Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through post-diapause quiescence.

Distinct differences in the temporal expression patterns of genes associated with pupal diapause were noted in the flesh fly, Sarcophaga crassipalpis. The first change observed was a decline in expression of the gene encoding heat shock protein 90 (hsp90) 2 days after pupariation (1 day before the pupa reaches the phanerocephalic stage characteristic of diapause). In contrast, hsp23 and hsp70 transcripts were undetectable in nondiapause samples and d1-d4 diapause-programmed pupae, but were up-regulated just after the start of diapause, 5 days after pupariation. An increase of glycerol content in diapausing pupae was also noted at the start of diapause. The gene encoding proliferating cell nuclear antigen (pcna) was diapause down-regulated, and this occurred in two phases, with the first decline in expression 7 days after pupariation and a second decline in the level of expression on day 14. For pupae held at 20 degrees C for 20 days and transferred to 10 degrees C, diapause ended after 90-100 days at the lower temperature. However, pupae remained in a state of post-diapause quiescence (d100-d150) and sustained diapause-like hsp and pcna expression patterns until adult development was initiated. Glycerol concentrations and survival declined during the post-diapause phase. This study suggests a distinct sequence in the pattern of gene expression at the onset of diapause, but the genes we have monitored do not contribute to the switch to covert developmental potential at the transition from diapause to post-diapause quiescence.

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons.

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.

Ceramide selectively inhibits apoptosis-associated events in NGF-deprived sympathetic neurons.

Ceramide manifests both neurotoxic and neuroprotective properties depending on the experimental system. Ito and Horigome previously reported that ceramide delays apoptosis in a classic model of developmental programmed cell death, i.e. sympathetic neurons undergoing NGF deprivation.1 Here, we investigated the actions of ceramide upon the biochemical and genetic changes that occur in NGF deprived neurons. We correlate ceramide's neuroprotective actions with the ability of ceramide to antagonize NGF deprivation-induced oxidative stress and c-jun induction, both of which contribute to apoptosis in this model. However, ceramide did not block NGF deprivation-induced declines in RNA and protein synthesis, suggesting that ceramide does not slow all apoptosis-related events. Overall, these results are significant in that they show that ceramide acts early in the death cascade to antagonize two events necessary for NGF-deprivation induced neuronal apoptosis. Moreover, these results dissociate declines in neuronal function, i.e. macromolecular synthesis, from the neuronal death cascade.

NADPH oxidase contributes directly to oxidative stress and apoptosis in nerve growth factor-deprived sympathetic neurons.

Reactive oxygen species (ROS) are necessary for programmed cell death (PCD) in neurons, but the underlying ROS-producing enzymes have not been identified. NADPH oxidase produces ROS, although the expression of its five subunits are thought to be restricted largely to non-neuronal cells. Here, we show that NADPH oxidase subunits are present in neurons. Moreover, both an NADPH oxidase inhibitor, diphenyleneiodonium, and NAPDH oxidase genetic deficiency inhibit apoptosis in a classic model of PCD, i.e., NGF-deprived sympathetic neurons. Overall, these results indicate that NADPH oxidase is unexpectedly present in neurons and can contribute to neuronal apoptosis.

Prostate apoptosis response-4 mediates trophic factor withdrawal-induced apoptosis of hippocampal neurons: actions prior to mitochondrial dysfunction and caspase activation.

Prostate apoptosis response-4 (Par-4) is the product of a gene up-regulated in prostate cancer cells undergoing apoptosis. We now report that Par-4 mRNA and protein levels rapidly and progressively increase 4-24 h following trophic factor withdrawal (TFW) in cultured embryonic rat hippocampal neurons. The increased Par-4 levels follow an increase of reactive oxygen species, and precede mitochondrial membrane depolarization, caspase activation, and nuclear chromatin condensation/fragmentation. Pretreatment of cultures with 17beta-estradiol, vitamin E, and uric acid largely prevented Par-4 induction and cell death following TFW, demonstrating necessary roles for oxidative stress and membrane lipid peroxidation in TFW-induced neuronal apoptosis. Par-4 antisense oligonucleotide treatment blocked Par-4 protein increases and attenuated mitochondrial dysfunction, caspase activation, and cell death following TFW. Collectively, our data identify Par-4 as an early and pivotal player in neuronal apoptosis resulting from TFW and suggest that estrogen and antioxidants may prevent apoptosis, in part, by suppressing Par-4 production.

