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.

Changes in synaptic plasticity required for memory formation are dynamically regulated through opposing excitatory and inhibitory neurotransmissions. To explore the potential contribution of NF-B/Rel to these processes, we generated transgenic mice conditionally expressing a potent NF-B/Rel inhibitor termed IB␣ superrepressor (IB␣-SR). Using the prion promoter-enhancer, IB␣-SR is robustly expressed in inhibitory GABAergic interneurons and, at lower levels, in excitatory neurons but not in glia. This neuronal pattern of IB␣-SR expression leads to decreased expression of glutamate decarboxylase 65 (GAD65), the enzyme required for synthesis of the major inhibitory neurotransmitter, ␥-aminobutyric acid (GABA) in GABAergic interneurons. IB␣-SR expression also results in diminished basal GluR1 levels and impaired synaptic strength (input/output function), both of which are fully restored following activity-based task learning. Consistent with diminished GAD65-derived inhibitory tone and enhanced excitatory firing, IB␣-SR ؉ mice exhibit increased late-phase long-term potentiation, hyperactivity, seizures, increased exploratory activity, and enhanced spatial learning and memory. IB␣-SR ؉ neurons also express higher levels of the activity-regulated, cytoskeleton-associated (Arc) protein, consistent with neuronal hyperexcitability. These findings suggest that NF-B/Rel transcription factors act as pivotal regulators of activity-dependent inhibitory and excitatory neuronal function regulating synaptic plasticity and memory.Stimulus-coupled changes in synaptic plasticity are required for the storage, retrieval, and removal of acquired information collectively referred to as memory formation (28,32,39). Such changes are facilitated by both modifications of existing synaptic effectors and the de novo synthesis of new gene products regulated by various transcriptional regulators. These processes are tightly controlled by the coordinated action of both excitatory and inhibitory neurotransmitters derived from glutamatergic neurons and GABAergic (where GABA is ␥-aminobutyric acid) interneurons, respectively (47, 54). While the vast majority of studies to date have focused on the cyclic AMPresponsive transcription factor (CREB) regulating excitatory neuron function (7, 32-34, 62, 72), more recently, other transcription factors, including members of the NF-B/Rel family of transcription factors, have been implicated in experiencebased synaptic adaptations (38,45,49,55). However, our understanding of their precise role in regulating synaptic plasticity remains rudimentary at best.Although NF-B/Rel factors were originally implicated as central regulators of the immune and inflammatory responses, both basal expression and stimulus-coupled induction of NF-B/Rel factors occur in neurons and glial cells (23,30,31,45,48,55). Activation of NF-B/Rel proceeds through the sitespecific phosphorylation, polyubiquitylation, and proteasomemediated degradation of the major NF-B/Rel inhibitor protein, IB␣ (41). The newly liberated NF-B/Rel complex ra...

Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 °C and in the intestinal tract of birds at close to 41 °C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30–42 °C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 °C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex.

No abstract

Activity of the transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to be necessary for maintaining neuronal viability. In cultured rat cerebellar granule neurons, trophic factor withdrawal induces NF-kappaB inactivation, resulting in cell death. The exact mechanism of this inactivation, however, has not been revealed. Here we report that trophic factor deprivation in cultured cerebellar granule neurons leads to a rapid and sustained increase in the level of IkappaBalpha and IkappaBbeta, the inhibitory proteins of NF-kappaB, causing prolonged NF-kappaB inactivation. Transient NF-kappaB activation resulting in new IkappaBalpha mRNA and protein synthesis gives rise to the rapid increase of IkappaBalpha level. The importance of elevated IkappaB level in neuronal apoptosis was confirmed in transfection experiments. Ectopic expression of a stabilized form of IkappaBalpha protein promoted neuronal death. Our findings suggest a novel mode of initiation of neuronal apoptosis wherein survival signal withdrawal induces NF-kappaB to lethally turn itself off.