Motor neuron diseases, a group of heterogeneous neurological disorders such as Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA), are characterized by varying degrees of motor neuron degeneration. In particular, SMA is an autosomal recessive motor neuron disease and the first genetic cause of infant mortality, characterized by the degeneration of motor neurons in the anterior horn of the spinal cord, resulting in muscular atrophy and weakness. SMA patients have a homozygous loss of the survival motor neuron 1 (SMN1) gene, but retain one or more copies of a nearly identical homolog, SMN2. Therefore this disease is the result of insufficient amounts of SMN protein and its levels are generally inversely correlated with the severity of the disease, hence making SMN2 copy number the predominant modifier of the neuromuscular phenotype (Monani and De Vivo, 2014). In SMA pathology, the most striking component is the loss of alpha motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. Despite the time that has elapsed since the SMA genes were identified, the currently available treatments for patients affected by the disease are palliative, based on symptomatic treatments and supportive care (Monani and De Vivo, 2014). Nowadays, the most interesting therapeutic strategies are represented by molecular, gene and stem cell-mediated approaches which are focused on activating SMN2 expression, modulating splicing of SMN2 or replacing SMN1 (Zanetta et al., 2014; d’Ydewalle and Sumner, 2015).

A promising therapeutic approach could be the non-viral gene therapy based on the use of atoxic C-terminal fragment of the tetanus toxin (TTC). Tetanus neurotoxin is a protein produced by Clostridium tetani that cause tetanus, a fatal condition characterized by painful and uncontrolled muscle contractions (Farrar et al., 2000). The toxin is synthesized as a single polypeptide and is posts-translationally modified to produce light and heavy chains linked by disulfide bonds (Turton et al., 2002). The catalytic domain of the toxin resides in the light chain, while the translocation and receptor-binding domains are present in the heavy chain (Montal, 2010). Moreover, the heavy chain consists of two non-toxic fragments, the N-terminal or translocation domain and the C-terminal or receptor-binding domain (Chen et al., 2012). The atoxic TTC heavy chain (hereafter called TTC) can be retrogradely transported to the central nervous system and may be linked to different molecules without apparent loss of biological activity. The ganglioside-binding properties of fragment C have demonstrated that the presence of polysialic acids within the gangliosides, such as GD1b (disialic acid residues attached to the internal galactose residue) and GT1b (disialic acid residues attached to the terminal galactose residue) in cell membranes are neccesary to enable the tetanus-toxin internalization and therefore the TTC internalization in neurons. Additionally, the neurotrophin receptor p75NTR and TrkB is also essential in the retrograde pathway of TTC, sharing with NGF and BDNF the same retrograde transport organelles (Calvo et al., 2012b). These features allow the use of TTC as a valuable biological carrier of therapeutic molecules such as reporter genes or neurotrophic factors to ameliorate the disease advances of neurodegenerative disorders (Toivonen et al., 2010). In ALS, the delivery of glial cell-derived neurotrophic factor (GDNF) or brain derived neurotrophic factor (BDNF) to the spinal cord is improved by conjugation with TTC after intramuscular administration (Ciriza et al., 2008; Calvo et al., 2011). In SMA, one therapeutic strategy could be based on increasing the levels of neuronal SMN, for this reason, the genetic fusion of SMN and TTC was applied to deliver exogenous SMN to the cytosolic compartment of motor neurons (Francis et al., 2004). Interestingly, in vitro and in vivo studies have also shown that TTC itself may well harbor neuroprotective properties (Toivonen et al., 2010; Calvo et al., 2012b). In this way, in the mouse model SOD1G93A of ALS, the intramuscular injection of naked DNA encoding for TTC ameliorated the decline of hind limb muscle innervation, significantly delayed the onset of symptoms and functional deficits, improved spinal motor neuron survival and prolonged lifespan (Moreno-Igoa et al., 2010).

