Research Communication Can proline-rich polypeptide complex mimic the effect of nerve growth factor?

Agnieszka Zabłocka1* Anna Urbaniak1 Marianna Kuropatwa2 Joanna Zyzak2 Joanna Rossowska3 Maria Janusz1

1

Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocł aw, Poland

2

Department of Microbiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocł aw, Poland

3

Laboratory of Glycobiology and Cellular Interactions, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocł aw, Poland

Abstract Naturally occurring compounds that can act as prosurvival factors and neurite formation stimulants in the conditions of reduced neurotrophins production are important both in neuronal protection and therapy of neurodegenerative disorders. Therefore, the role of proline-rich polypeptide complex (PRP) and its nonapeptide fragment (NP) in the promotion of pheochromocytoma cell line (PC12) survival and neurite outgrowth pathway is presented. It was shown that PRP/NP did not affect the neuronal nitric oxide synthase (nNOS) at the transcriptional and protein level. However, the activity of nNOS and intracellular nitric oxide (NO) concentration was markedly increased after treatment of PC12 cells with peptides. This reaction was inhibited by L-NAME—nNOS inhibitor. It was shown that PRP and NP induce the soluble guanylyl cyclase to release higher amount of cyclic GMP (cGMP), and subsequently, the

increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) is observed. This effect was abolished by both U0126 (inhibitor of ERK1/2) and also by L-NAME. Reduction of ERK1/2 activity observed in the presence of nNOS inhibitor suggests that its activation is NO-dependent. The presented results shed some light on the mechanism of action of PRP complex. PRP and NP can activate NO/cGMP/ERK1/2 signaling pathway, similarly to nerve growth factor (NGF). The prosurvival action and short fibers formation suggest the role of PRP and NP in neuroprotection and the initiation of neuritogenesis. They can also participate in the amplification of signals controlling the survival and differentiation of neurons effect C when the deficit of NGF takes place. V 2014 BioFactors, 40(5):501–512, 2014

Keywords: proline-rich polypeptide complex (PRP); nonapeptide (NP); nerve growth factor; nitric oxide; cyclic GMP; ERK1/2 kinases

1. Introduction C 2014 International Union of Biochemistry and Molecular Biology V

Volume 40, Number 5, September/October 2014, Pages 501–512 *Address for correspondence: Agnieszka Zabł ocka, Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocł aw, Poland. Tel.: 148 71 337 11 72; E-mail: [email protected]. Received 23 April 2014; accepted 26 June 2014 DOI 10.1002/biof.1174 Published online 7 July 2014 in Wiley Online Library (wileyonlinelibrary.com)

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Proline-rich proteins and peptides (PRPs, containing >20% proline residues) are widespread in microorganisms, plants, and animals. They play an important role by influencing a wide variety of biological functions, such as the regulation of cell signaling and protein interactions. The most important are the peptides showing antiviral, antibacterial, antitumor, or immunoregulatory activities. Immunologically active PRPs were identified in the hypothalamus and in biological fluids such as colostrum and milk. PRPs produced by bovine hypothalamic magnocellular cells act as neuroprotectors or function as putative neurotransmitters and immunomodulators and were identified in various brain structures of intact and

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trauma-injured rats [1]. Colostrum and milk—the first mammalian nourishment—are the richest reservoir of constituents of great importance in newborn development. They contain protective and supporting factors. One of them are proline-rich polypeptides (PRPs), which were found for the first time in our laboratory during studies on ovine and bovine serum and colostral IgG. It was found that ovine IgG2 is accompanied with a PRP fraction. Subsequently, similar polypeptides were found in human, bovine, and caprine colostra. Proline-rich polypeptide complex (PRP) complex isolated from ovine colostrum is a complex of peptides composed of over 50 peptides of various molecular masses, ranging from 500 to 3,000 Da. It contains a high proportion of hydrophobic amino acids (40%) and proline residues (25%), a low percentage of glycine, and no alanine, arginine, or cystine residues. Most of PRP peptides are derived from casein or a hypothetical b-casein homolog, annexin. Peptides with no significant homology to any specific protein in the current GenBank database have also been identified. PRP possesses immunoregulatory properties, including effects on adaptive (both humoral and cellular) and innate immune responses, and has also shown psychotropic properties. PRP demonstrates widespread efficacy as a cognitive enhancer effective in low doses, nontoxic, and not speciesspecific. Activities similar to whole PRP complex were reflected by a nonapeptide (NP) fragment, VESYVPLFP. Properties of PRP, its role in the development of the immune system, and cognitive function suggested its potential use in the treatment of neurodegenerative disorders. A beneficial effect of PRP complex in the form of sublingual tablets, ColostrininTM, was shown in double-blind placebo-controlled trials, in long-term open-label studies, and in multicenter clinical trials in the case of Alzheimer’s disease (AD). We can assume that these effects involve the modification of cytokine, reactive oxygen and reactive nitrogen species release, activity of superoxide dismutase, inhibition of inducible nitric oxide synthase (iNOS) activity/ expression, and the functional/phenotypic differentiation of cells. Another possibility to explain the therapeutic effect of PRP/Colostrinin is its effect on neurite outgrowth. Also, Colostrinin, as well as its constituent nonapeptide not only inhibits aggregation of amyloid b and formation of fibrillar structures but also can dissolve aggregates already formed, acting as a b-sheet breaker [1,2]. Nerve growth factor (NGF) is a neurotrophin that has been shown to exert a number of different, distinguishable effects on neurons, such as survival, growth of fibers, and differentiation of target neurons [3–6]. NGF plays a critical role in the cortical and hippocampal plasticity, and promotes the survival and functioning of cholinergic neurons. These neurons are important for memory processes, which are affected in AD. Also, NGF is able to increase neurogenesis of hippocampal neurons [7] and promotes survival of a new neurons in the adult brain [3,8,9]. In AD the decreased synthesis of NGF, the decreased level of TrkA receptors on the neuron surface, and decreased neuronal survival are observed [5,6,9–13]. These deficits can be ameliorated by application of neurotrophic fac-

