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Recent reports indicate that the activating transcription factor 5 (ATF5) is required for the survival of cancer cells but not for noncancer cells. However, the mechanisms by which ATF5 regulates genes and promotes cell survival are not clear. Using a cyclic amplification and selection of targets (CASTing) approach, we identified a novel ATF5 consensus DNA binding sequence. We show in C6 glioma and MCF-7 breast cancer cells that ATF5 occupies this sequence and that ATF5 activates reporter gene expression driven by this site. Conversely, reporter activity is diminished when ATF5 activity is blocked or when ATF5 expression is down-regulated by serum withdrawal. We further show that early growth response factor 1 (Egr-1), whose promoter contains two adjacent ATF5 consensus binding sites at a conserved promoter position in rat, mouse, and human, is targeted and regulated by ATF5 in C6 and MCF-7 cells. These data provide new insight on the mechanisms by which ATF5 promotes gene regulation and cancer-specific cell survival. (Mol Cancer Res 2009;7(6):933–43)

Supplementary Data from Identification of a Novel DNA Binding Site and a Transcriptional Target for Activating Transcription Factor 5 in C6 Glioma and MCF-7 Breast Cancer Cells

Suppl. fig. 1: Effect of CP-d/n-ATF5-S1 on ATF5 expression and stability Suppl. fig. 2: Effect of CP-d/n-ATF5-S1 on ASNS mRNA expression Suppl. fig. 3: Effect of CP-d/n-ATF5-S1 on cell viability in HL-60 cells Suppl. fig. 4: Pro-apoptotic activity of CP-d/n-ATF5-S1 in SF188 and GBM12 cells Suppl. fig. 5: Effect of CP-d/n-ATF5-S1 in HCT116 colorectal cancer Suppl. fig. 6: Effect of pan-caspase inhibition on CP-d/n-ATF5-S1 treatment Suppl. fig. 7: Effect of CP-d/n-ATF5-S1 on Bcl-2 family and Usp9X/Bag3 protein expression in A375 and PC3 Suppl. fig. 8: Effect of pan-caspase inhibition on CP-d/n-ATF5-S1-mediated down-regulation of Usp9X Suppl. fig. 9: Effect of Usp9X knock-down on apoptosis in LN229 Suppl. fig. 10: Isobologram for CP-d/n-ATF5-S1 and ABT263 Suppl. fig. 11: Effect of CP-d/n-ATF5-S1 and ABT263 on GBM12 cells Suppl. fig. 12: Knock-down of Mcl-1 sensitizes for ABT263-mediated apoptosis Suppl. fig. 13: Effects of CP-d/n-ATF5-S1 on U87MG xenograft model Suppl. fig. 14: Effects of CP-d/n-ATF5-S1 on PANC-1 and MDA-MB-231 xenograft model Suppl. fig. 15: Effect of CP-d/n-ATF5-S1 on organ toxicity

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Purpose: Despite significant progress in cancer research, many tumor entities still have an unfavorable prognosis. Activating transcription factor 5 (ATF5) is upregulated in various malignancies and promotes apoptotic resistance. We evaluated the efficacy and mechanisms of the first described synthetic cell-penetrating inhibitor of ATF5 function, CP-d/n-ATF5-S1.Experimental Design: Preclinical drug testing was performed in various treatment-resistant cancer cells and in vivo xenograft models.Results: CP-d/n-ATF5-S1 reduced the transcript levels of several known direct ATF5 targets. It depleted endogenous ATF5 and induced apoptosis across a broad panel of treatment-refractory cancer cell lines, sparing non-neoplastic cells. CP-d/n-ATF5-S1 promoted tumor cell apoptotic susceptibility in part by reducing expression of the deubiquitinase Usp9X and led to diminished levels of antiapoptotic Bcl-2 family members Mcl-1 and Bcl-2. In line with this, CP-d/n-ATF5-S1 synergistically enhanced tumor cell apoptosis induced by the BH3-mimetic ABT263 and the death ligand TRAIL. In vivo, CP-d/n-ATF5-S1 attenuated tumor growth as a single compound in glioblastoma, melanoma, prostate cancer, and triple receptor–negative breast cancer xenograft models. Finally, the combination treatment of CP-d/n-ATF5-S1 and ABT263 significantly reduced tumor growth in vivo more efficiently than each reagent on its own.Conclusions: Our data support the idea that CP-d/n-ATF5-S1, administered as a single reagent or in combination with other drugs, holds promise as an innovative, safe, and efficient antineoplastic agent against treatment-resistant cancers. Clin Cancer Res; 22(18); 4698–711. ©2016 AACR.

Developing novel therapeutics often follows three steps: target identification, design of strategies to suppress target activity and drug development to implement the strategies. In this review, we recount the evidence identifying the basic leucine zipper transcription factors ATF5, CEBPB, and CEBPD as targets for brain and other malignancies. We describe strategies that exploit the structures of the three factors to create inhibitory dominant-negative (DN) mutant forms that selectively suppress growth and survival of cancer cells. We then discuss and compare four peptides (CP-DN-ATF5, Dpep, Bpep and ST101) in which DN sequences are joined with cell-penetrating domains to create drugs that pass through tissue barriers and into cells. The peptide drugs show both efficacy and safety in suppressing growth and in the survival of brain and other cancers in vivo, and ST101 is currently in clinical trials for solid tumors, including GBM. We further consider known mechanisms by which the peptides act and how these have been exploited in rationally designed combination therapies. We additionally discuss lacunae in our knowledge about the peptides that merit further research. Finally, we suggest both short- and long-term directions for creating new generations of drugs targeting ATF5, CEBPB, CEBPD, and other transcription factors for treating brain and other malignancies.

