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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.

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.

The transcription factor ATF5 is expressed in cells of the embryonic and neonatal ventricular zone/subventricular zone (VZ/SVZ), and must be down-regulated for their differentiation into neurons and astrocytes. Here, we show that ATF5 plays a major role in directing oligodendrocyte development. ATF5 is expressed by oligodendrocyte precursors but is absent from mature oligodendroglia. Constitutively expressed ATF5 maintains SVZ cells and O4(+) oligodendrocyte precursors in cycle and inhibits their differentiation into oligodendrocytes in vitro and in vivo. In contrast, ATF5 loss-of-function (LOF; produced by a dominant-negative form of the protein) accelerates oligodendrocyte differentiation of O4(+) cells in vitro and of SVZ cells in vivo. Significantly, the accelerated oligodendrocyte differentiation promoted by ATF5 LOF in vivo results in aberrant migration. Thus, appropriately regulated expression of ATF5 is required for proper expansion of oligodendroglial progenitors as well as for their timely differentiation. Regulation of oligodendrocyte, astrocyte, and neuronal differentiation indicates that ATF5 operates as a general regulator of the timing of differentiation, independent of cell lineage.

E2F-mediated gene repression plays a key role in regulation of neuron survival and death. However, the key molecules involved in such regulation and the mechanisms by which they respond to apoptotic stimuli are largely unknown. Here we show that p130 is the predominant Rb family member associated with E2F in neurons, that its major partner for repression of pro-apoptotic genes is E2F4, and that the p130-E2F4 complex recruits the chromatin modifiers HDAC1 and Suv39H1 to promote gene silencing and neuron survival. Apoptotic stimuli induce neuron death by sequentially causing p130 hyperphosphorylation, dissociation of p130-E2F4-Suv39H1-HDAC complexes, altered modification of H3 histone and gene derepression. Experimental suppression of such events blocks neuron death while interference with the synthesis of E2F4 or p130, or with the interaction of E2F4-p130 with chromatin modifiers, induces neuron death. Thus, neuron survival and death are dependent on the integrity of E2F4-p130-HDAC/Suv39H1 complexes.

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 factors B- and C-myb are 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 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.

Simple Summary: Dpep is a novel cell-penetrating peptide that selectively promotes the death of multiple tumor cell types in vitro and in vivo, and that is a potential therapeutic option for cancer. To better understand how it kills cancer cells, we compared gene expression levels in each of six diverse cancer cell lines before and after peptide treatment. An analysis of the data suggested a mechanism in which Dpep initiates a cascade of perturbations in gene expression that are particular to each cancer cell type, but that represent shared pathways that regulate cell growth and survival. These findings provide insight as to how drugs such as Dpep are able to impact the survival of a wide variety of tumor cell types, and identify how it might be combined with other cancer drugs for optimal efficacy. Dpep is a cell-penetrating peptide targeting transcription factors ATF5, CEBPB, and CEBPD, and that selectively promotes the apoptotic death of multiple tumor cell types in vitro and in vivo. As such, it is a potential therapeutic. To better understand its mechanism of action, we used PLATE-seq to compare the transcriptomes of six cancer cell lines of diverse origins before and after Dpep exposure. This revealed a context-dependent pattern of regulated genes that was unique to each line, but that exhibited a number of elements that were shared with other lines. This included the upregulation of pro-apoptotic genes and tumor suppressors as well as the enrichment of genes associated with responses to hypoxia and interferons. Downregulated transcripts included oncogenes and dependency genes, as well as enriched genes associated with different phases of the cell cycle and with DNA repair. In each case, such changes have the potential to lie upstream of apoptotic cell death. We also detected the regulation of unique as well as shared sets of transcription factors in each line, suggesting that Dpep may initiate a cascade of transcriptional responses that culminate in cancer cell death. Such death thus appears to reflect context-dependent, yet shared, disruption of multiple cellular pathways as well as of individual survival-relevant genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 GABAB receptors (GABABRs). Knocking down ATF4 results in significant reduction of GABABR-induced GIRK currents and increased mIPSC frequency. Furthermore, reducing ATF4 significantly decreases expression of membrane-exposed, but not total, GABABR 1a and 1b subunits, indicating that ATF4 regulates GABABR trafficking. In contrast, ATF4 knockdown has no effect on surface expression of GABABR2s, several GABABR-coupled ion channels or β2 and γ2 GABAARs. Pharmacologic manipulations confirmed the relationship between GABABR 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 GABABR 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 GABABRs, which in turn modulates the excitability properties of neurons. [ABSTRACT FROM AUTHOR]

