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Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction.

Nishimune H, Valdez G, Jarad G, Moulson CL, Müller U, Miner JH, Sanes JR - J. Cell Biol. (2008)

Bottom Line: Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation.Another synaptically concentrated laminin receptor, Bcam, is dispensable.Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. hnishimune@kumc.edu

ABSTRACT
A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the alpha4, alpha5, and beta2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin alpha5 and arrested in mutants lacking both alpha4 and alpha5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.

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Muscle-specific deletion of laminin α5 leads to delayed neuromuscular synapse maturation. (A) Laminin α5 protein was undetectable at NMJs by P21 in Lama5M/M mice, but laminin α4 levels were unaltered. Muscle sections were stained with laminin antibodies (green) and Alexa 594–BTX to label AChRs (red). (B) En face views of NMJs in P21 control and Lama5M/M mice stained with antibodies to neurofilaments and SV2 (nerve, green), and Alexa 594–BTX (AChR, red). Motor nerve terminals fully occupied complex AChR clusters in controls. In Lama5M/M muscle, many AChR clusters had a simple morphology and were partially innervated. (C and D) Quantification of topological maturation of AChR clusters (C) and completeness of innervation of AChR clusters (D) in control (open circles) and Lama5M/M (closed squares) sternomastoid muscle. Counts are from two animals per age, >40 NMJs per animal, with the exception of controls at P21 and P40, which are from one animal. (E) Laminin chains present in synaptic BL (adapted from Patton et al., 1997). Bars, 10 μm.
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fig1: Muscle-specific deletion of laminin α5 leads to delayed neuromuscular synapse maturation. (A) Laminin α5 protein was undetectable at NMJs by P21 in Lama5M/M mice, but laminin α4 levels were unaltered. Muscle sections were stained with laminin antibodies (green) and Alexa 594–BTX to label AChRs (red). (B) En face views of NMJs in P21 control and Lama5M/M mice stained with antibodies to neurofilaments and SV2 (nerve, green), and Alexa 594–BTX (AChR, red). Motor nerve terminals fully occupied complex AChR clusters in controls. In Lama5M/M muscle, many AChR clusters had a simple morphology and were partially innervated. (C and D) Quantification of topological maturation of AChR clusters (C) and completeness of innervation of AChR clusters (D) in control (open circles) and Lama5M/M (closed squares) sternomastoid muscle. Counts are from two animals per age, >40 NMJs per animal, with the exception of controls at P21 and P40, which are from one animal. (E) Laminin chains present in synaptic BL (adapted from Patton et al., 1997). Bars, 10 μm.

Mentions: In wild-type mice, laminin α5 is present throughout the basal lamina of embryonic myotubes. Over the first three postnatal weeks, laminin α5 is lost from extrasynaptic basal lamina and becomes concentrated in synaptic basal lamina (Patton et al., 1997; Fig. 1 E). In Lama5M/M muscle, laminin α5 was barely detectable in extrasynaptic basal lamina at postnatal day 0; it was undetectable at 75% of NMJs at P0 and at 95% of NMJs by P10 (Fig. 1 A and not depicted). The low levels of laminin α5 present at birth may result from slow degradation of laminins inserted before gene deletion (see Cohn et al., 2002). In any case, the absence of laminin α5 from NMJs at later stages indicates that synaptic deposits of this subunit in postnatal muscle are contributed solely by the muscle and not by motor neurons or Schwann cells. Quantitative analysis of immunofluorescence indicated that levels of laminins α4 and β2 at the NMJ were unaffected by the absence of laminin α5 (Fig. 1 A and not depicted).


Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction.

Nishimune H, Valdez G, Jarad G, Moulson CL, Müller U, Miner JH, Sanes JR - J. Cell Biol. (2008)

Muscle-specific deletion of laminin α5 leads to delayed neuromuscular synapse maturation. (A) Laminin α5 protein was undetectable at NMJs by P21 in Lama5M/M mice, but laminin α4 levels were unaltered. Muscle sections were stained with laminin antibodies (green) and Alexa 594–BTX to label AChRs (red). (B) En face views of NMJs in P21 control and Lama5M/M mice stained with antibodies to neurofilaments and SV2 (nerve, green), and Alexa 594–BTX (AChR, red). Motor nerve terminals fully occupied complex AChR clusters in controls. In Lama5M/M muscle, many AChR clusters had a simple morphology and were partially innervated. (C and D) Quantification of topological maturation of AChR clusters (C) and completeness of innervation of AChR clusters (D) in control (open circles) and Lama5M/M (closed squares) sternomastoid muscle. Counts are from two animals per age, >40 NMJs per animal, with the exception of controls at P21 and P40, which are from one animal. (E) Laminin chains present in synaptic BL (adapted from Patton et al., 1997). Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2542479&req=5

fig1: Muscle-specific deletion of laminin α5 leads to delayed neuromuscular synapse maturation. (A) Laminin α5 protein was undetectable at NMJs by P21 in Lama5M/M mice, but laminin α4 levels were unaltered. Muscle sections were stained with laminin antibodies (green) and Alexa 594–BTX to label AChRs (red). (B) En face views of NMJs in P21 control and Lama5M/M mice stained with antibodies to neurofilaments and SV2 (nerve, green), and Alexa 594–BTX (AChR, red). Motor nerve terminals fully occupied complex AChR clusters in controls. In Lama5M/M muscle, many AChR clusters had a simple morphology and were partially innervated. (C and D) Quantification of topological maturation of AChR clusters (C) and completeness of innervation of AChR clusters (D) in control (open circles) and Lama5M/M (closed squares) sternomastoid muscle. Counts are from two animals per age, >40 NMJs per animal, with the exception of controls at P21 and P40, which are from one animal. (E) Laminin chains present in synaptic BL (adapted from Patton et al., 1997). Bars, 10 μm.
Mentions: In wild-type mice, laminin α5 is present throughout the basal lamina of embryonic myotubes. Over the first three postnatal weeks, laminin α5 is lost from extrasynaptic basal lamina and becomes concentrated in synaptic basal lamina (Patton et al., 1997; Fig. 1 E). In Lama5M/M muscle, laminin α5 was barely detectable in extrasynaptic basal lamina at postnatal day 0; it was undetectable at 75% of NMJs at P0 and at 95% of NMJs by P10 (Fig. 1 A and not depicted). The low levels of laminin α5 present at birth may result from slow degradation of laminins inserted before gene deletion (see Cohn et al., 2002). In any case, the absence of laminin α5 from NMJs at later stages indicates that synaptic deposits of this subunit in postnatal muscle are contributed solely by the muscle and not by motor neurons or Schwann cells. Quantitative analysis of immunofluorescence indicated that levels of laminins α4 and β2 at the NMJ were unaffected by the absence of laminin α5 (Fig. 1 A and not depicted).

Bottom Line: Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation.Another synaptically concentrated laminin receptor, Bcam, is dispensable.Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. hnishimune@kumc.edu

ABSTRACT
A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the alpha4, alpha5, and beta2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin alpha5 and arrested in mutants lacking both alpha4 and alpha5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.

Show MeSH
Related in: MedlinePlus