Activating Transcription Factor 4 (ATF4) has been postulated as a key regulator of learning and memory. We previously reported that specific hippocampal ATF4 down-regulation causes deficits in synaptic plasticity and memory and reduction of glutamatergic functionality. Here we extend our studies to address ATF49s role in neuronal excitability. We find that long-term ATF4 knockdown in cultured rat hippocampal neurons significantly increases the frequency of spontaneous action potentials. This effect is associated with decreased functionality of metabotropic GABA B receptors (GABA B Rs). Knocking down ATF4 results in significant reduction of GABA B R-induced GIRK-currents and increased mIPSCs frequency. Furthermore, reducing ATF4 significantly decreases expression of membrane-exposed, but not total, GABA B R 1a and 1b subunits, indicating that ATF4 regulates GABA B R trafficking. In contrast, ATF4 knockdown has no effect on surface expression of GABA B R2s, several GABA B R-coupled ion channels or β2 and Ɣ2 GABA A Rs. Pharmacologic manipulations confirmed the relationship between GABA B R functionality and action potential frequency in our cultures. Specifically, the effects of ATF4 down-regulation cited-above are fully rescued by transcriptionally active, but not by transcriptionally-inactive, shRNA-resistant, ATF4. We previously reported that ATF4 promotes stabilization of the actin-regulatory protein Cdc42 by a transcription-dependent mechanism. To test the hypothesis that this action underlies the mechanism by which ATF4 loss affects neuronal firing rates and GABA B R trafficking, we down-regulated Cdc42 and found that this phenocopies the effects of ATF4 knockdown on these properties. In conclusion, our data favor a model in which ATF4, by regulating Cdc42 expression, affects trafficking of GABA B Rs, which in turn modulates the excitability properties of neurons. Significance statement: GABA B receptors (GABA B Rs), the metabotropic receptors for the inhibitory neurotransmitter GABA, have crucial roles in controlling the firing rate of neurons. Deficits in trafficking/functionality of GABA B Rs have been linked to a variety of neurological and psychiatric conditions, including epilepsy, anxiety, depression, schizophrenia, addiction, and pain. Here we show that GABA B Rs trafficking is influenced by Activating Transcription Factor 4 (ATF4), a protein that has a pivotal role in hippocampal memory processes. We found that ATF4 down-regulation in hippocampal neurons reduces membrane-bound GABA B R levels and thereby increases intrinsic excitability. These effects are mediated by loss of the small GTPase Cdc42 following ATF4 down-regulation. These findings reveal a critical role for ATF4 in regulating the modulation of neuronal excitability by GABA B Rs.

Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin.

Mutations in the E3 ubiquitin ligase parkin are the most common known cause of autosomal recessive parkinsonism. Multiple types of stress decrease parkin protein levels, an effect that may be relevant to sporadic Parkinson's disease (PD), but the mechanism(s) involved in this loss remain largely unclear. We sought to elucidate these mechanisms using a PD-relevant stressor, L-DOPA, the precursor to dopamine, which forms reactive oxygen species (ROS) as well as toxic quinones via auto-oxidation. We find that L-DOPA causes parkin loss through both an oxidative stress-independent and an oxidative stress-dependent pathway. Characterization of the latter reveals that it requires both the kinase PINK1 and parkin's interaction with phosphorylated ubiquitin (phospho-Ub) and is mediated by proteasomal degradation. Surprisingly, mitochondrial parkin activity and autoubiquitination as well as mitophagy are not required for such loss. During stress induced by the oxidative stressor hydrogen peroxide or the metabolic uncoupler CCCP, parkin degradation also requires its association with phospho-Ub, indicating that this mechanism is broadly generalizable. As oxidative stress, metabolic dysfunction and phospho-Ub levels are all elevated in PD patients, we suggest that these changes may lead to the loss of parkin expression in PD.

The basic leucine zipper transcription factor ATF5 is overexpressed in many tumor types and interference with its expression or function inhibits cancer cell survival. As a potential therapeutic approach to exploit these findings, we created dominant-negative (DN) ATF5 forms lacking DNA-binding ability that retain the ATF5 leucine zipper, and thus associate with and sequester ATF5's requisite leucine zipper–binding partners. Preclinical studies with DN-ATF5, including a cell-penetrating form, show in vitro and in vivo efficacy in compromising cancer cell survival. However, DN-ATF5's targets, and particularly those required for tumor cell survival, have been unknown. We report that cells lacking ATF5 succumb to DN-ATF5, indicating that ATF5 itself is not DN-ATF5's obligate target. Unbiased pull-down assays coupled with mass spectrometry and immunoblotting revealed that DN-ATF5 associates in cells with the basic leucine zipper proteins CEBPB and CEBPD and coiled-coil protein CCDC6. Consistent with DN-ATF5 affecting tumor cell survival by suppressing CEBPB and CEBPD function, DN-ATF5 interferes with CEBPB and CEBPD transcriptional activity, while CEBPB or CEBPD knockdown promotes apoptotic death of multiple cancer cells lines, but not of normal astrocytes. We propose a two-pronged mechanism by which DN-ATF5 kills tumor cells. One is by inhibiting heterodimer formation between ATF5 and CEBPB and CDBPD, thus suppressing ATF5-dependent transcription. The other is by blocking the formation of transcriptionally active CEBPB and CEBPD homodimers as well as heterodimers with partners in addition to ATF5. Implications: This study indicates that the potential cancer therapeutic DN-ATF5 acts by associating with and blocking the transcriptional activities of CEBPB and CEBPD.

Activating transcription factor 4 (ATF4/CREB2), in addition to its well-studied role in stress responses, is proposed to play important physiologic functions in regulating learning and memory. However, the nature of these functions has not been well defined and is subject to apparently disparate views. Here, we provide evidence that ATF4 is a regulator of excitability during synaptic plasticity. We evaluated ATF49s role in mature hippocampal cultures subjected to a brief chemical-LTP (cLTP) induction protocol that results in changes in mEPSC properties and synaptic AMPA receptor density one hour later, with return to baseline by 24 hours. We find that ATF4 protein, but not its mRNA, is rapidly depleted by about 50% in response to cLTP induction via NMDA receptor activation. Depletion is detectable in dendrites within 15 min and in cell bodies by 1 hour and returns to baseline by 8 hours. Such changes correlate with a parallel depletion of phospho-eIF2a, suggesting that ATF4 loss is driven by decreased translation. To probe the physiologic role of cLTP-induced ATF4 depletion, we constitutively overexpressed the protein. Reversing ATF4 depletion by over-expression blocked the recovery of synaptic activity and AMPA receptor density to baseline values that would otherwise occur 24 hours after cLTP induction. This reversal was not reproduced by a transcriptionally inactive ATF4 mutant. These findings support ATF49s role as a required element in resetting baseline synaptic responsiveness after cLTP. Significance The mechanism(s) by which synaptic responsiveness is reset after LTP are not well understood. Resetting avoids LTP "saturation" and uncontrolled feed-forward potentiation and may play a part in synaptic "scaling”. In the work reported here we have found that the transcription factor ATF4 is translationally down-regulated following cLTP induction and acts as a regulator of long-term synaptic plasticity, resetting the synapse to the de-potentiated state. Our findings may serve to begin to reconcile the conflicting views regarding the role of ATF4 in synaptic plasticity and further illuminate the role of ATF4 in learning and memory.

