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Cell cycle regulators appear to play a paradoxical role in neuronal death. We have shown previously that cyclin-dependent kinases (CDKs), along with their downstream effectors, Rb (retinoblastoma) and E2F/DP1 (E2 promoter binding factor/deleted in polyposis 1), regulate neuronal death evoked by the DNA damaging agent camptothecin. However, the mechanism by which CDKs are activated in this model is unclear. The cell division cycle 25A (Cdc25A) phosphatase is a critical regulator of cell cycle CDKs in proliferating cells. In cortical neurons, we presently show that expression of Cdc25A promotes death even in the absence of DNA damage. Importantly, Cdc25A activity is rapidly increased during DNA damage treatment. Inhibition of Cdc25A blocks death and reduces cyclin D1-associated kinase activity and Rb phosphorylation. This indicates that endogenous Cdc25A activity is important for regulation of cell cycle-mediated neuronal death. We also examined how Cdc25A activity is regulated after DNA damage. Cultured embryonic cortical neurons have a significant basal activity of checkpoint kinase 1 (Chk1), a kinase that regulates cell cycle arrest. During camptothecin treatment of neurons, this activity is rapidly downregulated with a concomitant increase in Cdc25A activity. Importantly, expression of wild-type Chk1, but not kinase-dead Chk1, inhibits the camptothecin-induced increase in Cdc25A activity. In addition, Chk1 expression also promotes survival in the presence of the DNA-damaging agent. Together, our data suggest that a Chk1/Cdc25A activity participates in activation of a cell cycle pathway-mediated death signal in neurons. These data also define how a proliferative signal may be abnormally activated in a postmitotic environment.

Cyclin-dependent kinase 5 (cdk5) is a member of the cyclin-dependent kinase family whose activity is localized mainly to postmitotic neurons attributable to the selective expression of its activating partners p35 and p39. Deregulation of cdk5, as a result of calpain cleavage of p35 to a smaller p25 form, has been suggested to be a central component of neuronal death underlying numerous neurodegenerative diseases. However, the relevance of cdk5 in apoptotic death that relies on the mitochondrial pathway is unknown. Furthermore, evidence that cdk5 can also promote neuronal survival has necessitated a more complex understanding of cdk5 in the control of neuronal fate. Here we explore each of these issues using apoptotic and excitotoxic death models. We find that apoptotic death induced by the DNA-damaging agent camptothecin is associated with early transcription-mediated loss of p35 and with late production of p25 that is dependent on Bax, Apaf1, and caspases. In contrast, during excitotoxic death induced by glutamate, neurons rapidly produce p25 independent of the mitochondrial pathway. Analysis of the localization of p35 and p25 revealed that p35 is mainly cytoplasmic, whereas p25 accumulates selectively in the nucleus. By targeting a dominant-negative cdk5 to either the cytoplasm or nucleus, we show that cdk5 has a death-promoting activity within the nucleus and that this activity is required in excitotoxic death but not apoptotic death. Moreover, we also find that cdk5 contributes to pro-survival signaling selectively within the cytoplasm, and manipulation of this signal can modify death induced by both excitotoxicity and DNA damage.

The mechanisms involving neuronal death after ischemic/hypoxic insult are complex, involving both rapid (excitotoxic) and delayed (apoptotic-like) processes. Recent evidence suggests that cell cycle regulators such as cyclin-dependent kinases are abnormally activated in neuropathological conditions, including stroke. However, the function of this activation is unclear. Here, we provide evidence that inhibition of the cell cycle regulator, Cdk4, and its activator, cyclinD1, plays critical roles in the delayed death component of ischemic/hypoxic stress by regulating the tumor suppressor retinoblastoma protein. In contrast, the excitotoxic component of ischemia/hypoxia is predominately regulated by Cdk5 and its activator p35, components of a cyclin-dependent kinase complex associated with neuronal development. Hence, our data both characterize the functional significance of the cell cycle Cdk4 and neuronal Cdk5 signals as well as define the pathways and circumstances by which they act to control ischemic/hypoxic damage.

Recent evidence indicates that cyclin-dependent kinases (CDKs, cdks) may be inappropriately activated in several neurodegenerative conditions. Here, we report that cdk5 expression and activity are elevated after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that damages the nigrostriatal dopaminergic pathway. Supporting the pathogenic significance of the cdk5 alterations are the findings that the general cdk inhibitor, flavopiridol, or expression of dominant-negative cdk5, and to a lesser extent dominant-negative cdk2, attenuates the loss of dopaminergic neurons caused by MPTP. In addition, CDK inhibition strategies attenuate MPTP-induced hypolocomotion and markers of striatal function independent of striatal dopamine. We propose that cdk5 is a key regulator in the degeneration of dopaminergic neurons in Parkinson's disease.

