Analysis of Adhesion Molecules and Basement Membrane Contributions to Synaptic Adhesion at the Drosophila Embryonic NMJ

Synapse formation and maintenance crucially underlie brain function in health and disease. Both processes are believed to depend on cell adhesion molecules (CAMs). Many different classes of CAMs localise to synapses, including cadherins, protocadherins, neuroligins, neurexins, integrins, and immunoglobulin adhesion proteins, and further contributions come from the extracellular matrix and its receptors. Most of these factors have been scrutinised by loss-of-function analyses in animal models. However, which adhesion factors establish the essential physical links across synaptic clefts and allow the assembly of synaptic machineries at the contact site in vivo is still unclear. To investigate these key questions, we have used the neuromuscular junction (NMJ) of Drosophila embryos as a genetically amenable model synapse. Our ultrastructural analyses of NMJs lacking different classes of CAMs revealed that loss of all neurexins, all classical cadherins or all glutamate receptors, as well as combinations between these or with a Laminin deficiency, failed to reveal structural phenotypes. These results are compatible with a view that these CAMs might have no structural role at this model synapse. However, we consider it far more likely that they operate in a redundant or well buffered context. We propose a model based on a multi-adaptor principle to explain this phenomenon. Furthermore, we report a new CAM-independent adhesion mechanism that involves the basement membranes (BM) covering neuromuscular terminals. Thus, motorneuronal terminals show strong partial detachment of the junction when BM-to-cell surface attachment is impaired by removing Laminin A, or when BMs lose their structural integrity upon loss of type IV collagens. We conclude that BMs are essential to tie embryonic motorneuronal terminals to the muscle surface, lending CAM-independent structural support to their adhesion. Therefore, future developmental studies of these synaptic junctions in Drosophila need to consider the important contribution made by BM-dependent mechanisms, in addition to CAM-dependent adhesion

Congenital Myasthenic Syndrome Type 19 Is Caused by Mutations in COL13A1, Encoding the Atypical Non-fibrillar Collagen Type XIII α1 Chain

The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs∗71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals

Type XIII collagen:organization of the mouse gene, generation of three genetically engineered mouse lines by homologous recombination, and biochemical studies on the molecular properties of the type XIII collagen protein

Abstract Genomic clones covering the entire mouse type XIII collagen gene (Col13a1) were isolated, and the complete exon-intron organization was characterized. The gene was found to be about 135 kb in size and to locate in the mouse chromosome 10. Comparison of gene structures and promoter regions between man and mouse indicated high conservation between the two species. In order to understand the biological function of type XIII collagen, a mouse line that expresses type XIII collagen with replacement of the cytosolic and transmembrane domains by a short, non-descript sequence was generated using homologous recombination. Expression of this aminoterminally altered type XIII collagen led to mild but progressive muscular atrophy in mice. The integrity of muscle cells was disturbed and the basement membrane showed areas of detachment from the sarcolemma as well as clearly altered structure at myotendinous junctions. These phenotypical changes were, nevertheless, local, since the majority of the muscle was intact. The results show the importance of the membrane anchorage of the type XIII collagen protein in adhesion and, consequently in the maintenance of muscle integrity. To study the significance of various regions of type XIII collagen, wild-type and mutant forms of the protein were produced recombinantly in insect cells. The transmembrane domain and the adjacent region of ectodomain were found to be crucial for the formation of type XIII collagen molecules with all of the three collagenous domains in trimeric conformation. A previously characterized conserved membrane-proximal region of the ectodomain was predicted to harbour a coiled-coil conformation. This was suggested to begin in the transmembrane domain of type XIII collagen and in several other collagenous transmembrane proteins. Type XIII collagen lacking this coiled-coil sequence was correctly folded with respect to its central COL2 and carboxylterminal COL3 domains. Between them, in the NC3 domain, a second coiled-coil sequence was found, and this was suggested to function as a second association region. The second coiled-coil sequence was found to be conserved in the two other type XIII collagen-like molecules as well. To obtain precise information about the location and level of type XIII collagen expression, a reporter mouse line synthesizing a recombinant protein with the cytoplasmic and transmembrane portions of type XIII collagen linked in-frame with the β-galactosidase enzyme was generated. The reporter mice showed high expression of type XIII collagen at neuromuscular junctions and in the periosteum of bone. Interestingly, the growth of the reporter mice was reduced at puberty. Their long bones showed a decreased diameter and impaired mechanical properties. In addition, their peripheral nerves showed areas of detachment from muscle cells at neuromuscular junctions. These results provide further evidence for the role of type XIII collagen in cell adhesion. They also show the importance of proper adhesion conducted by type XIII collagen in signaling between the extracellular matrix and cells and in the cellular response