Synaptic cell adhesion molecule-2 and collapsin response mediator protein-2 are novel members of the matrix metalloproteinase-9 degradome

Abstract

The elucidation of entire sets of protease substrates (" proteodegradomes") is important for understanding proteolytic pathways, their networks, and their role in the regulation of cell function. Matrix metalloproteinase-9 (MMP-9) is an extracellularly operating protease that is expressed and released in the brain in response to enhanced neuronal activity. Under physiological conditions, MMP-9 is involved in neuronal plasticity, including long-term potentiation, learning, and memory. This function may be related to its activity at the synapse. Under pathological conditions (e.g., during excitotoxicity, stroke, and traumatic brain injury), when the concentration of glutamate is persistently increased, MMP-9 is detrimental to brain tissue. To assess the MMP-9 degradome, we used synaptoneurosomal fractions isolated from the hippocampus of wildtype and MMP-9 knockout mice. To induce MMP-9 activity, the synaptoneurosomal fractions were treated with 50 μM glutamate for 30 min at 37°C. To investigate MMP-9 targets, two-dimensional fluorescence difference gel electrophoresis was performed. This approach enabled the accurate analysis of differences in protein abundance between samples. The differential spots that contained potential MMP-9 substrates were excised from the gel, and proteins of interest were identified using mass spectrometry. Two novel MMP-9 targets were identified: synaptic cell adhesion molecule-2 and collapsin response mediator protein-2. The MMP-9-driven processing of the newly identified substrates was confirmed by western blot in primary hippocampal neurons after stimulation with either N-methyl-D-aspartate or glutamate or incubation with recombinant autoactivating MMP-9 and use of a specific inhibitor. Matrix metalloproteinase-9 is an extracellularly operating protease that is expressed and released in the brain in response to enhanced neuronal activity. In this study, two novel targets of MMP-9 degradome were identified: synaptic cell adhesion molecule-2 and collapsin response mediator protein-2, both of which are expressed in the brain and participate in different neuronal activities and signal transduction pathways that may play a role in synaptic plasticity. The search for MMP-9 targets at the synaptic level is needed to understand the functional role of the MMP-9 proteodegradome. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.