Activation of matrix metalloproteinase-9 via neuronal nitric oxide synthase contributes to NMDA-induced retinal ganglion cell death.
OBJECTIVE
Understanding the mechanism of neuronal cell death in retinal diseases like glaucoma is important for devising new treatments. One factor involves excitatory amino acid stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors, excessive Ca2+ influx, and formation of nitric oxide (NO) via neuronal NO synthase (nNOS). Another factor is the abnormal activation of matrix metalloproteinases (MMPs), in particular MMP-9, which triggers an extracellular signaling cascade leading to apoptosis. This study was designed to investigate the mechanism of excitotoxic retinal ganglion cell (RGC) death in vivo and its relationship to MMP activation.


METHODS
NMDA and glycine were injected into the vitreous of the eye in rats and in nNOS-deficient mice (nNOS-/-) versus control. Gelatinolytic activity of MMP-9 and MMP-2 by zymography and cellular localization by immunohistochemistry were examined, and the effect of MMP inhibition on NMDA-induced RGC death was tested.


RESULTS
NMDA was found to upregulate the proform of MMP-9 in the retina and to increase MMP-9 gelatinolytic activity. Retrograde labeling with aminostilbamidine to identify RGCs confirmed that MMP activity occurred only in these retinal neurons and not in glial or other retinal cell types after excitotoxic insult. Deconvolution fluorescence microscopy revealed that MMP activity colocalized with immunoreactive S-nitrosylated protein. NMDA-induced MMP activation was diminished in the retina of nNOS-/- mice, implying that S-nitrosylation of MMP had indeed occurred. In addition, the broad-spectrum MMP inhibitor GM6001 protected RGCs after intravitreal NMDA injection.


CONCLUSIONS
These findings suggest that an extracellular proteolytic pathway in the retina contributes to RGC death via NO-activated MMP-9.
Manabe
Shin-Ichi
S
Center for Neuroscience and Aging, Burnham Institute for Medical Research, La Jolla, California 92037, USA.
Gu
Zezong
Z
Lipton
Stuart A
SA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D019084:Fluorescent Antibody Technique, Indirect; D020868:Gene Silencing / Q000502:physiology; D007267:Injections; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D016202:N-Methylaspartate / Q000633:toxicity*; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism*; D015538:Nitrosation; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D020318:Rats, Long-Evans; D012165:Retinal Ganglion Cells / Q000201:enzymology / Q000473:pathology*; D015854:Up-Regulation; D014822:Vitreous Body
D004151:Dipeptides; D018690:Excitatory Amino Acid Agonists; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; D016202:N-Methylaspartate; D052248:Nitric Oxide Synthase Type I; D020780:Matrix Metalloproteinase 9