Biomedical subjects

Lloyd A Greene

Publications and source records attributed to Lloyd A Greene.

At least 19 recordsLinked to original sources

PubMed2006

Cell cycle molecules define a pathway required for neuron death in development and disease.

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.

Animals↗

PubMed2006

Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model.

Nix, a pro-apoptotic BH3-only protein, promotes apoptosis of non-neuronal cells, although the mechanisms involved remain incompletely understood. Using a yeast two-hybrid screen with POSH (plenty of SH3 domains, a scaffold involved in activation of the apoptotic JNK/c-Jun pathway) as the bait, we identified an interaction between POSH and Nix. Co-immunoprecipitation and in vitro binding studies confirmed a direct interaction between POSH and Nix in mammalian cells. When overexpressed in HEK293 cells, Nix promotes apoptosis along with enhanced phosphorylation/activation of JNKs and their target c-Jun. These effects appear to be dependent on POSH because Nix does not promote either JNK/c-Jun phosphorylation or apoptosis of 293 cells that do not express POSH. Nix and POSH appear to mutually stabilize one another and this effect could contribute to their promotion of death. Past work showed induction of Nix transcripts in a cellular model of Parkinson disease based on neuronal PC12 cells exposed to 6-hydroxydopamine. Here, we confirm elevation of Nix protein in this model and that Nix over-expression causes apoptotic death of PC12 cells by a mechanism dependent on c-Jun activation. Expression of s-Nix, a dominant-negative form of Nix, protects neuronal PC12 cells from 6-hydroxydopamine but not from nerve growth factor deprivation. These results indicate that Nix promotes cell death via interaction with POSH and activation of the JNK/c-Jun pathway and that Nix protein is induced and contributes to cell death in a cellular model of Parkinson disease.

Adaptor Proteins, Signal Transducing↗

PubMed2006

RTP801 is elevated in Parkinson brain substantia nigral neurons and mediates death in cellular models of Parkinson's disease by a mechanism involving mammalian target of rapamycin inactivation.

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.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Direct interaction of the molecular scaffolds POSH and JIP is required for apoptotic activation of JNKs.

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.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Activation of the apoptotic JNK pathway through the Rac1-binding scaffold protein POSH.

The JNKs (c-Jun N-terminal protein kinases) play important roles in a variety of physiological and pathological functions including induction of apoptosis. A major pathway by which JNKs are activated in response to apoptotic stimuli includes the GTP*Rac1-binding scaffold protein POSH (plenty of SH3s). POSH acts as a scaffold for binding and autoactivation of the MLK family of MKKK proteins, which in turn phosphorylate and activate the MKK family members MKK4 and 7, which in turn phosphorylate JNKs. In this chapter, we describe methods and techniques that have been successfully used to study the POSH-dependent apoptotic JNK pathway. Use of these techniques may lead to a better understanding of the components of this pathway and of how it is suppressed in viable cells and rapidly activated in response to apoptotic stimuli.

Apoptosis↗

PubMed2005

Siah1 interacts with the scaffold protein POSH to promote JNK activation and apoptosis.

Siah proteins are ubiquitin-protein isopeptide ligases (E3) that have been implicated in a variety of cellular actions, including promotion of apoptotic death. Here, we show that Siah1 is a binding partner for POSH (plenty of SH3s), a scaffold component of the apoptotic JNK pathway, and that Siah contributes to death of neurons and other cell types by activating the JNK pathway. Such proapoptotic activity requires the E3 ligase activity of Siah1. Moreover, apoptotic stimuli markedly elevate cellular Siah1 levels by a mechanism reliant on Siah1 protein stabilization. This stabilization requires JNK pathway activation and interaction with POSH and is enhanced by phosphorylation of SIAH1 at tyrosines 100 and 126. Depletion of intracellular Siah proteins via small interference RNA partially protects cells from death evoked by apoptotic stimuli such as trophic factor deprivation and DNA damage. These findings thus reveal a "loop" mechanism in which the JNK pathway promotes SIAH1 stabilization and in which SIAH1 in turn activates the JNK pathway and, ultimately, contributes to cell death.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Bim is a direct target of a neuronal E2F-dependent apoptotic pathway.

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.

Animals↗

PubMed2005

CHOP/GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism.

