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Restoring Laminin Polymerization Via a Chimeric Linker Protein Rescues Muscle Pathology of Congenital Muscular Dystrophy Type 1A

Stephanie Capizzi Crosson
B.A., Rutgers University - 2006

Thesis Advisors: Peter D. Yurchenco, M.D., Ph.D.
Graduate Program in Biochemistry

Robert Wood Johnson Medical School
Research Tower, Room V-10

Friday, June 6, 2014
2:00 p.m.


Laminin-2-deficient congenital muscular dystrophy (MDC1A), an incurable disease of skeletal muscle wasting with peripheral neuropathy, is caused by the reduction or absence of the 2-chain of laminin-211 in neuromuscular basement membranes. While there is an increase in laminin-4 (Lm-411) expression in MDC1A, it is thought that it cannot substitute for the absent subunit because it does not polymerize or bind well to receptors.
The goal of this project was to study the contributions from laminin polymerization and receptor-binding domains to basement membrane assembly, to explore differences among laminin isoforms, and to develop reparative proteins that may improve the disease phenotype in mouse models of MDC1A by modifying laminin-4 function.
A series of laminin-binding linker proteins were engineered to provide scaffold-forming and receptor-binding activities to recombinant laminins. Two of these proteins (LNNd and 1NtAFs1) were found to enable laminin polymerization and thereby increase basement membrane assembly of non-polymerizing laminins on cultured Schwann cells and myotubes. When LNNd was combined with mini-agrin (a laminin-binding linker to enhance receptor-binding) on myotubes or Schwann cells, or provided with both activities contained within a single protein (1shortmA), basement membrane assembly of laminin-411 was promoted beyond that seen with each activity alone.
To evaluate LNNd in dystrophic mouse muscle, LNNd was also transgenically expressed in muscle of two MDC1A mouse models, i.e. an N-terminally-truncated laminin-2 subunit (dy2J) and the laminin 2-knockout mouse (dy3K). Expression of LNNd in dy2J mice increased myofiber areas, decreased fibrosis and the fraction of regenerating myofibers, and shifted the N-terminally truncated laminin-211 in muscle homogenates from soluble to a matrix fraction. LNNd expression in dy3K mice doubled their life expectancy, increased weights and muscle strength, increased myofiber size, and decreased muscle fibrosis. However, the extent of repair was less in dy3K compared to dy2J. This led to the prediction that combining a polymerization and anchorage repair (LNNd + mini-agrin) will maximize muscle repair in laminin-2 deficient muscular dystrophies.
Overall the study validates a polymerization/anchorage model of basement membrane assembly, establishes that an engineered protein can enable laminin polymerization, and reveals that two models of MDC1A can benefit from transgenic expression of the protein.

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