Abstract
BackgroundAmyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases. Neurotrophic factors have been widely tested to counteract neurodegenerative conditions, despite their unspecific neuronal access. The non-toxic C-terminal fragment of the tetanus toxin (TTC) heavy chain has been studied not only as a carrier molecule to the CNS but also as a neuroprotective agent. Because the neurotrophic effects of BDNF have been demonstrated in vitro and in vivo, the question addressed in this work is whether a fusion molecule of BDNF-TTC may have a synergistic effect and enhance the neuroprotective properties of TTC alone in a mouse model of ALS.MethodsRecombinant plasmid constructs (pCMV-TTC and pCMV-BDNF-TTC) were injected into the quadriceps femoris and triceps brachialis muscles of SOD1G93A transgenic mice at 8 weeks of age. The hanging wire and rotarod tests were performed to assess motor coordination, strength and balance. Electrophysiological tests, morphological assays of spinal cord sections of L2 and L4 segments, and gene and protein expression analyses were performed. The Kaplan-Meier survival analysis test was used for comparisons of survival. Multiple comparisons of data were analyzed using a one-way analysis of variance (ANOVA).ResultsTreatment with the fusion-molecule BDNF-TTC and with TTC alone significantly delayed the onset of symptoms and functional deficits of SOD1G93A mice. Muscle innervation was partially preserved with these treatments, and the number of surviving motoneurons in L2 spinal cord segment was increased particularly by the fusion protein induction. Inhibition of pro-apoptotic protein targets (caspase-3 and Bax) and significant phosphorylation of Akt and ERK were also found in the spinal cord of treated mice.ConclusionsSignificant improvements in behavioral and electrophysiological results, motoneuron survival and anti-apoptotic/survival-activated pathways were observed with BDNF-TTC treatment. However, no synergistic effect was found for this fusion molecule. Although BDNF in the fusion molecule is capable of activating autocrine and neuroprotective pathways, TTC treatment alone yielded similar neuroprotection. Therefore, an accurate study of the neuroprotective effects of TTC fusion molecules should be performed to obtain a better understanding of its effects.
Highlights
Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases
RT-PCR revealed the presence of the TTC gene amplification in muscles inoculated with the TTC and brain derived neurotrophic factor (BDNF)-TTC encoding vectors, indicating that they were transcribed in the muscle cells
Because BDNF and TTC have already been described to be involved in the activation of 2 different survival signaling pathways in vitro, such as the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ ERK pathways [15], we investigated how treatment with the different plasmid constructs could affect these cascades in SOD1G93A mice by analyzing the phosphorylation state of Akt and extracellular signal-regulated kinase (ERK) proteins in the spinal cord
Summary
Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases. The non-toxic C-terminal fragment of the tetanus toxin (TTC) heavy chain has been studied as a carrier molecule to the CNS and as a neuroprotective agent. Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases, involving the progressive loss of motoneurons, muscle weakness and atrophy. C-fragment of the tetanus toxin (TTC) into the central nervous system (CNS) after an intramuscular injection of naked DNA or recombinant protein has been widely demonstrated and was originally used for the transport of active molecules because of the carrier properties of TTC [6,7,8]. In vitro studies have reported that TTC rescues degenerating cultured neurons from apoptotic death through the activation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) survival signaling pathways [9]. Our group has recently described that the intramuscular gene delivery of TTC in SOD1G93A mice exerts a positive effect on the prevention of neurodegeneration, improves motor function, inhibits apoptotic pathways and prolongs the survival of these animals [10]
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