José Aguilera

C-terminal fragment of tetanus toxin heavy chain activates Akt and MEK/ERK signalling pathways in a Trk receptor-dependent manner in cultured cortical neurons.

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.

Fragment C of tetanus toxin, more than a carrier. Novel perspectives in non-viral ALS gene therapy

The non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC) has been implicated in the activation of cascades responsible for trophic actions and neuroprotection by inhibition of apoptosis. Previous in vitro studies have described signalling pathways that underlie the administration of TTC to neurons. We investigated whether these properties were maintained in a mouse model of neurodegenerative disease. Naked DNA encoding for TTC was injected intramuscularly and neuromuscular function and clinical behaviour were monitored until endstage in the transgenic SOD1G93A mouse model that expresses a mutant variant of human superoxide dismutase 1 (SOD1). Our results indicate that TTC treatment ameliorated the decline of hindlimb muscle innervation, significantly delayed the onset of symptoms and functional deficits, improved spinal motor neuron survival, and prolonged lifespan. Furthermore, we found that caspase-1 and caspase-3 proapoptotic genes were down-regulated in the spinal cord of treated mice. Western blot analysis showed that the active form of caspase-3 was also down-regulated after TTC treatment and survival signals, such as the significant phosphorylation of serine/threonine protein kinase Akt, were also detected. These results suggest that fragment C of tetanus toxin, TTC, provides a potential therapy for neurodegenerative diseases.

The C‐terminal domain of the heavy chain of tetanus toxin rescues cerebellar granule neurones from apoptotic death: involvement of phosphatidylinositol 3‐kinase and mitogen‐activated protein kinase pathways

When cultured cerebellar granule neurones are transferred from a medium containing high extracellular potassium concentration ([K+]e) (25 mm) to one with lower [K+]e (5 mm), caspase‐3 activity is induced and cells die apoptotically. In contrast, if cells in non‐depolarizing conditions are treated with brain‐derived neurotrophic factor (BDNF), caspase‐3 activity, chromatin condensation and cell death are markedly diminished. In this study, we show that the C‐terminal domain of the tetanus toxin heavy‐chain (Hc‐TeTx) is able to produce the same neuroprotective effect, as assessed by reduction of tetrazolium salts and by chromatin condensation. Hc‐TeTx‐conferred neuroprotection appears to depend on phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase kinase, as is demonstrated by the selective inhibitors Wortmannin and PD98059, respectively. Hc‐TeTx also induces phosphorylation of the tyrosine kinase BDNF receptor, activation of p21Ras in its GTP‐bound form, and phosphorylation of the cascade including extracellular‐signal‐regulated kinases‐1/2 (ERK‐1/2), p90 ribosomal S6 kinase (p90rsk) and CREB (cAMP‐response‐element‐binding protein). On the other hand, activation of the Akt pathway is also detected, as well as inhibition of the active form of caspase‐3. These results point to an implication of both PI3K‐ and ERK‐dependent pathways in the promotion of cerebellar granule cell survival by Hc‐TeTx.

Activation of signal transduction pathways involving trkA, PLCγ-1, PKC isoforms and ERK-1/2 by tetanus toxin

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 Cγ‐1 (PLCγ‐1), and extracellular regulated kinases 1 and 2 (ERK‐1/2) by TeTx in a synaptosome‐enriched P2 fraction from rat brain is reported. TeTx induces clear translocation from the soluble (cytosolic) compartment to the particulate (membranous) compartment of PKC‐β, ‐γ and ‐δ isoforms, whereas PKC‐ϵ showed a slight decrease of its soluble fraction immunoreactivity. On the contrary, the PKC‐ζ isoform shows no consistent response, whereas down‐regulation of total PKC‐α immunoreactivity is shown. Immunoprecipitation assays against phosphotyrosine show an increase of trkA and PLCγ‐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.

Clostridium Neurotoxins Influence Serotonin Uptake and Release Differently in Rat Brain Synaptosomes

Abstract: Clostridium neurotoxins produce inhibition of both basal and K+‐evoked serotonin release in rat brain synaptosomes. To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37°C over a long interval (hours). This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2+‐metalloprotease inhibitors. Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium‐dependent, high‐affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A. Both Zn2+‐metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C‐terminal portion of the TeTx heavy chain (Hc‐TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose‐dependent manner. The IC50 of HC‐TeTx ranges from 0.62 to 2.08 nM. Binding of [3H]imipramine and [3H]serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high‐affinity site at the serotonin transporter or at other serotonin high‐affinity sites. These results could indicate that TeTx and HC‐TeTx bind to different targets than BoNT/A in the plasma membrane.

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

A recent report [Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182] describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn(2+)-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease. In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)gamma-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (H(C)-TeTx) is reported. The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-alpha, -beta, -gamma and -delta isoforms exists, whereas PKC-epsilon showed a slight decrease in its soluble fraction immunoreactivity. The PKC-zeta isoform showed no consistent response. Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCgamma-1 and ERK-1/2. The effects shown by the H(C)-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF. The trkA and ERK-1/2 activation were corroborated using phospho-specific antibodies against trkA phosphorylated on Tyr(490), and antibodies against Thr/Tyr phosphorylated ERK-1/2. Moreover, PLCgamma-1 phosphorylation was supported by its H(C)-TeTx-induced translocation to the membranous compartment, an event related to PLCgamma-1 activation. Since H(C)-TeTx is the domain responsible for membrane binding and lacks catalytic activity, the activations described here must be exclusively triggered by the interaction of TeTx with a membrane component.

