconic. They were housed two per cage in a standard animal room with a 12-hr light/dark cycle and given free access to food and water. All procedures were conducted in accordance with National Institutes of Health Guidelines for the Care and Use of