Frontiers Neuromuscular Junctions as Key Contributors and Therapeutic Targets in Spinal Muscular Atrophy Frontiers in Neuroanatomy sintomas de hernia discal lumbar l4 l5

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, representing the most common fatal pediatric pathology. Even though, classically and in a simplistic way, it is categorized as a motor neuron (MN) disease, there is an increasing general consensus that its pathogenesis is more complex than expected. In particular, neuromuscular junctions (nmjs) are affected by dramatic alterations, including immaturity, denervation and neurofilament accumulation, associated to impaired synaptic functions: these abnormalities may in turn have a detrimental effect on MN survival. Here, we provide a description of NMJ development/maintenance/maturation in physiological conditions and in SMA, focusing on pivotal molecules and on the time-course of pathological events.Sintomas de hernia discal lumbar l4 l5 moreover, since nmjs could represent an important target to be exploited for counteracting the pathology progression, we also describe several therapeutic strategies that, directly or indirectly, aim at nmjs.

Even though for SMA no effective treatment is currently available, the efforts of researchers are constantly oriented to discover and test new drugs or compounds, at least aiming to delay the disease progression. A therapeutic strategy aimed at improving neuromuscular transmission (for example by increasing the release and the half-life of ach) could be an effective and valid approach ( wadman et al., 2012).

Here we will focus on the therapeutic effects on nmjs, either when endplates represent the direct target of the treatment, or in case of systemic/less focused treatment also having an impact on nmjs.Sintomas de hernia discal lumbar l4 l5 additionally we will also mention still unexplored molecular targets in SMA. SMN overexpression

Due to the central role of SMN in the SMA pathogenesis, many groups have evaluated the effects of its overexpression in mouse models. Moreover, some authors displayed that the synaptic connectivity at the NMJ level strictly depends on the presence of SMN in mns, rather than in muscles: overexpression of SMN in mns re-established the presynaptic properties of quantal content and the probability of synaptic vesicle release, with the consequent increase of the endplate size, the NF reduction and the denervation decrease (tibialis anterior and splenius capitis muscles; martinez et al., 2012). Moreover the beneficial effects on rescuing endplate size and mitigating NMJ denervation status was obtained by restoring low levels of SMN ( paez-colasante et al., 2013).Sintomas de hernia discal lumbar l4 l5 however, selective overexpression of SMN in SMA neurons significantly reduced the NMJ defects (gastrocnemius, triceps and tibialis anterior muscles), but moderately improved motor performance and extended the lifespan of treated mice: despite the partially positive results, an ubiquitous restoration of SMN could have been more effective ( lee et al., 2012; paez-colasante et al., 2013). Moreover there is a time window in which SMN replacement is most beneficial to nmjs: indeed early P2 intracerebroventricular administration of scaav9-SMN in SMNΔ7 SMA mice completely preserved the integrity and innervation of nmjs (longissimus capitis), compared to late (P7) administration ( robbins et al., 2014). Therefore even high SMN levels are ineffective in rescuing the phenotype, once the disease has reached advanced stages.Sintomas de hernia discal lumbar l4 l5 antisense oligonucleotides (asos)

The use of asos represents another valid strategy to increase the levels of SMN, specifically restoring the SMN2 splicing pattern, activating the SMN2 promoter, and extending the half-life of SMN mrna or protein ( tsai, 2012). Valproic acid (VPA) is currently one of the most studied asos: VPA administration in SMA type III mice improved motor performance, reduced NMJ denervation and muscular atrophy ( tsai et al., 2008). Moreover, in vitro, co-culturing C2C12 cells (myoblast cell line) and SMA patient-specific ipscs resulted in a defective achr clustering, that was improved by VPA administration, in association with the increase of SMNΔ7 and SMN-FL mrna levels, but without NF decrease ( yoshida et al., 2015).

In a mouse model of the intermediate forms of SMA (burgheron mutant) showing a delay in NMJ maturation and a decrease in the number of functional neuromuscular units, the systemic administration of SMN-restoring asos at the age of onset could extend survival and rescue the neurological phenotypes ( bogdanik et al., 2015).Sintomas de hernia discal lumbar l4 l5

Similar positive results has been also induced by trichostatin A injection, which increased innervation in severely affected muscles (longissimus capitis, splenius capitis, serratus posterior inferior and semispinalis capitis) in SMNΔ7 SMA mice: indeed denervation was reduced of about 90% in the most vulnerable muscles ( ling et al., 2012). Other asos (2′-O-2-methoxyethyl–modified ASO, passini et al., 2011; 8-mer ASO, keil et al., 2014) induced significant improvements in the NMJ morphology and innervation, in muscle physiology, motor performance, and survival respectively in SMNΔ7 SMA mice (quadriceps and intercostal muscles) and in the milder 5058-hemi hybrid SMA mice (intercostal muscles).

