The signaling pathway from phosphoinositide 3-kinase to the protein kinase Akt controls organismal life-span in invertebrates and cell survival and proliferation in mammals by inhibiting the activity of members of the FOXO family of transcription factors. We show that mammalian FOXO3a also functions at the G2 to M checkpoint in the cell cycle and triggers the repair of damaged DNA. By gene array analysis, FOXO3a was found to modulate the expression of several genes that regulate the cellular response to stress at the G2-M checkpoint. The growth arrest and DNA damage response gene Gadd45a appeared to be a direct target of FOXO3a that mediates part of FOXO3a's effects on DNA repair. These findings indicate that in mammals FOXO3a regulates the resistance of cells to stress by inducing DNA repair and thereby may also affect organismal life-span.

The assembly of 6 -12 subunits of Ca 2؉ /calmodulin-dependent kinase II (CaM kinase II) into holoenzymes is an important structural feature of the enzyme and its postulated role as a molecular detector of Ca 2؉ oscillations. Using single cell reverse transcriptase-polymerase chain reaction, we show that ␣-and ␤-CaM kinase II mRNAs are simultaneously present in the majority of hippocampal neurons examined and that co-assembly of their protein products into heteromers is therefore possible. The subunit composition of CaM kinase II holoenzymes was analyzed by immunoprecipitation with subunit-specific monoclonal antibodies. Rat forebrain CaM kinase II consists of heteromers composed of ␣ and ␤ subunits at a ratio of 2:1 and homomers composed of only ␣ subunits. We examined the functional effect of the heteromeric assembly by analyzing the calmodulin dependence of autophosphorylation. Recombinant homomers of ␣-or ␤-CaM kinase II, as well as of alternatively spliced ␤ isoforms, have distinct calmodulin dependences for autophosphorylation based on differences in their calmodulin affinities. Half-maximal autophosphorylation of ␣ is achieved at 130 nM calmodulin, while that for ␤ occurs at 15 nM calmodulin. In CaM kinase II isolated from rat forebrain, however, the calmodulin dependence for autophosphorylation of the ␤ subunits is shifted toward that of ␣ homomers. This suggests that Thr 287 in ␤ subunits is phosphorylated by ␣ subunits present in the same holoenzyme. Once autophosphorylated, ␤-CaM kinase II traps calmodulin by reducing the rate of calmodulin dissociation.Multifunctional Ca 2ϩ /calmodulin-dependent protein kinase II (CaM kinase II) is a major mediator of Ca 2ϩ action whose activation and autophosphorylation appear to regulate numerous neuronal processes, including two forms of synaptic plasticity, long term potentiation and long term depression (1, 2). These two opposing changes in synaptic strength are differentially regulated by the frequency of stimulation of hippocampal neurons. Interestingly, CaM kinase II has been suggested to be a molecular detector of the Ca 2ϩ spike frequency, based on its unique structural and regulatory properties (3) (reviewed in Refs. 4 and 5). The neuronal CaM kinase II consists of two major subunits of 52 and 60 kDa that are encoded by ␣-and

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Programmed cell death (PCD) during neuronal development and disease has been shown to require de novo RNA synthesis. However, the time course and regulation of target genes is poorly understood. By using a brain-biased array of over 7,500 cDNAs, we profiled this gene expression component of PCD in cerebellar granule neurons challenged separately by potassium withdrawal, combined potassium and serum withdrawal, and kainic acid administration. We found that hundreds of genes were significantly regulated in discreet waves including known genes whose protein products are involved in PCD. A restricted set of genes was regulated by all models, providing evidence that signals inducing PCD can regulate large assemblages of genes (of which a restricted subset may be shared in multiple pathways).

Synaptic inhibition in the thalamus plays critical roles in sensory processing and thalamocortical rhythm generation. To determine kinetic, pharmacological, and structural properties of thalamic gamma-aminobutyric acid type A (GABA(A)) receptors, we used patch-clamp techniques and single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in neurons from two principal rat thalamic nuclei-the reticular nucleus (nRt) and the ventrobasal (VB) complex. Single-channel recordings identified GABA(A) channels with densities threefold higher in VB than nRt neurons, and with mean open time fourfold longer for nRt than VB [14.6 +/- 2.5 vs. 3.8 +/- 0.7 (SE) ms, respectively]. GABA(A) receptors in nRt and VB cells were pharmacologically distinct. Zn(2+) (100 microM) reduced GABA(A) channel activity in VB and nRt by 84 and 24%, respectively. Clonazepam (100 nM) increased inhibitory postsynaptic current (IPSC) decay time constants in nRt (from 44.3 to 77.9 ms, P < 0.01) but not in VB. Single-cell RT-PCR revealed subunit heterogeneity between nRt and VB cells. VB neurons expressed alpha1-alpha3, alpha5, beta1-3, gamma2-3, and delta, while nRt cells expressed alpha3, alpha5, gamma2-3, and delta. Both cell types expressed more subunits than needed for a single receptor type, suggesting the possibility of GABA(A) receptor heterogeneity within individual thalamic neurons. beta subunits were not detected in nRt cells, which is consistent with very low levels reported in previous in situ hybridization studies but inconsistent with the expected dependence of functional GABA(A) receptors on beta subunits. Different single-channel open times likely underlie distinct IPSC decay time constants in VB and nRt cells. While we can make no conclusion regarding beta subunits, our findings do support alpha subunits, possibly alpha1 versus alpha3, as structural determinants of channel deactivation kinetics and clonazepam sensitivity. As the gamma2 and delta subunits previously implicated in Zn(2+) sensitivity are both expressed in each cell type, the observed differential Zn(2+) actions at VB versus nRt GABA(A) receptors may involve other subunit differences.