Synaptic pathology at the neuromuscular junction in a mouse model of childhood motor neuron disease (spinal muscular atrophy)

Mounting evidence suggests that synaptic connections are early pathological targets in many neurodegenerative diseases, including motor neuron disease (Wishart et al., J Neuropathol Exp Neurol 65, 2006). A better understanding of synaptic pathology is therefore likely to be critical in order to develop effective therapeutic strategies. We have investigated synaptic pathology in the Smn1–/–;SMN2 mouse model of type I Spinal Muscular Atrophy (SMA: a childhood form of motor neuron disease). We quantified synaptic innervation at the neuromuscular junction in a range of late-symptomatic (P5-P6) Smn1–/–;SMN2 muscle groups. Neonatal mice were killed by intraperitoneal injection of sodium pentabarbitol and the transversus abdominis (TA) and levator auris longus (LAL) muscles dissected. Post-synaptic acetylcholine receptors were labelled by exposure to rhodamine-conjugated α-bungarotoxin, and neurons were labelled immunocytochemically with anti-150 kDa neurofilament antibodies. Synaptic pathology was quantified using a combination of fluorescence and confocal microscopy. There was a significant reduction in the number of fully innervated motor endplates in the LAL (∼20% reduction) and the TA (∼40% reduction) in Smn1–/–;SMN2 mice. We observed the presence of abnormal axon branching that was most prevalent in areas where synapse loss was less advanced. Significant intra-muscle variation existed for both synaptic loss and abnormal axon branching, in agreement with previous studies from adult-onset motor neuron disease (ALS). We also show that synapse loss in the wasted mouse model of early-onset motor neuron degeneration (which has a phenotype remarkably similar to Smn1–/–;SMN2 mice) proceeds in advance of axonal pathology, suggesting the presence of a distal axonopathy. Visualisation of synaptic ultrastructure in wasted mice indicated that synapses were loss via a mechanism distinct from Wallerian degeneration. We conclude that synaptic loss is a major pathological event in conditions characterized by early-onset motor neuron degeneration, including SMA.