Neurotrophic Properties of C-Terminal Domain of the Heavy Chain of Tetanus Toxin on Motor Neuron Disease

Abstract

The carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) exerts a neuroprotective effect in neurodegenerative diseases via the activation of signaling pathways related to neurotrophins, and also through inhibiting apoptotic cell death. Here, we demonstrate that Hc-TeTx preserves motoneurons from chronic excitotoxicity in an in vitro model of amyotrophic lateral sclerosis. Furthermore, we found that PI3-K/Akt pathway, but not p21ras/MAPK pathway, is involved in their beneficial effects under chronic excitotoxicity. Moreover, we corroborate the capacity of the Hc-TeTx to be transported retrogradely into the spinal motor neurons and also its capacity to bind to the motoneuron-like cell line NSC-34. These findings suggest a possible therapeutic tool to improve motoneuron preservation in neurodegenerative diseases such as amyotrophic lateral sclerosis.

Keywords: amyotrophic lateral sclerosis; carboxyl-terminal domain of the heavy chain of tetanus toxin; excitotoxicity; neuroprotection; spinal muscular atrophy.

Figure 1

Purification and fluorescence labeling methodology of the carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) protein. (A) Elution profile of the Hc-TeTx protein. Electrophoresis SDS-PAGE stained with comassie blue and fast protein liquid chromatography (FPLC) chromatogram. (B) Densometric quantification of the eluted lanes corresponding to Hc-TeTx fractions. (C) Detection of the Hc-TeTx protein purified using the Western blot technique with the anti-histidine antibody. (D) Labeling detection of the Hc-TeTx protein conjugated to the Alexa Fluor^®555 fluorochrome. Histogram and electrophoretic gel representing more than 30 purification processes carried out.

Figure 2

Internalization and retrograde transport of Hc-TeTx-Alexa⁵⁵⁵ protein on motoneurons. (A) Time-course of Hc-TeTx-Alexa⁵⁵⁵ internalization in the NSC-34 cells, a motoneuron-like cell line. Controls performed by adding the same amount of fluorochrome did not show fluorescence at the indicated times. The bar represents 10 μm. (B) The Hc-TeTx-Alexa⁵⁵⁵ (red) protein is detected in motor nerve endings identified with α-bungarotoxin (BGTX) (green) 24 h after a single intramuscular injection into the tibialis anterior (TA) muscle. The injection with the same amount of fluorocrom (Alexa⁵⁵⁵) does not emit fluorescence in the neuromuscular junction (NMJ). (C) The Hc-TeTx-Alexa⁵⁵⁵ protein can be detected in the injected muscle but does not spread to other muscles such as gastrocnemius muscles (GM) of the same leg (Left leg) or in the TA muscles and GM of the right leg, where there is no presence of Hc-TeTx-Alexa⁵⁵⁵. (D) At 24 h the Hc-TeTx-Alexa⁵⁵⁵ protein is located inside the NMJ without colocalizing with AChR receptors. The arrows indicate the formation of clusters inside the NMJ and presynaptic endings. Representative images of 3 independent experiments by condition. (E) Visualization of the Hc-TeTx-Alexa⁵⁵⁵ protein in the spinal cord at 24 h after intramuscular injection. The Hc-TeTx-Alexa⁵⁵⁵ protein (red) was visualized inside the soma of the motor neurons (green), which were marked with anti-SMI-32. The markup of Hc-TeTx-Alexa⁵⁵⁵ followed a vesicular pattern. The control injection with the same amount of fluorocrom (Alexa Fluor^®555) did not emit fluorescence in the spinal cord.

Figure 3

Evaluation of the neuroprotective effect of the Hc-TeTx protein against chronic excitotoxic damage caused by the addition of DL-threo-β-hydroxyaspartic acid (THA) (100 μM) in spinal cord organotypic cultures. Motoneurons (MNs) located in the ventral horn of the spinal cord were visualized by immunofluorescence with anti-SMI-32. The addition of THA (100 μM) at 7, 14, and 21 days in vitro (DIV) caused a loss of MNs (C) with respect to the control (A). The treatment with Hc-TeTx (10 nM) under conditions of chronic excitotoxicity allowed the viability of the MNs (D). There were no differences in the viability of the MNs with treatment with Hc-TeTx under controlled conditions (B). (E) Representative micrographs of the counting of positive SMI-32 cells in the ventral area of the spinal cord. The values are shown according to ± SEM with a minimum of 15 sections per treatment. On *** p < 0.001 and ** p < 0.01 with respect to THA (100 μM) according to an ANOVA analysis with the statistical test of Bonferroni. The bar represents 50 μm.

Figure 4

Hc-TeTx activates PI3-K/Akt signaling pathway in a chronic excitotoxicity model caused by the addition of DL-threo-β-hydroxyaspartic acid (THA). Organotypic cultures were treated with the glutamate transporter inhibitor (THA) at 100 μM and the Hc-TeTx protein at 10 nM. (A) Number of SMI-32⁺ cells countered after addition of the MEK inhibitor, PD98059 at 100μM. (B) Number of SMI-32⁺ cells counted after addition of the PI3-K inhibitor, LY294002 at 50μM. While the PI3-K inhibitor blocks the neuroprotective effect of Hc-TeTx protein, in the face of chronic excitotoxicity, the MEK inhibitor does not impede this ability. The values are shown according to ± SEM with a minimum of 15 sections per treatment. On *** p < 0.001, ** p < 0.01 and * p < 0.05 with respect to THA (100 μM) according to an ANOVA analysis with the statistical test of Bonferroni.