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tors. It is known that NGF is attractive candidate for therapeutic agents in chronic neurodegenerative diseases, but its therapeutic application is considerably restricted by its poor penetration of the blood–brain barrier and its short half-life [12]. Thus, there is a need to find small molecules that are safe, nontoxic, and can mimic the neurotrophic/neuroprotective action. They include antisense nucleotides, regulatory proteins, and peptides. It was shown that major signaling pathway promoting the survival and differentiation of neurons by NGF through TrkA receptor is connected with activation of NO–cGMP–PKG pathway. In the mechanisms underlying the early phases (neurogenesis) and the advanced phases (neuritogenesis) of neuronal differentiation the NO has been implicated [14,15]. It was shown that it plays both proliferative and antiproliferative roles [16]. It was also shown that activation of neuronal NOS (nNOS) and NO release is required for survival and induction of neuronal differentiation in response to NGF in PC12 cells. NO itself can induce neurite outgrowth by ERK activation through the NO–cGMP–PKG [cyclic GMP (cGMP)-dependent protein kinase] pathway in PC12 cells [17,18]. The aim of this study was to determine the possible role of PRP and NP in the promotion of neural cell survival and neurite outgrowth pathway in a model of PC12 (Tet On) cells, in comparison with NGF. The pheochromocytoma cells, PC12 (Tet On), derived from a transplantable chromaffin tumor are commonly used as a model system to study processes of survival and differentiation to a neuronal phenotype and are suitable to use in testing activity of biological materials such as PRP. In this study, it was shown that PRP and NP can activate NO–cGMP–ERK1/2 cellular pathway in PC12 (Tet On) cells, similarly to NGF, and are able to amplify the NGF-dependent signals controlling the survival or differentiation of neuronal cells.

2. Materials and Methods 2.1. Materials NGF was from Promega (Madison, WI). Nitrate reductase, bNADPH, L-glutamine, antibiotics (penicillin/streptomycin mixture), and inhibitor L-NAME were from Sigma-Aldrich (St. Louis, MO). N-(1-naphthyl)-ethylenediamine was from Serva Feinbiochemica (Heidelberg, Germany). Sulfanilamide, sodium nitrite, and orthophosphoric acid were from Polish Chemical Reagents (Gliwice, Poland). Fetal bovine serum (FBS) was from Gibco BRL (San Francisco, CA). Trizol reagent was from Invitrogen (CA). The RevertAidTM First Strand cDNA Synthesis Kit was from Fermentas (Life Sciences). 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and 1-methyl-3-isobutylxanthine (IBMX) were from Cayman Chemical (San Diego, CA). Monoclonal antibody: anti-nNOS, anti-ERK1/2, and anti-phospho-ERK1/ 2, and U0126-selective MEK 1/2 inhibitor were from Cell Signaling (Boston, MA). Subcellular Proteome Extraction Kit was from EMD Biosciences (La Jolla, CA). 5-Bromo-4-chloro-3indolyl phosphate disodium salt (BCIP) and nitroblue

PRP and NP Can Mimic NGF Effects

tetrazolium (NBT) were from Carl Roth (Germany). Phospho Safe Extraction Reagent was from Novagen (San Diego, CA). Molecular weight marker (1–250 kDa) was from Pierce (Rockford, IL). The PRP complex was prepared from ovine colostrum according to the procedure of Janusz et al. [19]. NP fragment of PRP: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro was obtained by chemical synthesis at Wrocław University (Poland).

2.2. Cell Culture PC12 (Tet On) rat pheochromocytoma cells was a gift from Prof. Janusz Matuszyk (Institute of Immunology and Experimental Therapy, PAN, Wrocław). It is a good model to examine the molecular mechanisms of nervous cells survival, proliferation, and differentiation. The cells were maintained under 5% CO2/95% humidified air at 37 C in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% horse serum and 10% FBS, antibiotics (penicillin and streptomycin), and 2 mM L-glutamine. The culture medium was changed every 3 days.

2.3. Reverse Transcription–Polymerase Chain Reaction The mRNA was isolated from peptide-stimulated or nonstimulated PC12 (Tet On) cells after 90 min of incubation using commercially available Trizol reagent following the manufacturer’s protocol. Synthesis of the first-strand cDNA was performed using the Revert AidTM First Strand cDNA Synthesis Kit. The reaction was also carried out in the absence of reverse transcriptase to ensure that the amplified material was derived from RNA. For nNOS two primers were used— (sense) 50 -GGCAC TGGCA TCGCA CCCTT-30 and (antisense) 50 CTTTG GCCGG TCCGG TTCCC-30 —which would be expected to produce a polymerase chain reaction (PCR) product of 213 base pairs (bp). The PCRs were performed in a total volume of 50 lL samples containing 4 lM of the specific oligonucleotide primers and 1.5 U of Taq DNA polymerase. After the initial denaturation at 94 C for 3 min, 35 cycles of amplification (30 s denaturing step at 94 C, 30 s annealing step at 57 C) were performed in a DNA Thermal Cycler (Bio-Rad). The products of amplification were separated on a 1.5% agarose gel in Trisacetic acid–EDTA (TAE) buffer, stained with ethidium bromide (0.5 lg/mL), visualized under ultraviolet light, and photographed.

