P. Saftig

Evaluation of the Contribution of Different ADAMs to Tumor Necrosis Factor α (TNFα) Shedding and of the Function of the TNFα Ectodomain in Ensuring Selective Stimulated Shedding by the TNFα Convertase (TACE/ADAM17)

Tumor necrosis factor-α (TNFα), a potent pro-inflammatory cytokine, is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-α converting enzyme (TACE; a disintegrin and metalloprotease17; ADAM17) is known to have a key role in the ectodomain shedding of TNFα in several cell types. However, because purified ADAMs 9, 10, and 19 can also cleave a peptide corresponding to the TNFα cleavage site in vitro, these enzymes are considered to be candidate TNFα sheddases as well. In this study we used cells lacking ADAMs 9, 10, 17 (TACE), or 19 to address the relative contribution of these ADAMs to TNFα shedding in cell-based assays. Our results corroborate that ADAM17, but not ADAM9, -10, or -19, is critical for phorbol ester- and pervanadate-stimulated release of TNFα in mouse embryonic fibroblasts. However, overexpression of ADAM19 increased the constitutive release of TNFα, whereas overexpression of ADAM9 or ADAM10 did not. This suggests that ADAM19 may contribute to TNFα shedding, especially in cells or tissues where it is highly expressed. Furthermore, we used mutagenesis of TNFα to explore which domains are important for its stimulated processing by ADAM17. We found that the cleavage site of TNFα is necessary and sufficient for cleavage by ADAM17. In addition, the ectodomain of TNFα makes an unexpected contribution to the selective cleavage of TNFα by ADAM17: it prevents one or more other enzymes from cleaving TNFα following PMA stimulation. Thus, selective stimulated processing of TNFα by ADAM17 in cells depends on the presence of an appropriate cleavage site as well as the inhibitory role of the TNF ectodomain toward other enzymes that can process this site.

Functions of the protease bace1 in the maturation of the nervous system

TAG-1 is a GPI-anchored cell adhesion molecule, expressed by neurons and myelinating glial cells. In the adult, TAG-1 controls axon–glia contact at the juxta-paranodal regions of myelinated fibers, contributing to the organization of axonal domains at the node of Ranvier. During development, the transient expression of TAG-1 by axons in several fascicles of the embryonic brain suggests a role for TAG-1 in axono-axonal and/or axono-glial interactions during development. To investigate this possible role of TAG-1, we focused our study on the embryonic optic nerve where astroglial and oligodendroglial cells develop in contact with the axons, and where TAG-1 is strongly expressed by retinal ganglion cells (RGCs) during the period of axonal growth and targeting. First, we examined the effect of TAG-1 on RGC axons and glial precursor cells in vitro. TAG-1 protein promoted RGC axon outgrowth, similar to other adhesion molecules such as L1 and NrCAM. Time-lapse videomicroscopy imaging of RGC axons growing on TAG-1 cells will allow us to discriminate between the possible effects of TAG1 on growth cone extension, formation of collaterals and axon fasciculation. The trophic and adhesive responses of developing glial cells were also changed in contact with TAG-1. Recombinant TAG-1 protein strongly promoted the survival and adhesion, but not the proliferation, of astroglial and oligodendroglial precursor cells. Secondly, we used TAG-1 deficient mice to examine the consequences of TAG1 loss on the morphology of axons and glial cells in the optic nerve. Electron-microscopy analysis of hetero and homozygous animals showed structural anomalies in axon compaction and the distribution of glial cells between the axonal bundles. The mapping of retino-collicular projections, as well as the analysis of glial cell differentiation in the optic nerve will address the final functional impact of TAG-1 loss on the visual pathway. Altogether our present findings indicate that TAG-1 acts on the development of both RGC axons and glial cells, and suggest that TAG-1 would mediate axon–glial cells interactions required for the proper development of the optic nerve.