Parkinson9s disease (PD) is characterized by the progressive loss of select neuronal populations, but the prodeath genes mediating the neurodegenerative processes remain to be fully elucidated. Trib3 (tribbles pseudokinase 3) is a stress-induced gene with proapoptotic activity that was previously described as highly activated at the transcriptional level in a 6-hydroxydopamine (6-OHDA) cellular model of PD. Here, we report that Trib3 immunostaining is elevated in dopaminergic neurons of the substantia nigra pars compacta (SNpc) of human PD patients. Trib3 protein is also upregulated in cellular models of PD, including neuronal PC12 cells and rat dopaminergic ventral midbrain neurons treated with 6-OHDA, 1-methyl-4-phenylpyridinium (MPP + ), or α-synuclein fibrils (αSYN). In the toxin models, Trib3 induction is substantially mediated by the transcription factors CHOP and ATF4. Trib3 overexpression is sufficient to promote neuronal death; conversely, Trib3 knockdown protects neuronal PC12 cells as well as ventral midbrain dopaminergic neurons from 6-OHDA, MPP + , or αSYN. Mechanism studies revealed that Trib3 physically interacts with Parkin, a prosurvival protein whose loss of function is associated with PD. Elevated Trib3 reduces Parkin expression in cultured cells; and in the SNpc of PD patients, Parkin levels are reduced in a subset of dopaminergic neurons expressing high levels of Trib3. Loss of Parkin at least partially mediates the prodeath actions of Trib3 in that Parkin knockdown in cellular PD models abolishes the protective effect of Trib3 downregulation. Together, these findings identify Trib3 and its regulatory pathways as potential targets to suppress the progression of neuron death and degeneration in PD. SIGNIFICANCE STATEMENT Parkinson9s disease (PD) is the most common neurodegenerative movement disorder. Current treatments ameliorate symptoms, but not the underlying neuronal death. Understanding the core neurodegenerative processes in PD is a prerequisite for identifying new therapeutic targets and, ultimately, curing this disease. Here, we describe a novel pathway involving the proapoptotic protein Trib3 in neuronal death associated with PD. These findings are supported by data from multiple cellular models of PD and by immunostaining of postmortem PD brains. Upstream, Trib3 is induced by the transcription factors ATF4 and CHOP; and downstream, Trib3 interferes with the PD-associated prosurvival protein Parkin to mediate death. These findings establish this new pathway as a potential and promising therapeutic target for treatment of PD.

Activating Transcription Factor 4 (ATF4) has been postulated as a key regulator of learning and memory. We previously reported that specific hippocampal ATF4 downregulation causes deficits in synaptic plasticity and memory and reduction of glutamatergic functionality. Here we extend our studies to address ATF4's role in neuronal excitability. We find that long-term ATF4 knockdown in cultured rat hippocampal neurons significantly increases the frequency of spontaneous action potentials. This effect is associated with decreased functionality of metabotropic GABA(B) receptors (GABA(B)Rs). Knocking down ATF4 results in significant reduction of GABA(B)R-induced GIRK currents and increased mIPSC frequency. Furthermore, reducing ATF4 significantly decreases expression of membrane-exposed, but not total, GABA(B)R 1a and 1b subunits, indicating that ATF4 regulates GABA(B)R trafficking. In contrast, ATF4 knockdown has no effect on surface expression of GABA(B)R2s, several GABA(B)R-coupled ion channels or β2 and γ2 GABA(A)Rs. Pharmacologic manipulations confirmed the relationship between GABA(B)R functionality and action potential frequency in our cultures. Specifically, the effects of ATF4 downregulation cited above are fully rescued by transcriptionally active, but not by transcriptionally inactive, shRNA-resistant, ATF4. We previously reported that ATF4 promotes stabilization of the actin-regulatory protein Cdc42 by a transcription-dependent mechanism. To test the hypothesis that this action underlies the mechanism by which ATF4 loss affects neuronal firing rates and GABA(B)R trafficking, we downregulated Cdc42 and found that this phenocopies the effects of ATF4 knockdown on these properties. In conclusion, our data favor a model in which ATF4, by regulating Cdc42 expression, affects trafficking of GABA(B)Rs, which in turn modulates the excitability properties of neurons. SIGNIFICANCE STATEMENT GABA(B) receptors (GABA(B)Rs), the metabotropic receptors for the inhibitory neurotransmitter GABA, have crucial roles in controlling the firing rate of neurons. Deficits in trafficking/functionality of GABA(B)Rs have been linked to a variety of neurological and psychiatric conditions, including epilepsy, anxiety, depression, schizophrenia, addiction, and pain. Here we show that GABA(B)Rs trafficking is influenced by Activating Transcription Factor 4 (ATF4), a protein that has a pivotal role in hippocampal memory processes. We found that ATF4 downregulation in hippocampal neurons reduces membrane-bound GABA(B)R levels and thereby increases intrinsic excitability. These effects are mediated by loss of the small GTPase Cdc42 following ATF4 downregulation. These findings reveal a critical role for ATF4 in regulating the modulation of neuronal excitability by GABA(B)Rs.

Activating transcription factor 4 (ATF4) plays important physiologic roles in the brain including regulation of learning and memory as well as neuronal survival and death. Yet, outside of translational regulation by the eIF2α-dependent stress response pathway, there is little information about how its levels are controlled in neurons. Here, we show that brain-derived neurotrophic factor (BDNF) promotes a rapid and sustained increase in neuronal ATF4 transcripts and protein levels. This increase is dependent on tropomyosin receptor kinase (TrkB) signaling, but independent of levels of phosphorylated eIF2α. The elevation in ATF4 protein occurs both in nuclei and processes. Transcriptome analysis revealed that ATF4 mediates BDNF-promoted induction of Sesn2 which encodes Sestrin2, a protector against oxidative and genotoxic stresses and a mTor complex 1 inhibitor. In contrast, BDNF-elevated ATF4 did not affect expression of a number of other known ATF4 targets including several with pro-apoptotic activity. The capacity of BDNF to elevate neuronal ATF4 may thus represent a means to maintain this transcription factor at levels that provide neuroprotection and optimal brain function without risk of triggering neurodegeneration.

