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Biomedical subjects

L A Greene

Publications and source records attributed to L A Greene.

At least 19 recordsLinked to original sources
PubMed2001

Regulation of neuronal survival and death by E2F-dependent gene repression and derepression.

Neuronal death induced by a variety of means requires participation of the E2F family of transcription factors. Here, we show that E2F acts as a gene silencer in neurons and that repression of E2F-responsive genes is required for neuronal survival. Moreover, neuronal death evoked by DNA damaging agents or trophic factor withdrawal is characterized by derepression of E2F-responsive genes. Such derepression, rather than direct E2F-promoted gene activation, is required for death. Among the genes that are derepressed in neurons subjected to DNA damage or trophic factor withdrawal are the transcription factors B- and C-myb. Overexpression of B- and C-myb is sufficient to evoke neuronal death. These findings support a model in which E2F-dependent gene repression and derepression play pivotal roles in neuronal survival and death, respectively.

Animals↗
PubMed2001

Death in the balance: alternative participation of the caspase-2 and -9 pathways in neuronal death induced by nerve growth factor deprivation.

The data presented here demonstrate that sympathetic neurons have the potential to activate two alternative caspase-dependent pathways either of which is capable of mediating death induced by NGF deprivation and that these neurons have the potential to switch from one pathway to the other. The presence of these two alternative pathways to trophic factor deprivation-induced death may have implications for ensuring the correct development of the nervous system. In wild-type neurons, a caspase-2-dependent pathway is required for death, and a caspase-9-dependent pathway appears to be suppressed by endogenous inhibitors of apoptosis proteins (IAPs). In contrast, for caspase-2-null neurons, death is dependent on the caspase-9 pathway. The mechanism underlying the shift is the result of a threefold compensatory elevation of caspase-9 expression and a doubling of levels of direct IAP binding protein with low pI/(DIABLO)/second mitochondria-derived activator of caspase (Smac), an IAP inhibitor, both at the mRNA and protein levels [corrected]. These findings resolve seemingly discrepant findings regarding the roles of various caspases after NGF deprivation and raise a cautionary note regarding the interpretation of findings with caspase-null animals. The choice of the death-mediating caspase pathway in the sympathetic neurons is thus dependent on the regulated relative expression of components of the pathways including those of caspases, IAPs, and IAP inhibitors.

Animals↗
PubMed2001

Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family.

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 IC(50) values 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.

Animals↗
PubMed2001

Characterization of a novel isoform of caspase-9 that inhibits apoptosis.

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.

Amino Acid Sequence↗
PubMed2001

Neuronal apoptosis at the G1/S cell cycle checkpoint.

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.

Animals↗
PubMed2001

Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells.

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.

Animals↗
PubMed2001

beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation.

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.

Alzheimer Disease↗
PubMed2001

The MLK family mediates c-Jun N-terminal kinase activation in neuronal apoptosis.

Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.

Animals↗
PubMed2000

Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles.

Melanin, the pigment in hair, skin, eyes, and feathers, protects external tissue from damage by UV light. In contrast, neuromelanin (NM) is found in deep brain regions, specifically in loci that degenerate in Parkinson's disease. Although this distribution suggests a role for NM in Parkinson's disease neurodegeneration, the biosynthesis and function of NM have eluded characterization because of lack of an experimental system. We induced NM in rat substantia nigra and PC12 cell cultures by exposure to l-dihydroxyphenylalanine, which is rapidly converted to dopamine (DA) in the cytosol. This pigment was identical to human NM as assessed by paramagnetic resonance and was localized in double membrane autophagic vacuoles identical to NM granules of human substantia nigra. NM synthesis was abolished by adenoviral-mediated overexpression of the synaptic vesicle catecholamine transporter VMAT2, which decreases cytosolic DA by increasing vesicular accumulation of neurotransmitter. The NM is in a stable complex with ferric iron, and NM synthesis was inhibited by the iron chelator desferrioxamine, indicating that cytosolic DA and dihydroxyphenylalanine are oxidized by iron-mediated catalysis to membrane-impermeant quinones and semiquinones. NM synthesis thus results from excess cytosolic catecholamines not accumulated into synaptic vesicles. The permanent accumulation of excess catechols, quinones, and catechol adducts into a membrane-impermeant substance trapped in organelles may provide an antioxidant mechanism for catecholamine neurons. However, NM in organelles associated with secretory pathways may interfere with signaling, as it delays stimulated neurite outgrowth in PC12 cells.

