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(Investigative Ophthalmology and Visual Science. 2005;46:933-938.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0766
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The Peptides ADNF-9 and NAP Increase Survival and Neurite Outgrowth of Rat Retinal Ganglion Cells In Vitro

Wolf A. Lagrèze,1 Amelie Pielen,1 Ruth Steingart,2 Günther Schlunck,3 Hans-Dieter Hofmann,4 Illana Gozes,2 and Matthias Kirsch4

1From the Eye Hospital and the 4Institute of Anatomy I, Albert-Ludwig-Universität, Freiburg, Germany; the 3Eye Hospital of the Julius-Maximilians-Universität Würzburg, Wurzburg, Germany; and the 2Department of Clinical Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

PURPOSE. Recent studies demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. We evaluated these findings in cultures of purified rat retinal ganglion cells (RGCs) using two such peptides: ADNF-9 and NAP. In a second step, the influence of these peptides on neurite outgrowth in retinal explants was investigated.

METHODS. Retinal ganglion cells (RGCs) were purified from newborn (postnatal day [P]0–P2) rat retina by immunopanning with antibodies against Thy1.1 and were cultured in serum-free N2 medium for 2 days. RGCs were treated with ADNF-9 and NAP at concentrations ranging from 10–18 to 10–10 M. Survival was quantified by counting viable cells by phase-contrast microscopy. Retinal explants from postnatal (P9–P11) rats were cultured in three-dimensional fibrin clots in serum-free medium for 3 days. Explants were treated with 1 µM NAP or 1 µM ADNF-9. Neurite outgrowth was visualized by staining with Sudan black and quantified by measuring axonal length.

RESULTS. Both peptides enhanced survival of RGCs in a dose-dependent manner. ADNF-9 showed a maximum effect at 0.1 pM with an increase in survival to 177% (95% confidence interval: 149–204) of the control level. The EC50 was 10.9 fM. NAP showed a maximum effect at 5 pM with an increase in survival to 167% (146–189) and an EC50 of 6.1 fM. In the explants, 1 µM ADNF-9 enhanced axonal outgrowth to 126% (118–133) and 1 µM NAP to 117% (98–137) compared with the control.

CONCLUSIONS. Both peptides, ADNF-9 and NAP, not only increase RGC survival in vitro but also support neurite outgrowth in retinal explants. These peptides deserve further attention as potential neuroprotective compounds in retinal and optic nerve diseases.






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