The ubiquitously expressed activating transcription factor 4 (ATF4) has been variably reported to either promote or inhibit neuronal plasticity and memory. However, the potential cellular bases for these and other actions of ATF4 in brain are not well-defined. In this report, we focus on ATF4's role in post-synaptic synapse development and dendritic spine morphology. shRNA-mediated silencing of ATF4 significantly reduces the densities of PSD-95 and GluR1 puncta (presumed markers of excitatory synapses) in long-term cultures of cortical and hippocampal neurons. ATF4 knockdown also decreases the density of mushroom spines and increases formation of abnormally-long dendritic filopodia in such cultures. In vivo knockdown of ATF4 in adult mouse hippocampal neurons also reduces mushroom spine density. In contrast, ATF4 over-expression does not affect the densities of PSD-95 puncta or mushrooom spines. Regulation of synaptic puncta and spine densities by ATF4 requires its transcriptional activity and is mediated at least in part by indirectly controlling the stability and expression of the total and active forms of the actin regulatory protein Cdc42. In support of such a mechanism, ATF4 silencing decreases the half-life of Cdc42 in cultured cortical neurons from 31.5 to 18.5 h while knockdown of Cdc42, like ATF4 knockdown, reduces the densities of mushroom spines and PSD-95 puncta. Thus, ATF4 appears to participate in neuronal development and plasticity by regulating the post-synaptic development of synapses and dendritic mushroom spines via a mechanism that includes regulation of Cdc42 levels. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a common neurodegenerative disorder, for which there are no effective disease-modifying therapies. The transcription factor ATF4 (activating transcription factor 4) is induced by multiple PD-relevant stressors, such as endoplasmic reticulum stress and oxidative damage. ATF4 may exert either protective or deleterious effects on cell survival, depending on the paradigm. However, the role of ATF4 in the pathogenesis of PD has not been explored. We find that ATF4 levels are increased in neuromelanin-positive neurons in the substantia nigra of a subset of PD patients relative to controls. ATF4 levels are also upregulated in neuronal PC12 cells treated with the dopaminergic neuronal toxins 6-hydroxydopamine (6-OHDA) and l-methyl-4-phenylpyridinium (MPP+). To explore the role of ATF4 in cell survival in PD-relevant contexts, we either silenced or overexpressed ATF4 in cellular models of PD. In neuronal PC 12 cells, silencing of ATF4 enhanced cell death in response to either 6-OHDA or MPP +. Conversely, overexpression of ATF4 reduced cell death caused by dopaminergic neuronal toxins. ATF4 was also protective against 6-OHDA-induced death of cultured mouse ventral midbrain dopaminergic neurons. We further show that parkin, a gene associated with autosomal recessive PD, plays a critical role in ATF4-mediated protection. After treatment with 6-OHDA or MPP+, parkin protein levels fall, despite an increase in mRNA levels. ATF4 silencing exacerbates the toxin-induced reduction of parkin, whereas ATF4 overexpression partially preserves parkin levels. Finally, parkin silencing blocked the protective capacity of ATF4. These results indicate that ATF4 plays a protective role in PD through the regulation of parkin. [ABSTRACT FROM AUTHOR]

The superior colliculus (SC)/optic tectum of the dorsal mesencephalon plays a major role in responses to visual input, yet regulation of neuronal differentiation within this layered structure is only partially understood. Here, we show that the zinc finger transcription factor Gata2 is required for normal SC development. Starting at embryonic day 15 (E15) (corresponding to the times at which neurons of the outer and intermediate layers of the SC are generated), Gata2 is transiently expressed in the rat embryonic dorsal mesencephalon within a restricted region between proliferating cells of the ventricular zone and the deepest neuronal layers of the developing SC. The Gata2- positive cells are postmitotic and lack markers of differentiated neurons, but express markers for immature neuronal precursors including Ascl1 and Pax3/7. In utero electroporation with Gata2 small hairpin RNAs at E16 into cells along the dorsal mesencephalic ventricle interferes with their normal migration into the SC and maintains them in a state characterized by retention of Pax3 expression and the absence of mature neuronal markers. Collectively, these findings indicate that Gata2 plays a required role in the transition of postmitotic neuronal precursor cells of the retinorecipient layers of the SC into mature neurons and that loss of Gata2 arrests them at an intermediate stage of differentiation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The inappropriate expression/activation of cell-cycle-related molecules is associated with neuron death in many experimental paradigms and human neuropathologic conditions. However, the means whereby this links to the core apoptotic machinery in neurons have been unclear. Here, we show that the pro-apoptotic Bcl-2 homology 3 domain-only molecule Bcl-2 interacting mediator of cell death (Bim) is a target of a cell-cycle-related apoptotic pathway in neuronal cells. Induction of Bim in NGF-deprived cells requires expression and activity of cyclin-dependent kinase 4 (cdk4) and consequent de-repression of E2 promoter binding factor (E2F)-regulated genes including members of the myb transcription factor family. The Bim promoter contains two myb binding sites, mutation of which abolishes induction of a Bim promoter-driven reporter by NGF deprivation or E2F-dependent gene de-repression. NGF deprivation significantly increases endogenous levels of C-myb and its occupancy of the endogenous Bim promoter. These findings support a model in which apoptotic stimuli lead to cdk4 activation, consequent de-repression of E2F-regulated mybs, and induction of pro-apoptotic Bim. [ABSTRACT FROM AUTHOR]

