SMN Is Physiologically Downregulated at Wild-Type Motor Nerve Terminals but Aggregates Together with Neurofilaments in SMA Mouse Models.

Survival motor neuron (SMN) is an essential and ubiquitously expressed protein that participates in several aspects of RNA metabolism. SMN deficiency causes a devastating motor neuron disease called spinal muscular atrophy (SMA). SMN forms the core of a protein complex localized at the cytoplasm and nuclear gems and that catalyzes spliceosomal snRNP particle synthesis. In cultured motor neurons, SMN is also present in dendrites and axons, and forms part of the ribonucleoprotein transport granules implicated in mRNA trafficking and local translation. Nevertheless, the distribution, regulation, and role of SMN at the axons and presynaptic motor terminals in vivo are still unclear. By using conventional confocal microscopy and STED super-resolution nanoscopy, we found that SMN appears in the form of granules distributed along motor axons at nerve terminals. Our fluorescence in situ hybridization and electron microscopy studies also confirmed the presence of ß-actin mRNA, ribosomes, and polysomes in the presynaptic motor terminal, key elements of the protein synthesis machinery involved in local translation in this compartment. SMN granules co-localize with the microtubule-associated protein 1B (MAP1B) and neurofilaments, suggesting that the cytoskeleton participates in transporting and positioning the granules. We also found that, while SMN granules are physiologically downregulated at the presynaptic element during the period of postnatal maturation in wild-type (non-transgenic) mice, they accumulate in areas of neurofilament aggregation in SMA mice, suggesting that the high expression of SMN at the NMJ, together with the cytoskeletal defects, contribute to impairing the bi-directional traffic of proteins and organelles between the axon and the presynaptic terminal.

Calcium is reduced in presynaptic mitochondria of motor nerve terminals during neurotransmission in SMA mice.

Spinal muscular atrophy (SMA) is an autosomal recessive degenerative motor neuron disease characterized by symmetrical muscle weakness and atrophy of limb and trunk muscles being the most severe genetic disease in children. In SMA mouse models, motor nerve terminals display neurotransmitter release reduction, endocytosis decrease and mitochondria alterations. The relationship between these changes is, however, not well understood. In the present study, we investigated whether the endocytosis impairment could be related to the functional alteration of the presynaptic mitochondria during action potential (AP) firing. To this aim, we generated a Synaptophysin-pHluorin (SypHy) transgenic mouse, crossed it with Taiwanese SMA mice, and recorded exo- and endocytosis and mitochondria Ca2+ signaling in real-time at ex vivo motor nerve terminals of Taiwanese-SypHy mice. The experiments were performed at the beginning of the motor symptoms to get an integrated view of the nerve terminal's functional state before degeneration. Our electrophysiological and live imaging results demonstrated that the mitochondria's capacity to increase matrix-free Ca2+ in SMA mice was significantly limited during nerve AP firing, except when the rate of Ca2+ entry to the cytosol was considerably reduced. These results indicate that both the mitochondrial Ca2+ signaling alterations and the secretion machinery defects are significant players in the dysfunction of the presynaptic terminal in SMA.

R-Roscovitine Improves Motoneuron Function in Mouse Models for Spinal Muscular Atrophy.

Neurotransmission defects and motoneuron degeneration are hallmarks of spinal muscular atrophy, a monogenetic disease caused by the deficiency of the SMN protein. In the present study, we show that systemic application of R-Roscovitine, a Cav2.1/Cav2.2 channel modifier and a cyclin-dependent kinase 5 (Cdk-5) inhibitor, significantly improved survival of SMA mice. In addition, R-Roscovitine increased Cav2.1 channel density and sizes of the motor endplates. In vitro, R-Roscovitine restored axon lengths and growth cone sizes of Smn-deficient motoneurons corresponding to enhanced spontaneous Ca2+ influx and elevated Cav2.2 channel cluster formations independent of its capability to inhibit Cdk-5. Acute application of R-Roscovitine at the neuromuscular junction significantly increased evoked neurotransmitter release, increased the frequency of spontaneous miniature potentials, and lowered the activation threshold of silent terminals. These data indicate that R-Roscovitine improves Ca2+ signaling and Ca2+ homeostasis in Smn-deficient motoneurons, which is generally crucial for motoneuron differentiation, maturation, and function.