The Peptides ADNF-9 and NAP Increase Survival and Neurite Outgrowth of Rat Retinal Ganglion Cells In Vitro

Abstract

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.

Differentiation and survival of neurons depend on neurotrophic polypeptides and proteins. These are endogenous substances, such as nerve growth factor, ¹ ciliary neurotrophic factor (CNTF), ² brain-derived neurotrophic factor (BDNF), ³ neurotrophin-3, ⁴ neurotrophin-4/5, ⁵ glial-derived neurotrophic factor, ⁶ pigment-epithelium derived factor, ⁷ and lens epithelium–derived growth factor. ⁸ Another endogenous substance with neurotrophic properties is vasoactive intestinal peptide (VIP), which is widely distributed in the nervous system ⁹ and retina. ¹⁰ Its neuroprotective properties were first described in spinal cord cell cultures with a maximum effect at concentrations as low as 0.1 nM. ¹¹ It has been shown that some of the neuroprotective action of VIP is mediated by two different glial-derived proteins: activity-dependent neurotrophic factor (ADNF) ¹² and activity-dependent neuroprotective protein (ADNP). ¹³ Their active sites have been identified and synthesized as the short peptides ADNF-9 (single letter code: SALLRISPA), ¹⁴ a 9-amino acid peptide, and NAP, an 8-amino acid peptide (single letter code: NAPVSIPQ), ¹³ Remarkably, both peptides act at concentrations in the femtomolar range. ^{15 16}  

The neuroprotective mechanisms of both peptides are not completely understood; however, several interactions with antiapoptotic pathways have been described. ADNF-9 increases the levels of heat shock protein 60 in rat cortical neurons. ¹⁷ It enhances nuclear factor κB-DNA binding ¹⁸ ; and ADNF-14, a larger variant of ADNF-9; activated protein kinase C; and mitogen-activated protein kinase. ¹⁹ After inhibiting protein kinase A or blocking the transcription factor cAMP response element binding protein (CREB) with antisense oligonucleotides, ADNF-14 no longer enhanced neurite outgrowth of rat dorsal root ganglion cells. ²⁰ NAP protects against apoptosis in vivo in a rat model of stroke as measured by the number of caspase 3–positive cells and fragmented DNA staining. ²¹ NAP has been further shown to act as a peptide chaperone protecting against toxic protein aggregation. ²² Other experiments have demonstrated that NAP crosses the plasma membrane and interacts directly with tubulin, the microtubule subunit, to induce microtubule reorganization and improve survival. ²³ NAP furthermore reduces the level of p53, a key regulator of cellular apoptosis. ²⁴ Chicken VIP stimulates cAMP production in chicken cortical slices ²⁵ and mammalian VIP and NAP stimulates cGMP production in rat cerebral cortical cultures at doses corresponding to the neuroprotective doses. ²⁶ ADNP has been found to be essential for brain formation during embryogenesis, as ADNP-knockout mice exhibited defective neuronal tube closure and a lack of PAX6-gene expression. ²⁷  

The apoptotic loss of retinal ganglion cells (RGCs) is a key feature of various optic nerve diseases. The most common one is glaucomatous optic neuropathy, defined by characteristic visual field defects and structural changes in the optic nerve head. In the United States, it is the second most common ²⁸ and in Europe the third most common cause of blindness. ²⁹ So far, glaucoma therapy is solely based on reduction of intraocular pressure, which is the most important risk factor, but other factors, which are not completely known yet, contribute to RGC death as well. ³⁰ Therefore, numerous in vitro and in vivo investigations attempting to prevent apoptosis of RGCs have been performed. The spectrum of tested substances comprises antagonists of excitatory amino acids, ^{31 32} openers of K_ATP-sensitive potassium channels, ³³ α₂-adrenoceptor agonists, ³⁴ caspase inhibitors, ¹³ free radical scavengers, ³⁵ and neurotrophic factors, such as BDNF. ³⁶  

The neuroprotective potential of neuropeptides has not been extensively evaluated in cultures of RGCs or animal models of optic nerve disease. In this study, we tested the effect of both peptides ADNF-9 and NAP on survival of isolated RGCs in vitro and neurite outgrowth in retinal explants. The cell death model used in this experiment is based on apoptosis induced by neurotrophic deprivation. It results from axotomy before RGC harvesting for cell cultures and by axon transsection during preparation of retinal explants. 