Neuron death during development and in Alzheimer’s disease (AD) is associated with aberrant regulation/induction of cell cycle proteins. However, the proximal events in this process are unknown. Cell cycle initiation requires dephosphorylation of cyclin-dependent kinases by cell division cycle 25A (Cdc25A). Here, we show that Cdc25A is essential for neuronal death in response to NGF deprivation or β-amyloid (Aβ) treatment and describe the mechanisms by which it is regulated in these paradigms. Cdc25A mRNA, protein and Cdc25A phosphatase activity were induced by NGF deprivation and Aβ treatment. Enhanced Cdc25A expression was also observed in rat brains infused with Aβ and in Aβ-overexpressing AβPPswe-PS1dE9 mice. In cultured neurons Cdc25A inhibition by chemical inhibitors or shRNA prevented cell death and neurite degeneration caused by NGF deprivation or Aβ. Additionally, Cdc25A inhibition diminished distal signaling events including Cdk-dependent elevation of phospho-pRb and subsequent caspase-3 activation. Mechanism studies revealed that Cdc25A induction by NGF deprivation and Aβ is mediated by activation of Forkhead transcription factors that in turn suppress miR-21, a negative regulator of Cdc25A. Our studies thus identify Cdc25A as a required upstream element of the apoptotic cell cycle pathway that is required for neuron death in response to trophic factor deprivation and to Aβ exposure and therefore as a potential target to suppress pathologic neuron death.

The mechanisms governing neuron death following NGF deprivation are incompletely understood. Here, we show that Trib3, a protein induced by NGF withdrawal, has a key role in such death via a loop involving the survival kinase Akt and FoxO transcription factors. Trib3 overexpression is sufficient to induce neuron death, and silencing of endogenous Trib3 strongly protects from death when NGF is withdrawn. Mechanism studies reveal that Trib3 interferes with phosphorylation/activity of Akt and contributes to Akt inactivation after NGF deprivation. FoxO1a, a direct Akt substrate, is dephosphorylated upon NGF withdrawal and consequently undergoes nuclear translocation and activates pro-apoptotic genes. We find that Trib3 is required for FoxO1a dephosphorylation and nuclear translocation after NGF deprivation. Conversely, Trib3 induction requires FoxO transcription factors, which show enhanced occupancy of the Trib3 promoter region following NGF withdrawal. Collectively, these findings support a mechanism in which NGF deprivation, Akt dephosphorylation/inactivation, FoxO dephosphorylation/activation and Trib3 induction are linked in a self-amplifying feed-forward loop that culminates in neuron death.

Neuron death during development and in Alzheimer’s disease (AD) is associated with aberrant regulation/induction of cell cycle proteins. However, the proximal events in this process are unknown. Cell cycle initiation requires dephosphorylation of cyclin-dependent kinases by cell division cycle 25A (Cdc25A). Here, we show that Cdc25A is essential for neuronal death in response to NGF deprivation or β-amyloid (Aβ) treatment and describe the mechanisms by which it is regulated in these paradigms. Cdc25A mRNA, protein and Cdc25A phosphatase activity were induced by NGF deprivation and Aβ treatment. Enhanced Cdc25A expression was also observed in rat brains infused with Aβ and in Aβ-overexpressing AβPPswe-PS1dE9 mice. In cultured neurons Cdc25A inhibition by chemical inhibitors or shRNA prevented cell death and neurite degeneration caused by NGF deprivation or Aβ. Additionally, Cdc25A inhibition diminished distal signaling events including Cdk-dependent elevation of phospho-pRb and subsequent caspase-3 activation. Mechanism studies revealed that Cdc25A induction by NGF deprivation and Aβ is mediated by activation of Forkhead transcription factors that in turn suppress miR-21, a negative regulator of Cdc25A. Our studies thus identify Cdc25A as a required upstream element of the apoptotic cell cycle pathway that is required for neuron death in response to trophic factor deprivation and to Aβ exposure and therefore as a potential target to suppress pathologic neuron death.

Purpose: Despite significant progress in cancer research, many tumor entities still have an unfavorable prognosis. Activating transcription factor 5 (ATF5) is upregulated in various malignancies and promotes apoptotic resistance. We evaluated the efficacy and mechanisms of the first described synthetic cell-penetrating inhibitor of ATF5 function, CP-d/n-ATF5-S1. Experimental Design: Preclinical drug testing was performed in various treatment-resistant cancer cells and in vivo xenograft models. Results: CP-d/n-ATF5-S1 reduced the transcript levels of several known direct ATF5 targets. It depleted endogenous ATF5 and induced apoptosis across a broad panel of treatment-refractory cancer cell lines, sparing non-neoplastic cells. CP-d/n-ATF5-S1 promoted tumor cell apoptotic susceptibility in part by reducing expression of the deubiquitinase Usp9X and led to diminished levels of antiapoptotic Bcl-2 family members Mcl-1 and Bcl-2. In line with this, CP-d/n-ATF5-S1 synergistically enhanced tumor cell apoptosis induced by the BH3-mimetic ABT263 and the death ligand TRAIL. In vivo, CP-d/n-ATF5-S1 attenuated tumor growth as a single compound in glioblastoma, melanoma, prostate cancer, and triple receptor–negative breast cancer xenograft models. Finally, the combination treatment of CP-d/n-ATF5-S1 and ABT263 significantly reduced tumor growth in vivo more efficiently than each reagent on its own. Conclusions: Our data support the idea that CP-d/n-ATF5-S1, administered as a single reagent or in combination with other drugs, holds promise as an innovative, safe, and efficient antineoplastic agent against treatment-resistant cancers. Clin Cancer Res; 22(18); 4698–711. ©2016 AACR.

This review discusses current knowledge about cell death in the developing enteric nervous system (ENS). It also includes findings about the molecular mechanisms by which such death is mediated. Additional consideration is given to trophic factors that contribute to survival of the precursors and neurons and glia of the ENS, as well to genes that, when mutated or deleted, trigger their death. Although further confirmation is needed, present observations support the view that enteric neural crest-derived precursor cells en route to the gut undergo substantial levels of apoptotic death, but that once these cells colonize the gut, there is relatively little death of precursor cells or of neurons and glia during the fetal period. There are also indications that normal neuron loss occurs in the ENS, but at times beyond the perinatal stage. Taken together, these findings suggest that ENS development is similar is some ways, but different in others from extra-enteric areas of the vertebrate central and peripheral nervous systems, in which large-scale apoptotic death of precursor neurons and glia occurs during the fetal and perinatal periods. Potential reasons for these differences are discussed such as a fetal enteric microenvironment that is especially rich in trophic support. In addition to the cell death that occurs during normal ENS development, this review discusses mechanisms of experimentally-induced ENS cell death, such as those that are associated with defective glial cell-line derived neurotrophic factor/Ret signaling, which are an animal model of human congenital megacolon (aganglionosis; Hirschsprung's disease). Such considerations underscore the importance of understanding cell death in the developing ENS, not just from a curiosity-driven point of view, but also because the pathophysiology behind many disorders of human gastrointestinal function may originate in abnormalities of the mechanisms that govern cell survival and death during ENS development.

