davunetide and Retinal-Diseases

NAP (NAPVSIPQ) is a short peptide derived from activity-dependent neuroprotective protein (ADNP) sequence, whose potent and direct neuroprotective capabilities have been widely accepted. However, due to the high risk and inconvenience of intraocular injections, NAP is difficult to be clinically administered as therapeutic agent in treating retinal diseases. Currently, stable transfection of this octapeptide into cells has not been reported, partly because of its small size and lacking of 5' signal sequence. Here, we have developed a novel NT4-NAP fusion gene by attaching the 5' nonfunctional preproregion of neurotrophin 4 (NT4) to NAP cDNA. Recombinant adeno-associated virus was established to introduce NT4-NAP construct into cultured rat retinal Müller cells (RMC), resulting in sustained high level NAP production from stable transfection. Functional analyses of RMC cells transfected with NAP revealed the remarkably reduced cytotoxicity and apoptosis of the cells under hypoxia. Furthermore, coculturing of transfected RMC-NAP cells with primary rat retinal neural cells offer marked protection to the latter against hypoxia induced cellular damages. Together our data indicate that stable transfection of NAP into retinal Müller cells with constant NAP production is possible. NAP produced from cellular transfection maintained its biological neuroprotective activities. This targeted gene expression may provide an effective treatment for retinal diseases in the near future.

Topics: Animals; Cell Hypoxia; Cell Proliferation; Cells, Cultured; Dependovirus; Gene Fusion; Genetic Therapy; Nerve Growth Factors; Neuroprotective Agents; Oligopeptides; Rats; Rats, Sprague-Dawley; Retina; Retinal Diseases; Retinal Neurons; Transfection

NAP, an 8-amino acid peptide (NAPVSIPQ=Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) derived from activity-dependent neuroprotective protein (ADNP), plays an important role in neuronal differentiation and the survival of neurons in different pathological situations. We already discovered that NAP increases the survival of retinal ganglion cells (RGC) in vitro, and supports neurite outgrowth in retinal explants at femtomolar concentrations. The aim of this study was to investigate the effects of NAP on RGC survival after transient retinal ischemia and optic nerve crush.. RGC of male Wistar rats were labelled retrogradely with 6 l FluoroGold injected stereotactically into both superior colliculi. Seven days later, retinal ischemia was induced by elevating the intraocular pressure to 120 mm Hg for 60 minutes or by crushing one optic nerve for 10 s after a partial orbitotomy. NAP was either injected intraperitoneally in the concentration of 100 microg/kg [corrected] 1 day before, directly after, and on the first and the second days after damage, or intravitreally (0.05 or 0.5 microg/eye) [corrected] directly after the optic nerve crush. Controls received the same concentrations of a control peptide. Densities of surviving RGC and activated microglial cells (AMC) were quantified in a masked fashion 10 days after damage by counting FluoroGold-labelled cells.. After retinal ischemia, intraperitoneal injections of NAP increased the number of surviving RGC by 40% (p < 0.005) compared to the control group. After optic nerve crush, NAP raised the number of surviving RGC by 31% (p = 0.07) when injected intraperitoneally and by 54% (p < 0.05) when administered intravitreally.. NAP acts neuroprotectively in vivo after retinal ischemia and optic nerve crush, and may have potential in treating optic nerve diseases.

Topics: Animals; Cell Count; Cell Survival; Disease Models, Animal; Injections, Intraperitoneal; Ischemia; Male; Nerve Crush; Neurites; Neuroprotective Agents; Oligopeptides; Optic Nerve Injuries; Rats; Rats, Wistar; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Retinal Vessels