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Developmental and pathological death of neurons requires activation of a defined pathway of cell cycle proteins. However, it is unclear how this pathway is regulated and whether it is relevantin vivo. A screen for transcripts robustly induced in cultured neurons by DNA damage identified Sertad1, a Cdk4 (cyclin-dependent kinase 4) activator. Sertad1 is also induced in neurons by nerve growth factor (NGF) deprivation and Aβ (β-amyloid). RNA interference-mediated downregulation of Sertad1 protects neurons in all three death models. Studies of NGF withdrawal indicate that Sertad1 is required to initiate the apoptotic cell cycle pathway since its knockdown blocks subsequent pathway events. Finally, we find that Sertad1 expression is required for developmental neuronal death in the cerebral cortex. Sertad1 thus appears to be essential for neuron death in trophic support deprivationin vitroandin vivoand in models of DNA damage and Alzheimer's disease. It may therefore be a suitable target for therapeutic intervention.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (Htt). Group I metabotropic glutamate receptors (mGluRs) are coupled to Gαqand play an important role in neuronal survival. We have previously demonstrated that mGluRs interact with Htt. Here we used striatal neuronal primary cultures and acute striatal slices to demonstrate that mGluR-mediated signaling pathways are altered in a presymptomatic mouse model of HD (HdhQ111/Q111), as compared to those of control mice (HdhQ20/Q20). mGluR1/5-mediated inositol phosphate (InsP) formation is desensitized in striatal slices fromHdhQ111/Q111mice and this desensitization is PKC-mediated. Despite of decreased InsP formation, (S)-3,5-dihydroxylphenylglycine (DHPG)-mediated Ca2+release is higher inHdhQ111/Q111than inHdhQ20/Q20neurons. Furthermore, mGluR1/5-stimulated AKT and extracellular signal-regulated kinase (ERK) activation is altered inHdhQ111/Q111mice. Basal AKT activation is higher inHdhQ111/Q111neurons and this increase is mGluR5 dependent. Moreover, mGluR5 activation leads to higher levels of ERK activation inHdhQ111/Q111than inHdhQ20/Q20striatum. PKC inhibition not only bringsHdhQ111/Q111DHPG-stimulated InsP formation toHdhQ20/Q20levels, but also causes an increase in neuronal cell death inHdhQ111/Q111neurons. However, PKC inhibition does not modify neuronal cell death inHdhQ20/Q20neurons, suggesting that PKC-mediated desensitization of mGluR1/5 inHdhQ111/Q111mice might be protective in HD. Together, these data indicate that group I mGluR-mediated signaling pathways are altered in HD and that these cell signaling adaptations could be important for striatal neurons survival.

DNA damage is an important initiator of neuronal apoptosis and activates signaling events not yet fully defined. Using the camptothecin-induced DNA damage model in neurons, we previously showed that cyclin D1-associated cell cycle cyclin-dependent kinases (Cdks) (Cdk4/6) and p53 activation are two major events leading to activation of the mitochondrial apoptotic pathway. With gene array analyses, we detected upregulation of Cited2, a CBP (cAMP response element-binding protein-binding protein)/p300 interacting transactivator, in response to DNA damage. This upregulation was confirmed by reverse transcription-PCR and Western blot. CITED2 was functionally important because CITED2 overexpression promotes death, whereas CITED2 deficiency protects. Cited2 upregulation is upstream of the mitochondrial death pathway (BAX, Apaf1, or cytochromecrelease) and appears to be independent of p53. However, inhibition of the Cdk4 blocked Cited2 induction. The Cited2 prodeath mechanism does not involve Bmi-1 or p53. Instead, Cited2 activates peroxisome proliferator-activated receptor-γ (PPARγ), an activity that we demonstrate is critical for DNA damage-induced death. These results define a novel neuronal prodeath pathway in which Cdk4-mediated regulation of Cited2 activates PPARγ and consequently caspase.

