Pituitary adenylate cyclase-activating peptide induces neurite outgrowth in cultured monkey trigeminal ganglion cells: Involvement of receptor PAC1

Experimental animals

Trigeminal nerves from rhesus monkeys (Macaca mulatta) were obtained at necropsy from the Oregon National Primate Research Center (Beaverton, OR) from experiments unrelated to the present studies. Experimental animals were handled in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and with the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80–23). Trigeminal nerves were obtained from 20 monkeys ranging in age from 0 to 15 years. The average time between death and dissection was less than 1 h.

Monkey primary trigeminal nerve cell culture

The method for cell culture of monkey TgN was modified from Geenen’s method for porcine trigeminal nerves [9]. Fresh trigeminal ganglia from one monkey were dissected and cut into four longitudinal and cross-sectional pieces before enzymatic digestion was started with 0.2% collagenase A (Roche Applied Science, Indianapolis, IN). Every 30 min, dissociated cells were harvested by centrifugation at 200 ×g for 5 min, and the collagenase solution was reused for further digestion of TgN. After the ganglia were fully digested, the collected cell suspension was centrifuged at 200 ×g for 5 min. The pellet was resuspended in culture medium (Neurobasal Medium supplemented with 2% B-27 and 2 mM L-glutamine; Life Technologies, Grand Island, NY), and the cells were plated on polylysine and laminin-coated 24-well tissue culture plates (BD Bioscience, Franklin Lakes, NJ) at 2,500 neuron cells/cm². Two trigeminal nerves from one monkey resulted in about 2.5×10⁵ TgN ganglion cells. One day after seeding, the cells were washed to remove non-adherent cells, and fresh medium was added. PACAP-27 (EMD Chemicals, Gibbstown, NJ), VIP (EMD Chemicals), and nerve growth factor (NGF; Sigma-Aldrich, St. Louis, MO) were used to induce neurite outgrowth. To investigate the mechanism of neurite outgrowth by PACAP-27, cells were treated with inhibitors 1 h before PACAP-27 or VIP was added. The N-terminus sequence of PACAP stimulates PACAP receptors, and the C-terminus sequence is involved in binding PACAP to receptors. Since deleting the N-terminal 5 amino acids inhibits PACAP-27-induced adenylate cyclase activation, PACAP6-27 (AnaSpec, Fremont, CA) was added to block the action of PACAP-27 [10,11]. PLC inhibitor U73122 (Sigma-Aldrich) and AC inhibitor 2’, 3′-dideoxyadenosine (ddA; Sigma-Aldrich) were used to determine downstream signal pathways induced by PACAP-27. Two days after the inducers were added, the cells were fixed for 30 min at room temperature in phosphate-buffered saline (PBS; Life Technologies) containing 10% formalin followed by immunocytochemistry. We did not observe obvious differences between tissues from young and adult monkeys during TgN cell culture or in axonal elongation studies.

Immunohistochemistry of monkey trigeminal nerve sections

Trigeminal nerves were fixed in 10% formaldehyde and embedded in paraffin. Four-micron sections were blocked with 10% fetal bovine serum (FBS) in PBS for 30 min at room temperature. The sections were then incubated 2 h at room temperature with primary antibodies for neurofilament (NF; Sigma-Aldrich), glial fibrillary acidic protein (GFAP; Santa Cruz Biotechnology, Santa Cruz, CA), or PAC1 receptor (Abcam, Cambridge, MA). After the sections were rinsed with PBS, they were incubated with Alexa-Fluor 546-labeled antimouse immunoglobulin (IgG; Life Technologies) for NF, Alexa-Fluor 488- or 546-labeled antirabbit IgG (Life Technologies) for GFAP, Alexa-Fluor 488-labeled antimouse IgG (Life Technologies) for GFAP, and 1 μg 4’,6-Diamidino-2-phenylindole, dihydrochloride (DAPI; Life Technologies)/ml for nuclei staining for 1 h at room temperature. Negative controls were treated in the same manner, but normal rabbit or mouse IgG (Santa Cruz) was used in place of the primary antibody. All of the primary, secondary, and IgG antibodies were used at a dilution of 1:1,000. Immunoblotting on HeLa cell lysates with the antibody for the PAC1 receptor (Abcam, # ab54980) shows, along with the expected binding to the 50 kDa PAC1 receptor, a cross-reaction with an unknown 75 kDa band protein. Our mass spectrographic analysis identified the 75 kDa band as possibly human Lamin-A/C protein (data not shown). Lamin has been used as a nuclear membrane marker because the lamina matrix is located next to the inner nuclear membrane. Thus, DAPI nuclei staining overlapping green staining was interpreted as the location of cross-reacting lamin fibers. The optically sectioned fluorescence images of the stained samples were photographed using an inverted microscope (Axiovert 200), equipped with an AxioCam MRm camera and an ApoTome slider (Carl Zeiss Vision Gmbh, Hallbergmoos, Germany). Images were compiled in ImageJ 1.42 software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD) and Adobe Photoshop (Adobe Systems, San Jose, CA).

