Recent studies have revealed a positive correlation between astrocyte apoptosis and rapid disease progression in persons with neurodegenerative diseases. Glycogen synthase kinase 3␤ (GSK-3␤) is a molecular regulator of cell fate in the central nervous system and a target of the phosphatidylinositol 3-kinase (PI-3K) pathway. We have therefore examined the role of the PI-3K pathway, and of GSK-3␤, in regulating astrocyte survival. Our studies indicate that inhibition of PI-3K leads to apoptosis in primary cortical astrocytes. Furthermore, overexpression of a constitutively active GSK-3␤ mutant (S9A) is sufficient to cause astrocyte apoptosis, whereas an enzymatically inactive GSK-3␤ mutant (K85M) has no effect. In light of reports on the interplay between GSK-3␤ and nuclear factor B (NF-B), and because of the antiapoptotic activity of NF-B, we examined the effect of GSK-3␤ overexpression on NF-B activation. These experiments revealed strong inhibition of NF-B activation in astrocytes upon overexpression of the S9A, but not the K85M, mutant of GSK-3␤. This was accompanied by stabilization of the NF-B-inhibitory protein, IB␣ and down-regulation of IB kinase (IKK) activity. These findings therefore implicate GSK-3␤ as a regulator of NF-B activation in astrocytes and suggest that the pro-apoptotic effects of GSK-3␤ may be mediated at least in part through the inhibition of NF-B pathway.

HIV-1 associated dementia is thought to be caused by neuronal damage and death in response to the production of soluble neurotoxic factors by virally infected mononuclear phagocytes. These neurotoxins include HIV-1 Tat. The ability of neurotrophins to promote cell survival prompted us to examine whether neurotrophins might also be capable of opposing the pro-apoptotic effects of Tat. Here, we show that Tat-induced neuronal apoptosis in primary cultures of rat cerebellar granule cells and in neuronally differentiated human SK-N-MC cells is profoundly inhibited by brain-derived neurotrophic factor, nerve growth factor and activity-dependent neurotrophic factor nonamer peptide. These neurotrophins activated the transcription factor NF-kB, and inhibition of NF-kB activation using a super-repressor IkB-a mutant was found to block the survival-promoting activity of the neurotrophins. Reporter gene assays and immunoblot experiments revealed that the neurotrophins also up-regulated the expression of Bcl-2, at both the transcriptional and protein levels. Overexpression of the super-repressor IkB-a mutant prevented this induction of Bcl-2 expression. Moreover, overexpression of either Bcl-2, alone, or the RelA subunit of NF-kB, alone, protected neurons from Tat-induced apoptosis. These ®ndings suggest that the activation of NF-kB by neurotrophic factors may promote survival of neurons exposed to Tat, via regulation of anti-apoptotic genes including Bcl-2.

The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3′ end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins—a 45% success rate. Modeling of detected versus non‐detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically‐significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification.

BackgroundPrenatal exposure of the developing brain to cocaine causes morphological and behavioral abnormalities. Recent studies indicate that cocaine-induced proliferation inhibition and/or apoptosis in neural progenitor cells may play a pivotal role in causing these abnormalities. To understand the molecular mechanism through which cocaine inhibits cell proliferation in neural progenitors, we sought to identify the molecules that are responsible for mediating the effect of cocaine on cell cycle regulation.Methods and FindingsMicroarray analysis followed by quantitative real-time reverse transcription PCR was used to screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line where cocaine exposure caused a robust anti-proliferative effect by interfering with the G1-to-S transition. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated human primary neural and A2B5+ progenitor cells, as well as in rat fetal brains exposed to cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine. Further, we found that cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A down-regulation by causing an endoplasmic reticulum (ER) stress response, as indicated by increased phosphorylation of eIF2α and expression of ATF4. In the developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of neural progenitor cell proliferation as well as down-regulation of cyclin A.ConclusionsOur results demonstrate that down-regulation of cyclin A underlies cocaine-induced proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N-oxidative metabolism by P450 inhibitors may provide a preventive strategy for counteracting the adverse effects of cocaine on fetal brain development.