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First published online 22 April 2008
doi: 10.1242/jcs.015354

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A single point mutation in the LN domain of LAMA2 causes muscular dystrophy and peripheral amyelination

¹ Center for Research on Occupational and Environmental Toxicology, Oregon Health and Sciences University, Portland, OR 97239, USA
² The Jackson Laboratory, Bar Harbor, ME 04609, USA

View larger version (37K): [in this window] [in a new window]   Fig. 1. The Lama2nmf417 mutation. (A-C) cDNA-sequence chromatograms from (A) wild-type, (B) heterozygous mutant nmf417 and (C) homozygous mutant nmf417 mice revealed mutation of T to C in the first position of codon 79 (arrowheads), which changes Cys79 to Arg (circled residue in C). Translation is shown above each chromatogram. (B) Intensities of the C- and T-containing sequences were similar in nmf417 heterozygotes, suggesting that the two transcripts were similarly abundant. (D) Alignment of laminin 2 peptide sequences from human, mouse and chicken, and their homologs in Drosophila and C. elegans, reveals phylogenetic conservation of Cys at this position (arrowhead). Residues identical to mouse are shown in red; peptide positions are shown at the right, provisional for the incomplete chick sequence (*). (E) Cys79 in 2 is conserved in all laminin chains containing an LN domain. (F) Laminin 2 is a heterotrimer of the 2, β1 and 1 orthologs, with N-terminal portions separated and C-terminal portions entwined in a coiled-coil. LN domains at each N-terminus mediate aggregation. The chain ends in a series of LG domains, which bind to cell receptors. The nmf417 mutation is in the 2 LN domain.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Laminin 2 association with nmf417 sarcolemma. (A-F) Cross-sections of quadriceps immunostained with antibodies against laminin 2, β1 and 1. These concentrated at similar levels in both normal and nmf417 homozygotes. Samples were stained in parallel and imaged with identical settings. (G) Immunoblotting of non-reduced BL-enriched matrix fractions from control and nmf417 muscles showed co-migration of 2, β1 and 1 at similar heterotrimer mobilities, and at similar relative abundance. Control and mutant samples contained equal total-protein content. Longer exposures (lanes 3B, 9B) showed no appreciable degradation of 2 in nmf417 muscle. 1- and 4-laminins were not significantly upregulated in nmf417 matrix. Variable immunoreactivity for 5 was noted in nmf417 samples (lane 12), but without a distinct band pattern and without corroboration by immunostaining beyond its normal distribution in blood vessels (see Fig. 8F). Reactivity for laminin β2, which did not change, probably reflects vascular BLs. Laminin 1 (Ln1), purified 1β11 protein. Values at left indicate relative molecular mass. (H) Reducing western blots on crude muscle homogenates indicated that 2 levels were not decreased in nmf417/nmf417 mutant muscle. The myosin heavy chain was used as a control. (I,J) Laminin 2 is enriched at the NMJ (arrowheads) over extrasynaptic regions (double arrows) in both control (I) and nmf417-homozygous (J) samples. (K,L) Medial sciatic nerve sections stained for 2 show similar expression by wild-type (K) and nmf417 (L) Schwann cells. (M) In nmf417/nmf417 nerves, laminin-2-positive immature Schwann cells (green) enwrap bundles of unmyelinated axons (arrowhead), whereas strong laminin 2 immunoreactivity is detected in the BL of myelinated axons (double arrow; laminin 2 in green, neurofilament in red). (N) Laminin 2 (green) colocalizes with other laminin 2 chains (β1 and 1) and with nidogen on immature Schwann cells (arrowheads). The muscle BL is noted (double arrowhead). Scale bar: 60 µm (A-F,K,L); 15 µm (I,J,M,N).

View larger version (76K): [in this window] [in a new window]   Fig. 3. Muscle pathology in Lama2 mutants. (A-D') Low (A-D) and high (A'-D')-magnification images of age-matched (P59) plantaris muscles stained with H&E. (A) Normal muscle fibers were closely aligned, had a narrow distribution of calibers and contained peripheral nuclei. (B) In nmf417-homozygote muscles, the myofiber population contained many smaller fibers, which were interposed by endomysial connective tissue and monocyte-rich lesions, and many fibers contained central nuclei. (C) Dystrophic changes in dy2J muscle were comparable to those in nmf417 muscle. (D) Dystrophic changes in dy-homozygote muscle were more severe than in nmf417, including increased fiber loss and fibrotic lesions. Scale bar: 300 µm (A-D); 75 µm (A'-D').