In the light of these preliminary results, the aim of the present study was to evaluate the possible therapeutic effect of intramuscular delivery of a TTC-encoding plasmid in the spinal cord and the skeletal muscle tissues of an intermediate mouse model of SMA, the SMNdelta7 mouse. In order to evaluate the neuroprotective effects under TTC treatment, we firstly registered the weight and the survival time, and the survival rate to further analyze the expression of several genes related to neurodegenerative process, in particular, autophagy, apoptosis and muscle atrophy.

The care and use of animals were performed accordingly with the Spanish Policy for Animal Protection RD53/2013, which meets the European Union Directive 2010/63/UE on the protection of animals used for experimental and other scientific purposes. The in-house Ethic Committees for Animal Experiments of the Universidad de Zaragoza and Universitat Autònoma de Barcelona approved all of the experimental procedures.

In recent years the neuroprotective effects of TTC have been described in relation to the antiapoptotic and survival pathways, suggesting a similar way of action as in the case of neurotrophins in animal models of several neurodegenerative diseases (Chaib-Oukadour et al., 2004). In this sense, it has been demonstrated in neurons of mSOD1 mouse model of ALS that the neuroprotective role of TTC was possibly due to its pro-survival and anti-apoptotic properties (Moreno-Igoa et al., 2010; Herrando-Grabulosa et al., 2013). A recent work in a mouse model intermediate of SMA showed that this disease in skeletal muscle emerges before pathology in spinal cord, including loss of motor neurons. (Fayzullina and Martin, 2014). This cellular loss may be mediated by apoptosis or autophagy, therefore therapeutic strategies based on modulating these molecular mechanisms may have potential beneficial effects in the SMA pathology or disease progression. Moreover, the increment of SMN observed after non-viral gene therapy treatment with pCMV-TTC could be improved the beneficial effects related to TTC.

Autophagy has come to the forefront in motor neuron diseases as a common molecular pathway altered in degenerating motor neurons. The loss of critical genes involved in the execution of the autophagy pathway in the central nervous system results in profound severe neurodegenerative diseases (Komatsu et al., 2006). Moreover, induction of autophagy has been reported in numerous models of neurodegenerative diseases, and may be a component of the cellular response to depleted SMN levels (Custer and Androphy, 2014). At P7, the SMA pups were still active and significantly smaller than their phenotypically normal siblings (2.17 ± 0.28 vs. 4.18 ± 0.11), but signs of muscle weakness were observed compared to their normal littermates. Moreover, their relative mobility insured that they were still gaining access to milk. The presence of milk in the stomach was also visible through the skin, preventing any potential autophagy induction due to poor nursing following the onset of major motor coordination losses. In a severe model of SMA, in vitro experiments showed a deregulated autophagy in spinal cord motor neurons (Garcera et al., 2013). Moreover, LC3-II and p62 protein levels were increased in lysates of spinal cord from a severe mouse model of SMA indicating that autophagy is dysregulated (Custer and Androphy, 2014). In accordance with these results, our data revealed a significant increase in the expression levels of all autophagy markers in spinal cord and skeletal muscle tissues and therefore an activation of this process. Although autophagy remained activated under TTC treatment in SMA mice, the gene expression profile observed in treated SMA mice tended to reach WT levels. In case of muscle, TTC treatment decreased Lc3 expression to WT ones. This reduction of Lc3 levels suggested that TTC was able to reduce the pathological autophagy until a constitutive autophagy, since Lc3 is considered the main marker of autophagosomes.