Methods

Cell Culture

All experiments were performed in accordance with the Principles of Laboratory Animal Care (NIH publication No. 85-23, rev. 1985), the OPRR Public Health Service Policy on the Human Care and Use of Laboratory Animals (revised 1986), the U.S. Animal Welfare Act as amended, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and our national and institutional guidelines for the care and use of animals in research. ³⁷ RGCs were purified by immunopanning with antibodies against Thy1.1 and cultured in serum-free N2 medium (Invitrogen, Karlsruhe, Germany). ³⁸ In brief, retinas were dissected from 0- to 2-day-old Sprague-Dawley rats and incubated at 37°C for 20 minutes in 0.125% trypsin in Ca²⁺/Mg²⁺-free Hanks’ balanced salt solution. Enzyme treatment was stopped by washing the tissue twice with Dulbecco’s minimum essential medium (DMEM) containing 10% horse serum, 2 mM l-alanyl-l-glutamine, 10 mM HEPES, 100 U/mL penicillin G (sodium salt), and 100 μg/mL streptomycin sulfate, followed by centrifugation at 140g for 2 minutes. To yield a suspension of single cells, the retinal tissue was triturated with a flame-narrowed glass pipette in DMEM containing 10% horse serum. 

Panning dishes (Falcon; BD Biosciences, San Diego, CA) were incubated with goat anti-mouse IgG antibodies (2 μg/mL; Sigma-Aldrich, Munich, Germany) in Tris-HCl buffer (pH 9.5) for 12 hours at 4°C. Dishes were then washed three times with phosphate-buffered saline (PBS) before each of the subsequent steps. Incubation with antibodies against Thy1.1 (1.25–2.5 μg/mL, mouse anti-rat CD90; Serotec, Düsseldorf, Germany) was performed for at least 2 hours at 4°C in PBS. To prevent nonspecific binding of cells, dishes were then incubated with 2 mg/mL bovine serum albumin in PBS for 20 minutes at room temperature. Approximately 35 × 10⁶ cells in 5 mL medium were added per dish and incubated for 20 minutes at 37°C. Dishes were gently swirled every 5 minutes to ensure access of all RGCs to the surface of the plate. To remove nonadherent cells, dishes were washed repeatedly with PBS and swirled moderately until only adherent cells remained. Washing was monitored under a microscope. 

RGCs were mechanically removed from the panning dish in DMEM containing 10% HS by using a cell scraper. After centrifugation at 140g for 5 minutes, purified RGCs were suspended in culture medium, and their density was determined by counting an aliquot in a hemocytometer. The cells were seeded in 96-well plates (Falcon; BD Biosciences) at a density of approximately 3000 cells per well and incubated for 48 hours at 37°C in a humidified atmosphere containing 5% CO₂. Plates had been previously coated with poly-l-lysine (0.1 mg/mL) followed by laminin (0.94 μg/cm²) in DMEM. Culture medium consisted of DMEM supplemented with N2 (500 μg/mL insulin, 10 mg/mL human transferrin, 0.63 μg/mL progesterone, 1611 μg/mL putrescine, and 0.52 μg/mL selenite), and 5 μM forskolin (Tocris Cookson Ltd., Avonmouth, UK). ADNF-9 or NAP were added to culture medium in various concentrations ranging between 1.6 aM and 0.1 nM. For positive controls some cultures were treated with 50 ng/mL each of BDNF and CNTF. ³⁸ After a culture period of 2 days, RGC cultures were fixed in 1% glutaraldehyde, rinsed with water and examined by phase-contrast microscopy at 400× magnification. The number of surviving RGCs was assessed by counting vital cells over the vertical and horizontal diameter of each well. Viable ganglion cells were morphologically identified by their phase bright appearance, intact cell bodies with smooth membranes, and neuritic processes. 