Precise regulation of neuroprogenitor cell proliferation and differentiation is required for successful brain development, but the factors that contribute to this are only incompletely understood. The transcription factor ATF5 promotes proliferation of cerebral cortical neuroprogenitor cells and its down-regulation permits their differentiation. Here, we examine the expression and regulation of ATF5 in cerebellar granule neuron progenitor cells (CGNPs) as well as the role of ATF5 in the transition of CGNPs to post-mitotic cerebellar granule neurons (GCNs). We find that ATF5 is expressed by proliferating CGNPs in both the embryonic and post-natal cerebellar external granule layer (EGL) and in the rhombic lip, the embryonic structure from which the EGL arises. In contrast, ATF5 is undetectable in post-mitotic GCNs. In highly enriched dissociated cultures of CGNPs and CGNs, ATF5 is expressed only in CGNPs. Constitutive ATF5 expression in CGNPs does not affect their proliferation or exit from the cell cycle. In contrast, in presence of sonic hedgehog (Shh), a mitogen for CGNPs, constitutively expressed ATF5 promotes CGNP proliferation and delays their cell cycle exit and differentiation. Conversely, ATF5 loss-of-function conferred by a dominant-negative form of ATF5, significantly diminishes Shh-stimulated CGNP proliferation and promotes differentiation. In parallel with its stimulation of CGNP proliferation, Shh enhances ATF5 expression by what appeared to be a post-transcriptional mechanism involving protein stabilization. These findings indicate a reciprocal interaction between ATF5 and Shh in which Shh stimulates ATF5 expression and in which ATF5 contributes to Shh-stimulated CGNP expansion.

Malignant gliomas have poor prognosis and urgently require new therapies. Activating Transcription Factor 5 (ATF5) is highly expressed in gliomas, and interference with its expression/function precipitates targeted glioma cell apoptosis in vitro and in vivo. We designed a novel deliverable truncated-dominant-negative (d/n) form of ATF5 fused to a cell-penetrating domain (Pen-d/n-ATF5-RP) that can be intraperitoneally/subcutaneously administered to mice harboring malignant gliomas generated; (1) by PDGF-B/sh-p53 retroviral transformation of endogenous neural progenitor cells; and (2) by human U87-MG xenografts. In vitro Pen-d/n-ATF5-RP entered into glioma cells and triggered massive apoptosis. In vivo, subcutaneously-administered Pen-d/n-ATF5-RP passed the blood brain barrier, entered normal brain and tumor cells, and then caused rapid selective tumor cell death. MRI verified elimination of retrovirus-induced gliomas within 8-21 days. Histopathology revealed growth-suppression of intracerebral human U87-MG cells xenografts. For endogenous PDGF-B gliomas, there was no recurrence or mortality at 6-12 months versus 66% mortality in controls at 6 months. Necropsy and liver-kidney blood enzyme analysis revealed no adverse effects on brain or other tissues. Our findings thus identify Pen-d/n-ATF5-RP as a potential therapy for malignant gliomas.

Glioblastomas are among the most incurable cancers. Our past findings indicated that glioblastoma cells, but not neurons or glia, require the transcription factor ATF5 (activating transcription factor 5) for survival. However, it was unknown whether interference with ATF5 function can prevent or promote regression/eradication of malignant gliomas in vivo. To address this issue, we created a mouse model by crossing a human glial fibrillary acidic protein (GFAP) promoter-tetracycline transactivator mouse line with tetracycline operon-dominant negative-ATF5 (d/n-ATF5) mice to establish bi-transgenic mice. In this model, d/n-ATF5 expression is controlled by doxycycline and the promoter for GFAP, a marker for stem/progenitor cells as well as gliomas. Endogenous gliomas were produced with high efficiency by retroviral delivery of platelet-derived growth factor (PDGF)-B and p53-short hairpin RNA (shRNA) in adult bi-transgenic mice in which expression of d/n-ATF5 was spatially and temporally regulated. Induction of d/n-ATF5 before delivery of PDGF-B/p53-shRNA virus greatly reduced the proportion of mice that formed tumors. Moreover, d/n-ATF5 induction after tumor formation led to regression/eradication of detectable gliomas without evident damage to normal brain cells in all 24 mice assessed.

The generation, differentiation, and migration of newborn neurons are critical features of normal brain development that are subject to both extracellular and intracellular regulation. However, the means of such control are only partially understood. Here, we show that expression of RTP801/REDD1, an inhibitor of mTOR (mammalian target of rapamycin) activation, is regulated during neuronal differentiation and that RTP801 functions to influence the timing of both neurogenesis and neuron migration. RTP801 levels are high in embryonic cortical neuroprogenitors, diminished in newborn neurons, and low in mature neurons. Knockdown of RTP801in vitroandin vivoaccelerates cell cycle exit by neuroprogenitors and their differentiation into neurons. It also disrupts migration of rat newborn neurons to the cortical plate and results in the ectopic localization of mature neurons. On the other hand, RTP801 overexpression delays neuronal differentiation. These findings suggest that endogenous RTP801 plays an essential role in temporal control of cortical development and in cortical patterning.

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.

Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apoptosis - nerve growth factor (NGF) deprivation and DNA damage - cellular levels of c-Cbl and Cbl-b fell well before the onset of cell death. NGF deprivation also induced rapid loss of tyrosine phosphorylation (and most likely, activation) of c-Cbl. Targeting c-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuronal death is by regulation of apoptotic c-Jun N-terminal kinase (JNK) signaling. We demonstrate that Cbl proteins interact with the JNK pathway components mixed lineage kinase (MLK) 3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK activation and on cell death.