Expression of the transcription factor c-Jun is induced in neurons of the central nervous system (CNS) in response to injury. Mechanical transection of the nigrostriatal pathway at the medial forebrain bundle (MFB) results in the delayed retrograde degeneration of the dopamine neurons in the substantia nigra pars compacta (SNc) and induces protracted expression and phosphorylation of c-Jun. However, the role of c-Jun after axotomy of CNS neurons is unclear. Here, we show that adenovirus-mediated expression of a dominant negative form of c-Jun (Ad.c-JunDN) inhibited axotomy-induced dopamine neuron death and attenuated phosphorylation of c-Jun in nigral neurons. Ad.c-JunDN also delayed the degeneration of dopaminergic nigral axons in the striatum after MFB axotomy. Taken together, these findings suggest that activation of c-Jun mediates the loss of dopamine neurons after axotomy injury.

The tumor suppressor, retinoblastoma (Rb), is involved in both terminal mitosis and neuronal differentiation. We hypothesized that activation of the Rb pathway would induce cell cycle arrest in primary neural precursor cells, independent of the proposed function of cyclin-dependent kinases 4/6 (CDK4/6) to sequester the CIP/KIP CDK inhibitors (CKIs) p21 and p27 from CDK2. We expressed dominant negative adenovirus mutants of CDKs 2, 4, and 6 (dnCDK2, dnCDK4, and dnCDK6) in neural progenitor cells derived from E12.5 wild type and Rb-deficient mouse embryos. In contrast to previous studies, our results demonstrate that in addition to dnCDK2, the dnCDK4/6 mutants can induce growth arrest. Moreover, the dnCDK4/6-mediated inhibition is Rb-dependent. The dnCDK2 partially inhibited cell growth in Rb-deficient cells, suggesting that CDK2 may have additional targets. A previously proposed function of CDK4/6 is CKI sequestration, thereby preventing the resulting inhibition of CDK2, believed to be the key regulator of cell cycle. However, our immunoprecipitations revealed that the dominant negative CDK mutants could arrest cell growth despite their interaction with p21 and p27. Taken together, our results demonstrate that both CDK2 and CDK4/6 are crucial for cell cycle regulation. Furthermore, our data underscore the importance of the Rb regulatory pathway in neuronal development and cell cycle regulation, independent of CKI sequestration.

Recombinant adenovirus vectors have provided a major advance in gene delivery systems for post-mitotic neurons. However, the use of these first generation vectors has been limited due to the onset of virally mediated effects on cellular function and viability. In the present study we have used primary cultures of cerebellar granule neurons to examine the efficacy and cytotoxic effects of a helper-dependent adenovirus vector (hdAd) in comparison with a first generation vector. Our results demonstrate that the hdAd system provides equally efficient infectivity with significantly reduced toxicity in comparison to first generation vectors. Neurons transduced with a high titre of a first generation vector exhibited a time-dependent shut down in global protein synthesis and impaired physiological function as demonstrated by a loss of glutamate receptor responsiveness. This was followed by an increase in the fraction of TUNEL-positive cells and a loss of neuronal survival. In contrast, hdAds could be used at titres that transduce >85% of neurons with little cytotoxic effect: cellular glutamate receptor responses and rates of protein synthesis were indistinguishable from uninfected controls. Furthermore, cell viability was not significantly affected for at least 7 days after infection. At excessive viral titres, however, infection with hdAd did cause moderate but significant changes in cell function and viability in primary neuronal cultures. Thus, while these vectors are remarkably improved over first generation vectors, these also have limitations with respect to viral effects on cellular function and viability. Gene Therapy (2000) 7, 1200-1209.

An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in acute and chronic neurodegenerative conditions. Extensive ER stress can trigger neuronal apoptosis, but the signaling pathways that regulate this cell death remain unclear. In the present study, we demonstrate that PUMA, a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family, is transcriptionally activated in cortical neurons by ER stress and is essential for ER-stress-induced cell death. PUMA is known to be a key transcriptional target of p53, but we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism mediated by the ER-stress-inducible transcription factor ATF4 (activating transcription factor 4). Specifically, we demonstrate that ectopic expression of ATF4 sensitizes mouse cortical neurons to ER-stress-induced apoptosis and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP (C/EBP homologous protein) and that CHOP in turn activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER-stress-induced neuronal death involving ATF4–CHOP-mediated transactivation of the proapoptotic Bcl-2 family member PUMA. We propose that this pathway may be an important therapeutic target relevant to a number of neurodegenerative conditions.