The mechanisms that regulate neural progenitor cell differentiation are primarily unknown. The transcription factor activating transcription factor 5 (ATF5) is expressed in neural progenitors of developing brain but is absent from mature astrocytes and neurons. Here, we demonstrate that ATF5 regulates the conversion of ventricular zone (VZ) and subventricular zone (SVZ) neural progenitors into astrocytes. Constitutive ATF5 expression maintains neural progenitor cell proliferation and blocks their in vitro and in vivo differentiation into astrocytes. Conversely, loss of ATF5 function promotes cell-cycle exit and allows astrocytic differentiation in vitro and in vivo. CNTF, a promoter of astrocytic differentiation, downregulates endogenous ATF5, whereas constitutively expressed ATF5 suppresses CNTF-promoted astrocyte genesis. Unexpectedly, constitutive ATF5 expression in neonatal SVZ cells both in vitro and in vivo causes them to acquire properties and anatomic distributions of VZ cells. These findings identify ATF5 as a key regulator of astrocyte formation and potentially of the VZ to SVZ transition. [ABSTRACT FROM AUTHOR]

Simple Summary: The gene-regulatory factors ATF5, CEBPB and CEBPD promote survival, growth, metastasis and treatment resistance of a range of cancer cell types. Presently, no drugs target all three at once. Here, with the aim of treating cancers, we designed novel cell-penetrating peptides that interact with and inactivate all three. The peptides Bpep and Dpep kill a range of cancer cell types in culture and in animals. In animals with tumors, they also significantly increase survival time. In contrast, they do not affect survival of non-cancer cells and have no apparent side effects in animals. The peptides work in combination with other anti-cancer treatments. Mechanism studies of how the peptides kill cancer cells indicate a decrease in survival proteins and increase in death proteins. These studies support the potential of Bpep and Dpep as novel, safe agents for the treatment of a variety of cancer types, both as mono- and combination therapies. Transcription factors are key players underlying cancer formation, growth, survival, metastasis and treatment resistance, yet few drugs exist to directly target them. Here, we characterized the in vitro and in vivo anti-cancer efficacy of novel synthetic cell-penetrating peptides (Bpep and Dpep) designed to interfere with the formation of active leucine-zipper-based dimers by CEBPB and CEBPD, transcription factors implicated in multiple malignancies. Both peptides similarly promoted apoptosis of multiple tumor lines of varying origins, without such effects on non-transformed cells. Combined with other treatments (radiation, Taxol, chloroquine, doxorubicin), the peptides acted additively to synergistically and were fully active on Taxol-resistant cells. The peptides suppressed expression of known direct CEBPB/CEBPD targets IL6, IL8 and asparagine synthetase (ASNS), supporting their inhibition of transcriptional activation. Mechanisms by which the peptides trigger apoptosis included depletion of pro-survival survivin and a required elevation of pro-apoptotic BMF. Bpep and Dpep significantly slowed tumor growth in mouse models without evident side effects. Dpep significantly prolonged survival in xenograft models. These findings indicate the efficacy and potential of Bpep and Dpep as novel agents to treat a variety of cancers as mono- or combination therapies. [ABSTRACT FROM AUTHOR]

Bim is a pro-apoptotic member of the Bcl-2 family that is induced and contributes to neuron death in response to nerve growth factor (NGF) deprivation. Past work has revealed that Bim is downstream of multiple independent transcriptional pathways in neurons, including those culminating in activation of the c-Jun, FoxO, and Myb transcription factors. This study addresses the issue of whether the three signaling pathways are redundant with respect to Bim induction or whether they act cooperatively. Examination of the proximal Bim promoter reveals binding sites for FoxO, Mybs, and, as shown here, c-Jun. We find that mutation of any one of these types of sites abolishes induction of a Bim promoter-driven reporter in response to NGF deprivation. Moreover, down-regulation of either c-Jun, FoxOs, or Mybs by short hairpin RNAs blocks induction of Bim promoter-reporter activity triggered by withdrawal of NGF, This was the case for reporters driven by either the proximal promoter or a promoter that also includes additional regulatory elements in the first intron of the Bim gene. Such short hairpin RNAs also suppressed the induction of endogenous Bim protein. These findings thus indicate that the Bim promoter acts as a coincidence detector that optimally responds to the simultaneous activation of three different pro-apoptotic transcriptional pathways. Such a mechanism provides a ‘fail-safe’ that prevents neurons from dying by accidental activation of any single pathway. It also permits neurons to utilize individual pathways such as JNK signaling for other purposes without risk of demise. [ABSTRACT FROM AUTHOR]