Nuclear factor B (NF-B) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo, rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-B in the induction of apoptosis-related genes and gene products was evaluated. QA administration induced timedependent NF-B nuclear translocation. The nuclear NF-B protein after QA treatment was comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein were markedly increased at the time of QA-induced NF-B nuclear translocation. Immunohistochemical analysis showed that c-Myc and p53 induction occurred in the excitotoxin-sensitive medium-sized striatal neurons. NF-B nuclear translocation was blocked in a dosedependent manner by the cell-permeable recombinant peptide NF-B SN50, but not by the NF-B SN50 control peptide. NF-B SN50 significantly inhibited the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-B SN50, but not the control peptide, also substantially reduced the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-B may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating an unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis.

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)-kappa B may be involved in excitotoxicity. To further analyze the contribution of NF kappa B to excitotoxic neuronal death in vivo, changes in binding activities of NF kappa B and other transcription factors as well as the consequences of inhibiting NF kappa B 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. NF kappa B binding activity increased several-fold 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 NF kappa B, 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 NF kappa B SN50, a cell-permeable recombinant peptide that blocks NF kappa B 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 NF kappa B activation contributes to the excitotoxin-induced death of striatal neurons.

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Abstract:The present study evaluated whether nuclear factor-B (NF-B) activation contributes to the apoptoticlike death of striatal neurons induced by kainic acid (KA) receptor stimulation. Intrastriatally infused KA (1.25-5.0 nmol) produced substantial neuronal loss as indicated by an 8 -73% decrease in 67-kDa glutamic acid decarboxylase ( p Ͻ 0.05). KA (1.25-5.0 nmol) elicited internucleosomal DNA fragmentation that was inhibited by the AMPA/KA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dibenzo[f]quinoxaline-7-sulfonamide) but not by the NMDA receptor antagonist MK-801. A decrease in I B-␣ protein levels, which was accompanied by an increase in NF-B binding activity, was found from 6 to 72 h after KA (2.5 nmol) infusion. NF-B was composed mainly of p65 and c-Rel as revealed by supershift assay. In addition, c-Myc and p53 increased from five-to sevenfold from 24 to 72 h after KA (2.5 nmol) administration. Immunohistochemistry revealed high levels of c-Myc and p53 immunoreactivity, mainly in medium-sized striatal neurons. Pretreatment with the cell-permeable recombinant peptide NF-B SN50 (5-20 g) blocked NF-B nuclear translocation, but had no effect on AP-1 binding. NF-B SN50 also inhibited the KA-induced up-regulation of c-Myc and p53, as well as internucleosomal DNA fragmentation. The apoptotic-like destruction of rat striatal neurons induced by KA receptor stimulation thus appears to involve biochemical mechanisms similar to those mediating the excitotoxic response to NMDA receptor stimulation. The present results provide additional support for the view that NF-B activation contributes to c-Myc and p53 induction and subsequent apoptosis in an excitotoxic model of Huntington's disease. Key Words: Kainic acidApoptosis-Neurodegeneration-Nuclear transcription factor-B (NF-B)-I B-␣-NF-B p65-c-Myc-p53. J. Neurochem. 74, 647-658 (2000).The excitotoxic destruction of neurons within the mammalian CNS occurs, at least in part, by an apoptotic mechanism (Bonfoco et al., 1995;Portera-Cailliau et al., 1995;Qin et al., 1996). To date, most research has focused on neuronal death induced by the excitation of glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype (Choi, 1988;Olney, 1989). At the molecular level, NMDA receptor-mediated cell death reportedly can involve several critical apoptotic modulators, including p53, Bcl-2, Bax, and caspase-3 (Montpied et al., 1993;Hughes et al., 1996;Du et al., 1997). More recently, evidence has arisen to suggest that the destruction of rat striatal neurons induced by the selective NMDA receptor agonist, quinolinic acid (QA), involves activation of nuclear factor-B (NF-B) (Qin et al., 1998). Unique to other transcription factors, NF-B normally resides in the cytoplasm bound to I B family proteins (Baeuerle, 1991;Thanos and Maniatis, 1995).QA exposure now appears to promote the selective degradation of I B-␣ by a caspase-3-dependent mechanism, thus releasing NF-B from its cytoplasmic sequestration sites (Z.-H. Qin et al., submitted). Nuclear translocation of NF-B lead...