Animals↗
PubMed2000

Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling.

Neurotrophic factors such as nerve growth factor (NGF) promote a wide variety of responses in neurons, including differentiation, survival, plasticity, and repair. Such actions often require changes in gene expression. To identify the regulated genes and thereby to more fully understand the NGF mechanism, we carried out serial analysis of gene expression (SAGE) profiling of transcripts derived from rat PC12 cells before and after NGF-promoted neuronal differentiation. Multiple criteria supported the reliability of the profile. Approximately 157,000 SAGE tags were analyzed, representing at least 21,000 unique transcripts. Of these, nearly 800 were regulated by 6-fold or more in response to NGF. Approximately 150 of the regulated transcripts have been matched to named genes, the majority of which were not previously known to be NGF-responsive. Functional categorization of the regulated genes provides insight into the complex, integrated mechanism by which NGF promotes its multiple actions. It is anticipated that as genomic sequence information accrues the data derived here will continue to provide information about neurotrophic factor mechanisms.

14-3-3 Proteins↗
PubMed2000

NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR.

The low affinity neurotrophin receptor p75NTR can mediate cell survival as well as cell death of neural cells by NGF and other neurotrophins. To elucidate p75NTR-mediated signal transduction, we screened p75NTR-associated proteins by a yeast two-hybrid system. We identified one positive clone and named NADE (p75NTR-associated cell death executor). Mouse NADE has marked homology to the human HGR74 protein. NADE specifically binds to the cell-death domain of p75NTR. Co-expression of NADE and p75NTR induced caspase-2 and caspase-3 activities and the fragmentation of nuclear DNA in 293T cells. However, in the absence of p75NTR, NADE failed to induce apoptosis, suggesting that NADE expression is necessary but insufficient for p75NTR-mediated apoptosis. Furthermore, p75NTR/NADE-induced cell death was dependent on NGF but not BDNF, NT-3, or NT-4/5, and the recruitment of NADE to p75NTR (intracellular domain) was dose-dependent. We obtained similar results from PC12 cells, nnr5 cells, and oligodendrocytes. Taken together, NADE is the first signaling adaptor molecule identified in the involvement of p75NTR-mediated apoptosis induced by NGF, and it may play an important role in the pathogenesis of neurogenetic diseases.

Amino Acid Sequence↗
PubMed2000

Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage.

Neuronal death evoked by DNA damage requires cyclin-dependent kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6 phosphorylates retinoblastoma protein (pRb) family members that then modulate the transcriptional activity of E2F/DP1 complexes, we examined the involvement of these components in DNA damage-evoked neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and p107. The CDK inhibitor flavopiridol suppressed pRb and p107 phosphorylation and loss, implicating CDK activity in these events. Moreover, the loss of pRb and p107 appeared to be mediated by caspases because it was blocked by general caspase inhibitors. The role of phosphorylation and pRb and p107 loss in the death pathway was indicated by observations that virally mediated expression of pRb mutated at sites of phosphorylation, including the Cdk4/6 site, inhibited death. Finally, expression of dominant-negative versions of DP1, known to compromise E2F transcriptional activity, protects cortical neurons from death induced by camptothecin and sympathetic neurons from death evoked by UV treatment. Taken together, these results implicate the CDK-pRb/E2F/DP pathway as a required element in the neuronal death evoked by DNA damage.

Animals↗