MMP-2 and MT1-MMP as axonal outgrowth-promoting molecules in the neuroretina

Abstract

Purpose: Intensive research efforts focus on elucidating mechanisms that can enhance a regenerative capacity in the adult CNS. Over the past years considerable knowledge was obtained from studying optic nerve regeneration in adult animals. As matrix metalloproteinases (MMPs) are upregulated during CNS repair, reduce glial scar formation and potentially promote axonal regrowth, MMPs or their underlying molecules likely form potent regenerative molecules. Here, we investigate the possible role of specific MMPs in axonal outgrowth of injured retinal ganglion cells (RGCs).

Methods: RGC neurite outgrowth was analysed using ex vivo culturing of retinal explants from neonatal mice. Adult zebrafish were subjected to optic nerve crush (ONC) and axonal regeneration was followed using biocytin tracing. Immunohistochemistry (IHC), Western blotting (WB) and gel zymography were used to investigate MMP expression. MMP function was studied by using MMP deficient animals or MMP-inhibiting compounds.

Results: Broad-spectrum MMP inhibition reduces neurite extension of RGCs from retinal explants, implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific inhibitors and MMP deficient mice, disclosed that MMP-2 and MT1-MMP, but not MMP-9, are involved in this process. Furthermore, administration of a novel antibody to MT1-MMP that selectively blocks proMMP-2 activation, revealed a functional co-involvement of these proteinases in determining RGC outgrowth. Subsequent immunostainings showed expression of both MMPs in/on RGC axons and glial cells and gel zymography revealed the presence of active MMP-2 in retinal explants. In the retina of adult zebrafish subjected to ONC, WB and IHC confirmed a restricted time-dependent upregulated MMP-2 and MT1-MMP expression in both Müller glia and RGC axons during axonal regrowth. Furthermore, broad-spectrum MMP inhibition in the retina greatly reduced the regenerative response after ONC. Currently, more specific MMP inhibition/knockdown is being applied in vivo in the zebrafish eye to determine their effects on RGC survival, glial reactivity, axonal regeneration and tectal reinnervation.

Conclusions: Overall, our results suggest that MT1-MMP activates proMMP-2 at the axolemma to exert its axonal outgrowth-promoting function. These observations are currently being validated in a mouse ONC model, characterized by partial regeneration of RGC axons.

Keywords: 531 ganglion cells • 687 regeneration • 699 retinal glia