2.4. Western Blotting Analysis PC12 (Tet On) cells (5 3 105/mL) were seeded onto 60-mm culture dishes and maintained at 37 C with PRP (0.1 lg/mL), NP (0.1 mg/mL), and NGF (0.1 lg/mL) for 6, 24, and 48 h for nNOS stimulation, and 5, 10, 30, and 60 min for ERK1/2. The cells pretreated with the nonselective nNOS inhibitor L-NAME (1 mM, 60 min) and U0126, and selective MEK 1/2 inhibitor (20 lM, 30 min) were used as a negative control. Next, the cells were collected and washed in PBS, and then kept at 4 C in 100 mL of lysis buffer from Proteome Extraction Kit. The protein concentration in lysates was estimated by the bicinchoninic assay. Protein samples were separated on gradient (4– 15%) sodium dodecyl sulfate (SDS)-polyacrylamide gel (MW

marker 10–250 kDa) and next transferred to nitrocellulose membrane. The membrane was blocked (Tris–HCl buffer, pH 7.0, 5% Tween 20, and 5% nonfat dried milk) for 1 h at room temperature and then probed overnight at 4 C with mouse monoclonal anti-nNOS antibody 1:1,000. For ERK1/2 detection, rabbit monoclonal antibody anti-ERK1/2 and rabbit monoclonal antibody anti-phospho-ERK1/2 (1:1,000) were used. After three washes with TBS/Tween 20, the membrane was exposed to alkaline phosphatase conjugated with goat anti-mouse IgG antibody (1:10,000) for 1 h at room temperature. As a reference sample for nNOS, whole-brain lysate was used. Immunocomplexes were visualized using a NBT/BCIP substrate and analyzed in Molecular Imager ChemiDoc MP Imaging System with Image Lab Software (BioRad).

2.5. Nitrite/Nitrate Generation PC12 (Tet On) cells, plated onto a 48-well plate at a density of 1 3 106 cells per well, were cultured in Dulbecco’s modified medium for cell culture. PRP and NP (in the most effective inducible dose 0.1 lg/mL) were added to the cells as potential inducers of NO. NGF (0.1 lg/mL) was used as a positive control, while untreated PC12 cells were used as a negative control. Also, nonselective nNOS inhibitor L-NAME (1 mM) was used. After 24 h of incubation the cells were lysed, and the intracellular level of NO was determined.

2.6. Nitrite/Nitrate Determination Nitrite and nitrate levels were measured after reduction of nitrate to nitrite with NADPH nitrate reductase with some modifications [20]. In brief, 100 lL samples of cell lysates were incubated for 45 min at 37 C with nitrate reductase (25 mU/ sample) in 20 mM Tris buffer, pH 7.6 and b-NADPH (80 lM). The total volume of the reaction mixture was 300 lL. After the enzymatic conversion, nitrite concentration in the sample was measured using Griess reagent [0.1% N-(1-naphthyl)-ethylenediamine and 1% sulfanilamide in 5% phosphoric acid]. After 10-min incubation at room temperature, the absorbance at 550 nm was measured. The concentration of nitrite was calculated from a NaNO2 standard curve.

2.7. Cyclic GMP Determination PC12 cells (1 3 106/mL) were plated onto a 48-well plate in a Dulbecco’s medium without serum. Before the experiment, endogenous phosphatases were inhibited by addition of 10 lM IBMX inhibitor for 15 min. Then cells were treated with PRP or NP (0.1 lg/mL). NGF (0.1 lg/mL) was used as a positive control. As a negative control, ODQ (0.3 lM), the selective inhibitor of soluble guanylyl cyclase (sGC), and L-NAME (1 mM), nonselective nNOS inhibitor, were used. Cells were incubated with inducers for 3 h at 37 C. Intracellularly accumulated cGMP was determined using competitive immunoenzymatic assay with use of polyclonal antibodies highly specific for cGMP. The assay was performed in 96-well microplates (MaxiSorp, Nunc) coated with conjugates of thyroglobulin and cGMP in PBS (0.5 lg/mL) and blocked with 0.2% casein in PBS. Samples were preincubated with antiserum for 2 h at 4 C, then transferred

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into microplate wells, and incubated for 1 h at 4 C. After this the supernatants were removed, the plates were washed, and next incubated for 1 h at room temperature with secondary antibodies, goat anti-rabbit IgG conjugated with horseradish peroxidase. The color reaction was developed using tetramethylbenzidine as a substrate and the absorbance was measured in a Dynatech MR5000 plate reader. The amount of cGMP in the sample was calculated from a calibration curve [21].

2.8. Cell Viability Assay Cell viability was evaluated by MTT assay [22]. PC12 cells were seeded in 96-well plates (1 3 104 cells per well) and incubated for 48 h with inducers: NGF (0.1 lg/mL), PRP/NP (0.1, 1, and 10 lg/mL), and fibrillar amyloid b1–42 (40 lg/mL)). The cell viability was expressed as the percentage of living cells incubated with inducers (NGF, PRP, NP, and amyloid b1–42) versus control.

2.9. Analysis of Neurite Outgrowth PC12 cells (1 3 104 cells per well) were plated onto poly-Llysine-coated chamber slides (Nunc) and cultured in medium for cell culture with 1% horse serum. PRP and NP (at concentration range 0.1–10 lg/mL) were added to the cells as potential inducers of neuritogenesis. NGF (0.1 lg/mL) was used as a positive control, while untreated PC12 (Tet On) cells were used as a negative control. In some experiments the cells were cultured in the presence of U0126 inhibitors. PC12 cells were maintained at 37 C in a humidified atmosphere of 95% air, 5% CO2 for 3–6 days in the presence of all tested substances. Cells were observed by phase-contrast microscopy and the number of neurite-positive cells was counted.