Immunocytochemistry of monkey trigeminal nerve cells

Fixed cultured cells were blocked with 10% FBS in PBS for 30 min at room temperature. Fluorescent cell staining was performed as above. The confocal fluorescence images of stained samples were acquired using a Leica TCS SP laser scanning confocal microscope with an inverted Leica IRBE (Leica Microsystems, Heidelberg, Germany) with an HCX PL APO 40.0×1.25 OIL UV objective (NA 1.25 and zoom 2.0); with an HCX PL APO 40.0×1.25 OIL UV objective (NA 1.25 and zoom 1.0); with an HCX PL APO 40.0×1.25 OIL UV objective (NA 1.25 and zoom 2.0); and with a PL APO 100.0×1.40 OIL UV objective (NA 1.4 and zoom 3.0). The 361-nm line of a UV argon laser was used to excite DAPI, the 488-nm line of an argon laser to excite Alexa-Fluor 488, and the 568-nm line of a krypton laser to excite Alexa-546. Images were compiled in ImageJ and Adobe Photoshop.

RNA extraction and reverse transcription

Total RNA from dissociated monkey trigeminal nerve cells, lacrimal acinar cells, and cultured trigeminal cells treated with PACAP was extracted using TRIzol reagent (Life Technologies) following the manufacturer’s instructions. After phase separation, RNA was precipitated with 75% ethyl alcohol, bound to a glass fiber filter, washed, and collected using an RNA extraction kit (RNEasy Plus Universal Mini Kit; Qiagen, Valencia, CA). RNA quality was determined on a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Concentration of total RNA was determined with RiboGreen reagent (Life Technologies). RNA was reverse transcribed at a concentration of 50 ng/μl with 6 U/μl SuperScript II Reverse Transcriptase following the manufacturer’s instructions with 0.25 U SUPERase-In/μl, 12.5 ng random primers/μl, and 0.5 mM deoxynucleotide triphosphates (dNTPs; Life Technologies). No DNase reaction was performed since all probes were designed to cross exon boundaries.

Quantitative real-time polymerase chain reaction

Custom primers and probes for Rhesus macaque PAC1, VPAC1, and VPAC2 were developed by Life Technologies using the Custom TaqMan Gene Expression Assays (Table 1). Assays for follistatin (Rh01121161_m1) were performed with the TaqMan Gene Expression Assay (Life Technologies). The quantitative real-time polymerase chain reaction (qPCR) probe for the PAC1 gene aligns to all four known transcript variants. All assays contained 250 nM probe, 900 nM each primer, 1X PCR Master Mix (Life Technologies), and 50 ng cDNA in 20 µl total volume. Following initial uracil DNA glycosylase (UDG) activation at 50 °C for 2 min and 95 °C for 10 min, PCR reactions were run for 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Fluorescence measurements were taken at each cycle following extension (StepOnePlus, Life Technologies).

Quantification of PACAP receptor gene transcripts in the trigeminal nerve was determined using an absolute method relative to a standard dilution of synthetic oligonucleotides. PCR efficiencies and limit of detection (LoD) for each assay were determined from standard curves. LoDs varied among genes for PAC1, VPAC1, and VPAC2 at 8.81, 232, and 343 copies per 20 μL reaction, respectively, due to variances in qPCR efficiencies and stochastic effects between different primer sets when used for measuring low copy numbers. Normalized expression to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was converted to absolute copy numbers by comparison to a standard curve made from serial dilutions of a quantified (DNAQuant), polyacrylamide gel electrophoresis–purified oligomer. Relative ΔΔG expression analysis was used for follistatin qPCR. Transcript levels were well within the LoD and normalized by the geometric mean for the GAPDH and HPRT1 expression levels. Sequences for the primers and probes for GAPDH and HPRT1 are shown in Table 1.

