Abstract
In neuronal/glial cocultures, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) prevented neuronal death induced by gp120, lipopolysaccharide (LPS), or other toxic agents, but the dose response of the neuroprotective effect is bimodal, with a peak at a subpicomolar concentration and another peak at a subnanomolar to nanomolar concentration. Although the signaling cascade involved in neuroprotection by nanomolar concentration of the peptide has been shown to be mediated by activation of cAMP-dependent protein kinase and subsequent activation of mitogen-activated protein kinase (MAPK), the mechanism for neuroprotection by a subpicomolar level of PACAP38 remains elusive. In the present study, the signaling involved in neuroprotection by subpicomolar PACAP38 was studied in rat neuronal/glial cocultures. Addition of PACAP38 stimulated expression and activation of extracellular signal-related kinase-type MAPK with a peak response at 10−13 M; greater concentrations of the peptide induced lesser response. cAMP production also increased at subpicomolar levels of PACAP38, but the level remained unchanged at a level four to five times higher than the base level at concentration below 10−11 M. cAMP then started increasing again dose-dependently in a range >10−11 MPACAP38. Lipopolysaccharide (LPS)-induced neuronal death, indicated by increased release of neuronspecific enolase, was suppressed by PACAP38 in a bimodal fashion. Neuroprotection by 10−12 M PACAP38 was completely abolished by a MAPK kinase-1 inhibitor, PD98059, and also partially suppressed by Rp-cAMP, a cAMP-dependent protein kinase inhibitor. Moreover, neuroprotection by a nanomolar level of PACAP38 was completely suppressed by Rp-cAMP but not affected by PD98059. We conclude that neuroprotection by subpicomolar PACAP38 is mainly mediated by the signaling pathway involving MAPK activation and partially regulated by cAMP-dependent protein kinase activation. Furthermore, PACAP38 stimulated expression of activity-dependent neuroprotective protein (ADNP), with a peak at 10−13 M. Greater doses of the peptide induced lesser response. However, 10−13 M PACAP38-stimulated expression of ADNP was not affected by PD98059. This suggests that neuroprotection by subpicomolar PACA38 might be mediated partially by expression of ADNP, but the major events for neuroprotection by subpicomolar PACAP38 remain to be identified.
Similar content being viewed by others
References
Arimura A., Somogyvari-Vigh A., Weill C., Fiore R. C., Tatsuno I., Bay V., and Brenneman D. E. (1994) PACAP functions as a neurotrophic factor. Ann. N. Y. Acad. Sci. 739, 228–243.
Ashur-Fabian O., Giladi E., Brenneman D. E., and Gozes I. (1997) Identification of VIP/PACAP receptors on rat astrocytes using antisense oligodeoxynucleotides. J. Mol. Neurosci. 9, 211–222.
Barrie A. P., Clohessy A. M., Buensuceso C. S., Rogers M. V., and Allen J. M. (1997) Pituitary adenylyl cyclaseactivating peptide stimulates extracellular signalregulated kinase 1 or 2 (ERK1/2) activity in a Rasindependent, mitogen-activated protein kinase/ERK kinase 1 or 2-dependent manner in PC12 cells. J. Biol. Chem. 272, 19666–19671.
Bassan M., Zamostiano R., Davidson A., Pinhasov A., Giladi E., Perl O., et al. (1999) Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide. J. Neurochem. 72, 1283–1293.
Bassan M., Zamostiano R., Giladi E., Davidson A., Wollman Y., Pitman J., et al. (1998) The identification of secreted heat shock 60-like protein from rat glial cells and a human neuroblastoma cell line. Neurosci. Lett. 250, 37–40.
Beaudet M. M., Braas K. M., and May V. (1998) Pituitary adenylate cyclase activating polypeptide (PACAP) expression in sympathetic preganglionic projection neurons to the superior cervical ganglion. J. Neurobiol. 36, 325–336.
Beni-Adani L., Gozes I., Cohen Y., Assaf Y., Steingart R. A., Brenneman D. E., et al. (2001) A peptide derived from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice. J. Pharmacol. Exp. Ther. 296, 57–63.
Boeshore K. L., Schreiber R. C., Vaccariello S. A., Sachs H. H., Salazar R., Lee J., et al. (2004) Novel changes in gene expression following axotomy of a sympathetic ganglion: a microarray analysis. J. Neurobiol. 59, 216–235.
