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Presynaptic localization of Smn and hnRNP R in axon terminals of embryonic and postnatal mouse motoneurons.

Dombert B, Sivadasan R, Simon CM, Jablonka S, Sendtner M - PLoS ONE (2014)

Bottom Line: Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo.We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons.These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University Hospital Wuerzburg, Wuerzburg, Germany.

ABSTRACT
Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

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Colocalization of Smn and hnRNP R invivo in E18 motoneurons and axon terminals.(A) Representative cross section from E18 spinal cord stained against Smn, hnRNP R and ChAT (scale bar: 10 µm). Superimposed colocalizing points are highlighted in white. Smn signals were mainly found in the cytosol, with very few positive spots in the nuclei. HnRNP R immunoreactivity was observed in the nucleus and in the cytosol. Colocalization of Smn and hnRNP R was detected in the cytosol, especially in axonal initiation segments (PCC 0.27±0.03, MOC 0.81±0.01, N = 8). (B) Whole mount preparations from Diaphragm muscles from E18 mouse embryos stained against Smn, hnRNP R, ω-BTX and DAPI (scale bar: 2 µm). Both Smn and hnRNP R immunoreactivity were detected at these defined sites showing partial overlap (PCC 0.24±0.04, MOC 0.54±0.02, N = 6).
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pone-0110846-g005: Colocalization of Smn and hnRNP R invivo in E18 motoneurons and axon terminals.(A) Representative cross section from E18 spinal cord stained against Smn, hnRNP R and ChAT (scale bar: 10 µm). Superimposed colocalizing points are highlighted in white. Smn signals were mainly found in the cytosol, with very few positive spots in the nuclei. HnRNP R immunoreactivity was observed in the nucleus and in the cytosol. Colocalization of Smn and hnRNP R was detected in the cytosol, especially in axonal initiation segments (PCC 0.27±0.03, MOC 0.81±0.01, N = 8). (B) Whole mount preparations from Diaphragm muscles from E18 mouse embryos stained against Smn, hnRNP R, ω-BTX and DAPI (scale bar: 2 µm). Both Smn and hnRNP R immunoreactivity were detected at these defined sites showing partial overlap (PCC 0.24±0.04, MOC 0.54±0.02, N = 6).

Mentions: Based on these results we studied distribution and colocalization of Smn and hnRNP R in spinal cord cross sections from E18 mouse embryos (Fig. 5A) which correlates with the developmental stage of primary motoneurons isolated at E13.5 and cultured for 5DIV. Motoneurons were identified by choline acetyltransferase (ChAT) staining. Again, Smn immunoreactivity was mostly found in the cytosol and in proximal axonal processes, whereas nuclei appeared relatively spared revealing only distinct Gem-like immunoreactive structures. In contrast, hnRNP R was detected both in the nucleus and in the cytosol. In particular, perinuclear cytoplasm and proximal axons showed an overlap of hnRNP R and Smn signals (PCC 0.27±0.03; MOC 0.81±0.01; N = 8) (Fig. 5A) which is similar to the data obtained by immunofluorescence in isolated embryonic motoneurons (see Fig. 2) and Western blot analyses of coimmunoprecipitation from cytosolic fractions (see Fig. 3).


Presynaptic localization of Smn and hnRNP R in axon terminals of embryonic and postnatal mouse motoneurons.

Dombert B, Sivadasan R, Simon CM, Jablonka S, Sendtner M - PLoS ONE (2014)

Colocalization of Smn and hnRNP R invivo in E18 motoneurons and axon terminals.(A) Representative cross section from E18 spinal cord stained against Smn, hnRNP R and ChAT (scale bar: 10 µm). Superimposed colocalizing points are highlighted in white. Smn signals were mainly found in the cytosol, with very few positive spots in the nuclei. HnRNP R immunoreactivity was observed in the nucleus and in the cytosol. Colocalization of Smn and hnRNP R was detected in the cytosol, especially in axonal initiation segments (PCC 0.27±0.03, MOC 0.81±0.01, N = 8). (B) Whole mount preparations from Diaphragm muscles from E18 mouse embryos stained against Smn, hnRNP R, ω-BTX and DAPI (scale bar: 2 µm). Both Smn and hnRNP R immunoreactivity were detected at these defined sites showing partial overlap (PCC 0.24±0.04, MOC 0.54±0.02, N = 6).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206449&req=5

pone-0110846-g005: Colocalization of Smn and hnRNP R invivo in E18 motoneurons and axon terminals.(A) Representative cross section from E18 spinal cord stained against Smn, hnRNP R and ChAT (scale bar: 10 µm). Superimposed colocalizing points are highlighted in white. Smn signals were mainly found in the cytosol, with very few positive spots in the nuclei. HnRNP R immunoreactivity was observed in the nucleus and in the cytosol. Colocalization of Smn and hnRNP R was detected in the cytosol, especially in axonal initiation segments (PCC 0.27±0.03, MOC 0.81±0.01, N = 8). (B) Whole mount preparations from Diaphragm muscles from E18 mouse embryos stained against Smn, hnRNP R, ω-BTX and DAPI (scale bar: 2 µm). Both Smn and hnRNP R immunoreactivity were detected at these defined sites showing partial overlap (PCC 0.24±0.04, MOC 0.54±0.02, N = 6).
Mentions: Based on these results we studied distribution and colocalization of Smn and hnRNP R in spinal cord cross sections from E18 mouse embryos (Fig. 5A) which correlates with the developmental stage of primary motoneurons isolated at E13.5 and cultured for 5DIV. Motoneurons were identified by choline acetyltransferase (ChAT) staining. Again, Smn immunoreactivity was mostly found in the cytosol and in proximal axonal processes, whereas nuclei appeared relatively spared revealing only distinct Gem-like immunoreactive structures. In contrast, hnRNP R was detected both in the nucleus and in the cytosol. In particular, perinuclear cytoplasm and proximal axons showed an overlap of hnRNP R and Smn signals (PCC 0.27±0.03; MOC 0.81±0.01; N = 8) (Fig. 5A) which is similar to the data obtained by immunofluorescence in isolated embryonic motoneurons (see Fig. 2) and Western blot analyses of coimmunoprecipitation from cytosolic fractions (see Fig. 3).

Bottom Line: Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo.We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons.These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University Hospital Wuerzburg, Wuerzburg, Germany.

ABSTRACT
Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

Show MeSH
Related in: MedlinePlus