Complementary research in mammals and fish indicates MMP-2 as a pleiotropic contributor to optic nerve regeneration

Matrix metalloproteinases (MMPs) are members of the metzincin superfamily named after the zinc ion and the conserved methionine residue at the active site. In addition to their role in extracellular matrix (ECM) remodeling, these proteinases (in)activate many signaling molecules such as growth factors, adhesion molecules and cytokines; and even exert some intracellular functions. Hence, they are long-proven regulators of neurogenesis, axonal outgrowth, guidance and myelinogenesis during vertebrate central nervous system (CNS) development and considered crucial for maintaining normal functioning of the adult CNS (Verslegers et al., 2013). However, next to their involvement in a wide range of physiological processes, uncontrolled MMP activities have been associated with the onset of various neurological disorders and CNS injuries. Nonetheless, recent research indicates MMPs as benefactors in the repair and regeneration of the adult mammalian CNS. This paper then also reviews the attributed roles of MMPs, with a focus on MMP-2, in vertebrate axonal outgrowth and mammalian axonal regrowth, and renders in vivo proof for a regulatory function of MMP-2 in zebrafish optic nerve regeneration. Moreover, this paper provides novel evidence that the use of zebrafish (successful) and mouse (unsuccessful) regeneration models can be applied as a two-pronged approach to examine how manipulation of MMPs, or other potential targets, can be used to promote/inhibit axonal regeneration in the injured adult mammalian CNS. During development of retinofugal projections within the visual system – which is a widely used model in axonal outgrowth studies because of its accessibility and well-known morphology – MMPs, and gelatinases (MMP-2 and -9) in particular, have been implicated as promoters of retinal ganglion cell (RGC) axonal outgrowth and modulators of guidance, both in mammals and anamniotes. Indeed, the first in vivo evidence was provided by a study on retinotectal development in Xenopus embryos, which showed that administration of broad-spectrum MMP inhibitors or more specific gelatinase inhibitors to the bathing medium of developing embryos, disrupted axonal guidance cues at low concentrations, whereas higher concentrations reduced axonal outgrowth (Hehr et al., 2005). Due to high sequence conservation among gelatinases, and MMPs in general, the production of inhibitors which act specifically and solely on one MMP remains a challenge. However, over time, inhibitors more specific to MMP-2, that show minimal activity to MMP-9, have been generated. Through use of those more potent MMP-2 inhibitors and by application of genetic loss-of-function techniques, our research group provided primary proof for MMP-2 as a main player in RGC axonal outgrowth in developing vertebrates and for regulatory interactions between MT1-MMP and MMP-2 herein (Janssens et al., 2013; Gaublomme et al., 2014). In zebrafish embryos - a powerful model system to study retinotectal development due to its transparency and conservation of retinal anatomy - single knockdown of Mt1-mmp, a membrane-bound proteinase, significantly decreased the RGC axon innervation area in the optic tectum (OT). Intriguingly, additional Mmp-2 knockdown further reduced OT innervation as compared to single Mt1-mmp knockdown, indicating a potential co-involvement for both proteinases in RGC axonal outgrowth (Janssens et al., 2013). Indeed, mammalian MT1-MMP has been repeatedly reported as an efficient MMP-2-activator (Visse and Nagase, 2003). Likewise, Mt1-mmp knockdown also resulted in reduced Mmp-2 activity levels in zebrafish embryos, suggestive of Mt1-mmp being a major in vivo activator of Mmp-2 in zebrafish retinotectal development (Janssens et al., 2013). A reduced RGC axonal outgrowth was also observed after application of broad-spectrum and more specific MMP-2 inhibitors to ex vivo postnatal mouse retinal explants. Moreover, utilization of an antibody that specifically blocks the MMP-2 activating ability of MT1-MMP reduced axonal outgrowth to the same extent as a general MT1-MMP neutralizing antibody, indicating that MT1-MMP mainly contributes to mouse RGC axonal development through activation of MMP-2, similar as in developing zebrafish. Furthermore, explants of MMP-2 deficient, but not of MMP-9 deficient mice, showed a reduced neurite outgrowth as compared to wild-type explants, thereby confirming a specific role for this gelatinase in RGC axonal growth (Gaublomme et al., 2014). Despite this established role for MMP-2 in axonal outgrowth in the developing CNS, its underlying targets remain largely undefined. In general, MMPs have been implicated in the release of ECM-bound growth factors, like NGF, and modification of adhesion molecules, like NCAM and ICAM5, which are known to stimulate neurite outgrowth (Verslegers et al., 2013). Within the optic system, activated MMP-2 has been suggested to interact with β1-integrin, a transmembrane cell adhesion receptor that affects neurite outgrowth of RGCs (Gaublomme et al., 2014). These data are supported by developmental expression studies, which, besides a macroglial localization, localized MMP-2 in/on outgrowing RGC axons and their growth cones in vertebrates (Figure 1A, left panel) (Janssens et al., 2013; Verslegers et al., 2013; Gaublomme et al., 2014). Altogether, these data ascribe an intrinsic function to MMP-2, serving as an activator of axonal growth stimulating factors, which are in close contact with, or directly located inside or on RGC axons or growth cones (Figure 1A, left panel).