Biol. treatment rendered neurons resistant to myelin-associated inhibitors. Launch of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin 6-Acetamidohexanoic acid in an NgR1-dependent manner. and promotes regeneration following CNS trauma (3). Further studies have suggested that NgR1 plays a protective role in Alzheimer disease (4), that NgR1 variants contribute to increased risk of schizophrenia (5), and that NgR1 limits plasticity in the hippocampus (6) and in the visual cortex (7). Understanding the mechanisms that regulate NgR1 will have important implications for CNS plasticity, disease, and regeneration. NgR1 shed from the cell surface is a soluble fragment spanning amino acids 1C358 (8). This fragment is similar to NgREcto (amino acids 1C310), a dominant-negative protein that promotes axonal regeneration and by antagonizing MAI signaling (9, 10). The shed NgR1 product can be detected in human cerebral spinal fluid, indicative of a physiological role for this 6-Acetamidohexanoic acid fragment. NgR1 shedding is blocked by the metalloproteinase inhibitor PKF226C967. Metalloproteinases are a family of zinc-dependent proteases that include ADAM (a disintegrin and metalloproteinases) and secreted 6-Acetamidohexanoic acid or membrane-type matrix metalloproteinases (MT-MMPs) (11). MT1-MMP (MMP-14), MT2-MMP (MMP-15), MT3-MMP (MMP-16), and MT5-MMP (MMP-24) anchor to the cell surface with a transmembrane domain name, whereas MT4-MMP (MMP-17) and MT6-MMP (MMP-25) attach with a GPI anchor. MMPs have been widely studied for their capacity to degrade the extracellular matrix and to promote invasion and migration of tumor cells; however, it is also known that they can cleave a wide range of ligands and receptors. In fact, p75NTR is processed by the ADAM protein TNF–converting enzyme (TACE) (12). MT1-MMP cleaves the MAI NogoA (13), and chondroitin sulfate proteoglycans are processed by MT1-, 6-Acetamidohexanoic acid MT2-, MT3-, MT5-, and MT6-MMPs (14). Thus, MMPs are interesting candidates to modulate inhibitory activity in the CNS in the context of disease, injury, and plasticity. NgR1 shedding is blocked by endogenous tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3, but not by TIMP-1 (8). The distinct inhibitory profiles of the TIMPs suggest that the metalloproteinase responsible for NgR1 shedding is an MT-MMP or TACE (15). Recombinant TACE has been reported to enhance NgR1 cleavage (16), but the physiological regulator of NgR1 shedding has not been identified. Through a loss-of-function approach, we identified MT3-MMP as a regulator of basal NgR1 cleavage in cortical neurons and demonstrate that metalloproteinase cleavage of NgR1 attenuates growth cone collapse in response to myelin in an NgR1-dependent fashion. Our data demonstrate that specific MT-MMPs regulate NgR1-dependent processing, a finding that is likely to bear on recovery from a spectrum of CNS disorders and injuries. EXPERIMENTAL PROCEDURES Plasmids and Antibodies Constructs for human MT-MMPs were provided by Dr. M. Seiki (University of Tokyo) (17). The human TACE construct was provided by Dr. P. Barker (McGill University). Constructs for soluble human MT1-MMP and MT2-MMP were described previously (18). Recombinant active Rabbit Polyclonal to KLRC1 human TACE was purchased from R&D Systems (Minneapolis, MN). Constructs for the production of soluble recombinant human MT3-MMP and MT5-MMP were generated by deleting the transmembrane domain name (truncated at Asp-533 for MT3-MMP and Asn-570 for MT5-MMP) and subcloning into the psecTAGhygro A vector (Invitrogen). Antibodies were directed against mouse NgR1 (R&D Systems), rat NgR1 (Dr. R. J. Giger, University of Michigan), human MT1-MMP (Abcam, Cambridge, MA), human MT5-MMP (Dr. E. B. Ziff, New York University), TACE and TrkA (Dr. P. Barker), FLAG (Sigma), and the human EGF receptor (Santa Cruz Biotechnology). Anti-N-cadherin antibody was developed by M. Takeichi and H. Matsunami and was obtained from the Developmental Studies.