Desiccation elicits heat shock protein transcription in the flesh fly, Sarcophaga crassipalpis, but does not enhance tolerance to high or low temperatures.

Although heat shock protein (hsp) production has been noted in response to multiple environmental stresses, no link has been previously established between desiccation and hsps. Following a nonlethal desiccation at 0% relative humidity (R.H.) for up to 48 h, two heat shock protein transcripts, hsp23 and hsp70, were upregulated in pupae of the flesh fly, Sarcophaga crassipalpis. The transcripts were nearly undetectable in control pupae, but within 24 h after being placed at 0% R.H. high transcript expression was observed. This suggests that protection against desiccation may be linked to the general stress response employed by flesh flies against other environmental stressors such as low or high temperature. Adaptive cross-tolerance to cold shock and heat shock following an hsp-inducing desiccation pretreatment was also tested. Although the two hsp transcripts were upregulated in response to desiccation, the upregulation was less dramatic than the upregulation elicited by heat shock, and desiccation failed to generate tolerance to high or low temperatures.

Cloning and sequencing of proliferating cell nuclear antigen (PCNA) from the flesh fly, Sarcophaga crassipalpis, and its expression in response to cold shock and heat shock.

We report the isolation and sequencing of a 1326bp cDNA fragment encoding the cell-cycle proliferation protein ScPCNA from the flesh fly, Sarcophaga crassipalpis. The amino acid (aa) sequence shows 91% and 79% identity to Drosophila melanogaster and Bombyx mori proliferating cell nuclear antigen (PCNA), respectively. The coding sequence is interrupted by a single intron of 60 bp, resulting in a deduced aa sequence of 260 residues. The gene is transcribed as a single mRNA (approx. 1.2 ) as determined by Northern blot hybridization. Following a cold shock at -10 degrees C for 1 h, expression of ScPCNA decreased in S. crassipalpis whole-body mRNA, suggesting a possible cell-cycle arrest in response to a cold shock. One hour after removal from cold shock, ScPCNA transcript levels were restored to the control level. By contrast, a 1 h heat shock at 45 degrees C did not alter expression of ScPCNA.

G0/G1 cell cycle arrest in the brain of Sarcophaga crassipalpis during pupal diapause and the expression pattern of the cell cycle regulator, proliferating cell nuclear antigen.

During pupal diapause in the flesh fly, Sarcophaga crassipalpis, the cells of the brain are arrested in the G0/G1 phase of the cell cycle. When diapause is terminated with a topical application of hexane, cell cycling is evident within 12 hours. Four G1 and S phase regulatory genes were examined by Northern blot analysis to evaluate their expression patterns in relation to this cell cycle arrest. A distinction between diapausing and nondiapausing individuals was noted only for Proliferating Cell Nuclear Antigen (PCNA). PCNA was highly expressed after diapause was terminated but not during diapause. In contrast, cyclin E, p21, and p53 were expressed equally at all times. In situ hybridization using PCNA probes further indicated a correlation between PCNA transcription (expression) in the brain and cell cycling. Our evidence thus suggests a potential role for PCNA as an important regulator of cell cycle arrest during diapause.

Diapause-specific gene expression in pupae of the flesh fly Sarcophaga crassipalpis.

Several cDNAs isolated from brains of diapausing pupae of the flesh fly, Sarcophaga crassipalpis, show expression patterns unique to diapause. To isolate such cDNAs a diapause pupal brain cDNA library was screened by using an elimination hybridization technique, and cDNAs that did not hybridize with cDNA probes constructed from the RNA of nondiapausing pupae were selected for further screening. The 95 clones that did not hybridize in the initial library screen were selected for further characterization. These clones were then screened against diapause and nondiapause pupal poly(A)+ Northern blots. The secondary screen identified 4 diapause-up-regulated clones, 7 diapause-down-regulated clones, 8 clones expressed equally in both diapause and nondiapause, and 75 clones without detectable expression. The diapause-up-regulated and down-regulated clones were further characterized by partial DNA sequencing and identity searches by using GenBank. Identities between our cloned cDNAs and other genes included those linked to cell cycle progression, stress responses, and DNA repair processes. The results suggest that insect diapause is not merely a shutdown of gene expression but is a unique, developmental pathway characterized by the expression of a novel set of genes.