Spinal muscular atrophy (SMA), a progressive neurodegenerative disease affecting motor neurons, is caused by mutations or deletions of the SMN1 gene encoding the survival of motor neuron (SMN) protein. In immortalized non-neuronal cell lines, SMN has been shown to form a ribonucleoprotein (RNP) complex with Gemin proteins, which is essential for the assembly of small nuclear RNPs (snRNPs). An additional function of SMN in neurons has been hypothesized to facilitate assembly of localized messenger RNP complexes. We have shown that SMN is localized in granules that are actively transported into neuronal processes and growth cones. In cultured motor neurons, SMN granules colocalized with ribonucleoprotein Gemin proteins but not spliceosomal Sm proteins needed for snRNP assembly. Quantitative analysis of endogenous protein colocalization in growth cones after three-dimensional reconstructions revealed a statistically nonrandom association of SMN with Gemin2 (40%) and Gemin3 (48%). SMN and Gemin containing granules distributed to both axons and dendrites of differentiated motor neurons. A direct interaction between SMN and Gemin2 within single granules was indicated by fluorescence resonance energy transfer analysis of fluorescently tagged and overexpressed proteins. High-speed dual-channel imaging of live neurons depicted the rapid and bidirectional transport of the SMN-Gemin complex. The N terminus of SMN was required for the recruitment of Gemin2 into cytoplasmic granules and enhanced Gemin2 stability. These findings provide new insight into the molecular composition of distinct SMN multiprotein complexes in neurons and motivation to investigate deficiencies of localized RNPs in SMA.
"Moreover, axonal defects in Smn-knocked down zebrafish embryos are corrected by overexpression of mutant SMNs which are incapable of snRNP assembly . The core protein components of snRNPs, the Sm proteins, do not colocalize with SMN in neuronal processes . Taken together, these observations suggest that SMN may have a unique function in neurons that is independent of snRNP biogenesis. "
[Show abstract][Hide abstract] ABSTRACT: Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2+/+;mSmn-/-) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3,094 upregulated and 6,964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs.
PLoS ONE 09/2014; 9(9):e106818. DOI:10.1371/journal.pone.0106818 · 3.23 Impact Factor
"Against this backdrop, the SMN complex has been implicated in two key non-canonical functions. In neuronal processes, the SMN complex is present in large stationary and small actively-transported granules devoid of Sm proteins , , , , . On the postsynaptic side, the Drosophila SMN complex has been reported to localise to the sarcomeric Z-disc , . "
[Show abstract][Hide abstract] ABSTRACT: Membership of the survival motor neuron (SMN) complex extends to nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and Unrip. The best-characterised function of this macromolecular machine is the assembly of the Sm-class of uridine-rich small nuclear ribonucleoprotein (snRNP) particles and each SMN complex member has a key role during this process. So far, however, only little is known about the function of the individual Gemin components in vivo. Here, we make use of the Drosophila model organism to uncover loss-of-function phenotypes of Gemin2, Gemin3 and Gemin5, which together with SMN form the minimalistic fly SMN complex. We show that ectopic overexpression of the dead helicase Gem3(ΔN) mutant or knockdown of Gemin3 result in similar motor phenotypes, when restricted to muscle, and in combination cause lethality, hence suggesting that Gem3(ΔN) overexpression mimics a loss-of-function. Based on the localisation pattern of Gem3(ΔN), we predict that the nucleus is the primary site of the antimorphic or dominant-negative mechanism of Gem3(ΔN)-mediated interference. Interestingly, phenotypes induced by human SMN overexpression in Drosophila exhibit similarities to those induced by overexpression of Gem3(ΔN). Through enhanced knockdown we also uncover a requirement of Gemin2, Gemin3 and Gemin5 for viability and motor behaviour, including locomotion as well as flight, in muscle. Notably, in the case of Gemin3 and Gemin5, such function also depends on adequate levels of the respective protein in neurons. Overall, these findings lead us to speculate that absence of any one member is sufficient to arrest the SMN-Gemins complex function in a nucleocentric pathway, which is critical for motor function in vivo.
PLoS ONE 12/2013; 8(12):e83878. DOI:10.1371/journal.pone.0083878 · 3.23 Impact Factor
"Motor neurons for axial and proximal muscles supply a large number of terminal motor units in a complex tridimensional fashion. Since SMN is likely involved in axonal transport of relevant molecules [12,48], this function might be more easily challenged in motor neurons with extensive axonal branching that support many different terminal motor units. "
[Show abstract][Hide abstract] ABSTRACT: Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.
PLoS ONE 12/2013; 8(12):e82654. DOI:10.1371/journal.pone.0082654 · 3.23 Impact Factor
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