In relation to apoptosis, the anti-apoptotic properties of TTC were evidenced by in vitro studies in cultured neurons in which TTC preserve mitochondrial function decreased nuclear fragmentation and reduced activation of pro-Casp3 (Chaib-Oukadour et al., 2004). Similarly, in vivo experiments in a model of Parkinson’s (Mendieta et al., 2009) or ALS’s (Moreno-Igoa et al., 2010) diseases, showed an anti-apoptotic effect of TTC. In regard to apoptosis and neurodegenerative disorders, a deregulation of this process is associated with a long list of pathologies (Marino et al., 2014). In SMA disease, genetic studies in mice (Kerr et al., 2000; Anderton et al., 2013) support a role for programmed neuronal death. Thus, in the central nervous system of SMA mouse models, elevated levels of pro-apoptotic genes and an enhancement of apoptosis have been observed (Tsai et al., 2006). Moreover, a significant loss of large motor neurons was observed in the spinal cord from SMNdelta7 mouse at P7 (Baumer et al., 2009), and 2 days later this loss was over 50% (Edens et al., 2014). Furthermore, in the skeletal muscle of severe SMA model mice, the presence of apoptotic cell death signs was detected (Dachs et al., 2011). In connection with these results, we observed that the pro-apoptotic gen Bax was upregulated as well as the gen Casp3. Both genes encode for the “executioner” proteins in apoptosis, suggesting that apoptosis was activated. In relation to the skeletal muscle, a recent work using the “Taiwanese” SMA mouse model, detected at birth DNA damage that was getting worse until P6 when the muscle exhibited cell death (Fayzullina and Martin, 2014). However, in our SMA mice, the upregulation of Bax together with the lack of upregulation of Casp3 suggested an absence of apoptotic cell death in untreated SMA mice, which is in accordance with the results obtained by Hayhurst and co-workers that revealed normal proportion of apoptotic cells. After TTC treatment, the expression levels of Bax and Casp3 in spinal cord and Casp3 in skeletal muscle were downregulated untreated SMA mice, suggesting that TTC favored the normalization of the expression levels of both genes and therefore played a relevant role in the modulation of apoptosis in this animal model.

Although the importance of motor neuron pathology is well-established in SMA, a cumulative body of work supports the involvement of other cell types, including myocytes (Hamilton and Gillingwater, 2013; Martinez-Hernandez et al., 2013; Iascone et al., 2015). In SMA disease, muscle weakness and atrophy are also principal pathological hallmarks. In this way, in mouse models of the disease, several works reported an abnormal skeletal muscle development (Boyer et al., 2013).

Consequently, our next step was the evaluation of the expression of several genetic biomarkers related to muscular atrophy, previously characterized by our group (Calvo et al., 2012a). TTC administration significantly reduced the transcriptional level of Myod1, an early marker of myogenesis (Manzano et al., 2011), and Col19a1 which increasing levels are related to the regenerative response to muscle damage (Sumiyoshi et al., 2001), shifting their expression toward the wild-type levels. Additionally, after TTC treatment the expression levels of Ankrd1, a marker of muscle damage (Laure et al., 2009), and Nogo A that accelerates the progressive failure of motor neuron innervation (Pradat et al., 2007) decreased to reach WT ones, suggesting that TTC could ameliorate muscle atrophy. Finally, TTC treatment modified the expression of Mt2, Calm1 and Sln genes which are also associated to muscle atrophy (Hyldahl et al., 2010; Casas et al., 2013).

In summary, non-viral gene therapy based on TTC improved the expression levels of main genes related to autophagy and apoptosis in SMA mice. In particular, in response to the neurodegenerative progression in SMA mice, TTC treatment modifies the expression of autophagy and apoptotic genes. Additionally, TTC reduced the expression of autophagy markers and pro-apoptotic genes in spinal cord while in skeletal muscle TTC was able to downregulate the expression of the main marker of autophagy (Lc3) to WT levels, as well as the expression of the apoptosis effector, Casp3. Furthermore, in the skeletal muscle tissue of treated SMA mice, TTC showed a compensatory effect in the expression of genes involved in muscle damage response, oxidative stress and calcium homeostasis. These preliminary findings provide new insights into the effect of TTC in the spinal cord and the skeletal muscle tissues in SMA disease and suggest the need for further experiments to accurate study the effect of TTC in this disorder.

Conceived and designed the experiments: SO, ACC, RO, and JA. Performed the experiments: SO, AR, MH-G, and RM. Analyzed data: SO, ACC, and AR. Contributed reagents/materials/analysis tools: JA, PZ, and RO. Wrote the manuscript: SO, ACC, EFT, and RO.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.