Tetanus Toxin Hc Fragment Induces the Formation of Ceramide Platforms and Protects Neuronal Cells against Oxidative Stress

Abstract

Tetanus toxin (TeTx) is the protein, synthesized by the anaerobic bacteria Clostridium tetani, which causes tetanus disease. TeTx gains entry into target cells by means of its interaction with lipid rafts, which are membrane domains enriched in sphingomyelin and cholesterol. However, the exact mechanism of host membrane binding remains to be fully established. In the present study we used the recombinant carboxyl terminal fragment from TeTx (Hc-TeTx), the domain responsible for target neuron binding, showing that Hc-TeTx induces a moderate but rapid and sustained increase in the ceramide/sphingomyelin ratio in primary cultures of cerebellar granule neurons and in NGF-differentiated PC12 cells, as well as induces the formation of ceramide platforms in the plasma membrane. The mentioned increase is due to the promotion of neutral sphingomyelinase activity and not to the de novo synthesis, since GW4869, a specific neutral sphingomyelinase inhibitor, prevents neutral sphingomyelinase activity increase and formation of ceramide platforms. Moreover, neutral sphingomyelinase inhibition with GW4869 prevents Hc-TeTx-triggered signaling (Akt phosphorylation), as well as the protective effect of Hc-TeTx on PC12 cells subjected to oxidative stress, while siRNA directed against nSM2 prevents protection by Hc-TeTx of NSC-34 cells against oxidative insult. Finally, neutral sphingomyelinase activity seems not to be related with the internalization of Hc-TeTx into PC12 cells. Thus, the presented data shed light on the mechanisms triggered by TeTx after membrane binding, which could be related with the events leading to the neuroprotective action exerted by the Hc-TeTx fragment.

Trk receptors need neutral sphingomyelinase activity to promote cell viability

“…The breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide increases proteolysis (in a mechanism also known as 'ectodomain shedding') of another TACE substrate, such as the interleukin-6 receptor (IL-6R) [46]. Thus, according to published data, a model in which the nSMase-activated TACE contributes to Akt activation arises, which is in agreement with the nSMase-dependent phosphorylation of Akt exerted by the BDNF action in CGN, shown in the present work, as well as in previous work from our group [42].…”

“…The increase in ceramide levels is supposed to change membrane configuration, increasing the content in ceramide platforms, which are considered to be a subset of the so‐called ‘lipid rafts’, which are membrane microdomains rich in cholesterol and sphingolipids. Recently, it has been demonstrated that the fragment Hc from tetanus toxin induces the formation of ceramide platforms and protects cells from oxidative stress in a nSMase‐dependent manner [42]. Regarding the role of SMase activity in cytoprotective outcome, some metabolites derived from sphingolipid processing have been involved in Akt signaling, pointing to S1P as a crucial effector molecule in some events leading to Akt activation [43].…”

“…Cerebellar granule neuron (CGN) cultures were prepared as described [42]. PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% horse serum, 5% fetal calf serum, 50 U/ml penicillin and 50 μg/ml streptomycin, at 37 °C in a humidified 5% CO 2 atmosphere.…”

“…Recently, it has been demonstrated that the fragment Hc from tetanus toxin induces the formation of ceramide platforms and protects cells from oxidative stress in a nSMase-dependent manner [42]. Regarding the role of SMase activity in cytoprotective outcome, some metabolites derived from sphingolipid processing have been involved in Akt signaling, pointing to S1P as a crucial effector molecule in some events leading to Akt activation [43].…”

Cargo-Delivery Platforms for Targeted Delivery of Inhibitor Cargos Against Botulism

“…Several fusion proteins have been tested and reported to have some effects on neuronal cells, including Bcl-xL-TTC [116], BDNF-TTC [117], and GDNF-TTC [118]. However, the TTC delivery platform itself without a fusion cargo also stimulated signaling pathways [117, 119], which complicated the interpretation of the results.…”