Although B-amyloid (AB) is suggested to play an important role in Alzheimer's disease, the mechanisms that control AB-evoked toxicity are unclear. We demonstrated previously that the cell cycle-related cyclin-dependent kinase 4/6/retinoblastoma protein pathway is required for AB-mediated death. However, the downstream target(s) of this pathway are unknown. We show here that neurons lacking E2F1, a transcription factor regulated by the retinoblastoma protein, are significantly protected from death evoked by AB. Moreover, p53 deficiency does not protect neurons from death, indicating that E2F1-mediated death occurs independently of p53. Neurons protected by E2F1 deficiency have reduced Bax-dependent caspase 3-like activity. However, protection afforded by E2F1, Bax, or caspase 3 deficiency is transient. In the case of E2F1, but not with Bax or caspase 3 deficiency, delayed death is accompanied by DEVD-AFC cleavage activity. Taken together, these results demonstrate the required role of E2F1, Bax, and caspase 3 in AB evoked death, but also suggest the participation of elements independent of these apoptosis regulators.Alzheimer's disease (AD) 1 is a neurodegenerative disorder characterized by memory loss and cognitive impairment (1). Although the etiology of AD is not fully understood, an increasing body of evidence suggests the importance of B-amyloid in the initiation/progression of the disease. B-amyloid (AB), a 39 -43-amino acid peptide, assembles into insoluble aggregates forming plaques characteristic of AD (2, 3). AB is formed by alternative proteolysis from an integral membrane-localized B-amyloid precursor protein (4), and its importance in AD is underscored by the identification of mutations in B-amyloid precursor protein in some familial cases of AD (5, 6). In addition, studies with in vitro cultured neurons treated with toxic forms of aggregated AB protein (7-9) as well as in vivo studies utilizing transgenic mice expressing AB (10) demonstrated neuronal loss by an apoptotic pathway. However, the mechanism by which AB causes neuronal apoptosis is not well understood.Recent evidence has suggested that cell cycle molecules that normally control cell proliferation play an important required role in some forms of neuronal death. Of particular relevance to AD, numerous reports demonstrate abnormal up-regulation of a variety of cell cycle proteins/activity in brains from AD patients. These include cyclin B, D, and E, cdc2, and Cdk4 (11-13). In addition, phosphoepitopes, including phosphorylated tau, common to both mitotic cells and degenerating AD neurons, have been described (12). In support of the potential importance of cell cycle in AD, we have shown that flavopiridol, a pharmacological inhibitor of Cdks, as well as expression of dominant negative Cdk4/6 but not 2 or 3, protects cultured cortical neurons from death evoked by AB (14). This evidence supports the idea that Cdk4/6 activation plays an obligatory role in death of neurons evoked by AB.These previous observations raise the question of the relev...

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Growing evidence suggests that certain cell cycle regulators also mediate neuronal death. Of relevance, cyclin D1-associated kinase activity is increased and the retinoblastoma protein (Rb), a substrate of the cyclin D1-Cdk4/6 complex, is phosphorylated during K ؉ deprivation-evoked death of cerebellar granule neurons (CGNs). Cyclin-dependent kinase (CDK) inhibitors block this death, suggesting a requirement for the cyclin D1/ Cdk4/6-Rb pathway. However, the downstream target(s) of this pathway are not well defined. The transcription factor E2F-1 is regulated by Rb and is reported to evoke death in proliferating cells when overexpressed. Accordingly, we examined whether E2F-1 was sufficient to evoke death of CGNs and whether it was required for death evoked by low K ؉ . We show that adenovirus-mediated expression of E2F-1 in CGNs results in apoptotic death, which is independent of p53, dependent upon Bax, and associated with caspase 3-like activity. In addition, we demonstrate that levels of E2F-1 mRNA and protein increase during K ؉ deprivation-evoked death. The increase in E2F-1 protein is blocked by the CDK inhibitor flavopiridol. Finally, E2F-1-deficient neurons are modestly resistant to death induced by low K ؉ . These results indicate that E2F-1 expression is sufficient to promote neuronal apoptosis and that endogenous E2F-1 modulates the death of CGNs evoked by low K ؉ .

Collapsin response mediator proteins (CRMPs) mediate growth cone collapse during development, but their roles in adult brains are not clear. Here we report the findings that the full-length CRMP-3 (p63) is a direct target of calpain that cleaves CRMP-3 at the N terminus (ϩ76 amino acid). Interestingly, activated calpain in response to excitotoxicity in vitro and cerebral ischemia in vivo also cleaved CRMP-3, and the cleavage product of CRMP-3 (p54) underwent nuclear translocation during neuronal death. The expression of p54 was colocalized with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive nuclei in glutamatetreated cerebellar granule neurons (CGNs) and in ischemic neurons located in the infarct core after focal cerebral ischemia, suggesting that p54 might be involved in neuronal death. Overexpression studies showed that p54, but not p63, caused death of human embryonic kidney cells and CGNs, whereas knock-down CRMP-3 expression by selective small interfering RNA protected neurons against glutamate toxicity. Collectively, these results reveal a novel role of CRMP-3 in that calpain cleavage of CRMP-3 and the subsequent nuclear translocation of the truncated CRMP-3 evokes neuronal death in response to excitotoxicity and cerebral ischemia. Our findings also establish a novel route of how calpain signals neuron death.