Growing evidence highlights a role for mitochondrial dysfunction and oxidative stress as underlying contributors to Parkinson’s disease (PD) pathogenesis. DJ-1 (PARK7) is a recently identified recessive familial PD gene. Its loss leads to increased susceptibility of neurons to oxidative stress and death. However, its mechanism of action is not fully understood. Presently, we report that DJ-1 deficiency in cell lines, cultured neurons, mouse brain and lymphoblast cells derived from DJ-1 patients display aberrant mitochondrial morphology. We also show that these DJ-1-dependent mitochondrial defects contribute to oxidative stress-induced sensitivity to cell death since reversal of this fragmented mitochondrial phenotype abrogates neuronal cell death. Reactive oxygen species (ROS) appear to play a critical role in the observed defects, as ROS scavengers rescue the phenotype and mitochondria isolated from DJ-1 deficient animals produce more ROS compared with control. Importantly, the aberrant mitochondrial phenotype can be rescued by the expression of Pink1 and Parkin, two PD-linked genes involved in regulating mitochondrial dynamics and quality control. Finally, we show that DJ-1 deficiency leads to altered autophagy in murine and human cells. Our findings define a mechanism by which the DJ-1-dependent mitochondrial defects contribute to the increased sensitivity to oxidative stress-induced cell death that has been previously reported.

Mechanical transection of the nigrostriatal dopamine pathway at the medial forebrain bundle (MFB) results in the delayed degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We have previously demonstrated that c-Jun activation is an obligate component of neuronal death in this model. Here we identified the small GTPase, cdc42, and mixed lineage kinases (MLKs) as upstream factors regulating neuronal loss and activation of c-Jun following MFB axotomy. Adenovirus-mediated expression of a dominant-negative form of cdc42 in nigral neurons blocked MFB axotomy-induced activation (phosphorylation) of MAP kinase kinase 4 (MKK4) and c-Jun, resulting in attenuation of SNpc neuronal death. Pharmacological inhibition of MLKs, MKK4-activating kinases, significantly reduced the phosphorylation of c-Jun and abrogated dopaminergic neuronal degeneration following MFB axotomy. Taken together, these findings suggest that death of nigral dopaminergic neurons following axotomy can be attenuated by targeting cell signaling events upstream of c-Jun N-terminal mitogen-activated protein kinase/c-Jun.

Thep53tumor suppressor gene has been implicated in the regulation of apoptosis in a number of different neuronal death paradigms. Because of the importance of p53 in neuronal injury, we questioned the mechanism underlying p53-mediated apoptosis in neurons. Using adenoviral-mediated gene delivery, reconstitution experiments, and mice carrying a knock-in mutation in the endogenousp53gene, we show that the transactivation function of p53 is essential to induce neuronal cell death. Although p53 possesses two transactivation domains that can activate p53 targets independently, we demonstrate that the first activation domain (ADI) is required to drive apoptosis after neuronal injury. Furthermore, the BH3-only proteins Noxa and PUMA exhibit differential regulation by the two transactivation domains. Here, we show that Noxa can be induced by either activation domain, whereas PUMA induction requires both activation domains to be intact. Unlike Noxa, the upregulation of PUMA alone is sufficient to induce neuronal cell death. We demonstrate, therefore, that the first transactivation domain of p53 is indispensable for the induction of neuronal cell death.

The p53 tumor suppressor gene is believed to play an important role in neuronal cell death in acute neurological disease and in neurodegeneration. The p53 signaling cascade is complex, and the mechanism by which p53 induces apoptosis is cell type-dependent. Using DNA microarray analysis, we have found a striking induction of the proapoptotic gene, SIVA. SIVA is a proapoptotic protein containing a death domain and interacts with members of the tumor necrosis factor receptor family as well as anti-apoptotic Bcl-2 family proteins. SIVA is induced following direct p53 gene delivery, treatment with a DNA-damaging agent camptothecin, and stroke injury in vivo. SIVA up-regulation is sufficient to initiate the apoptotic cascade in neurons. Through isolation and analysis of the SIVA promoter, we have identified response elements for both p53 and E2F1. Like p53, E2F1 is another tumor suppressor gene involved in the regulation of apoptosis, including neuronal injury models. We have identified E2F consensus sites in the promoter region, whereas p53 recognition sequences were found in intron1. Sequence analysis has shown that these consensus sites are also conserved between mouse and human SIVA genes. Electrophoretic mobility shift assays reveal that both transcription factors are capable of binding to putative consensus sites, and luciferase reporter assays reveal that E2F1 and p53 can activate transcription from the SIVA promoter. Here, we report that the proapoptotic gene, SIVA, which functions in a broad spectrum of cell types, is a direct transcriptional target for both tumor suppressors, p53 and E2F1.

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.