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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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Localization of Smn and hnRNP R at neuromuscular junctions from E18, P4 and adult Diaphragm.Whole mount preparations from Diaphragm muscles from developmental (E18) (A, C, left panels), postnatal (P4) (A, C, middle panels) and adult (3 months) (A, C, right panels) stages were performed (scale bar: 2 µm (C, left panel), 5 µm). (A) Muscles were stained against ω-BTX, SynPhys, DAPI and Smn protein. (A, left panel) At E18 Smn was highly enriched in presynaptic structures identified by SynPhys immunoreactivity. Few spots appeared in postsynaptic nuclei. (A, middle panel) Smn-positive signals were also detected in P4 motor endplates coresiding with SynPhys staining. Postsynaptic nuclei showed faint Smn immunoreactivity. (A, right panel) In 3 month old mice (adult stage) less Smn-positive signals were noticed as described before [53], [56]. The few immunoreactive particles were predominantly located in presynaptic structures visualized by SynPhys staining. (B) Single optical slices of the P4 neuromuscular synapse highlighted the co-occurring SynPhys and Smn signals (scale bar: 5 µm). (C) Muscles were stained against ω-BTX, SynPhys, DAPI and hnRNP R. HnRNP R was codistributed with SynPhys in presynaptic compartments at E18 (left panel), P4 (middle panel) and adult stage (right panel). HnRNP R was also detected in postsynaptic structures revealing stronger immunoreactivity at these sites in comparison to Smn. (D) Single optical slices of the P4 motor endplate emphasized the presynaptic localization of hnRNP R (scale bar: 5 µm).
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pone-0110846-g006: Localization of Smn and hnRNP R at neuromuscular junctions from E18, P4 and adult Diaphragm.Whole mount preparations from Diaphragm muscles from developmental (E18) (A, C, left panels), postnatal (P4) (A, C, middle panels) and adult (3 months) (A, C, right panels) stages were performed (scale bar: 2 µm (C, left panel), 5 µm). (A) Muscles were stained against ω-BTX, SynPhys, DAPI and Smn protein. (A, left panel) At E18 Smn was highly enriched in presynaptic structures identified by SynPhys immunoreactivity. Few spots appeared in postsynaptic nuclei. (A, middle panel) Smn-positive signals were also detected in P4 motor endplates coresiding with SynPhys staining. Postsynaptic nuclei showed faint Smn immunoreactivity. (A, right panel) In 3 month old mice (adult stage) less Smn-positive signals were noticed as described before [53], [56]. The few immunoreactive particles were predominantly located in presynaptic structures visualized by SynPhys staining. (B) Single optical slices of the P4 neuromuscular synapse highlighted the co-occurring SynPhys and Smn signals (scale bar: 5 µm). (C) Muscles were stained against ω-BTX, SynPhys, DAPI and hnRNP R. HnRNP R was codistributed with SynPhys in presynaptic compartments at E18 (left panel), P4 (middle panel) and adult stage (right panel). HnRNP R was also detected in postsynaptic structures revealing stronger immunoreactivity at these sites in comparison to Smn. (D) Single optical slices of the P4 motor endplate emphasized the presynaptic localization of hnRNP R (scale bar: 5 µm).

Mentions: To characterize the localization of Smn and hnRNP R at neuromuscular junctions in more detail, confocal microscopy at different developmental stages was performed with synaptophysin (SynPhys) as a marker for presynaptic terminals (Fig. 6). Postsynaptic nuclei were visualized by DAPI staining. At E18, Smn was strongly enriched in presynaptic compartments (Fig. 6A, left panel). Smn-positive signals were also detected in presynaptic terminals at postnatal day 4 (Fig. 6A, middle panel, 6B, Fig. S2A) and in the adult (Fig. 6A, right panel). However, levels of Smn immunoreactivity were lower at the latter stage, which corresponds to decreased Smn expression in spinal cord of adult mice [53]. At these analyzed neuromuscular junctions postsynaptic nuclei and the postsynaptic space labeled by BTX contained few Smn-positive signals at any developmental stage which confirms muscular expression and localization [54]–[58]. We also performed cryostat sections of ventral roots of the gastrocnemic muscle of adult mice and observed both Smn- and hnRNP R-positive signals in motor axons of sciatic nerves at this stage invivo (Fig. S2C).


