q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 287±297 287 Journal of Neurochemistry, 2001, 78, 287±297 The transcription factor E2F1 promotes dopamine-evoked neuronal apoptosis by a mechanism independent of transcriptional activation Sheng T. Hou, Emily Cowan, Teena Walker, Nick Ohan, Mike Dove, Ingrid Rasqinha and John P. MacManus Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada Abstract The E2F1 transcription factor plays an important role in promoting neuronal apoptosis; however, it is not clear how E2F1 does this. Here we show that E2F1 is involved in dopamine (DA)-evoked apoptosis in cerebellar granule neurons (CGNs). E2F1 ±/± CGNs and CGNs expressing an antisense E2F1 cDNA were signi®cantly protected from DA-toxicity relative to controls. The neuronal protection was accompanied by signi®cantly reduced caspase 3 activity. E2F1-mediated neuronal apoptosis did not require activation of gene transcription because: (1) ectopic expression of E2F1 or its mutants lacking the transactivation domain induced neuronal apoptosis, whereas an E2F1 mutant lacking the DNA-binding domain did not; (2) under all of these conditions, known E2F1 target genes including cyclin A, cdc2 and p19 ARF were not induced; and (3) DA-evoked neuronal apoptosis was associated with up-regulated E2F1, but not transcription of its target genes. Finally, E2F1-mediated neuronal apoptosis was associated with reduced nuclear factor (NF)-kB DNA-binding activity. Taken together, these data suggest that E2F1 promotes DA-evoked caspase 3-dependent neuronal apopto- sis by a mechanism independent of gene transactivation, and this may possibly occur through inhibition of anti-apoptotic genes including NF-kB. Keywords: adenovirus, caspase 3, dopamine, E2F1, neuronal apoptosis, p19 ARF . J. Neurochem. (2001) 78, 287±297. Dopamine (DA)-induced neuronal apoptosis contributes to the etiology of neurodegenerative disorders such as Parkinson's disease (Stokes et al. 1999; Cohen 2000). The toxicity of dopamine mainly comes from its oxidative metabolism. Enzymatic oxidation of DA and non-enzymatic auto-oxidation of DA in the presence of transition metals produces metabolic intermediates including reactive oxygen species (ROS). Increased oxidative stress through ROS is a primary cause of neurotoxicity (Lai and Yu 1997; Stokes et al. 1999; Lewen et al. 2000). Through the activity of ROS, DA activates several death signal transduction pathways. Examination of gene expres- sion pro®les by differential display has revealed that a mitosis-related gene, cyclin B2, is activated in cultured chick sympathetic neurons following DA treatment (Shirvan et al. 1997). This ®nding coincides with accumulating evidence suggesting that cell cycle components such as cyclin D and cyclin-dependent kinases (CDK) contribute to the induction of apoptosis in cerebellar granule neurons (CGNs) under depolarizing conditions (K 1 -withdrawal), trophic factor deprivation, or b-amyloid toxicity (Freeman et al. 1994; Padmanabhan et al. 1999; Giovanni et al. 2000; Osuga et al. 2000). The activity of CDK inhibitors, such as p27, decreases during neuronal death. Pharmacological inhibitors of CDKs, such as ¯avopiridol, inhibit CDK activity, prevent neuronal death and reduce ischemic damage to the brain (Osuga et al. 2000). One of the cellular targets of CDKs is pRb, the product of the retinoblastoma tumor suppressor RB gene. Increased phosphorylation of pRb by CDKs leads to the release of its binding partner Resubmitted manuscript received April 5, 2001; accepted April 12, 2001. Address correspondence and reprint requests to S. T. Hou, Apoptosis Research Group, Institute for Biological Sciences, National Research Council Canada, Montreal Road Laboratories, Building M-54, Ottawa, Ontario K1A 0R6, Canada. E-mail: sheng.hou@nrc.ca Abbreviations used: CFDA, 5-(6)-carboxy¯uorescein diacetate; CGNs, cerebellar granule neurons; DA, dopamine; DMEM, Dubecco's modi®ed Eagle's medium; GFP, green ¯uorescent protein; MEF, mouse embryonic ®broblasts; MOI, multiplicity of infection; NF-kB, nuclear factor kB; 6-OHDA, 6-hydroxydopamine.