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A synaptic nidogen: developmental regulation and role of nidogen-2 at the neuromuscular junction.

Fox MA, Ho MS, Smyth N, Sanes JR - Neural Dev (2008)

Bottom Line: All four core components of the basal lamina have synaptically enriched isoforms.Together, they form a highly specialized synaptic cleft material.Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA 02138, USA. mafox@vcu.edu

ABSTRACT

Background: The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.

Results: In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.

Conclusion: All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.

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Differential distribution of nidogens at the neuromuscular junction (NMJ). (A) Schematic representation of the three cellular components of the NMJ (motor nerve terminal (NT), peri-synaptic Schwann cell process (SC), and skeletal muscle fibers (M)) and the basal laminas (BLs) that coat them (dashed lines). Dashed lines: red, synaptic BL; blue, extrasynaptic BL; green, Schwann cell BL. (B-C") Young adult NMJ cross-sections costained with α-bungarotoxin (BTX) and either anti-nidogen-1 (B) or anti-nidogen-2 (C). Nidogen-1 is present in synaptic, extrasynaptic (arrows in B', B") and Schwann cell BLs (arrowheads in B', B"). Nidogen-2 is absent extrasynaptically, but is enriched in synaptic and Schwann cell BLs (arrowheads in C', C"). (D-D") Confocal, en face image of an NMJ double-stained with BTX and anti-nidogen-2. Regions of co-localization demonstrate nidogen-2-rich synaptic BL. Tube-like structures overlaying synaptic sites (arrowheads) represent Schwann cell BL. Nidogen-2-positive structures near synaptic sites are capillaries (asterisks). (E-E") Nidogen-2 localized to acetylcholine receptor-rich (BTX-stained) sites in C2C12 myotubes. Scale bars are 5 μm in (B, C) and 20 μm in (D, E).
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Figure 2: Differential distribution of nidogens at the neuromuscular junction (NMJ). (A) Schematic representation of the three cellular components of the NMJ (motor nerve terminal (NT), peri-synaptic Schwann cell process (SC), and skeletal muscle fibers (M)) and the basal laminas (BLs) that coat them (dashed lines). Dashed lines: red, synaptic BL; blue, extrasynaptic BL; green, Schwann cell BL. (B-C") Young adult NMJ cross-sections costained with α-bungarotoxin (BTX) and either anti-nidogen-1 (B) or anti-nidogen-2 (C). Nidogen-1 is present in synaptic, extrasynaptic (arrows in B', B") and Schwann cell BLs (arrowheads in B', B"). Nidogen-2 is absent extrasynaptically, but is enriched in synaptic and Schwann cell BLs (arrowheads in C', C"). (D-D") Confocal, en face image of an NMJ double-stained with BTX and anti-nidogen-2. Regions of co-localization demonstrate nidogen-2-rich synaptic BL. Tube-like structures overlaying synaptic sites (arrowheads) represent Schwann cell BL. Nidogen-2-positive structures near synaptic sites are capillaries (asterisks). (E-E") Nidogen-2 localized to acetylcholine receptor-rich (BTX-stained) sites in C2C12 myotubes. Scale bars are 5 μm in (B, C) and 20 μm in (D, E).

Mentions: To localize nidogen-2 within the NMJ, we examined cross- and en face-sectioned NMJs, allowing us to distinguish three distinct domains in BL near synaptic sites: synaptic BL, which occupies the synaptic cleft; extrasynaptic BL, in directly adjoining stretches of the muscle fiber surface; and Schwann cell BL, atop Schwann cell processes that themselves cap nerve terminals (Figure 2A). Nidogen-1 was present in all three of these domains, whereas nidogen-2 was present in both synaptic and Schwann cell BLs but absent from extrasynaptic BL (Figures 2B–D).


A synaptic nidogen: developmental regulation and role of nidogen-2 at the neuromuscular junction.

Fox MA, Ho MS, Smyth N, Sanes JR - Neural Dev (2008)

Differential distribution of nidogens at the neuromuscular junction (NMJ). (A) Schematic representation of the three cellular components of the NMJ (motor nerve terminal (NT), peri-synaptic Schwann cell process (SC), and skeletal muscle fibers (M)) and the basal laminas (BLs) that coat them (dashed lines). Dashed lines: red, synaptic BL; blue, extrasynaptic BL; green, Schwann cell BL. (B-C") Young adult NMJ cross-sections costained with α-bungarotoxin (BTX) and either anti-nidogen-1 (B) or anti-nidogen-2 (C). Nidogen-1 is present in synaptic, extrasynaptic (arrows in B', B") and Schwann cell BLs (arrowheads in B', B"). Nidogen-2 is absent extrasynaptically, but is enriched in synaptic and Schwann cell BLs (arrowheads in C', C"). (D-D") Confocal, en face image of an NMJ double-stained with BTX and anti-nidogen-2. Regions of co-localization demonstrate nidogen-2-rich synaptic BL. Tube-like structures overlaying synaptic sites (arrowheads) represent Schwann cell BL. Nidogen-2-positive structures near synaptic sites are capillaries (asterisks). (E-E") Nidogen-2 localized to acetylcholine receptor-rich (BTX-stained) sites in C2C12 myotubes. Scale bars are 5 μm in (B, C) and 20 μm in (D, E).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Differential distribution of nidogens at the neuromuscular junction (NMJ). (A) Schematic representation of the three cellular components of the NMJ (motor nerve terminal (NT), peri-synaptic Schwann cell process (SC), and skeletal muscle fibers (M)) and the basal laminas (BLs) that coat them (dashed lines). Dashed lines: red, synaptic BL; blue, extrasynaptic BL; green, Schwann cell BL. (B-C") Young adult NMJ cross-sections costained with α-bungarotoxin (BTX) and either anti-nidogen-1 (B) or anti-nidogen-2 (C). Nidogen-1 is present in synaptic, extrasynaptic (arrows in B', B") and Schwann cell BLs (arrowheads in B', B"). Nidogen-2 is absent extrasynaptically, but is enriched in synaptic and Schwann cell BLs (arrowheads in C', C"). (D-D") Confocal, en face image of an NMJ double-stained with BTX and anti-nidogen-2. Regions of co-localization demonstrate nidogen-2-rich synaptic BL. Tube-like structures overlaying synaptic sites (arrowheads) represent Schwann cell BL. Nidogen-2-positive structures near synaptic sites are capillaries (asterisks). (E-E") Nidogen-2 localized to acetylcholine receptor-rich (BTX-stained) sites in C2C12 myotubes. Scale bars are 5 μm in (B, C) and 20 μm in (D, E).
Mentions: To localize nidogen-2 within the NMJ, we examined cross- and en face-sectioned NMJs, allowing us to distinguish three distinct domains in BL near synaptic sites: synaptic BL, which occupies the synaptic cleft; extrasynaptic BL, in directly adjoining stretches of the muscle fiber surface; and Schwann cell BL, atop Schwann cell processes that themselves cap nerve terminals (Figure 2A). Nidogen-1 was present in all three of these domains, whereas nidogen-2 was present in both synaptic and Schwann cell BLs but absent from extrasynaptic BL (Figures 2B–D).

Bottom Line: All four core components of the basal lamina have synaptically enriched isoforms.Together, they form a highly specialized synaptic cleft material.Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA 02138, USA. mafox@vcu.edu

ABSTRACT

Background: The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.

Results: In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.

Conclusion: All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.

Show MeSH
Related in: MedlinePlus