2.10. Statistical Analysis All data were expressed as the mean 6 SD. Statistical differences were determined by an analysis by Student’s t-test (to compare two treated groups).

3. Results 3.1. Effect of PRP/NP on NO Production It was shown in Fig. 1a that NGF-upregulated nNOS activity causes the increased level of intracellular NO (5.9 vs. 4.85 lM in control cells). The presence of PRP or NP, at a dose of 0.1 mg/mL, significantly increased the intracellular level of NO in

FIG 1

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(a) Effect of NGF on NO production. PC12 cells were stimulated with NGF (0.1 lg/mL) without or with LNAME inhibitor (1 mM) applied to the cells 1 h before peptide application. After 24-h incubation, intracellular nitrite level was measured using Griess reagent (for details see Materials and Methods). The data are the mean 6 SD (n 5 3–5). *P  0.05, statistically significant difference between NGF-treated and nontreated control cells; **P  0.05, statistically significant difference in value between NGF 1 L-NAME and NGF (Student’s t-test for dependent samples). (b) Effect of PRP on NO production. PC12 cells were stimulated with PRP (0.1 lg/mL) without or with LNAME inhibitor (1 mM) applied to the cells 1 h before peptide application. After 24-h incubation, intracellular nitrite level was measured using Griess reagent (for details see Materials and Methods). The data are the mean 6 SD (n 5 3–5). *P  0.05, statistically significant difference between PRP-treated and nontreated control cells; **P  0.05, statistically significant difference in value between PRP 1 L-NAME and PRP (Student’s t-test for dependent samples). (c) Effect of NP on NO production. PC12 cells were stimulated with NP (0.1 lg/mL) without or with LNAME inhibitor (1 mM) applied to the cells 1 h before peptide application. After 24-h incubation, intracellular nitrite level was measured using Griess reagent (for details see Materials and Methods). The data are the mean 6 SD (n 5 3–5). *P  0.05, statistically significant difference between NP-treated and nontreated control cells; **P  0.05, statistically significant difference in value between NP 1 L-NAME and NP (Student’s t-test for dependent samples).

PRP and NP Can Mimic NGF Effects

the PC12 cells. After PRP treatment, 7.5 lM of NO is released (Fig. 1b), and NP generates the increase of NO amount to 7.7 lM versus 4.85 lM in control cells (Fig. 1c). The statistically significant induction of nNOS activity in response to NGF, PRP, and NP was abolished in the presence of the nonselective nNOS inhibitor L-NAME (1 mM) (Figs. 1a–1c).

3.2. Effect of PRP/NP on Expression and Protein Level of nNOS To explain the reason of the increased NO level observed in response to PRP/NP, the nNOS was analyzed both at the expression and protein level in the PC12 cells. No changes in the nNOS expression and in the nNOS protein level were observed in PRP- and NP-treated cells (data not shown). However, increased intracellular NO level observed after 24-h PRP/

NP stimulation confirms their ability to enhance the nNOS activity.

3.3. Effect of PRP/NP on sGC Activation and cGMP Release It has been previously reported that NGF-dependent increase of nNOS activity provides an increase of the intracellular NO level. Upon exposure to endogenously produced NO the soluble form of the guanylyl cyclase is activated. In consequence, the second messenger, cGMP, is produced [23–26]. Therefore, the ability of PRP and NP to activate sGC and cGMP release in the PC12 cells was examined. An enhancement of cGMP release, 80% in the presence of NGF (Fig. 2a) and also PRP (Fig. 2b), and 110% in the presence of NP (Fig. 2c), in relation to the control cells was noticed. Furthermore, a decrease of cGMP level after pretreatment of the cells with 10 lM ODQ, a specific

FIG 2

(a) Effect of NGF on level of cGMP released by PC12 cells. PC12 cells were placed in a medium without serum. Before the experiment, endogenous phosphatases were inhibited by addition of IBMX inhibitor (10 lM). Then cells were incubated with NGF (0.1 lg/ mL/106cells) for 3 h at 37 C. ODQ (0.3 lM) was used as selective inhibitor of sGC, and was added to the medium 30 min before NGF as indicated. cGMP level was examined using a competitive cGMP enzyme immunoassay. The data are the mean 6 SD (n 5 4). *P  0.05, statistically significant difference in the value between NGF-treated and nontreated control cells; **P  0.05, statistically significant difference in the value between NGF 1 ODQ/NGF 1 L-NAME and NGF (Student’s t-test for dependent samples). (b) Effect of PRP on level of cGMP released by PC12 cells. PC12 cells were placed in a medium without serum. Before the experiment, endogenous phosphatases were inhibited by addition of IBMX inhibitor (10 lM). Then cells were incubated with PRP (0.1 lg/ mL/106 cells) for 3 h at 37 C. ODQ (0.3 lM) was used as selective inhibitor of sGC, and was added to the medium 30 min before PRP as indicated. cGMP level was examined using a competitive cGMP enzyme immunoassay. The data are the mean 6 SD (n 5 4). *P  0.05, statistically significant difference in the value between PRP-treated and nontreated control cells; **P  0.05, statistically significant difference in the value between PRP 1 ODQ/PRP 1 L-NAME and PRP (Student’s t-test for dependent samples). (c) Effect of NP on level of cGMP released by PC12 cells. PC12 cells were placed in a medium without serum. Before the experiment, endogenous phosphatases were inhibited by addition of IBMX inhibitor (10 lM). Then cells were incubated with NP (0.1 lg/mL/ 106cells) for 3 h at 37 C. ODQ (0.3 lM) was used as selective inhibitor of sGC, and was added to the medium 30 min before NP as indicated. cGMP level was examined using a competitive cGMP enzyme immunoassay. The data are the mean 6 SD (n 5 4). *P  0.05, statistically significant difference in the value between NP-treated and nontreated control cells; **P  0.05, statistically significant difference in the value between NP 1 ODQ/NP 1 L-NAME and NP (Student’s t-test for dependent samples).