Despite the impressive results offered by asos, translation to human patients still needs additional steps, in order to better establish timing, volume, and location of dosing, and to avoid toxic side effects ( porensky and burghes, 2013).Sintomas de hernia discal lumbar l4 l5 neurotrophic factors and stem cells

Impairment of achr clustering in SMA is due to a delay in the switch from fetal gamma-subunit to the adult epsilon-subunit, normally occurring by 2 weeks after birth ( missias et al., 1996), and depending on neurotrophic factor release ( kues et al., 1995). Among neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) can especially support functional maturation of nmjs ( wang et al., 1995). For this reason their replacement could be a valid therapeutic approach. Indeed in several experimental models (MN diseases as well as trauma), neurotrophic factors can exert general positive effects, including reduction of MN death, stimulation of axon regrowth, preservation of peripheral functional connections, NMJ maturation ( wyatt and keirstead, 2010).Sintomas de hernia discal lumbar l4 l5 in 1997, adenovirus-mediated gene transfer of NT-3 induced substantial therapeutic effects in a murine model of progressive motor neuronopathy, increasing animal survival and improving NMJ function ( haase et al., 1997). More recently, stem cell-derived mns, transplanted in a murine model of SMA with respiratory distress type 1, appeared well integrated into the host, with axons reaching ventral roots and muscles, and forming new functional nmjs: this positive outcome can be ascribed to the neuroprotective effects exerted by graft, which even modulated inflammation ( corti et al., 2009). Also mesenchymal stem cells (mscs) could be good candidates for MN disease cell therapy, since they display a great plasticity, secrete several growth factors [such as vascular endothelial growth factor, glial cell-derived neurotrophic factor (GDNF) and BDNF], modulate host immune system ( mazzini et al., 2009), and delay atrophy in denervated muscles ( jiang et al., 2012): allogenic mscs have been administered to SMA type I patients, inducing improvements of general physical condition during treatment, without side effects ( villanova and bach, 2015), but reference to NMJ feature was not reported.Sintomas de hernia discal lumbar l4 l5

On the other hand, the administration of a recombinant AAV vector encoding human insulin-like growth factor 1 (IGF-1) into the deep cerebellar nucleus of a SMA type III mouse model significantly reduced MN death, although without a correlated correction of muscle pathology or improvement of motor functions: in fact, analysis of the gastrocnemius nmjs revealed that the treatment did not rescue the denervation, since probably the survived mns were not really functional and able to efficiently innervate the muscle ( tsai et al., 2012). However in our experience ( valsecchi et al., 2015), and in agreement with the literature ( D’amico et al., 2011), gastrocnemius, as a distal muscle, is generally less affected by SMA. Nevertheless the authors suggested to support such approach with SMN therapy, reinforcing the idea that SMN can positively influence the NMJ functionality ( tsai et al., 2012).Sintomas de hernia discal lumbar l4 l5 mirnas

Other promising therapeutic targets are represented by mirnas, whose involvement in MN diseases is gradually emerging. Notably, SMN complex is involved in mirna biogenesis and/or function, and increasing evidence demonstrated that mirna dysregulation (regarding cell cycle, development, metabolism, and synaptic plasticity) affects SMA neurons and muscles ( kye and gonçalves ido, 2014; valsecchi et al., 2015), even at embryonic age in SMNΔ7 SMA mice ( luchetti et al., 2015). In particular some mirnas (mirna-310, mirna-125, let-7, mirna-124) seem important players for both NMJ formation/function and synaptic homeostasis, in invertebrate and mammalian models ( kye and gonçalves ido, 2014). However, due to the relative novelty of such discoveries, few therapeutic approaches have been currently evaluated in SMA.Sintomas de hernia discal lumbar l4 l5

For example, mirna-9 is down-regulated in mns derived from SMN1 mut mescs ( haramati et al., 2010) and in skin fibroblast cells of SMA patients ( wang et al., 2014): mirna-9 is located downstream of SMN1 and can regulate the NF expression, suggesting its involvement in NF defects reported in SMA ( haramati et al., 2010). Additionally its expression seems to be related to SMA severity, making it a promising tool for SMA prognosis ( wang et al., 2014). An experimental modulation of the expression of mirna-9 (gain- and loss-of-function studies) is still lacking, but should help in clarifying its role in SMA NF.

