Protection of retinal ganglion cells by caspase substrate-binding peptide IQACRG from N-methyl-D-aspartate receptor-mediated excitotoxicity.
OBJECTIVE
This study investigated whether the enzymatically inactive caspase mimetic IQACRG protects rat retinal ganglion cells (RGCs) from excitotoxic insults. Minimally invasive delivery of the peptide to the retina was explored, and the mechanisms of neuroprotection were elucidated.


METHODS
IQACRG was linked to penetratin (P-IQACRG) to facilitate cellular uptake. RGC labeling by biotinylated-P-IQACRG delivered via intravitreal or subconjunctival injection was demonstrated by avidin-biotin chemistry. The authors used histologic and electrophysiological measures to evaluate the neuroprotective potential of P-IQACRG against RGC death induced by N-methyl-D-aspartate (NMDA) in vitro and in vivo. In addition, they monitored activity of an enzyme that is downstream of caspase-1, matrix metalloproteinase-9 (MMP-9), and protein levels of the caspase-3/7 substrate, myocyte enhancer factor 2C (MEF2C), to determine the effectiveness of IQACRG in blocking excessive caspase activity.


RESULTS
IQACRG significantly reduced NMDA-induced RGC death in culture and in vivo. Ex vivo electrophysiological recording of the retina on multielectrode arrays demonstrated functional rescue of RGCs by IQACRG. The authors also found that delivery of IQACRG to the retina inhibited NMDA-triggered MMP-9 activity and prevented cleavage of MEF2C protein that would otherwise have been engendered by caspase activation preceding RGC death. Strikingly, subconjunctival injection of P-IQACRG was very effective in preventing NMDA-induced RGC death in vivo.


CONCLUSIONS
These data demonstrate that IQACRG protects RGCs from excitotoxicity in vitro and in vivo. The positive results with subconjunctival administration of P-IQACRG suggest that in the future this treatment may be useful clinically in diseases such as glaucoma and retinal ischemia.
Seki
Masaaki
M
Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Burnham Institute for Medical Research, La Jolla, California 92037, USA.
Soussou
Walid
W
Manabe
Shin-ichi
S
Lipton
Stuart A
SA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D061945:Caspase Inhibitors; D002470:Cell Survival; D002478:Cells, Cultured; D004594:Electrophysiology; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D019084:Fluorescent Antibody Technique, Indirect; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D007267:Injections; D064326:MEF2 Transcription Factors; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000378:metabolism; D016202:N-Methylaspartate / Q000633:toxicity*; D018696:Neuroprotective Agents / Q000008:administration & dosage / Q000494:pharmacology*; D009842:Oligopeptides / Q000008:administration & dosage / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D012164:Retinal Diseases / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D014822:Vitreous Body
D061945:Caspase Inhibitors; D018690:Excitatory Amino Acid Agonists; C547097:IQACRG peptide; D064326:MEF2 Transcription Factors; C578042:MEF2C protein, rat; D018006:Myogenic Regulatory Factors; D018696:Neuroprotective Agents; D009842:Oligopeptides; D016194:Receptors, N-Methyl-D-Aspartate; D016202:N-Methylaspartate; C505410:Casp3 protein, rat; D053148:Caspase 3; D053179:Caspase 7; D020780:Matrix Metalloproteinase 9