There is increasing evidence that neuron death in neurodegenerative diseases, such as Parkinson's disease, is due to the activation of programmed cell death. However, the upstream mediators of cell death remain largely unknown. One approach to the identification of upstream mediators is to perform gene expression analysis in disease models. Such analyses, performed in tissue culture models induced by neurotoxins, have identified up-regulation of CHOP/GADD153, a transcription factor implicated in apoptosis due to endoplasmic reticulum stress or oxidative injury. To evaluate the disease-related significance of these findings, we have examined the expression of CHOP/GADD153 in neurotoxin models of parkinsonism in living animals. Nuclear expression of CHOP protein is observed in developmental and adult models of dopamine neuron death induced by intrastriatal injection of 6-hydroxydopamine (6OHDA) and in models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). CHOP is a mediator of neuron death in the adult 60HDA model because a null mutation results in a reduction in apoptosis. In the chronic MPTP model, however, while CHOP is robustly expressed, the null mutation does not protect from the loss of neurons. We conclude that the role of CHOP depends on the nature of the toxic stimulus. For 6OHDA, an oxidative metabolite of dopamine, it is a mediator of apoptotic death.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2005

Downregulation of activating transcription factor 5 is required for differentiation of neural progenitor cells into astrocytes.

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.

Activating Transcription Factors↗

PubMed2005

Regulation of neuron survival and death by p130 and associated chromatin modifiers.

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.

Animals↗

PubMed2005

You can't go home again: transcriptionally driven alteration of cell signaling by NGF.

Here we review findings indicating that neurotrophins such as NGF promote changes in gene transcription that in turn influence the ways that cells subsequently respond to trophic factors. As a result, initial responses of "naïve" cells to NGF and other trophic agents differ from those of cells with prior NGF exposure. We discuss specific examples based on reports in the literature as well as on data derived from a serial analysis of gene expression (SAGE) study of NGF-promoted transcriptional changes in PC12 pheochromocytoma cells.

14-3-3 Proteins↗

PubMed2005

ATF5 regulates the proliferation and differentiation of oligodendrocytes.

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.

Animals↗

PubMed2005

Analysis of gene expression changes in a cellular model of Parkinson disease.

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.

Animals↗

PubMed2005

Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop.

A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death.

Animals↗

PubMed2004

Highly efficient small interfering RNA delivery to primary mammalian neurons induces MicroRNA-like effects before mRNA degradation.

The study of protein function in neurons has been hindered by the lack of highly efficient, nontoxic methods of inducing RNA interference in such cells. Here we show that application of synthetic small interfering RNA (siRNA) linked to the vector peptide Penetratin1 results in rapid, highly efficient uptake of siRNA by entire populations of cultured primary mammalian hippocampal and sympathetic neurons. This treatment leads to specific knock-down of targeted proteins within hours without the toxicity associated with transfection. In contrast to current methods, our technique permits study of protein function across entire populations with minimal disturbance of complex cellular networks. Using this technique, we found that protein knock-down (evident after 6 hr) precedes any decrease in targeted message (evident after 24 hr), suggesting an early, translational repression by perfectly targeted siRNAs.

Animals↗

PubMed2004

B-myb and C-myb play required roles in neuronal apoptosis evoked by nerve growth factor deprivation and DNA damage.

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.

Animals↗

PubMed2004

Malignant pheochromocytoma: current status and initiatives for future progress.

Pheochromocytomas are rare catecholamine-producing neuroendocrine tumors that are usually benign, but which may also present as or develop into a malignancy. Predicting such behavior is notoriously difficult and there are currently no curative treatments for malignant tumors. This report follows from a workshop at the Banbury Conference Center, Cold Spring Harbor, New York, on the 16th-18th November 2003, held to review the state of science and to facilitate future progress in the diagnosis and treatment of malignant pheochromocytoma. The rarity of the tumor and the resulting fragmented nature of studies, typically involving small numbers of patients, represent limiting factors to the development of effective treatments and diagnostic or prognostic markers for malignant disease. Such development is being facilitated by the availability of new genomics-based tools, but for such approaches to succeed ultimately requires comprehensive clinical studies involving large numbers of patients, stringently collected clinical data and tumor samples, and interdisciplinary collaborations among multiple specialist centers. Nevertheless, the well-characterized hereditary basis and the unique functional nature of these neuroendocrine tumors provide a useful framework that offers advantages for establishing the pathways of tumorigenesis and malignancy. Such findings may have relevance for understanding the basis of other more common malignancies where similar frameworks are not available. As the relevant pathways leading to pheochromocytoma are established it should be possible to take advantage of the new generation of drugs being developed to target specific pathways in other malignancies. Again the success of this will require well-designed and coordinated multi-center studies.

Adrenal Gland Neoplasms↗

PubMed2003

Regulated expression of ATF5 is required for the progression of neural progenitor cells to neurons.

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.

Activating Transcription Factors↗