Also, deletion of mirna-206 may accelerate ALS progression (G93A-SOD1 mice; williams et al., 2009) and worsen dystrophic condition in an experimental model of duchenne muscular dystrophy ( liu et al., 2012); mirna-206 is also necessary to induce NMJ regeneration after sciatic nerve crush ( williams et al., 2009).Sintomas de hernia discal lumbar l4 l5 it can effectively repress histone deacetylase 4 (HDAC4, known to inhibit muscle reinnervation; bruneteau et al., 2013), resulting in the activation of FGF binding protein 1 (FGFBP1) which in turn boosts the FGF action during reinnervation ( williams et al., 2009). We have recently demonstrated that the same pathway is activated in SMNΔ7 SMA mice ( valsecchi et al., 2015): as in ALS, mirna-206 may play a compensatory role in reinnervation, acting as a survival endogenous mechanism, although not sufficient to prevent the disease progression ( williams et al., 2009; liu et al., 2012; valsecchi et al., 2015). Finally it is worth noting that, although other mirnas could contribute to formation of NMJ and muscle reinnervation (mirna-133b and mirna-1, that together with mirna-belong to the so called “myomir” network; van rooij et al., 2008), the only mirna-206 seems essential in maintaining and repairing endplates ( valdez et al., 2014).Sintomas de hernia discal lumbar l4 l5 indeed the delivery of mirna-206 mimics or the modulation of its downstream targets could be beneficial for SMA and similar neuromuscular diseases. Agrin

Agrin is one of the fundamental molecules in stabilizing synaptic contacts and controlling axon growth ( witzemann, 2006). Interestingly SMNΔ7 SMA mns lack neuronal (Z+) agrin protein at P1, and are completely deficient at P3 ( zhang et al., 2013). Moreover cultured skeletal muscle cells isolated from SMA type I patients show abnormal fusion mechanisms and impaired agrin-induced achr aggregation, proving that nmjs cannot be correctly built ( arnold et al., 2004). Therefore agrin could be an interesting therapeutic target. Agrin fragment administration determined NMJ stabilization and phenotype amelioration in SARCO mice (experimental model of sarcopenia, characterized by age-related muscle wasting), and accelerated reinnervation after sciatic nerve crush, by activating the agrin/lrp4/musk system ( hettwer et al., 2014).Sintomas de hernia discal lumbar l4 l5 moreover recently it has been demonstrated, in co-coltures of myotubes and PC12 cells, that muscular innervation (functional NMJ formation) was triggered when cells were treated with a combination of agrin and laminin, rather than only agrin, probably due to different signaling pathways underlying achr clustering ( zhang et al., 2015). Additional in vivo studies may help in clarifying which therapeutic approach could be more efficient for SMA or similar neuromuscular diseases, in order to support functional neuromuscular development/restoration. Exercise

Even though historically physical activity was not recommended to neuromuscular disorder patients (including SMA) dreading the risk of further muscle damage ( bennett and knowlton, 1958), nowadays strong evidence reversed this opinion.Sintomas de hernia discal lumbar l4 l5 exercise can exert beneficial effects on muscle and NMJ morphology/function, during aging and in case of peripheral nerve injury or degenerative pathologies. As reviewed by nishimune et al. (2014), exercise can induce the activation of several molecules, including extracellular matrix molecules (laminin), neuregulins (neuregulin-1, PGC-1a) and neurotrophic factors (BDNF, GDNF, IGF), and modulate mirna expression (mirna-206), positively influencing NMJ morphology and transmission, their maintenance and regeneration. Indeed, neuromuscular system is strongly influenced by exercise, able to induce both structural and functional modifications; physical activity can also favorably support achr assembly ( ferraiuolo et al., 2009). Therefore, using exercise as a trigger of these molecular pathways could represent a valid therapeutic approach for MN/neuromuscular diseases or age-related atrophic pathologies.Sintomas de hernia discal lumbar l4 l5 for example, old mice undergone wheel running training (in association with diet caloric restriction) displayed a reduced number of fragmented and denervated nmjs in the tibialis anterior, gracilis and gastrocnemius muscles ( valdez et al., 2010). Moreover, trained (running) type II SMA-like mice exhibited improved motor performance, diminished muscle atrophy and reduced MN death, compared to sedentary mice ( grondard et al., 2005). Better understanding the molecular pathways activated by exercise at the muscular and NMJ level in SMA could be extremely useful. Indeed physical activity should be associated to other therapies, to obtain a synergistic effect. Conclusion

However divergent data are often present in the literature, and a more extensive analysis is therefore needed to better clarify the molecular, functional and temporal defects affecting nmjs, and consequently muscles and mns.Sintomas de hernia discal lumbar l4 l5

As shown, numerous therapeutic approaches (stem cells, mirnas, asos, etc.) have been tested either directly in SMA or in other similar neuromuscular/atrophic diseases. The obtained results are generally promising, but additional studies are necessary in view of a translational approach.

Indeed many crucial points still need to be unravelled, concerning the overall SMN function, the selective vulnerability of proximal muscles and mns, the precise time-course of pathological events, the correlation among NMJ, muscle and neuron defects. Only the answers to these critical questions will allow to identify the best and most efficient therapy/ies for SMA. Author contributions

This work was supported by funds from RF-2009-1475235 (italian ministry of health), girotondo/ONLUS and smarathon-ONLUS associations to AV, and by university of turin (ex 60% linea B 2013) grant to MB.Sintomas de hernia discal lumbar l4 l5 conflict of interest statement