Fragment C of Tetanus Toxin: New Insights into Its Neuronal Signaling Pathway

When Clostridium tetani was discovered and identified as a Gram-positive anaerobic bacterium of the genus Clostridium, the possibility of turning its toxin into a valuable biological carrier to ameliorate neurodegenerative processes was inconceivable. However, the non-toxic carboxy-terminal fragment of the tetanus toxin heavy chain (fragment C) can be retrogradely transported to the central nervous system; therefore, fragment C has been used as a valuable biological carrier of neurotrophic factors to ameliorate neurodegenerative processes. More recently, the neuroprotective properties of fragment C have also been described in vitro and in vivo, involving the activation of Akt kinase and extracellular signal-regulated kinase (ERK) signaling cascades through neurotrophin tyrosine kinase (Trk) receptors. Although the precise mechanism of the molecular internalization of fragment C in neuronal cells remains unknown, fragment C could be internalized and translocated into the neuronal cytosol through a clathrin-mediated pathway dependent on proteins, such as dynamin and AP-2. In this review, the origins, molecular properties and possible signaling pathways of fragment C are reviewed to understand the biochemical characteristics of its intracellular and synaptic transport.

Inhibition by tetanus toxin of sodium-dependent, high-affinity [3H]5-hydroxytryptamine uptake in rat synaptosomes

Tetanus toxin (TeTx) is a powerful clostridial neurotoxin that inhibits Ca2+-dependent neurotransmitter secretion as do the botulinum neurotoxins (BoNTs). We found that TeTx (but not BoNT/A) produced a specific time- and dose-dependent inhibition of Na+-dependent [3H]5-hydroxytryptamine (serotonin, 5-HT) uptake in rat CNS synaptosomes. This effect was found in all CNS tryptaminergic areas, being maximal in the hippocampus and occipital cortex. TeTx produced the maximum reduction in [3H]5-HT uptake after 30 min of preincubation, being significant also at lower doses (10(-12) M) or shorter incubation times (10 min). Serotonin transport inhibitors such as fenfluramine (IC50, 11.0 +/- 0.9 microM), paroxetine (IC50, 33.5 +/- 0.1 microM), and imipramine (IC50, 89.9 +/- 5.7 microM) were 3 or 4 orders of magnitude less potent than TeTx (IC50, 8.7 +/- 1.0 nM). Of the two fragments of TeTx, (the C-terminal portion of the neurotoxin heavy chain, which is responsible for the binding to the nerve tissue) was consistently more effective than the L-H(N) fragment (the light neurotoxin chain disulfide linked to the N-terminal portion of the heavy chain, which is responsible for the toxic metalloprotease action) as inhibitor of [3H]5-HT uptake in synaptosomal preparations (56 +/- 5% and 95 +/- 3% with respect to control, respectively). Antagonism of the toxin-induced [3H]5-HT uptake blockade could not be reversed by zinc chelators but did have the ability to antagonize the TeTx inhibition of basal and K+-evoked [3H]5-HT release in rat synaptosomes. The reduction in serotonin accumulation induced by TeTx could be responsible for some tetanic symptoms that have been related to the serotonergic system.

Tetanus toxin enhances protein kinase C activity translocation and increases polyphosphoinositide hydrolysis in rat cerebral cortex preparations.

Abstract: Tetanus toxin (TeTx) has been recently demonstrated to be a Zn2+‐dependent endopeptidase 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 (≤ 10−8M) caused PKC activity translocation in a manner similar to that produced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). TPA (≤ 10−7M) 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 α and β 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 water‐soluble 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 (10−8M) increased to the same degree the water‐soluble inositol phosphate levels as did serotonin (10−5M) or carbachol (10−6M). It 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.

The carboxyl-terminal domain of the heavy chain of tetanus toxin prevents dopaminergic degeneration and improves motor behavior in rats with striatal MPP^+-lesions

Recently it has been shown that the C-terminus fragment of the tetanus toxin (Hc-TeTx) is transported retrogradely and had shown neuroprotective effects, preventing neuronal death by apoptosis. This could be a new alternative preventing ongoing cell death and restoring the motor function in Parkinsons disease (PD), which is characterized by dopaminergic neurodegeneration. Our aim was to evaluate the effects of local administration of Hc-TeTx on motor behavior and the dopamine (DA) levels in the striatum of MPP(+)-treated rats. In the rotational behavior task, the Hc-TeTx [2 microM]+MPP(+) group had a decreased number of contralateral rotations and the cylinder test improved for both forelimb-use asymmetry compared to the MPP(+) group. The staircase test showed that the Hc-TeTx+MPP(+) group had an improvement of fine motor skills compared to the same limb performance of the MPP(+) group. The group of animals with Hc-TeTx+MPP(+) had higher DA and metabolite levels compared to the MPP(+) group. Our study clearly shows that Hc-TeTx improves different motor behavior strongly, which favors the hypothesis of the Hc-TeTx fragment enhancing survival pathways that result in amelioration of the dopaminergic system of rats with a dopaminergic lesion.