We review recent findings regarding the properties of ATF5 and the major roles that this transcription factor plays in development of the nervous system and in survival of neural tumors. ATF5 is a widely expressed basic leucine zipper protein that has been subject to limited characterization. It is highly expressed in zones of neuroprogenitor cell proliferation. In vitro and in vivo studies indicate that it functions there to promote neuroprogenitor cell expansion and to suppress their differentiation into neurons or glia. ATF5 expression is down-regulated by trophic factors and this is required for their capacity to promote neuroprogenitor cell cycle exit and differentiation into either neurons, oligodendroglia or astrocytes. ATF5 is also highly expressed in a number of tumor types, including neural tumors such as neuroblastomas, medulloblastomas and glioblastomas. Examination of the role of ATF5 in glioblastoma cells indicates that interference with its expression or activity causes them to undergo apoptotic death. In contrast, normal astrocytes and neurons do not appear to require ATF5 for survival, indicating that it may be a selective target for treatment of glioblastomas and other neural neoplasias. Further studies are needed to identify the transcriptional targets of ATF5 and the mechanisms by which its expression is regulated in neuroprogenitors and tumors.

Previously, we reported that RTP801, a stress regulated protein, is induced in multiple cellular models of Parkinson's disease (PD), in an animal model of PD and in dopaminergic neurons of PD patients. In cellular PD models, RTP801 is both sufficient and necessary for death. We further showed that RTP801 and PD mimetics such as 6-OHDA trigger neuron death by suppressing activation of the key kinase mammalian target of rapamycin (mTOR). Here, we report that as a consequence of mTOR signaling blockade, 6-OHDA suppresses the phosphorylation and activation of Akt, a major supporter of neuron survival. This effect is mediated by RTP801 and appears to underlie neuron death induced by 6-OHDA. Examination of postmortem dopaminergic neurons reveals a consistent depletion of phospho-Akt, but not of total Akt in PD patients. These observations support a sequential mechanism in which PD-associated stresses induce RTP801, suppress mTOR signaling, deplete phosphorylated/activated Akt and permit neuron degeneration and death.

We review here evidence defining a molecular pathway that includes cell cycle-related molecules and that appears to play a required role in neuron death during normal development as well as in disease and trauma. The pathway starts with inappropriate activation of cyclin dependent kinase 4 (Cdk4) in neurons which leads to hyper-phosphorylation of the pRb family member p130. This in turn results in dissociation of p130 and its associated chromatin modifiers Suv39H1 and HDAC1 from the transcription factor E2F4. Dissociation of this complex results in de-repression of genes with E2F binding sites including those encoding the transcription factors B- and C-Myb. Once elevated in neurons, B- and C-Myb proteins bind to the promoter for the pro-apoptotic BH3-only protein Bim and promote its induction. Bim then interacts with the core cellular apoptotic machinery, leading to caspase activation and apoptotic death. This pathway is supported by a variety of observations and experimental findings that implicate it as a required element for neuron loss in development and in many nervous system traumas and disorders. The components of this pathway appear to represent potential therapeutic targets for prevention of disease-associated neuron death.

Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (+/- NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and approximately 50-80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk-associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity with Trk NGF receptors.

The molecules underlying neuron loss in Parkinson's disease (PD) are essentially unknown, and current therapies focus on diminishing symptoms rather than preventing neuron death. We identified RTP801 as a gene whose transcripts were highly induced in a cellular model of PD in which death of neuronal catecholaminergic PC12 cells was triggered by the PD mimetic 6-OHDA. Here, we find that RTP801 protein is also induced in this and additional cellular and animal PD models. To assess the relevance of these observations to PD, we used immunohistochemistry to compare RTP801 expression in postmortem brains from PD and control patients. For all PD brains examined, expression was highly elevated within neuromelanin-containing neurons of the substantia nigra but not in cerebellar neurons. Evaluation of the potential role of RTP801 induction in our cellular model revealed that RTP801 overexpression is sufficient to promote death but does not further elevate death caused by 6-OHDA. Furthermore, RTP801 induction is requisite for death in our cellular PD models and in 6-OHDA-treated cultured sympathetic neurons in that its knockdown by short hairpin RNAs (shRNAs) is protective. The mechanism by which 6-OHDA and RTP801 induce neuron death appears to involve repression of mammalian target of rapamycin (mTOR) kinase activity, and such death is inhibited by shRNAs targeting TSC2 (tuberous sclerosis complex), a protein with which RTP801 interacts to block mTOR activation. Our findings thus suggest that the elevation of RTP801 we detect in PD substantia nigral neurons may mediate their degeneration and death and that RTP801 and its signaling cascade may be novel potential therapeutic targets for the disease.

A sequential pathway (the JNK pathway) that includes activation of Rac1/Cdc42, mixed lineage kinases, MAP kinase kinases 4 and 7, and JNKs plays a required role in many paradigms of apoptotic cell death. However, the means by which this pathway is assembled and directed toward apoptotic death has been unclear. Here, we report that propagation of the apoptotic JNK pathway requires the cooperative interaction of two molecular scaffolds, POSH and JIPs. POSH (plenty of SH3s) is a multidomain GTP-Rac1-interacting protein that binds and promotes activation of mixed lineage kinases. JIPs are reported to bind MAP kinase kinases 4/7 and JNKs. We find that POSH and JIPs directly associate with one another to form a multiprotein complex, PJAC (POSH-JIP apoptotic complex), that includes all of the known kinase components of the pathway. Our observations indicate that this complex is required for JNK activation and cell death in response to apoptotic stimuli.

Caspase 2 has been implicated in trophic deprivation-induced neuronal death. We have shown that overexpression of the caspase 2-binding protein RAIDD induces neuronal apoptosis, acting synergistically with trophic deprivation. Currently, we examine the role of endogenous RAIDD in apoptosis of PC12 cells and sympathetic neurons. Expression of a truncated caspase recruitment domain-only form of caspase 2, which presumably disrupts the RAIDD interaction with endogenous caspase 2, attenuated trophic deprivation-induced apoptosis. Furthermore, downregulation of RAIDD by small interfering RNA led to inhibition of trophic deprivation-induced death, whereas death induced by DNA damage, which is not caspase 2-mediated, was not inhibited. Therefore, RAIDD, likely through interaction with caspase 2, is involved in trophic deprivation-induced neuronal apoptosis. This is the first demonstration of the involvement of RAIDD in apoptosis, and provides further support for the idea that apoptotic pathways in the same system may differ depending on the initiating stimulus.