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)

Localization of Smn and hnRNP R at neuromuscular junctions from E18, P4 and adult Diaphragm.Whole mount preparations from Diaphragm muscles from developmental (E18) (A, C, left panels), postnatal (P4) (A, C, middle panels) and adult (3 months) (A, C, right panels) stages were performed (scale bar: 2 µm (C, left panel), 5 µm). (A) Muscles were stained against ω-BTX, SynPhys, DAPI and Smn protein. (A, left panel) At E18 Smn was highly enriched in presynaptic structures identified by SynPhys immunoreactivity. Few spots appeared in postsynaptic nuclei. (A, middle panel) Smn-positive signals were also detected in P4 motor endplates coresiding with SynPhys staining. Postsynaptic nuclei showed faint Smn immunoreactivity. (A, right panel) In 3 month old mice (adult stage) less Smn-positive signals were noticed as described before [53], [56]. The few immunoreactive particles were predominantly located in presynaptic structures visualized by SynPhys staining. (B) Single optical slices of the P4 neuromuscular synapse highlighted the co-occurring SynPhys and Smn signals (scale bar: 5 µm). (C) Muscles were stained against ω-BTX, SynPhys, DAPI and hnRNP R. HnRNP R was codistributed with SynPhys in presynaptic compartments at E18 (left panel), P4 (middle panel) and adult stage (right panel). HnRNP R was also detected in postsynaptic structures revealing stronger immunoreactivity at these sites in comparison to Smn. (D) Single optical slices of the P4 motor endplate emphasized the presynaptic localization of hnRNP R (scale bar: 5 µm).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206449&req=5

pone-0110846-g006: Localization of Smn and hnRNP R at neuromuscular junctions from E18, P4 and adult Diaphragm.Whole mount preparations from Diaphragm muscles from developmental (E18) (A, C, left panels), postnatal (P4) (A, C, middle panels) and adult (3 months) (A, C, right panels) stages were performed (scale bar: 2 µm (C, left panel), 5 µm). (A) Muscles were stained against ω-BTX, SynPhys, DAPI and Smn protein. (A, left panel) At E18 Smn was highly enriched in presynaptic structures identified by SynPhys immunoreactivity. Few spots appeared in postsynaptic nuclei. (A, middle panel) Smn-positive signals were also detected in P4 motor endplates coresiding with SynPhys staining. Postsynaptic nuclei showed faint Smn immunoreactivity. (A, right panel) In 3 month old mice (adult stage) less Smn-positive signals were noticed as described before [53], [56]. The few immunoreactive particles were predominantly located in presynaptic structures visualized by SynPhys staining. (B) Single optical slices of the P4 neuromuscular synapse highlighted the co-occurring SynPhys and Smn signals (scale bar: 5 µm). (C) Muscles were stained against ω-BTX, SynPhys, DAPI and hnRNP R. HnRNP R was codistributed with SynPhys in presynaptic compartments at E18 (left panel), P4 (middle panel) and adult stage (right panel). HnRNP R was also detected in postsynaptic structures revealing stronger immunoreactivity at these sites in comparison to Smn. (D) Single optical slices of the P4 motor endplate emphasized the presynaptic localization of hnRNP R (scale bar: 5 µm).
Mentions: To characterize the localization of Smn and hnRNP R at neuromuscular junctions in more detail, confocal microscopy at different developmental stages was performed with synaptophysin (SynPhys) as a marker for presynaptic terminals (Fig. 6). Postsynaptic nuclei were visualized by DAPI staining. At E18, Smn was strongly enriched in presynaptic compartments (Fig. 6A, left panel). Smn-positive signals were also detected in presynaptic terminals at postnatal day 4 (Fig. 6A, middle panel, 6B, Fig. S2A) and in the adult (Fig. 6A, right panel). However, levels of Smn immunoreactivity were lower at the latter stage, which corresponds to decreased Smn expression in spinal cord of adult mice [53]. At these analyzed neuromuscular junctions postsynaptic nuclei and the postsynaptic space labeled by BTX contained few Smn-positive signals at any developmental stage which confirms muscular expression and localization [54]–[58]. We also performed cryostat sections of ventral roots of the gastrocnemic muscle of adult mice and observed both Smn- and hnRNP R-positive signals in motor axons of sciatic nerves at this stage invivo (Fig. S2C).

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