Oxidative stress has been implicated as a key trigger of neuronal apoptosis in stroke and neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The Bcl-2 homology 3 (BH3)-only subfamily of Bcl-2 genes consists of multiple members that can be activated in a cell-type- and stimulus-specific manner to promote cell death. In the present study, we demonstrate that, in cortical neurons, oxidative stress induces the expression of the BH3-only members Bim, Noxa, and Puma. Importantly, we have determined thatPuma−/− neurons, but notBim−/− orNoxa−/− neurons, are remarkably resistant to the induction of apoptosis by multiple oxidative stressors. Furthermore, we have determined that Bcl-2-associated X protein (Bax) is also required for oxidative stress induced cell death and that Puma plays a dominant role in regulating Bax activation. Specifically, we have established that the induction of Puma, but not Bim or Noxa, is necessary and sufficient to induce a conformational change in Bax to its active state, its translocation to the mitochondria and mitochondrial membrane permeabilization. Finally, we demonstrate that whereas both Puma and BimELcan bind to the antiapoptotic family member Bcl-XL, only Puma was found to associate with Bax. This suggests that in addition to neutralizing antiapoptotic members, Puma may play a dominant role by complexing with Bax and directly promoting its activation. Overall, we have identified Puma as a dominant regulator of oxidative stress induced Bax activation and neuronal apoptosis, and suggest that Puma may be an effective therapeutic target for the treatment of a number of neurodegenerative conditions.

Previous studies have shown that DNA damage-evoked death of primary cortical neurons occurs in a p53 and cyclin-dependent kinase-dependent (CDK) manner. The manner by which these signals modulate death is unclear. Nuclear factor-kappaB (NF-kappaB) is a group of transcription factors that potentially interact with these pathways. Presently, we show that NF-kappaB is activated shortly after induction of DNA damage in a manner independent of the classic IkappaB kinase (IKK) activation pathway, CDKs, ATM, and p53. Acute inhibition of NF-kappaB via expression of a stable IkappaB mutant, downregulation of the p65 NF-kappaB subunit by RNA interference (RNAi), or pharmacological NF-kappaB inhibitors significantly protected against DNA damage-induced neuronal death. NF-kappaB inhibition also reduced p53 transcripts and p53 activity as measured by the p53-inducible messages, Puma and Noxa, implicating the p53 tumor suppressor in the mechanism of NF-kappaB-mediated neuronal death. Importantly, p53 expression still induces death in the presence of NF-kappaB inhibition, indicating that p53 acts downstream of NF-kappaB. Interestingly, neurons cultured from p65 or p50 NF-kappaB-deficient mice were not resistant to death and did not show diminished p53 activity, suggesting compensatory processes attributable to germline deficiencies, which allow p53 activation still to occur. In contrast to acute NF-kappaB inhibition, prolonged NF-kappaB inhibition caused neuronal death in the absence of DNA damage. These results uniquely define a signaling paradigm by which NF-kappaB serves both an acute p53-dependent pro-apoptotic function in the presence of DNA damage and an anti-apoptotic function in untreated normal neurons.

p53 is a pivotal molecule regulating the death of neurons both after acute injury and during development. The molecular mechanisms by which p53 induces apoptosis in neuronal cells, however, are not well understood. We have shown previously that adenovirus-mediated p53 gene delivery to neurons was sufficient to induce apoptosis. In the present study we have examined the molecular mechanism by which p53 evokes neuronal cell death. Adenovirus-mediated delivery of p53 to cerebellar granule neurons resulted in caspase-3 (CPP32) activation followed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and loss of viability as determined by an MTT survival assay. To determine whether Bax is essential for caspase-3 activation, p53 was expressed in Bax-deficient cells. Bax null neurons did not exhibit caspase-3 activation in response to p53 and were protected from apoptosis. To determine whether Bax-dependent caspase-3 activation was required in p53-mediated neuronal cell death, caspase-3-deficient neurons were examined. Our results indicate that caspase-3-deficient neurons exhibit a remarkable delay in apoptosis and a dramatic decrease in TUNEL-positive cells. These studies demonstrate that p53-induced cell death in postmitotic neurons involves a Bax-dependent caspase-3 activation, suggesting that these molecules are important determinants in neuronal cell death after injury.