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inhibitor of the sGC, and L-NAME, a nonselective nNOS inhibitor, was observed (Figs. 2a–2c). This results suggest that activation of sGC and cGMP release depends on NO release occurred as a result of increased nNOS activation.

3.4. PRP/NP-Induced NO and cGMP Stimulate Phosphorylation and Activation of ERK1/2 Kinases In the activation of MAPK cascade, cGMP-dependent phosphorylation of ERK by MEK 1/2 plays an essential role. Therefore, the effect of PRP and NP on the phosphorylation of ERK1/2 kinases was examined. Induction of ERK phosphorylation in PRP- and NP-treated PC12 cells was noticed as early after 5 min of stimulation (data not shown), but the most effective result was observed after 30-min treatment and the phosphorylation value returned to basal level within 60 min (data not shown). In the presence of NGF increased level of phosphorylated ERK1/2 was also observed. It is known that NOdependent increase of cGMP level induces the activation of MEK1/2, which then phosphorylates ERK1/2 kinases. Therefore, the MEK1,2 inhibitor U0126 and nNOS inhibitor, LNAME, were used to control the specificity of the reaction. Pretreatment of PC12 cells for 30 min with U0126 and L-NAME abolished the PRP/NP-induced phosphorylation of ERK1/2 (Figs. 3a–3c). The significant inhibitory effect of U0126 was also observed for NGF-treated cells. However, L-NAME application did not reduce NGF-dependent ERK1/2 activation.

3.5. Effect of PRP and NP on PC12 Cell Viability The PC12 cell viability was evaluated by MTT assay. It was shown that both PRP and NP at doses 0.1, 1, and 10 lg/mL are not cytotoxic to PC12 cells, similarly to NGF used as a positive control (Figs. 4a and 4b). When aggregated amyloid beta peptide 1–42 (Ab1–42, 40lg/mL) was used, 20% reduction of viability was observed. In the presence of PRP and NP, comparable to NGF, the tendency to improve cell survival was observed (20 and 40%, respectively) (Fig. 4c).

3.6. Effect of PRP and NP on Neurite Extension PC12 cells were incubated on poly-L-lysine-coated chamber slides (Nunc) with various concentrations of PRP or NP (0.1, 1, and 10 mg/mL). NGF (0.1 lg/mL) was used as a positive control. After cultivation (5–7 days) the number of cells with neurites was estimated among 50 cells in a phase-contrast microscope field. As shown in Fig. 5, PC12 control cells were round without neurites. Neurite outgrowth was observed in at least 80% of NGF-treated cells; 20% of cells treated with PRP (0.1 mg/mL, 5–7 days) induced short neurites. No neuritogenic effect was observed after NP application. The neurite creation was abolished in the presence of ERK1/2 inhibitor—U0126.

4. Discussion Neurodegenerative disorders are accompanied by progressive impairment of neuronal cell function [6,9,27]. Neurotrophins are a family of polypeptide growth factors that play an important role in maintaining the balance between cell survival and

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death [4]. Alterations in levels of neurotrophins and their specific receptors have been implicated in the pathogenesis of neurodegenerative diseases including AD [6]. The best characterized neurotrophin is NGF, which promotes the growth and differentiation of neurons during development and supports neuronal survival in the adult brain. NGF plays a major role in signaling pathways that mediate survival and differentiation [5,11,28]. These pathways, initiated through tyrosine kinase receptor (TrkA), activate protein kinases and adaptor proteins in multiple signaling pathways, regulate transcription factors and other cellular proteins, and mediate survival, growth, and phenotypic and morphological differentiation of neuronal cells [7,10,29,30]. The effect of NGF on neuritogenesis promotes synthesis and activation of nNOS. In consequence, an increase of NO release takes place [14]. NO participates in development, learning, and memory processes, and in neurotransmitter release. It can modulate axon outgrowth, neural sympathetic plasticity, and neural precursor proliferation. NO at low physiological concentration can also act as a prosurvival factor in certain neural cells (e.g., hippocampal and sympathetic system cells and model PC12 cells) [29,31]. NO produced by nNOS at low physiological concentration activates sGC to release higher amounts of the intermediate effector—cGMP. cGMP exhibits diverse cellular and physiological processes in the cells, and directly regulates the activities of its downstream effectors such as MAPK kinases. Phosphorylated ERK1/2 kinases participate in at least two cascades: ERK1/2 may translocate into the nucleus, where they phosphorylate the transcription factor Elk1 encoding prosurvival proteins such as Bcl2, or may participate in the phosphorylation of transcription factor CREB regulating, for example, BDNF, initiating the differentiation of neural cells [5,32–34]. During aging a decreased level of released NGF and also reduced expression of TrkA receptor on the cell surface is observed. It leads to a decrease of NGF-induced signals, as a result of which reduced plasticity, memory loss, and cognitive impairment take place [10,11]. Regulation of the NO-cGMPMAPK signaling system activated by NGF appears to be an important tool for the control of regenerative therapies [35,36]. The importance of NGF-dependent signaling in aging processes and in the etiopathogenesis of neurodegenerative diseases prompts the search for new strategies of treatment. Drugs that can cross the blood–brain barrier are one of the greatest, most promising challenges in medicine nowadays. Naturally occurring substances such as proteins and peptides possessing regulatory properties appear to be very promising. One of them is colostrum-derived PRP complex. A PRP complex and its NP fragment possess the ability to modulate the innate and adaptive immune responses, and to modify cytokine production, release of free oxygen species, and functional/phenotypic differentiation of cells. Activity of PRP suggests its therapeutic use in the case of diseases in whose pathogenesis changes in innate immunity play a role. PRP/Colostrinin as well as NP besides its immunoregulatory