To confirm the neuronal identity of the cultured cells as being RGCs, cultures were immunofluorescence labeled with a rabbit pan-neurofilament antibody cocktail (Biomol, Hamburg, Germany) using a standard immunolabeling protocol with Cy3-conjugated secondary antibodies. It has been shown that this procedure labels only neurons of the RGC layer and horizontal cells. ³⁹  

Retinal Explants

To assess whether both peptides not only support RGC survival but also enhance outgrowth of dissected axons, we performed the following set of experiments. Explants were obtained and evaluated as published. ⁴⁰ In brief, Sprague-Dawley rats were decapitated between postnatal day (P)9 and P11 and eyes were enucleated under semisterile conditions. Eyes were washed in 70% ethanol followed by serum-free Leibowitz L-15 medium (Sigma-Aldrich), containing 20 mM HEPES, 2 mM l-alanyl-l-glutamine, 100 U/mL penicillin G (sodium salt), and 100 μg/mL streptomycin sulfate (Invitrogen-Gibco, GmbH, Karlsruhe, Germany). Eyes were dissected and retinas prepared and collected in L-15 medium. Retinal explants were punched out with a sharpened syringe needle (diameter 400 μm) and collected before plating in L-15 medium. 

The explants were cultured in a fibrin gel (fibrinogen concentration of approximately 3 mg/mL) in DMEM, supplemented with 0.5 mg/mL ε -amino-n-caproic acid (Sigma-Aldrich) as a plasmin inhibitor to prevent destruction of the fibrin gel, 100 U/mL penicillin G, and 100 μg/mL streptomycin sulfate. A 20-μL drop of fibrinogen was placed on a coverslip (10-mm diameter), which had been precoated with 15 μL (3 NIH-units) thrombin (Topostasin; Hoffmann La Roche, Basel, Switzerland). The explants were placed in gel just before coagulation. The peptides ADNF-9 (n = 23) or NAP (n = 8) were diluted in culture medium to a resultant concentration of 1 μM and added to the culture. Coverslips were placed in 24-well culture plates (Falcon; BD Biosciences) filled with 1 mL serum-free culture medium and cultured at 37°C in humidified atmosphere containing 5% CO₂ for 72 hours. Again, as a positive control, some cultures (n = 6) received 50 ng/mL BDNF plus 50 ng/mL CNTF. 

After 3 days, the explant cultures were fixed in 2% glutaraldehyde by microwave-irradiation (2 minutes, 175 W), dehydrated in ethanol, stained with Sudan black (2 minutes, microwave irradiation), and embedded in Kaiser’s gelatin (see Fig. 2 ). Neurite outgrowth from the RGCs was assessed with a camera-lucida projection onto concentric circles (radii corresponding to multiples of 50 μm) centered on the middle of the explants. Outgrowth was quantified by counting the number of neurites intersecting the farthest concentric circle. Because the individual RGC bodies could not be visualized in the explants, we could not identify the origin of each neurite. 

Statistics

Results are given as a percentage of the control cultures (i.e., all values were normalized to the control, which received no drug treatment). Results are given as means with their 95% confidence intervals (CI₉₅). ^{41 42} The dose–responses were analyzed by nonlinear regression analysis, using the following transformation: normalized cell count = 1 + E _max · 10^lg[peptide]/(10^−pEC50 + 10^lg[peptide]) with normalized cell count as the dependent and peptide as the independent variable, respectively. Parameters to be estimated were E _max, representing the maximum effect of the peptides, and pEC₅₀, the negative logarithm of the peptide concentration that yields the half-maximum effect (i.e., the inflection point of a semilogarithmic concentration-response curve). Statistical differences in the explants were evaluated with an unpaired t-test. 

Results

Cell Culture

Phase contrast photographs from two wells containing RGCs are shown in Figure 1 : one under control conditions, and one with additional BDNF and CNTF. Some of the cells formed fine neurites with branching growth cones, thus fulfilling the criteria for being counted as viable RGCs. According to the literature, ³⁸ the combination of BDNF and CNTF has the strongest effect on RGC survival published so far and has therefore been used to assess and compare the relative potency of ADNF-9 and NAP as shown in Figures 2 and 3 . 

Figure 4shows cells being immunofluorescently labeled with antibodies against neurofilament proteins, known to label only neurons from the RGC layer and horizontal cells. ³⁹ As the latter do not express Thy1, they are not selected by the panning procedure. This stain has been performed to further confirm the nature of cultured cell as being RGCs. 