We employed Serial Analysis of Gene Expression to identify transcriptional changes in a cellular model of Parkinson Disease (PD). The model consisted of neuronally differentiated PC12 cells compared before and after 8 hours' exposure to 6-hydroxydopamine. Approximately 1200 transcripts were significantly induced by 6-OHDA and approximately 500 of these are currently matched to known genes. Here, we categorize the regulated genes according to known functional activities and discuss their potential roles in neuron death and survival and in PD. We find induction of multiple death-associated genes as well as many with the capacity for neuroprotection. This suggests that survival or death of individual neurons in PD may reflect an integrated response to both protective and destructive gene changes. Our findings identify a number of regulated genes as candidates for involvement in PD and therefore as potential targets for therapeutic intervention. Such intervention may include both inhibiting the induction/activity of death-promoting genes and enhancing those with neuroprotective activity.

Activation of cell cycle elements plays a required role in neuronal apoptosis associated with both development and neurodegenerative disorders. We demonstrated previously that neuron survival requires gene repression mediated by the cell cycle transcription factor E2F (E2 promoter binding factor) and that apoptotic stimuli lead to de-repression of E2F-regulated genes and consequent death. However, the downstream mediators of such death have been unclear. The transcription factorsB- andC-mybare E2F-regulated genes that are induced in neurons by apoptotic stimuli. Here, we examine the role of B- and C-myb induction in neuron death. Antisense and siRNA constructs that effectively block the upregulation of B- and C-myb provide substantial protection against death of cultured neuronal PC12 cells, sympathetic neurons, and cortical neurons elicited by either NGF withdrawal or DNA damage. There is also significant protection from death induced by direct E2F-dependent gene de-repression. Our findings thus establish required roles for B- and C-myb in neuronal apoptosis.

An important milestone in brain development is the transition of neuroprogenitor cells to postmitotic neurons. We report that the bZIP transcription factor ATF5 plays a major regulatory role in this process. In developing brain ATF5 expression is high within ventricular zones containing neural stem and progenitor cells and is undetectable in postmitotic neurons. In attached clonal neurosphere cultures ATF5 is expressed by neural stem/progenitor cells and is undetectable in tau-positive neurons. In PC12 cell cultures nerve growth factor (NGF) dramatically downregulates endogenous ATF5 protein and transcripts, whereas exogenous ATF5 suppresses NGF-promoted neurite outgrowth. Such inhibition requires the repression of CRE sites. In contrast, loss of function conferred by dominant-negative ATF5 accelerates NGF-promoted neuritogenesis. Exogenous ATF5 also suppresses, and dominant-negative ATF5 and a small-interfering RNA targeted to ATF5 promote, neurogenesis by cultured nestin-positive telencephalic cells. These findings indicate that ATF5 blocks the differentiation of neuroprogenitor cells into neurons and must be downregulated to permit this process to occur.

We report that the multidomain protein POSH (plenty of SH3s) acts as a scaffold for the JNK pathway of neuronal death. This pathway consists of a sequential cascade involving activated Rac1/Cdc42, mixed-lineage kinases (MLKs), MAP kinase kinases (MKKs) 4 and 7, c-Jun N-terminal kinases (JNKs) and c-Jun, and is required for neuronal death induced by various means including nerve growth factor (NGF) deprivation. In addition to binding GTP-Rac1 as described previously, we find that POSH binds MLKs both in vivo and in vitro, and complexes with MKKs 4 and 7 and with JNKs. POSH overexpression promotes apoptotic neuronal death and this is suppressed by dominant-negative forms of MLKs, MKK4/7 and c-Jun, and by an MLK inhibitor. Moreover, a POSH antisense oligonucleotide and a POSH small interfering RNA (siRNA) suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal. Thus, POSH appears to function as a scaffold in a multiprotein complex that links activated Rac1 and downstream elements of the JNK apoptotic cascade.

Bim is a proapoptotic, BH3-domain-only member of the Bcl-2 family that plays a role in death of trophic factor-deprived sympathetic neurons as well as in other paradigms of apoptotic death. We report here that nerve growth factor (NGF) leads to both a slow down-regulation of Bim expression in neuronal PC12 cells and rapid Bim phosphorylation. Both effects appear to be mediated by the MEK/MAPK pathway. An assay for Bim-mediated death revealed that NGF-promoted phosphorylation suppresses the proapoptotic activity of Bim. The phosphorylation sites responsible for this effect in the extra long form of rBim were identified as Ser-109 and Thr-110. Thus, NGF protects neurons from the proapoptotic effects of Bim both by acute phosphorylation and the longer term repression of expression.

Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim− cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim− cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim− cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.

We used serial analysis of gene expression to identify new NGF-responsive immediate early genes (IEGs) with potential roles in neuronal differentiation. Among those identified was MafK, a small Maf family basic region and leucine zipper transcriptional repressor and coactivator expressed in immature neurons. NGF treatment elevates the levels of both MafK transcripts and protein. In contrast, there is no effect on expression of the closely related MafG. Unlike many other NGF-responsive IEGs, MafK regulation shows selectivity and is unresponsive to epidermal growth factor, depolarization, or cAMP derivatives. Inhibitor studies indicate that NGF-promoted MafK regulation is mediated by an atypical isoform of PKC but not by mitogen-activated kinase kinase, phospholipase Cgamma, or phosphoinositide 3'-kinase. Interference with MafK expression or activity by small interfering RNA and dominant negative strategies, respectively, suppresses NGF-promoted outgrowth and maintenance of neurites by PC12 cells and neurite outgrowth by immature telencephalic neurons. Our findings support a role for MafK as a novel regulator of neuronal differentiation.