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FIG 3

(a) Effect of PRP on ERK1/2 activation. For ERK1/2 activation PC12 cells were treated with PRP (0.1 lg/mL) and NGF (0.1 lg/mL) for 30 min at 37 C. The cells pretreated with the U0126, selective MEK 1/2 inhibitor (20 lM, 30 min), were used as a negative control. Lysates from control and compound-treated cells were separated in SDS–PAGE, and then transferred to nitrocellulose membrane. Phosphorylated and nonphosphorylated forms of ERK1/2 kinase were detected with the use of specific monoclonal antibodies anti-ERK1/2 and anti-phospho-ERK1/2. Immunocomplexes were visualized using a NBT/BCIP substrate and analyzed in Molecular Imager ChemiDoc MP Imaging System with Image Lab Software (BioRad). The representative immunoblot obtained after 30 min of incubation with PRP is presented. *P  0.1 0.05, statistically significant difference in the value between PRP/NGF-treated and nontreated control cells; **P  0.1  0.05, statistically significant difference in the value between NGF 1 U0126 and NGF; ***P  0.1  0.05, statistically significant difference in the value between PRP 1 U0126 and PRP (Student’s t-test for dependent samples). (b) Effect of NP on ERK1/2 activation. For ERK1/2 activation PC12 cells were treated with NP (0.1 mg/mL) and NGF (0.1 mg/mL) for 30 min at 37 C. The cells pretreated with the U0126, selective MEK 1/2 inhibitor (20 lM, 30 min), were used as a negative control. Lysates from control and compound-treated cells were separated in SDS–PAGE, and then transferred to nitrocellulose membrane. Phosphorylated and nonphosphorylated forms of ERK1/2 kinase were detected with the use of specific monoclonal antibodies anti-ERK1/2 and anti-phospho-ERK1/2. Immunocomplexes were visualized using a NBT/BCIP substrate and analyzed in Molecular Imager ChemiDoc MP Imaging System with Image Lab Software (BioRad). The representative immunoblot obtained after 30 min of incubation with NP is presented. *P  0.1  0.05, statistically significant difference in the value between NP/NGF-treated and nontreated control cells; **P  0.1  0.05, statistically significant difference in the value between NGF 1 U0126 and NGF, ***P  0.1  0.05, statistically significant difference in the value between NP 1 U0126 and NP (Student’s t-test for dependent samples). (c) Effect of PRP/NP on ERK1/2 activation. For ERK1/2 activation PC12 cells were treated with PRP (0.1 lg/mL), NP (0.1 mg/mL), and NGF (0.1 mg/mL) for 30 min at 37 C. The cells pretreated with L-NAME, nNOS inhibitor (1 mM, 60 min), were used as a negative control. Lysates from control and compoundtreated cells were separated in SDS–PAGE, and then transferred to nitrocellulose membrane. Phosphorylated and nonphosphorylated forms of ERK1/2 kinase were detected with the use of specific monoclonal antibodies anti-ERK1/2 and anti-phosphoERK1/2. Immunocomplexes were visualized using a NBT/BCIP substrate and analyzed in Molecular Imager ChemiDoc MP Imaging System with Image Lab Software (BioRad). The representative immunoblot obtained after 30 min of incubation with PRP/ NP/NGF is presented. *P  0.1  0.05, statistically significant difference in the value between PRP/NP/NGF-treated and nontreated control cells; **P  0.1  0.05, statistically significant difference in the value between PRP 1 L-NAME and PRP; ***P  0.1 0.05, statistically significant difference in the value between NP 1 L-NAME and NP (Student’s t-test for dependent samples).

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properties affect the lifespan, improve memory, and facilitate learning (see ref. 1). A significant feature of the PRP complex and NP is their ability to inhibit the aggregation of amyloid b peptides and also dissolve aggregates already existing (see ref. R, 1). Hence, PRP in the form of sublingual tablets, ColostrininV was proposed for the treatment of AD. The positive therapeutic effects were shown for the first time in preliminary clinical studies [37] and confirmed in multicenter clinical trials [38]. However, the molecular mechanisms of action of PRP are still under consideration. It was previously reported that a PRP complex can activate neuronal differentiation in a p53/p21wAF-dependent pathway without involving TrkA receptor [39]. There is some evidence indicating that NO-dependent signaling leading to sGC and ERK1/2 activation in PC12 cells is also involved in the control of survival and differentiation processes in neurons [40].

