RNAi Double-stranded RNAs (dsRNAs) were made using gld-2 cDNAs (pJK830, exons 2-8 or pJK831, exons 16-18) as templates. Young adults were either injected with 2 mg ml 21 gld-2 dsRNA or soaked in 10 ml of 2 mg ml 21 gld-2 dsRNA for 12 h at 20 8C or mock-treated by injection with M9 buffer. Embryos were collected at defined intervals after treatment and processed together. Poly(A) polymerase assayProteins were in vitro translated using the TNT coupled transcription-translation system (Promega), and assayed using buffer conditions essentially as described 26 . For scintillation counting, poly(A) (Roche) was used as substrate. For gel assays, we used RNA oligo, C 35 A 10 (Dharmacon), a 45-nucleotide and supplemental 1 mM MgCl 2 . Products were analysed on 12% sequencing gels.

Forkhead transcription factors of the FOXO class are negatively regulated by PKB/c-Akt in response to insulin/IGF signalling, and are involved in regulating cell cycle progression and cell death. Here we show that, in contrast to insulin signalling, low levels of oxidative stress generated by treatment with H 2 O 2 induce the activation of FOXO4. Upon treatment of cells with H 2 O 2 , the small GTPase Ral is activated and this results in a JNK-dependent phosphorylation of FOXO4 on threonine 447 and threonine 451. This Ral-mediated, JNK-dependent phosphorylation is involved in the nuclear translocation and transcriptional activation of FOXO4 after H 2 O 2 treatment. In addition, we show that this signalling pathway is also employed by tumor necrosis factor a to activate FOXO4 transcriptional activity. FOXO members have been implicated in cellular protection against oxidative stress via the transcriptional regulation of manganese superoxide dismutase and catalase gene expression. The results reported here, therefore, outline a homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI-3K/PKB) and positive (Ras/Ral) inputs.

Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAGinduced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1 T112A ), which prevents its PKCdependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca 2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect pairedpulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1 Δ109-116 ), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.P resynaptic strength changes rapidly during repetitive stimulation [short-term plasticity (STP)] and activation of intracellular signal transduction pathways (1, 2). The diacylglycerol (DAG)/protein kinase C (PKC) cascade is one of the most potent pathways at the presynaptic terminal. Its activation leads to 50-100% potentiation of spontaneous and action potential (AP)-evoked release (3-5), and is critical for multiple forms of presynaptic plasticity (6-8). DAG directly activates the vesicle priming factor Munc13-1 (9, 10) and indirectly activates downstream effectors via PKC. Activation of both Munc13-1 and PKC is essential for this pathway to operate (Fig. 1A) (8, 11). We previously identified Munc18-1 as an essential PKC substrate, because a nonphosphorylatable Munc18-1 mutant completely inhibits PKC-dependent STP (8, 12). Importantly, a phosphomimetic mutation of Munc18-1 cannot fully bypass the requirement for PKC activation, indicating that other PKC substrates must contribute to this form of plasticity (8). These substrates have not been identified to date.Among other presynaptic PKC substrates is synaptotagmin-1 (Syt1, ref. 13). Syt1 is the vesicular Ca 2+ sensor that mediates fast AP-evoked release in the hippocampus (14) and drives a large fraction of spontaneous release (15). In the latter case, Syt1 competes with alternative sensors, in particular Doc2s, for SNARE binding ...

Neuronal dense-core vesicles (DCVs) transport and secrete neuropeptides necessary for development, plasticity and survival, but little is known about their fusion mechanism. We show that -null mutant (SNAP-25 KO) neurons, previously shown to degenerate after 4 days (DIV), contain fewer DCVs and have reduced DCV fusion probability in surviving neurons at DIV14. At DIV3, before degeneration, SNAP-25 KO neurons show normal DCV fusion, but one day later fusion is significantly reduced. To test if other SNAP homologs support DCV fusion, we expressed SNAP-23, SNAP-29 or SNAP-47 in SNAP-25 KO neurons. SNAP-23 and SNAP-29 rescued viability and supported DCV fusion in SNAP-25 KO neurons, but SNAP-23 did so more efficiently. SNAP-23 also rescued synaptic vesicle (SV) fusion while SNAP-29 did not. SNAP-47 failed to rescue viability and did not support DCV or SV fusion. These data demonstrate a developmental switch, in hippocampal neurons between DIV3 and DIV4, where DCV fusion becomes SNAP-25 dependent. Furthermore, SNAP-25 homologs support DCV and SV fusion and neuronal viability to variable extents - SNAP-23 most effectively, SNAP-29 less so and SNAP-47 ineffectively.