View larger version (135K): [in this window] [in a new window]   Fig. 4. Amyelination in Lama2 mutants. Resin sections of the fifth lumbar ventral root taken at P59 were stained with Toluidine Blue to assess myelination (dark rings) of motor axons. (A) All motor axons are separated and uniformly myelinated in normal mice. (B) In nmf417 homozygotes, myelinated axons are largely replaced by bundles of unmyelinated axons. Immature Schwann cells associated with bundle surfaces. Defects in myelination in nmf417/nmf417 were similar in form and degree to that observed in homozygotes for the dy2J allele (C) and the dy allele (D). (E) In nmf417, premyelinating Schwann cells (SC) were only loosely associated with axons (arrowheads) and were not invasive. (F) In dy2J, premyelinating Schwann cells associated more tightly with axon bundles than in nmf417. (G) Premyelinating Schwann cells in dy mutants had tight axon associations, similar to dy2J. Invasive processes were sometimes observed to engulf an axon in dy (asterisk) and dy2J mutants without proceeding to myelination. All images show spinal ventral roots. Scale bars: 72 µm (A-D); 2 µm (E-G).

View larger version (96K): [in this window] [in a new window]   Fig. 5. Ultrastructure of muscle BLs. (A) In metal-stained ultrathin sections, myofiber membranes in wild-type mice are covered by a continuous BL, consisting of a transparent lamina rara adjacent to the plasma membrane (PM), a lamina densa (arrowheads) and an external reticular lamina that is rich in collagen fibrils (col). (B) In dy homozygotes, the BL is typically collapsed, with a thin lamina densa enmeshed with collagen fibrils adjacent to the sarcolemma. (C) In nmf417, myofiber BLs retain a layered structure, although the lamina densa is often thicker than normal. Scale bar: 200 nm.

View larger version (203K): [in this window] [in a new window]   Fig. 6. Schwann cell BL formation and axon interactions in 2 mutants. (A) Normal Schwann cells form continuous BLs (arrowheads) adjacent to the plasma membrane (PM), with collagen fibrils between cells (Col). (B) In dy2J, BLs on myelinating cells contained gaps (white arrowheads) and were comparatively indistinct. (C) In dy, myelinating Schwann cell BL is very sparse (black arrowheads) and discontinuous. (D) In nmf417, BLs on myelinating Schwann cells were continuous (arrowheads), but less compact. (E) BLs were absent from premyelinating Schwann cells in nmf417 (arrowheads). AX, axons. (F) In dy2J, premyelinating Schwann cells also lacked BLs. (G) Premyelinating Schwann cells in dy also lacked BLs (arrowheads). All images show L5 ventral roots. Scale bar: 250 nm (A-D); 500 nm (E-G).

View larger version (76K): [in this window] [in a new window]   Fig. 7. Composition of Schwann cell BLs. Medial sciatic nerve sections from control and nmf417-mutant littermates were stained with antibodies against the indicated BL components. (A-D) Normal Schwann cells (A,C) produce little laminin (Ln) 1 and 5, and these were not upregulated in nmf417 nerves (B,D). (E,F) Non-myelinating Schwann cells normally express high levels of laminin 4, whereas myelinating Schwann cells express 4 at low levels. There was no increase in 4 on myelinating Schwann cells in nmf417 nerves. (G-N) The other primary components of endoneurial BLs, including laminin 1 (G,H), nidogen (I,J), collagen IV (K,L) and perlecan (M,N), were also present at comparable levels and with normal localization in the mutant nerves. Scale bar: 60 µm.

View larger version (115K): [in this window] [in a new window]   Fig. 8. Composition of muscle BLs. (A-T) Cross-sections of quadriceps from P59 nmf417 and littermate controls were immunostained with antibodies against the indicated proteins and imaged with identical settings. Normal muscle fiber BL was rich in nidogen-1, collagen IV and perlecan, and contained low levels of laminin β2, but contained little or no laminin 1, 4 or 5. Laminins 4, 5 and β2 were concentrated in blood-vessel and perineurial BLs. Cell-surface proteins interacting with laminins were also examined, including β1 integrin, and β-sarcoglycan (βSG) and β-dystroglycan (βDG) were used as indicators of the DGC in muscle. There was little change in BL composition or cell-surface proteins in nmf417-mutant muscle. Notably, laminin 4 immunoreactivity increased on a subset of small-diameter fibers, but was absent from the majority of nmf417 myofibers. Scale bar: 60 µm.

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