Abstract
Normal brain development depends on tight temporal and spatial regulation of connections between cells. Mutations in L1cam, a member of the immunoglobulin (Ig) superfamily that mediate cell-cell contacts through homo- and heterophilic interactions, are associated with several developmental abnormalities of the nervous system, including mental retardation, limb spasticity, hydrocephalus, and corpus callosum aplasia. L1cam has been reported to be shed from the cell surface, but the significance of this during different phases of brain development is unknown. We here show that ADAM10-mediated shedding of L1cam is regulated by its fibronectin type III (FNIII) domains. Specifically, the third FNIII domain is important for maintaining a conformation where access to a membrane proximal cleavage site is restricted. To define the role of ADAM10/17/BACE1-mediated shedding of L1cam during brain development, we used a zebrafish model system. Knockdown of the zebrafish, l1camb, caused hydrocephalus, defects in axonal outgrowth, and myelination abnormalities. Rescue experiments with proteinase-resistant and soluble L1cam variants showed that proteolytic cleavage is not required for normal axonal outgrowth and development of the ventricular system. In contrast, metalloproteinase-mediated shedding is required for efficient myelination, and only specific fragments are able to mediate this stimulatory function of the shedded L1cam.
Highlights
Development of the central nervous system depends on tightly regulated cellular connections facilitated by dynamic changes in interactions between adhesion molecules
HEK293T cells were co-transfected with plasmids encoding the L1cam variants and ADAM10, and the amount of shedded L1cam fragments present in the culture media and the amount of full length proteins in cell lysates was assessed by Western blotting
Neither deletion of the 4th nor the 5th fibronectin type III (FNIII) domain had any effect on ADAM10-mediated shedding (Fig. 1b,c), indicating that cleavage does not depend on a specific recognition motif in either of these modules
Summary
Development of the central nervous system depends on tightly regulated cellular connections facilitated by dynamic changes in interactions between adhesion molecules. Soluble variants containing different elements of the extracellular part of L1cam have in vitro been reported to induce neuronal outgrowth[11], to enhance cell migration[10,11], and to stimulate myelination[12,13]. Based on these experiments it has been suggested that soluble L1cam is shed from the cell surface and incorporated into the extracellular matrix to act as an attractant during cell migration or axonal outgrowth[9,14]. We here aim to determine the role of L1cam cleavage in vivo, and to assess the involvement of proteolytic fragments in specific processes of brain development
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