Basement membranes are thin extracellular matrices that separate epithelial and mesenchymal cells, and surround cells such as endothelial, muscular, and neural cells. Basement membranes are the first extracellular matrix to appear during development, and are critical for organ development and tissue repair. They not only provide the scaffold for cells and cell layers, but they also play an essential role in morphogenesis, which affects cell adhesion, migration, proliferation, and differentiation. Basement membranes consist of laminin, collagen IV, perlecan, nidogen/entactin, and other molecules, which interact with one another to form the supramolecular structure. Laminins are multifunctional trimeric glycoproteins. There are 5 alpha (alpha1/LAMA1- alpha5/LAMA5), 3 beta (beta1/LAMB1- beta 3/ LAMB3), and 3 gamma (gamma1/LAMC1 - gamma3/LAMC3) chains, and at least 16 laminin isoforms can be formed by various combinations of each subunit. Laminin isoforms show tissue- and developmental stage-specific expression and play important roles in the structures and functions of basement membranes. The interactions of laminins with cells are essential for laminin-mediated biological activities in tissue development and adult tissue functions. Integrins, alpha-dystroglycan (alphaDG), and proteoglycans (PGs) such as syndecans have been identified as major cellular receptors for laminins. Neurite outgrowth is a key event in the differentiation of neuronal cells, and is regulated by extracellular environment factors such as nerve growth factor (NGF) and cell adhesion molecules such as laminins. Laminin-1 (LAM-111), which is composed of alpha1, beta1 and gamma 1 chains, is crucial for early basement membrane assembly, and embryonic implantation and development. In the nervous system, laminin-1 is expressed in the brain and peripheral nervous system during development. The binding of laminin-1 activates integrins via a conformational change, which promotes neurite outgrowth. Monosialoganglioside GM1, one of the major components of lipid rafts, also plays important roles in neurogenesis, such as facilitation of neurite formation, axon guidance, and synapse formation. However, the role of GM1 in laminin-1-induced neurite outgrowth is not clear. In collaboration with Dr. Eri Arikawa-Hirasawa's group, we found that laminin-1 bound to GM1 through a carbohydrate moiety and dependent on the conformation of GM1, it induced focal formation of large clusters of GM1, and it enhanced relocation of NGF receptor TrkA into the lipid rafts of dorsal root ganglion (DRG) and PC12 cells. We also found that laminin-1-mediated clustering of GM1 caused translocation and enrichment of the beta1 integrin in the lipid rafts. There, TrkA was colocalized, and it activated Lyn, Akt, and MAPK for promotion of neurite outgrowth. These results suggest that the binding of laminin-1 to GM1 facilitates the formation of a focal microdomain in the lipid rafts, and enhances signal transduction for neurite outgrowth by linking and cooperating with NGF-TrkA and laminin-integrin signaling pathways. Perlecan (Perl) is a major heparan sulfate proteoglycan in basement membranes and in cartilage. Perl interacts with many extracellular molecules and cell surface receptors, and is implicated in many biological functions in tissue homeostasis and diseases. We previously created Perl-/- mice, which developed a lethal chondrodysplasia, and we identified mutations of the perlecan gene in two human diseases, dyssegmental dysplasia, Silverman-Handmaker type (DDSH), which ischaracterized by lethal chondrodysplasia, and Schwartz-Jampel syndrome (SJS), characterized by myotonia and milder chondrodysplasia. During our perlecan project, we created several transgenic mouse lines expressing recombinant perlecan. We found that one of the lines developed severe tremors in the hindlimbs about 4 weeks after birth. The tremor phenotype was unique to this line. This suggests that the insertion of the transgene disrupts the expression of an endogenous gene and causes the tremor phenotype. The tremor is observed primarily in hindlimbs when the mice are walking, suggesting that a sensory problem causes the phenotype. Because of this unique tremor phenotype, this mouse line should be a useful animal model for understanding essential tremor, in which tremors occur when a person is moving. Therefore, we wanted to characterize the mutant mice. We subsequently found that teneurin-4 (Ten-4/Odz4) was deficient in mutant mice because of the transgene insertion, which caused the tremor. Ten-4 is a transmembrane protein that is highly expressed in neurons and oligodendrocytes, but its function is unknown. We observed severe hypomyelination and axon degeneration in the white matter of the cerebellum, spinal cord, and corticospinal tract of the brain stem of 4-week-old mutant mice. Myelin basic protein (MBP) staining in the spinal cord of 2-week-old mutant mice was reduced, while there was no significant difference in neurofilament staining, suggesting that hypomyelination is an earlier defect than axon degeneration. We also found that the number of mature oligodendrocytes (OLs) was dramatically decreased in 2-week-old mutant mice. Suppression of Ten-4 expression in the OL progenitor cell line CG-4 by shRNA reduced CG-4 cell differentiation with concomitant decreased phosphorylation of FAK, one of key regulators of OL differentiation and myelination. These results suggest that teneurin-4 is required for OL differentiation and maintenance, and is essential for myelination in the CNS. This mouse model should be useful in the development of therapeutic reagents for human tremors.
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