Spinal muscular atrophy (SMA) is a fatal paediatric genetic disease, characterized by motoneuron death, leading to progressive amyotrophic paralysis, respiratory failure, and, in more severe cases, to death. SMA is due to the deletion or mutation of the telomeric survival motoneuron gene (SMN1). Its homologous, SMN2 gene, encodes a truncated protein which can modulate SMA severity. Abnormalities at the neuromuscular junction (NMJ) have been reported in SMA, including neurofilament (NF) accumulation at presynaptic terminals, smaller and immature endplates, reduced transmitter release, and finally muscle denervation. We have studied the effects of manipulating the agrin/neurotrypsin system, fundamental in NMJ formation, in SMA delta7 mice: agrin is a synaptic organizer, responsible for NMJ development, i.e. for insertion of a nerve terminal, clustering of AChRs, NMJ maturation. We treated SMA and WT mice from birth with a modified 44kD C terminal agrin fragment (CAF) retaining synaptogenic properties (supplied by Neurotune AG) s.c., using vehicle for controls, which were analysed for behaviour, muscle and NMJ histology, and survival. Motor behavior evaluated by righting reflex and tail suspension tests was significantly improved by treatment in SMA mice. Survival was significantly extended in CAF-treated SMA by 2.5 days (percentage of improvement 16.5%; P = 0.001, Kaplan-Mayer analysis). Histological analysis was performed on P10 mice. Muscle histology was performed on the quadriceps, a proximal muscle early involved in SMA. In H/E stained muscles, CAF treatment increased cross-sectional area of whole muscle (3083940 μm2 in treated SMA vs 2595276 μm2 in control SMA) as well as of single muscle fibers (141.18 μm2 in treated SMA vs 127.39 μm2 control SMA), thus demonstrating reduced muscle atrophy. NMJ morphology was evaluated in whole mount diaphragm preparations: treated SMA mice showed more mature NMJs and a reduced NF accumulation, compared to controls. On the contrary, no differences were found in the number of lumbar motoneurons between treated and control mice. Therefore, manipulating the agrin/neurotrypsin system in SMA has beneficial effects on muscle trophism and NMJ maturation: this agrin-derived biological restores the crosstalk between muscle and motoneurons, delaying muscular/NMJ atrophy, improving motor performance and finally extending survival. Supported by Girotondo Onlus grant.

MANIPULATING THE AGRIN/NEUROTRYPSIN SYSTEM IMPROVES MUSCLE TROPHISM IN SMA.

BOIDO, Marina Maria;VERCELLI, Alessandro
2013

Abstract

Spinal muscular atrophy (SMA) is a fatal paediatric genetic disease, characterized by motoneuron death, leading to progressive amyotrophic paralysis, respiratory failure, and, in more severe cases, to death. SMA is due to the deletion or mutation of the telomeric survival motoneuron gene (SMN1). Its homologous, SMN2 gene, encodes a truncated protein which can modulate SMA severity. Abnormalities at the neuromuscular junction (NMJ) have been reported in SMA, including neurofilament (NF) accumulation at presynaptic terminals, smaller and immature endplates, reduced transmitter release, and finally muscle denervation. We have studied the effects of manipulating the agrin/neurotrypsin system, fundamental in NMJ formation, in SMA delta7 mice: agrin is a synaptic organizer, responsible for NMJ development, i.e. for insertion of a nerve terminal, clustering of AChRs, NMJ maturation. We treated SMA and WT mice from birth with a modified 44kD C terminal agrin fragment (CAF) retaining synaptogenic properties (supplied by Neurotune AG) s.c., using vehicle for controls, which were analysed for behaviour, muscle and NMJ histology, and survival. Motor behavior evaluated by righting reflex and tail suspension tests was significantly improved by treatment in SMA mice. Survival was significantly extended in CAF-treated SMA by 2.5 days (percentage of improvement 16.5%; P = 0.001, Kaplan-Mayer analysis). Histological analysis was performed on P10 mice. Muscle histology was performed on the quadriceps, a proximal muscle early involved in SMA. In H/E stained muscles, CAF treatment increased cross-sectional area of whole muscle (3083940 μm2 in treated SMA vs 2595276 μm2 in control SMA) as well as of single muscle fibers (141.18 μm2 in treated SMA vs 127.39 μm2 control SMA), thus demonstrating reduced muscle atrophy. NMJ morphology was evaluated in whole mount diaphragm preparations: treated SMA mice showed more mature NMJs and a reduced NF accumulation, compared to controls. On the contrary, no differences were found in the number of lumbar motoneurons between treated and control mice. Therefore, manipulating the agrin/neurotrypsin system in SMA has beneficial effects on muscle trophism and NMJ maturation: this agrin-derived biological restores the crosstalk between muscle and motoneurons, delaying muscular/NMJ atrophy, improving motor performance and finally extending survival. Supported by Girotondo Onlus grant.
23° CONVEGNO NAZIONALE DEL GRUPPO ITALIANO PER LO STUDIO DELLA NEUROMORFOLOGIA (G.I.S.N.)
Cagliari
22-23 Novembre 2013
57
3s
1
28
EUROPEAN JOURNAL OF HISTOCHEMISTRY
http://www.ejh.it/index.php/ejh/issue/view/142
M. Boido; E. De Amicis; S. Hettwer; R. Fariello; A. Vercelli
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/153700
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