Cell counting and statistical analysis

For quantitative measurement of neurite outgrowth, the total cell numbers of neurofilament stained cells were counted in each well (10–17 areas, 3.35×2.50 mm) as described previously [4]. Briefly, a cell with a neurite was defined as a cell with a neurofilament-stained cell body and a process extending at least twice the diameter of the cell body. Data are expressed as the percentage of cells with a neurite compared to the total stained cells.

Statistical analyses of the data were performed with Dunnett’s t test (JMP 8.0.1 software; SAS Institute, Cary, NC). p<0.05 was considered statistically significant.

Monkey lacrimal acinar cell culture and measurement of secreted tear proteins

Lacrimal acinar cells were isolated as described previously [12]. Briefly, lacrimal glands isolated from rhesus monkeys were rinsed in DMEM/F12 (Life Technologies) containing 0.1 mg soybean trypsin inhibitor (STI; Sigma-Aldrich) /ml and then minced with scissors. After centrifugation, cells were obtained by dissociation. Hanks balanced salt solution (HBSS; Life Technologies) containing 0.76 mg/ml EDTA was added and incubated for 15 min to activate trypsin. The fragments were collected by centrifugation and then incubated for 15 min in DMEM/F12 containing 200 U/ml collagenase (Roche Applied Science), 698 U/ml hyaluronidase (Worthington Biochemical Corporation, Lakewood, NJ) and 10 U/ml DNase I (Roche Applied Science) (CHD). The dissociation of cells was repeated one more time, and then DMEM/F12 containing 20% FBS was added to stop the enzyme reaction. Dissociated cells were obtained by filtration using 100 μm and 40 μm nylon meshes, and the cells were segregated by centrifugation with a discontinuous 10, 30, and 60% Percoll gradient (Sigma-Aldrich). Acinar cells were collected from the 30-60% interface. The cells were plated at 1×10⁵ cells/cm² on rat collagen I (0.01 mg/cm²; BD Bioscience) coated plates with the culture medium (DMEM/F12; Life Technologies) supplemented with 10 ng dexamethasone (Sigma-Aldrich)/ml, 50 ng epidermal growth factor (Life Technologies)/ml, 10 μg glutathione (Sigma-Aldrich)/ml, 1X ITS (Sigma-Aldrich), 25 μg L-ascorbic acid (Sigma-Aldrich)/ml, 1 mM putrescine (Sigma-Aldrich), and 50 μg gentamicin (Life Technologies)/ml. After overnight culture, the acinar cells were rinsed, pretreated with supplement-free medium for 30 min, and then incubated with PACAP-27 or VIP for 15 min at 37 °C. After 15 min induction, the cultured media were collected, and detached cells were removed with centrifugation. The collected media were concentrated and subjected to immunoblotting.

Immunoblotting for secreted tear proteins from cultured monkey lacrimal acinar cells

The concentrated media were separated by 4%–12% NuPAGE Bis-Tris gels with 2-(N-morpholino)ethanesulfonic acid (MES) buffer (Life Technologies). The gels were then electrotransferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA) for immunoblotting. Membranes were blocked with 0.5% skim milk in Tris-buffered saline with 0.05% Tween-20 (TTBS) for 30 min at room temperature, and incubated with primary antibodies for lactoferrin (1:5,000 dilution, Sigma-Aldrich) in 1% bovine serum albumin in TTBS overnight at 4 °C. Membranes were then rinsed in TTBS and incubated for 1 h at room temperature with horseradish peroxide-conjugated goat antirabbit secondary antibody (1:10,000–5,000, Santa Cruz). Membranes were developed with chemiluminescence (ECL Plus; GE Healthcare BioSciences, Piscataway, NJ), and images were captured with a FluorChem FC2 imager (ProteinSimple, Santa Clara, CA). Band intensities were measured with ImageJ. Statistical analyses of the data were performed as described above.

Monkey trigeminal nerve cell culture

Immunohistochemistry of whole monkey TgN showed ganglion cells (red) surrounded by glial cells (Schwann cells, green; Figure 1A). After dissociation, primary culture of the monkey TgN cells was established and similarly showed ganglion cells (red) and Schwann cells (green; Figure 1B). In these unstimulated cells at 3 days, extended axons from soma were only occasionally observed. This TgN culture also contained unknown cells that were negative to neurofilament, glial-fibrillary acidic protein, and fibroblast staining (data not shown). The mean cell proportions were TgN ganglion cells 3.5%, Schwann cells 76.1%, and unknown cells 20.4%.

