Abstract
Spinal Muscular Atrophy (SMA) is a severe autosomal recessive disease characterized by selective motor neuron degeneration, caused by disruptions of the Survival of Motor Neuron 1 (Smn1) gene. The main product of SMN1 is the full-length SMN protein (FL-SMN), that plays an established role in mRNA splicing. FL-SMN is also involved in neurite outgrowth and axonal transport. A shorter SMN isoform, axonal-SMN or a-SMN, displays a more specific axonal localization and has remarkable axonogenic properties in NSC-34. Introduction of known SMA mutations into the a-SMN transcript leads to impairment of axon growth and morphological defects similar to those observed in SMA patients and animal models. Although there is increasing evidence for the relevance of SMN axonal functions in SMA pathogenesis, the specific contributions of FL-SMN and a-SMN are not known yet. This work aimed to analyze the differential roles of FL-SMN and a-SMN in axon outgrowth and in neuronal homeostasis during differentiation of neurons into a mature phenotype. We employed primary cultures of hippocampal neurons as a well-defined model of polarization and differentiation. By analyzing subcellular localization, we showed that a-SMN is preferentially localized in the growing axonal compartment. By specifically silencing FL-SMN or a-SMN proteins, we demonstrated that both proteins play a role in axon growth, as their selective down-regulation reduces axon length without affecting dendritic arborization. a-SMN silencing, and in minor extent FL-SMN silencing, resulted in the growth of multi-neuritic neurons, impaired in the differentiation process of selecting a single axon out of multiple neurites. In these neurons, neurites often display mixed axonal and dendritic markers and abnormal distribution of the axonal initial segment protein Ankirin G, suggesting loss of neuronal polarity. Our results indicate that a-SMN and FL-SMN are needed for neuronal polarization and organization of axonal and dendritic compartments, processes that are fundamental for neuronal function and survival.
Highlights
Spinal Muscular Atrophy or SMA is a severe autosomal recessive disease and the leading genetic cause of infant mortality
To allow proper visualization of fine cellular details, cultured neurons were prepared from E18 Sprague-Dawley rats and co-labeled with 5-DTAF (Fig 1A1–A4) and #553 rat-specific anti-axonal SMN (a-SMN) antibodies (Fig 1B1–B4). # 553 antibody specficity was verified by western blot analysis on rat E14 and E16 embryo cortex and hippocampus lysates, where # 553 antibody evidenced a specific immunoreactive band of approximately 29 KDa, distinct from the band corresponding to full-length SMN protein (FL-SMN) (~37kDa) recognized by the BD Bioscience anti-SMN antibody (S1 Fig)
SMA is characterized by spinal motor neuron degeneration, leading to progressive denervation of skeletal and intercostal muscles, muscle weakness, paralysis, and eventual death due to respiratory failure [100]
Summary
Spinal Muscular Atrophy or SMA is a severe autosomal recessive disease and the leading genetic cause of infant mortality. SMA is primarily characterized by motor neuron degeneration leading to progressive amyotrophic paralysis, respiratory failure, and, in more severe cases, death. Classical neuropathologic studies and recent data from both affected patients and murine SMA models already demonstrated that other cell types such as pyramidal neurons and glia cells are affected in SMA [14,15,16,17,18,19,20,21] as well as abnormalities in non-neuronal systems [22,23,24,25, 18, 13, 26, 27], suggesting multiple pathogenetic pathways leading to the SMA phenotype
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