Figure 2shows the dose-dependent effect of ADNF-9 on RGC survival as estimated from seven concentration steps ranging from 1.6 aM to 0.1 nM (n = 12, number of wells per concentration). The EC₅₀ was 10.9 fM. Saturation was reached at 5 pM with a cell survival rate of 146% (CI₉₅ 123–169) compared with the control. According to the counting strategy, control wells yielded a mean of 23.9 cells with intact neurites (CI₉₅ 21.1–26.7). If the CI₉₅ of two groups does not overlap, the difference between the means can be assumed to be statistically significant. Hence, significant neuroprotection occurred at a concentration of 0.25 pM compared with the control. The combination of 50 ng/mL BDNF and CNTF lead to survival rates of 170% (CI₉₅ 131–209). To verify these findings, a second set of experiments (n = 12) with a narrowed concentration range from 24 aM to 0.1 pM was performed. They yielded a survival increase of up to 177% (CI₉₅ 149 - 204) at an ADNF-9 concentration of 0.1 pM. 

The respective data for NAP are shown in Figure 3(n = 17, number of wells per concentration). The EC₅₀ was 6.1 fM with an E _max of 167% (CI₉₅ 125 - 170) at 5 pM compared with the control, which contained a mean of 14.1 viable cells (CI₉₅ 10.8–17.4). A significant difference from the control was reached at a NAP concentration of 12.5 fM. In these experiments, the combination of 50 ng/mL BDNF and CNTF led to a significantly higher survival rate than NAP. Here too, a second set of experiments with a narrowed NAP concentration range yielded an E _max of 147% (CI₉₅ 135–160) at 0.1 pM. 

When tested together with 50 ng/mL BDNF plus CNTF, neither 10 pM ADNF-9 nor 10 pM NAP led to a statistically significant increase in cell survival compared with BDNF plus CNTF alone. 

Retinal Explants

Compared with the control, ADNF-9, NAP, and BDNF+CNTF stimulated neurite outgrowth in retinal explants obtained from postnatal rats. Figure 5shows examples of these four conditions. The explants themselves appear in the panels as black objects. Centrifugally growing neurites are visible as fine dark lines. The longest neurites were found with ADNF-9 or NAP added to the medium. 

The analysis of all explants showed that the combination of BDNF and CNTF, both at a concentration of 50 ng/mL, had a significantly stronger effect on outgrowth than ADNF-9 or NAP (Fig. 6) . ADNF-9 at 1 μM enhanced outgrowth to 126% (CI₉₅ 118–133, n = 23) and 1 μM NAP to 117% (CI₉₅ 98–137, n = 8). BDNF plus CNTF stimulated outgrowth to 201% (CI₉₅ 191–213, n = 6). 

Discussion

This article describes the neuroprotective effect of the ADNF- and ADNP-derived short peptides, ADNF-9 and NAP, on the survival and neurite formation of RGCs. In a first set of experiments, we used a culture system in which cell death results from neurotrophic deprivation due to previous transsection of RGC axons. The outstanding feature of those peptides is that they acted at very low concentrations—namely, in the femtomolar range. Both, ADNF-9 and NAP reduced RGC death in a dose-dependent manner. The maximum increase in cell survival was 177% compared with the control. Other neurotrophic factors, such as the combination of BDNF and CNTF, showed efficacy similar to that of the peptides; however, the potency of BDNF and CNTF was lower than that of the peptides. In second set of experiments using retinal explants both peptides also enhanced neurite outgrowth. Because a single peptide concentration was tested in this assay, the relative potency of the peptides in outgrowth stimulation cannot be estimated. The assay is hampered by the limited predictability of local peptide concentrations, because the fibrin clot poses an ill-defined diffusion barrier. 

Our findings are in line with those in other in vitro studies on the neurotrophic effects of ADNF and ADNP as well as their shorter fragments. ⁴³ ADNF-9 enhanced survival of embryonic hippocampal neurons against cell death induced by FeSO₄ with an EC₅₀ of approximately 0.1 fM. ¹⁵ ADNF prevented neuronal death associated with antiserum to ADNF in rat cerebral cortical cultures with an EC₅₀ of 10 fM. ⁴⁴ In cultures of dorsal root ganglion cells, ADNF-14 doubled the number of cells with neurites at doses between 1 and 100 aM. ²⁰ NAP protected against NMDA-induced excitotoxicity at a concentration as small as 0.1 fM. ¹³ Furthermore, it protected cerebral cortical neurons from transient glucose deprivation ⁴⁵ and cerebral cortical cells against the toxic envelope protein of HIV and the Alzheimer dementia neurotoxin. ⁴ Previously, both NAP and ADNF-9 have been shown to promote axonal elongation in primary rat hippocampal and cortical cultures ⁴⁶ and ADNF-9 was shown to promote synapse formation. ⁴⁷  