Pituitary adenylate cyclase-activating peptide with 27 residues enhanced neurite outgrowth in cultured trigeminal nerve cells

PACAP-27 and positive control NGF enhanced neurite outgrowth in cultured TgN ganglion cells (Figures 2A-D). The effect of PACAP-27 on elongation was quantified and compared to VIP, a well-studied and homologous signal transduction peptide. PACAP-27 and VIP (Figure 2E) increased neurite outgrowth in a dose-dependent manner, but VIP required 100-fold higher concentrations than PACAP-27. Receptor activation by PACAP-27 in rat superior cervical ganglion cells was reported to be between 10⁻⁹ and 10⁻⁸ M [13]. However, our results showed that 10⁻⁷ to 10⁻⁶ M PACAP-27 was required to cause statistically significant neurite outgrowth. PACAP showed a trend toward dose-dependent increase of neurite outgrowth at 10⁻⁹ M to 10⁻⁸ M, but this was not statistically significant. (Our impression was that the total TgN ganglion cell numbers, although not quantified, were not affected by treatment with NGF, PACAP-27, or VIP.)

Enhancement of neurite outgrowth by pituitary adenylate cyclase-activating peptide with 27 residues through the PAC1 receptor

Note that the receptor antagonist PACAP6-27 [10,11] almost completely inhibited PACAP-27-induced neurite outgrowth (Figure 2D). Further, all three PACAP binding receptors, PAC1, VPAC1, and VPAC2, were expressed in TgN cells from monkeys, but at fairly low levels (Figure 3A). For comparison, VPAC1 receptor expression was 20-fold higher in cultured monkey lacrimal gland (LG) acinar cells than in TgN cells, while PAC1 and VPAC2 receptor expression levels were at or below detection limits (Figure 3B).

Immunocytostaining was positive for the PAC1 receptor in TgN ganglion (Figure 3C) and Schwann cells (Figure 3D). Two staining patterns were observed: 1) orange resulting from the green PAC1 receptor staining overlaying the red cell marker protein staining and 2) blue DAPI nuclei staining overlapping green protein staining, interpreted as probable staining for nuclear lamin-A/C proteins (see Materials and Methods).

At higher magnification (Figure 3E), PAC1 receptor-positive staining was observed as green dots on the red TgN neuronal axons. Staining for both bands was blocked when the anti-PACAP receptor antibody was neutralized with a peptide epitope based on the N-terminal 50 amino acids of the PACAP receptor (GenWay Biotech, San Diego, CA; data not shown). When the PAC1 receptor antibody was preabsorbed with the receptor peptide, all staining on the neuronal body, axons, and Schwann cells was eliminated (Figure 3F). Thus, except the nuclei, the green dot staining indicated the presence of the PAC1 receptor on neuronal bodies, axons, and Schwann cells. Positive staining for the NF and the PAC1 receptor was eliminated when the primary antibodies for the NF and the PAC1 receptor were omitted (Figure 3G).

Downstream pathways in pituitary adenylate cyclase-activating peptide–induced neurite outgrowth

The PAC1 receptor activates AC and PLC-dependent signaling cascades via G proteins [7,8]. In our monkey TgN cells, PACAP-induced neurite outgrowth was significantly inhibited by the PLC inhibitor (U73122) and by the AC inhibitor (ddA) in a dose-dependent manner (Figure 4A). Higher concentrations of ddA or U73122 alone completely inhibited neurite outgrowth.

Activation of AC and PLC pathways by PACAP could also upregulate other relevant genes. In our cultured monkey TgN cells, PACAP-27 upregulated expression of mRNA for the neuronal differentiation gene, follistatin, which showed statistically significant increases at 4 and 8 h and then decreased gradually (Figure 4B).

Effect of pituitary adenylate cyclase-activating peptide on tear protein secretion

As described above, neuronal stimulation of the cornea is coupled to lacrimal gland tear flow. We next used lacrimal acinar cells, where the VPAC1 receptor is dominant, to evaluate if there is a difference between PACAP-27 and VIP in the ability to induce tear protein secretion. In LG acinar cells, in contrast to TgN, PACAP-27 and VIP showed similar, dose-dependent stimulation of tear protein secretion (lactoferrin) into the medium (Figure 5).

