The peptides ADNF-9 and NAP increase survival and neurite outgrowth of rat retinal ganglion cells in vitro.
OBJECTIVE
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 microM NAP or 1 microM 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 EC(50) was 10.9 fM. NAP showed a maximum effect at 5 pM with an increase in survival to 167% (146-189) and an EC(50) of 6.1 fM. In the explants, 1 microM ADNF-9 enhanced axonal outgrowth to 126% (118-133) and 1 microM 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.
Lagrèze
Wolf A
WA
Eye Hospital, Albert-Ludwig-Universität, Freiburg, Germany. mail@lagreze.de
Pielen
Amelie
A
Steingart
Ruth
R
Schlunck
Günther
G
Hofmann
Hans-Dieter
HD
Gozes
Illana
I
Kirsch
Matthias
M
D000818:Animals; D000831:Animals, Newborn; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000494:pharmacology; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D008858:Microscopy, Phase-Contrast; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D016501:Neurites / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000187:drug effects*
D019208:Brain-Derived Neurotrophic Factor; D020934:Ciliary Neurotrophic Factor; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D009842:Oligopeptides; C425904:davunetide