Therefore, we decided to check the ability of PRP and NP to induce intracellular NO synthesis and to activate the NOdependent signaling pathway in the rat pheochromocytoma cell line PC12 Tet On. This line is useful in studies on the role of proteins involved in neuronal differentiation and signal transduction. We observed that nNOS activity was increased by 37 and 58% after PRP and NP treatment, respectively. Although NO production was inhibited by L-NAME (Figs. 1b and 1c), a nonselective inhibitor of nNOS, no statistically significant inhibition by SMIU, a selective iNOS inhibitor, was observed (data not shown). Thus, it can be assumed that in the NO production in PRP/NP-treated PC12 cells the main role is played by the neuronal form of NOS. In RT-PCT and Western blotting experiments, the presence of nNOS in PC12 cells was observed, so we suppose that PRP/NP can regulate NO production via modulation of nNOS activity. No changes at the expression level of nNOS were shown. Also, the level of nNOS protein detected by monoclonal antibody anti-nNOS was not changed after PRP/NP stimulation of PC12 cells. The results indicate that neither PRP nor NP influenced the nNOS expression and the protein level of nNOS. However, increased release of intracellular NO observed after 24 h of PRP/NP stimulation argues in favor of their ability to enhance nNOS activity (Figs. 1b and 1c). The effect of PRP/NP on activation of nNOS and NO release indicates that both peptides are able to modify nNOS function. Similar modifying functions were also observed by Oh et al. [41].

FIG 4

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(a) Effect of PRP on viability of PC12 cells. PC12 cells were exposed to various concentrations of PRP (0.1, 1, and 10 lg/mL) for 48 h at 37 C. NGF (0.1 mg/mL) was used as relative sample. Cell viability was evaluated using MTT assay. The data are means 6 SEM of three independent experiments. *P  0.05, statistically significant differences between compoundtreated and nontreated control cells (Student’s t-test for dependent samples). (b) Effect of NP on viability of PC12 cells. PC12 cells were exposed to various concentrations of NP (0.1, 1, and 10 lg/mL) for 48 h at 37 C. NGF (0.1 mg/mL) was used as relative sample. Cell viability was evaluated using MTT assay. The data are means 6 SEM of three independent experiments. *P  0.05, statistically significant differences between compound-treated and nontreated control cells (Student’s t-test for dependent samples). (c) Effect of NGF, PRP, and NP on viability of PC12 cells treated with amyloid b1–42. PC12 cells were treated with NGF (0.1 lg/mL), PRP (10 lg/mL), and NP (10 lg/mL) applied to the cells simultaneously with aggregated ab1–42 (40 lg/mL). The cells were incubated for 48 h at 37 C. Cell viability was evaluated using MTT assay. The data are means 6 SEM of three independent experiments. *P  0.05, statistically significant differences between ab1–42-treated cells and control cells. **P  0.05, statistically significant differences between ab1–42-treated and NGF/ PRP/NP-treated samples applied simultaneously with ab (Student’s t-test for dependent samples).

PRP and NP Can Mimic NGF Effects

FIG 5

Effect of PRP on neurite extension of PC12 cells. PC12 cells were exposed to PRP (0.1 lg/mL) and NGF (0.1 lg/mL) for 5–7 days. Media, PRP, and NGF were replenished every 48h. After this the number of cells with neurites was counted and phasecontrast photomicrographs with 3400 magnification were taken (Axiovert, Zeiss). Treatment of cells with 0.1 lg/mL dose of PRP induced neurite extension. The experiments were performed in triplicate.

It is well established that intracellular NO can activate sGC to produce cGMP. cGMP plays a role as an important secondary messenger in the brain. cGMP is involved in the regulation of survival and differentiation of neurons, and also participates in the learning and memory processes. A decrease in cGMP level in the brain associated with aging and neurodegeneration has been observed [34,42,43]. Therefore, it was important to investigate the effect of PRP and NP on sGC activity. It was found that PC12 cells treated with PRP and NP synthesize increased levels of intracellular cGMP, comparable to

those produced by cells stimulated with NGF (Figs. 2a–2c) This process was inhibited by ODQ, a specific inhibitor of NOactivated sGC, and also by L-NAME, a nonselective nNOS inhibitor. Taking these results into consideration, it is suggested that PRP and NP, similarly to NGF, increase the cGMP production via activation of nNOS and release of NO. This indicates that both PRP and NP can trigger the NO-cGMP signaling pathway, similarly to other naturally occurring substances, for example, neuropeptide Y [44], genipin [45,46], or curcuminoids [47,48].

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It is well established that cGMP-dependent protein kinases (PKG) mediate phosphorylation of ERK1/2 kinases in neurons. Activation of ERK1/2 kinases is an essential step in the signaling pathway leading to the induction of neurite outgrowth and enhanced survival of neural cells [5,32,34]. It was observed in our preliminary experiments that NGF induced sustained activation of MAPK. In the presence of U0126, a selective inhibitor of MEK, activity of ERK1/2 was reduced by about 80%. Similar effects were observed in the presence of PRP or NP. Thirty minutes after PRP and NP application, phosphorylation of ERK1/2 was significantly increased. This process was inhibited in the presence of U0126 (Figs. 3a and 3b) and L-NAME application (Fig. 3c). Unexpectedly, no inhibitory effect of L-NAME in NGF-treated cells was observed. It is possible that the effect of NGF binding to TrkA receptor initiates at the same time directly ERK activation and nNOS-dependent response. The results obtained prompt us to postulate that nNOS activation and stimulation of the NO-dependent soluble GC by PRP and NP trigger the MAPK signaling cascade. Similar mechanisms of action have been observed in other naturally occurring substances, for example, neuropeptide Y [44], genipin [45,46], artemisinin [49], curcuminoids [47,48], and auraptene [50]. It is known that phosphorylated ERK1/2 kinases participate in at least two cascades: ERK1/2 may be translocated into the nucleus, where they phosphorylate the transcription factor Elk1, and control the expression of prosurvival proteins such as Bcl2. They can also participate in the activation of transcription factor CREB initiating the differentiation of neural cells, or encoding BDNF and its receptor TrkB. It is also possible that the NO-cGMP-ERK cellular signaling pathway activated in the presence of NGF and also PRP and NP leads to different final results, which are connected with activation of transcription factors and gene transcription. We suppose that PRP/NP-initiated activation of the NO/cGMP/ERK1/2 pathway may be connected with the effect of peptides on survival and neuritogenesis of PC12 cells. As shown by MTT assay, in the presence of PRP and NP, survival rate and proliferation are promoted. In PC12 cells treated with aggregates of amyloid beta 1–42 alone, less than 20% of cells survive, whereas simultaneous application of PRP or NP abolishes cell mortality and keeps the neural cells alive at a level comparable to control cells. The same neuroprotective and prosurvival effect was observed when cells were cultured in the presence of NGF. So, it is possible that the NO-cGMP-ERK1/2 pathway initiated by NGF, and also PRP and NP, can lead to activation of transcription factors such as Elk1 or CREB, which are responsible for Bcl2 or BDNF expression, respectively [51–54]. Similarly, norepinephrine-stimulated NO signaling in neural cells provokes cGMP-dependent ERK activation and finally activation of the prosurvival signaling pathway [55]. In PC12 cells treated with NGF, extensive neurite outgrowth was observed. More than 80% of cells develop neurite extensions greater than one cell body length within 5 days of treatment (Fig. 5). This process was inhibited by 30% after LNAME treatment. Reduction of outgrowth by 80% and reduced mortality of cells were observed after 48-h incubation with