These data encourage further experiments on the survival of RGCs in in vivo models of optic nerve disease. Experiments performed in other regions of the central nervous system yielded promising results. Subcutaneous injection of NAP reduced mortality and improved recovery in a mouse model of closed head trauma. Brain edema was reduced by 70% in NAP treated mice. ⁴⁸ In pregnant mice exposed to alcohol, fetal demise was inhibited by a single injection of NAP. ⁴⁹ A single intravenous injection of NAP reduced infarct size and protected against apoptosis in middle cerebral artery occlusion in rats. ²¹ Intranasal application of NAP improved memory performance in a water-maze task tested in middle-aged rats. ⁵⁰ The intranasal administration of either (stearyl-norleucine ¹⁸ ) VIP or NAP prevented impairments of spatial learning in rats pretreated with a cholinergic blocker simulating Alzheimer’s disease. ⁵¹  

Previous reports have shown that coadministration of ADNF-9 and NAP inhibits growth retardation in a model of fetal alcohol syndrome, suggestive of an additive effect of the two peptides. ⁴⁹ Here, whereas NAP seems to be slightly more potent in neuroprotection, ANDF-9 seems to be slightly more efficacious, suggesting different mechanisms of action. 

As indicated earlier, NAP can be delivered by intranasal application as well as by subcutaneous, intraperitoneal, or intravenous routes of administration. ⁴³ The time course of distribution in the organism has been studied by radioactively labeled NAP. Thirty minutes after intranasal administration it was demonstrated in the liver and intestine. The brain showed one fifth to one tenth of the concentration measured in the intestine, which is still comparable to brain tissue levels found after intraperitoneal injections. ⁴³ The fact that NAP does not induce cell division ⁴³ makes it a particularly interesting candidate for further drug testing, because other neurotrophic factors such as nerve growth factor have mitogenic potential and may induce cancer. ⁵² The use of low-dose ADNP-derived peptides as optic nerve neuroprotectants seems an exciting possibility that warrants further investigation. 

 Submitted for publication June 30, 2004; revised October 19, 2004; accepted November 16, 2004.

 Disclosure: W.A. Lagrèze, None; A. Pielen, None; R. Steingart, None; G. Schlunck, None; H.D. Hofmann, None; I. Gozes, None; M. Kirsch, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

RGCs fixed after 48 hours in culture and photographed under phase-contrast microscopy. (A) One RGC with intact neurites cultured in medium without peptides or other neurotrophic factors. (B) Two viable RGCs cultured in control medium plus 50 ng/mL BDNF and CNTF as a positive control. ( ) RGCs deemed vital.

Figure 2.

 

Effect and dose–response curve of ADNF-9 in comparison to the control and BDNF plus CNTF. The logarithmic concentration of ADNF-9 is shown on the x-axis. The survival rate of RGC is given on the y-axis in percentage of the control that received no ADNF-9. Control data are shown on the left. A nonlinear function is fitted to the mean value of each ADNF-9 concentration (n = 12). For comparison, the single data point on the right reflects the survival rate of RGCs treated with 50 ng/mL BDNF plus 50 ng/mL CNTF. Error bars, CI₉₅.

Figure 3.

 

Effect and dose–response curve of NAP in comparison with the control and BDNF plus CNTF. The logarithmic concentration of NAP is shown on the x-axis. The survival rate of RGCs is given on the y-axis in percentage of the control that received no NAP. Control data are shown on the left. A nonlinear function is fitted to the mean value of each NAP concentration (n = 17). For comparison, the single data point on the right reflects the survival rate of RGCs treated with 50 ng/mL each of BDNF and CNTF. Error bars: CI₉₅.

Figure 4.

 

Neurofilament immunostaining of cultured retinal neurons after 2 days in vitro. The neurofilament-expressing cells exhibit a differentiated ganglion-cell–like morphology with elongated neurites.

Figure 5.

 

Neurites growing from the edges of retinal explants stained with Sudan black after 72 hours of incubation. ( ) longest neurite visible. (A) Control condition; (B) 50 ng/mL each of BNDF and CNTF; (C) 1 μM ADNF-9; (D) 1 μM NAP. The diffuse, dark objects are debris outside the focal plane.

Figure 6.

 

Mean length of neurites growing from retinal explants of postnatal rats under different conditions indicated in the histograms. Error bars, CI₉₅. *Statistically significant difference from the control (P < 0.05).