Characteristics of monkey trigeminal nerve cells in culture

Culture of adult monkey trigeminal ganglion neurons was first reported in 1993 [14], and the present study provided further characterization of the TgN ganglion and Schwann cells in such cultures. Neuronal cells cultured for 3 days possess axons (Figure 1B, red), which innervate the cornea in vivo. PACAP induced monkey trigeminal cells to produce elongated axons (Figure 2). The ganglion cell bodies were 20 to 30 times larger than the Schwann cells that surrounded the ganglion. Schwann cells also formed fiber-like structures (Figure 1B, green) extending to the neuronal cell bodies and axons. This may mimic myelination by Schwann cells in vivo, and emphasizes the relevance of this monkey model for testing possible human medications such as PACAP-27.

Mechanism of pituitary adenylate cyclase-activating peptide–induced neurite outgrowth through the PAC1 receptor

Although PAC1 expression was less than VPAC1, PAC1 is probably most directly responsible for PACAP-induced neurite outgrowth because of the following: 1) Outgrowth was inhibited by the inhibitor of PACAP-27, PACAP6-27 [10,11] (Figure 2D). 2) PAC1 displays two to three orders of magnitude greater affinity for PACAP than for VIP [15]. 3) In our experiments, the bioactivity of PACAP-27 toward neurite outgrowth in TgN cells had a 100-fold stronger effect than VIP. 4) High concentrations of the VPAC2 receptor-specific agonist, Ro 25–1553, caused only minimal neurite outgrowth in human SH-SY5Y neuroblastoma cells after 4 days of culture [16], suggesting that VPAC receptors are not major factors for neurite outgrowth. 5) In contrast to TgN cells, PACAP-27 and VIP stimulated tear lactoferrin secretion at the same rate in LG acinar cells (Figure 5), where VPAC1 receptor expression was dominant. 6) The PAC1 receptor was expressed not only on the cultured TgN cell bodies but also on the neuronal axons themselves (Figure 3). These data suggested PACAP binding to PAC1 may occur directly on peripheral neuronal axons in the cornea to stimulate neurite outgrowth.

Activation of phospholipase C/protein kinase C and adenylate cyclase/protein kinase A pathways by pituitary adenylate cyclase-activating peptide with 27 residues through PAC1 during neurite outgrowth

PAC1 is coupled to AC and PLC, which induce production of cyclic adenosine monophosphate (cAMP) and increase intracellular Ca²⁺ subsequent to activation of protein kinase A (PKA) and protein kinase C (PKC) [7,8]. In our present studies, PLC inhibitor U73122 and AC inhibitor ddA completely inhibited PACAP-induced neurite outgrowth (Figure 4A). This indicates cross talk between the AC/PKA and PLC/PKC pathways during PACAP-induced neurite outgrowth. Note also that with 10⁻⁵ M U73122, neurite outgrowth was inhibited more than in the vehicle group (Figure 4A), suggesting that normal physiologic neurite outgrowth may be regulated via the PLC pathway. These conclusions could be further tested by axonal elongation studies with PKA and mitogen-activated protein kinase inhibitors using the present monkey model.

The AC/PKA pathway might also cause neurite growth by influencing the expression of other genes. We found that the expression level of the neuronal differentiation gene follistatin was upregulated by PACAP-27 throughout the culture period (Figure 4B). Follistatin was also upregulated 3.37 fold in chicken TgN cells cultured with PACAP-27 for 15 min [17]. Follistatin stimulated neurite outgrowth of the neuroblastoma cell line IMR-32 [18]. These data suggested that PACAP-27-induced follistatin expression may be one of the mechanisms stimulating neurite outgrowth in monkey TgN cells due to activation of the AC/PKA and PLC/PKC pathways confirmed by PACAP-induced TgN neurite outgrowth.

Stimulation of tear protein secretion

In addition to neurite outgrowth, PACAP-induced protein secretion from monkey lacrimal acinar cells was observed (Figure 5). PACAP null mice showed dry eye-like symptoms, including tear reduction and corneal keratinization [19]. Topical administration of PACAP stimulated tear secretion via the cAMP/PKA cascade. In lacrimal acinar cells, PACAP also stimulated translocation of the water transporter, aquaporin 5 (AQP5), from the cytosol to the membrane and induced tear secretion [19]. Thus, an additional advantage of PACAP may be its ability to promote tear protein secretion from lacrimal gland cells through the VPAC1 receptor.

Conclusion

These studies in a novel, relevant monkey model suggest that treatment of dry eye with PACAP-27 may be beneficial because of its stimulating effect on TgN neurite outgrowth to the cornea and because PACAP-27 stimulates tear protein secretion from the lacrimal gland.