Cyclin-dependent kinases and P53 pathways are activated independently and mediate Bax activation in neurons after DNA damage.

DNA damage has been implicated as one important initiator of cell death in neuropathological conditions such as stroke. Accordingly, it is important to understand the signaling processes that control neuronal death induced by this stimulus. Previous evidence has shown that the death of embryonic cortical neurons treated with the DNA-damaging agent camptothecin is dependent on the tumor suppressor p53 and cyclin-dependent kinase (CDK) activity and that the inhibition of either pathway alone leads to enhanced and prolonged survival. We presently show that p53 and CDKs are activated independently on parallel pathways. An increase in p53 protein levels, nuclear localization, and DNA binding that result from DNA damage are not affected by the inhibition of CDK activity. Conversely, no decrease in retinoblastoma protein (pRb) phosphorylation was observed in p53-deficient neurons that were treated with camptothecin. However, either p53 deficiency or the inhibition of CDK activity alone inhibited Bax translocation, cytochrome c release, and caspase-3-like activation. Taken together, our results indicate that p53 and CDK are activated independently and then act in concert to control Bax-mediated apoptosis.

Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage.

Neuronal death evoked by DNA damage requires cyclin-dependent kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6 phosphorylates retinoblastoma protein (pRb) family members that then modulate the transcriptional activity of E2F/DP1 complexes, we examined the involvement of these components in DNA damage-evoked neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and p107. The CDK inhibitor flavopiridol suppressed pRb and p107 phosphorylation and loss, implicating CDK activity in these events. Moreover, the loss of pRb and p107 appeared to be mediated by caspases because it was blocked by general caspase inhibitors. The role of phosphorylation and pRb and p107 loss in the death pathway was indicated by observations that virally mediated expression of pRb mutated at sites of phosphorylation, including the Cdk4/6 site, inhibited death. Finally, expression of dominant-negative versions of DP1, known to compromise E2F transcriptional activity, protects cortical neurons from death induced by camptothecin and sympathetic neurons from death evoked by UV treatment. Taken together, these results implicate the CDK-pRb/E2F/DP pathway as a required element in the neuronal death evoked by DNA damage.

Involvement of cell cycle elements, cyclin-dependent kinases, pRb, and E2F x DP, in B-amyloid-induced neuronal death.

Previous evidence by others has indicated that a variety of cell cycle-related molecules are up-regulated in brains of Alzheimer's disease patients. The significance of this increase, however, is unclear. Accordingly, we examined the obligate nature of cyclin-dependent kinases and select downstream targets of these kinases in death of neurons evoked by B-amyloid (AB) protein. We present pharmacological and molecular biological evidence that cyclin-dependent kinases, in particular Cdk4/6, are required for such neuronal death. In addition, we demonstrate that the substrate of Cdk4/6, pRb/p107, is phosphorylated during AB treatment and that one target of pRb/p107, the E2F x DP complex, is required for AB-evoked neuronal death. These results provide evidence that cell cycle elements play a required role in death of neurons evoked by AB and suggest that these elements play an integral role in Alzheimer's disease-related neuronal death.

E2F1 mediates death of B-amyloid-treated cortical neurons in a manner independent of p53 and dependent on Bax and caspase 3.

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.

Involvement of caspase 3 in apoptotic death of cortical neurons evoked by DNA damage.

Previous reports have shown that DNA-damage-evoked death of embryonic cortical neurons is delayed by general caspase inhibitors and is accompanied by an increase in DEVD-AFC cleavage activity. We show here that this cleavage activity is lacking in camptothecin-treated caspase 3-deficient neurons. Moreover, we report that death of camptothecin-treated caspase 3-deficient neurons cultured from E16 embryos is delayed and that no significant increase in survival is observed with cotreatment with the general caspase inhibitor BAF. These results indicate that caspase-dependent death of camptothecin-treated cortical neurons requires caspase 3 activity. The delay in death is accompanied by impairment of DNA fragmentation. However, Bax-dependent cytochrome c release still occurs in camptothecin-treated caspase 3-deficient cortical neurons. Accordingly, we hypothesize that the delayed death which occurs in the absence of caspase 3 activity may be due to mitochondrial dysfunction. Finally, we show that the delay in death observed with E16 caspase 3-deficient neurons does not occur in neurons cultured from E19 embryos. This suggests that the requirement for caspase 3 in death of neurons evoked by DNA damage may differ depending upon the developmental state of the cell.