Tetanus Toxin Enhances Protein Kinase C Activity Translocation and Increases Polyphosphoinositide Hydrolysis in Rat Cerebral Cortex Preparations

Abstract

Abstract:Tetanus toxin (TeTx) has been recently demonstrated to be a Zn 2~-dependentendopeptidase that cleaves synaptobrevin, a protein in part responsible for neurotransmitter release. Nevertheless, certain aspects of TeTx action, for example, the causal relationship between TeTx and protein kinase C (PKC; EC 2.7.1.37) activity cannot be explained by this cleavage alone. In the present study, primary neurons from fetal rat brain, synaptosomes, and whole slices have been used to examine this issue. Low doses of TeTx ( 10_8 M) caused PKC activity translocation in a manner similar to that produced by 1 2-O-tetradecanoylphorbol 13-acetate (TPA). TPA ( 10M) caused sustained PKC activity translocation, whereas TeTx produced translocation followed by relocation, depending on the dose and time of exposure. Immunoidentification with a monoclonal antibody recognizing both a and /3 isoforms revealed that TeTx induced moderate losses of PKC in the cytosolic fraction, without a comparable increase in the particulate fraction. Although moderate losses of activity were also noticed in the cytosolic fraction, the inconsistency with respect to activity translocation may be explained by translocation of additional PKC isoforms that are not identified by the antibody. Comparable levels of water-soluble inositol phosphate-labeled intermediates were obtained after treatment of cerebral cells and/or cortical brain slices with TeTx. Significant increases of 19 and 114% in the watersoluble myo-[2-3H]inositol-labeled inositol phosphate metabolites were found in cerebral cell culture and brain slices, respectively, after treatment with 10~8M TeTx. TeTx (10_8 M) increased to the same degree the watersoluble inositol phosphate levels as did serotonin (10-M ) or carbachol (10-6 M). lt is suggested that part of the signaling cascade of TeTx consists of a component involving inositol phospholipid hydrolysis, which is associated with PKC activity translocation. Key Words: Tetanus toxin-Protein kinase C-Phorbol ester-Neuronal cultures-Polyphosphoinositide hydrolysis-Phospholipase C-Synaptosomes.

HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction

“…Moreover, this PLCγ-1 phosphorylation is in accord with enhancement of polyphosphoinositide hydrolysis by TeTx observed in primary neuron cultures from fetal rat brain and in slices from adult rat cerebral cortex [11]. The increase in PLC activity, which is modest but significant, is very similar to that exerted by NGF in rat brain synaptosomes [29].…”

“…NGF is a signalling molecule, which acts through specific PLCγ activation by means of its association with trkA, autophosphorylated on tyrosine residues [30]. Thus the modest increase in polyphosphoinositide hydrolysis in synaptosomes by NGF and TeTx described by our group, when compared with the muscarinic agonist carbachol [11,29], can be related to isoformspecific PLC activation. On the other hand, PLCγ-1 translocation has been described in several studies, and this event has been related to its phosphotyrosine content and to the activation of tyrosine kinase receptors [31].…”

“…Since PKC activity is the sum of various molecular PKC entities, a detailed investigation into which PKC isoforms are affected by the H C -TeTx fragment was performed and is described in the present paper. In previous work by our group, it was determined that the H C -TeTx fragment inhibits 5-hydroxytryptamine uptake in rat brain synaptosomes, an action similar to that of TeTx in its whole di-chain form, as well as in the single-chain form [27,11]. This inhibition of 5-hydroxytryptamine transport is directly related to the TeTx-induced phosphorylation of serine residues in the 5-hydroxytryptamine transporter by PKC [28].…”

“…Later, our group described the TeTxinduced activation of PKC in the perinatal rat brain, which paralleled an elevation in 5-hydroxytryptamine levels [12]. The findings that intraventricular administration of TeTx into adult rats induced translocation and down-regulation of PKC [9], and that treatment of primary neuron cultures from fetal rat brain enhanced translocation of PKC activity and increased polyphosphoinositide hydrolysis [11] pointed in the same direction. Since PKC activity is the sum of various molecular PKC entities, a detailed investigation into which PKC isoforms are affected by the H C -TeTx fragment was performed and is described in the present paper.…”

“…PKC translocation appeared before the classic symptoms of tetanus neurotoxicity in the perinatal rat [10]. The increase in polyphosphoinositide hydrolysis in rat cerebral cortex preparations treated with TeTx indicates activation of phospholipases, an effect directly related to PKC activation [11]. This PKC activation is paralleled by an increase in 5-hydroxytryptamine levels [12], an effect which has recently been related to inhibition of the 5-hydroxytryptamine re-uptake produced by TeTx and by its H C fragment [13].…”