Abstract
Purpose: :
The adherence of cells to extracellular matrix (ECM) provides important survival signals through the activation of integrin receptors and Focal Adhesion Kinase (FAK) activation. Matrix Metalloproteinases (MMPs) are a group of enzymes that degrade ECM, and induce apoptosis. We examined the role of MMPs in RGC apoptosis after axotomy, and whether MMP inhibition could extend the therapeutic window of neurotrophic factors such as GDNF and Neurturin.
Methods: :
Optic nerve transection was performed in adult rats. Animals received intraocular injections of: MMP-2/9 inhibitor ((2R)-2-[(4-Biphenylylsulfonyl)amino]-3-phenylpropionic acid), MMP-3 inhibitor (N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid), MMP-8 inhibitor ((3R)-(+)-[2-(4-Methoxy-benzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-hydroxamate]), GM6001 (broad spectrum MMP inhibitor), FAK inhibitors (PF-573228, FAK-I-14), GDNF, or Neurturin, at 3 and 10 days postaxotomy. RGC survival was quantified in fixed flat-mounted retinas at 7, 14 or 21 days postaxotomy. Western blots, in situ fluorescent zymography, and immunohistochemistry were used to evaluate MMP activation after axotomy. Constitutively-active or dominant-negative FAK encoding plasmids were injected into the transected optic nerve in order to study the role of FAK in RGC apoptosis.
Results: :
We observed increased levels of MMP-2 and -9, coincident with increased MMP activity in the inner retina after axotomy. MMP-3 was localized to Müller glia, and MMP-9 and -12 were both localized to injured RGCs and cells in the inner nuclear layer. Intraocular delivery of an MMP-2/9 inhibitor, MMP-3 inhibitor, MMP-8 inhibitor, or GM6001 significantly enhanced RGC survival by 2 to 3-fold at 14 days postaxotomy (p<0.001). Transfection of axotomized RGCs with constitutively-active FAK significantly increased RGC survival while dominant-negative FAK decreased viability. FAK inhibitors (PF-573228 or FAK-I-14) reduced RGC survival at 7 days postaxotomy. Dominant-negative FAK abolished RGC rescue after co-delivery with GM6001. GM6001 prolonged the anti-apoptotic effect of GDNF or Neurturin treatment from 14 to 21 days postaxotomy.
Conclusions: :
ECM degradation by MMPs plays an important role in the degeneration of axotomized RGCs. MMP inhibition prolongs the therapeutic window of GDNF and Neurturin suggesting that FAK activity, concurrent with neurotrophic-dependent signals, is required for long-term RGC survival after injury.
Keywords: ganglion cells • apoptosis/cell death • neuroprotection