Cell cycle molecules are mostly dormant in differentiated neurons that are post-mitotic and in the G0 state of the cell cycle. However, a wealth of evidence strongly suggests that in response to a wide variety of apoptotic stimuli, including trophic factor deprivation, exposure to β-amyloid (Aβ) and DNA damage, neurons emerge from theG0 state with aberrant expression/activation of cell cycle proteins.1 This emergence is characterized by a consistent set of events related to the cell cycle that culminate in neuron death. Initial responses include activation of G1/S cyclin-dependent kinases (Cdks), such as Cdk4 that in turn phosphorylate retinoblastoma (pRb) family proteins and lead to dissociation of repressor complexes comprising E2F and pRb proteins, so that E2F-binding genes are de-repressed. Among genes that are de-repressed by loss of E2F-Rb family complexes are the B- and C-myb transcription factors that in turn transactivate Bim, a pro-apoptotic protein that promotes caspase activation and subsequent neuron death.1–4 This set of events has been termed the ‘apoptotic cell cycle pathway’.Cell division cycle 25A (Cdc25A), a member of a family comprising Cdc25A, B and C, is a dual specificity phosphatase that dephosphorylates inhibitory phosphates on adjacent threonine and tyrosine residues of Cdks such as Cdk4.5 This step is essential for initiation of cell cycle in proliferating cells. However, it was not known whether in the non-dividing neurons, the same events would activate the apoptotic cell cycle pathway. In our recent paper published in Cell Death Discovery,6 we report several novel findings regarding the potential role of Cdc25A in neuron death. First, Cdc25A is required for activation of the apoptotic cell cycle pathway and neuron death in response to nerve growth factor (NGF) deprivation and Aβ treatment. Second, Cdc25A acts upstream of Cdk-mediated Rb phosphorylation and caspase-3 cleavage. Third, NGF deprivation and Aβ lead to rapid increases in Cdc25A mRNA and protein levels. NGF withdrawal causes an increase in Cdc25A activity as well. These events occur at about the same time that apoptotic insults lead to Cdk4 activation and Rb phosphorylation in our experimental systems and well precede evident signs of neuron death.

Mutations in the alpha-synuclein gene have recently been identified in families with inherited Parkinson's disease and the protein product of this gene is a component of Lewy bodies, indicating that alpha-synuclein is involved in Parkinson's disease pathogenesis. A role for normal alpha-synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein-1, the rat homolog of human alpha-synuclein, is highly and selectively up-regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein-1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1-2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein-1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor-treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein-1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein-1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for alpha-synuclein in plasticity-maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.

Alpha-synuclein mutations have been identified in certain families with Parkinson's disease (PD), and alpha-synuclein is a major component of Lewy bodies. Other genetic data indicate that the ubiquitin-dependent proteolytic system is involved in PD pathogenesis. We have generated stable PC12 cell lines expressing wild-type or A53T mutant human alpha-synuclein. Lines expressing mutant but not wild-type alpha-synuclein show: (1) disruption of the ubiquitin-dependent proteolytic system, manifested by small cytoplasmic ubiquitinated aggregates and by an increase in polyubiquitinated proteins; (2) enhanced baseline nonapoptotic death; (3) marked accumulation of autophagic-vesicular structures; (4) impairment of lysosomal hydrolysis and proteasomal function; and (5) loss of catecholamine-secreting dense core granules and an absence of depolarization-induced dopamine release. Such findings raise the possibility that the primary abnormality in these cells may involve one or more deficits in the lysosomal and/or proteasomal degradation pathways, which in turn lead to loss of dopaminergic capacity and, ultimately, to death. These cells may serve as a model to study the effects of aberrant alpha-synuclein on dopaminergic cell function and survival.

Apoptosis is a fundamental and essential process in development and tissue homeostasis of multicellular organisms. Roughly half of all the neurons produced during neurogenesis die apoptotically before the nervous system matures. Apoptosis is also involved in various neurodegenerative disorders such as Alzheimer's disease and neuronal trauma. Investigation of the mechanisms underlying neuronal apoptosis led to an unexpected discovery that in many cases revival of the quiescent and dormant cell cycle machinery is a common theme. Recent data suggest that uncoordinated expression of cell cycle molecules and the consequent breach of cell cycle checkpoints could be one of the primary mechanisms by which postmitotic neurons undergo apoptotic death. Evidence indicates that upregulation of cyclin-D-CDK4/6 activity and deregulation of E2F transcription factors mark key events in early stages of neuronal apoptosis. Active E2F repression by Rb family members is required for the survival of neurons. Apoptotic signals promote successive phosphorylation and dysfunction of Rb family members, resulting in sequential E2F derepression and expression of selective E2F-responsive genes. Thus, expression of derepressed E2F-responsive genes may be instrumental in propagating and amplifying the apoptotic signals instructing neuronal cells to carry out the apoptotic program.

We have identified a novel isoform of rat caspase-9 in which the C terminus of full-length caspase-9 is replaced with an alternative peptide sequence. Casp-9-CTD (where CTD is carboxyl-terminal divergent) is expressed in multiple tissues, with the relative highest expression observed in ovary and heart. Casp-9-CTD was found primarily in the cytoplasm and was not detected in the nucleus. Structural predictions suggest that in contrast to full-length caspase-9, casp-9-CTD will not be processed. Our model is supported by reduced protease activity of casp-9-CTD preparations in vitro and by the lack of detectable processing of casp-9-CTD proenzyme or the induction of cell death following transfection into cells. Both neuronal and non-neuronal cell types transfected with casp-9-CTD were resistant to death evoked by trophic factor deprivation or DNA damage. In addition, cytosolic lysates prepared from cells permanently expressing exogenous casp-9-CTD were resistant to caspase induction by cytochrome c in reconstitution assays. Taken together, our observations indicate that casp-9-CTD acts as a dominant-negative variant. Its expression in various tissues indicates a physiological role in regulating cell death.

beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c-Jun N-terminal kinases (JNKs) and of their substrate, c-Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, we examined the effects of CEP-1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP-1347 (100--300 nM) in the culture medium effectively blocked the increases in cellular JNK activity caused by A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant-negative c-Jun construct also conferred protection from A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-stimulated activation of caspase-2 and -3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by A beta and hence identify it as a potential therapeutic target in AD.

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC50values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

Excitotoxic stress is potentially an important component of disorders such as stroke and neurodegenerative diseases. Its toxic effects appear to be transduced through mechanisms that result in both acute and delayed forms of death. We examined here whether cyclin dependent kinases (CDKs), molecules normally associated with cell cycle control, may be involved in delayed excitotoxic death in two different excitotoxin models. We show that nuclear localized cyclin D1, an activator of Cdk4/6, is upregulated during kainic acid evoked death of CA3/CA1 neurons and that this upregulation is associated with increased phosphorylation of a critical CDK substrate, pRb. In addition, we find that the CDK inhibitor, flavopiridol blocks the delayed death of cultured cortical neurons evoked by 3-nitroproprionic acid, an inhibitor of the mitochondrial electron transport chain, treatment and that the NMDA antagonist, MK801 provides short term protection in this model. Full, long-term protection occurs when both flavopiridol and MK-801 are present. Taken together, these data support a role for cell cycle regulators in neuronal death evoked by excitotoxic stress and indicate a potential therapeutic target for treatment of excitotoxicity-related disorders.