Previous publications from our group [Gil, Chaib, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182; Gil, Chaib, Blasi and Aguilera (2001) Biochem. J. 356, 97-103] have reported the activation, in rat brain synaptosomes, of several phosphoproteins, such as neurotrophin tyrosine kinase (Trk) A receptor, phospholipase Cgamma-1, protein kinase C (PKC) isoforms and extracellular-signal-regulated kinases 1 and 2 (ERK-1/2). In the present study, we examined, by means of phospho-specific antibodies, the activation of the signalling cascades involving neurotrophin Trk receptor, Akt kinase and ERK pathway, in cultured cortical neurons from foetal rat brain, by tetanus toxin (TeTx) as well as by the C-terminal part of its heavy chain (H(C)-TeTx). TeTx and H(C)-TeTx induce fast and transient phosphorylation of Trk receptor at Tyr(674) and Tyr(675), but not at Tyr(490), although the potency of TeTx in this action was higher when compared with H(C)-TeTx action. Moreover, H(C)-TeTx and TeTx also induced phosphorylation of Akt (at Ser(473) and Thr(308)) and of ERK-1/2 (Thr(202)/Tyr(204)), in a time- and concentration-dependent manner. The detection of TeTx- and H(C)-TeTx-induced phosphorylation at Ser(9) of glycogen synthase kinase 3beta confirms Akt activation. In the extended analysis of the ERK pathway, phosphorylation of the Raf, mitogen-activated protein kinase kinase (MEK)-1/2 and p90Rsk kinases and phosphorylation of the transcription factor cAMP-response-element-binding protein were detected. The use of tyrphostin AG879, an inhibitor of Trk receptors, demonstrates their necessary participation in the H(C)-TeTx-induced activation of Akt and ERK pathways, as well as in the phosphorylation of phospholipase Cgamma-1. Furthermore, both pathways are totally dependent on phosphatidylinositol 3-kinase action, and they are independent of PKC action, as assessed using wortmannin and Ro-31-8220 as inhibitors. The activation of PKC isoforms was determined by their translocation from the cytosolic compartment to the membranous compartment, showing a clear H(C)-TeTx-induced translocation of PKC-alpha and -beta, but not of PKC- epsilon.

When cultured cerebellar granule neurones are transferred from a medium containing high extracellular potassium concentration ( Abbreviations used: Akt/PKB, Akt/protein kinase B; BDNF, brainderived neurotrophic factor; CGN, cerebellar granule neurones; CREB, cAMP-response-element-binding protein; DAPI, 4¢,6-diamino-2-phenylindole; ERK, extracellular signal-regulated kinase; GSK3b, glycogen synthase kinase 3b; GST, glutathione S-transferase; Hc-TeTx, C-terminal domain of the heavy-chain of tetanus toxin; [K + ] e , extracellular potassium concentration; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NGF, nerve growth factor; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLCc-1, phospholipase Cc-1; p90rsk, p90 ribosomal S6 kinase; RBD, Ras-binding domain of Raf-1; TeTx, tetanus toxin; Trk, high-affinity tyrosine kinase receptor for neurotrophins.

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Previous reports have demonstrated that tetanus toxin (TeTx) induces activation and down-regulation of protein kinase C (PKC). In the present work the differential activation of PKC isoforms and of signal transduction pathways, including nerve growth factor receptor trkA, phospholipase CQ Q-1 (PLCQ Q-1), and extracellular regulated kinases 1 and 2 (ERK-1/2) by TeTx in a synaptosome-enriched P 2 fraction from rat brain is reported. TeTx induces clear translocation from the soluble (cytosolic) compartment to the particulate (membranous) compartment of PKC-L L, -Q Q and -N N isoforms, whereas PKC-O O showed a slight decrease of its soluble fraction immunoreactivity. On the contrary, the PKC-j j isoform shows no consistent response, whereas down-regulation of total PKC-K K immunoreactivity is shown. Immunoprecipitation assays against phosphotyrosine show an increase of trkA and PLCQ Q-1 phosphorylation. Moreover, trkA activation is corroborated using phospho-specific antibodies against phosphorylated trkA. On the other hand, TeTx-induced stimulation of mitogen-activated protein (MAP) kinase activity is observed, this event also being detected by Western analysis using phospho-specific antibodies against ERK-1/2. ß

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