510

ERK1/2 inhibitor U0126. This observation indicated that MAPK activity is sufficient for neurotrophin-mediated survival and differentiation of neural cells. We also evaluated the ability of PRP and NP to induce neuritogenesis in PC12 cells. It was observed that PRP, at low doses, plays a role in the initiation of neurite outgrowth in neural cells. We observed formation of short fibers in about 20% of the PRP-treated population. This effect of PRP was inhibited only partially with L-NAME and completely with U0126 inhibitors. No neuritogenic effect was observed when NP was used. It is quite likely that for initiation of neurite creation a component other than NP may be responsible or several PRP constituent components are required. The presented results shed some light on the mechanism of action of PRP complex. PRP/NP can amplify the prosurvival and neuritogenic effect of neurotropic factor NGF without activation of TrkA receptor. We have demonstrated that PRP and NP, similarly to NGF, significantly activate the NO/cGMP/ERK1/ 2 signaling pathway. The short fiber formation observed in the presence of PRP may suggest the role of PRP in the initiation of neuritogenesis and its participation in amplification of the NGF effect when a deficit of NGF and/or TrkA receptor in neurodegenerative disorders takes place. NO produced by PC12 cells in response to PRP/NP also mediates the neuroproliferative effect (Figs. 4a and 4b), similarly to neuropeptide Y [44]. The ability of PRP and its NP fragment to activate nNOS and induce the NO-dependent mechanism in PC12 Tet on cells indicates their bioavailability. However, the mechanism of transport of this peptide complex into cells is unknown. It was found that hydrophobic peptides containing proline residues, as in the case of PRP and NP, can freely penetrate the cell membrane, enter and activate the cells, and affect their function. Peptides can also be delivered into cells by peptidetransporting proteins [56,57]. The possibility of interaction of polypeptide complex PRP with other proteins can be facilitated by the presence of block sequences of proline residues, which may be recognized by proteins containing SH3 domains [58]. Previous results showed that PC12 cells pretreated with antiTrkA antibodies, and next stimulated with PRP, were able to create neurites [39]. Therefore, we can postulate that PRP and NP can stimulate the NO-cGMP-ERK1/2 pathway without activation of TrkA. We also speculate that the effect of PRP and NP on NO production may indicate that peptides interacting with the enzyme can modify its function. Activation of nNOS and release of NO play a crucial role in sustaining neuronal PC12 cell proliferation, survival, and differentiation [14,17,47]. A better understanding of the NGF-dependent signaling in aging processes is very important for planning the therapeutic and/or preventive strategy. Naturally occurring substances such as proteins and peptides that can cross the blood–brain barrier are very promising candidates. One of them is colostrum-derived PRP complex and, also easy to obtain by chemical synthesis, its constituent peptide NP. Comparison of the neurotrophin signaling pathway of NGF with action of PRP complex PRP/NP may help to better understand its function and to discuss the generated diversity of possible mechanisms

PRP and NP Can Mimic NGF Effects

References

SCH 1

PRP/NP mediate survival and differentiation of neural cells through NO/cGMP/ERK1/2-dependent pathway.

of action of a wider variety of growth factors participating in the regulation of cellular survival, morphology, and growth in the central nervous system. The NGF-dependent signaling in aging processes and in the etiopathogenesis of neurodegenerative diseases is very important. Difficulties connected with the use of recombinant NGF in the therapy of neurodegenerative diseases prompted the search for new therapeutic strategies. Drugs that can cross the blood–brain barrier are one of the greatest, most promising challenges in medicine. Naturally occurring substances such as proteins and peptides possessing regulatory properties appear to be very promising. One of them is colostrum-derived PRP complex and alternatively its NP fragment, stable and easy to obtain in chip chemical synthesis. The effects of PRP/NP on the NO-cGMP-ERK1/2 pathway could be one of the aspects of the therapeutic effects of PRP/ Colostrinin in AD.

5. Conclusion PRP and NP can activate NO/cGMP/ERK1/2 signaling pathway, similarly to NGF (Scheme 1). The prosurvival action and short fibers formation suggest the role of PRP in the initiation of neuritogenesis. PRP and NP can also participate in the amplification of signals controlling the survival/differentiation of neurons effect when the deficit of NGF takes place.

6. Acknowledgements This work was supported by the Ministry of Science and Higher Education of Poland (No. N N302 217838).

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PRP and NP Can Mimic NGF Effects

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