Bouschet T., Perez V., Fernandez C., Bockaert J., Eychene A., and Journot L. (2003) Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras, protein kinase C, and protein kinase A in neuronal cells. J. Biol. Chem. 278, 4778–4785.
Brenneman D. E. and Gozes I. (1996) A femtomolar-acting neuroprotective peptide. J. Clin. Invest. 97, 2299–2307.
Brenneman D. E., Hauser J., Neale E., Rubinraut S., Fridkin M., Davidson A., and Gozes I. (1998) Activity-dependent neurotrophic factor: structure-activity relationships of femtomolar-acting peptides. J. Pharmacol. Exp. Ther. 285, 619–627.
Brenneman D. E., Hauser J. M., Spong C., and Phillips T. M. (2002) Chemokine release is associated with the protective action of PACAP-38 against HIV envelope protein neurotoxicity. Neuropeptides 36, 271–280.
Brenneman D. E., Neale E. A., Foster G. A., d’Autremont S. W., and Westbrook G. L. (1987) Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide. J. Cell Biol. 104, 1603–1610.
Brenneman D. E., Spong C. Y., and Gozes I. (2000) Protective peptides derived from novel glial proteins. Biochem. Soc. Trans. 28, 452–455.
Brenneman D. E., Westbrook G. L., Fitzgerald S. P., Ennist D. L., Elkins K. L., Ruff M. R., and Pert C. B. (1988) Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide. Nature 335, 639–642.
Busca R., Abbe P., Mantoux F., Aberdam E., Peyssonnaux C., Eychene A., et al. (2000) Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes. EMBO J. 19, 2900–2910.
Delgado M., Leceta J., and Ganea D. (2003) Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit the production of inflammatory mediators by activated microglia. J. Leukoc. Biol. 73, 155–164.
DiCicco-Bloom E., Deutsch P. J., Maltzman J., Zhang J., Pintar J. E., Zheng J., et al. (2000) Autocrine expression and ontogenetic functions of the PACAP ligand/receptor system during sympathetic development. Dev. Biol. 219, 197–213.
Dugan L. L., Kim J. S., Zhang Y., Bart R. D., Sun Y., Holtzman D. M., and Gutmann D. H. (1999) Differential effects of cAMP in neurons and astrocytes. Role of B-raf. J. Biol. Chem. 274, 25842–25848.
Frechilla D., Garcia-Osta A., Palacios S., Cenarruzabeitia E., and Del Rio J. (2001) BDNF mediates the neuroprotective effect of PACAP-38 on rat cortical neurons. Neuroreport 12, 919–923.
Furman S., Hill J. M., Vulih I., Zaltzman R., Hauser J. M., Brenneman D. E., and Gozes I. (2005) Sexual dimorphism of activity-dependent neuroprotective protein in the mouse arcuate nucleus. Neurosci. Lett. 373, 73–78.
Gonzalez B. J., Basille M., Vaudry D., Fournier A., and Vaudry H. (1997) Pituitary adenylate cyclase-activating polypeptide promotes cell survival and neurite outgrowth in rat cerebellar neuroblasts. Neuroscience 78, 419–430.
Gottschall P. E., Katsuura G., Dahl R. R., Hoffmann S. T., and Arimura A. (1988) Discordance in the effects of interleukin-1 on rat granulosa cell differentiation induced by follicle-stimulating hormone or activators of adenylate cyclase. Biol. Reprod. 39, 1074–1085.
Gottschall P. E., Tatsuno I., and Arimura A. (1994) Regulation of interleukin-6 (IL-6) secretion in primary cultured rat astrocytes: synergism of interleukin-1 (IL-1) and pituitary adenylate cyclase activating polypeptide (PACAP). Brain Res. 637, 197–203.
Gozes I., Bassan M., Zamostiano R., Pinhasov A., Davidson A., Giladi E., et al. (1999) A novel signaling molecule for neuropeptide action: activity-dependent neuroprotective protein. Ann. N. Y. Acad. Sci. 897, 125–135.
Gozes I., Giladi E., Pinhasov A., Bardea A., and Brenneman D. E. (2000) Activity-dependent neurotrophic factor: intranasal administration of femotomolar-acting peptides improve performance in a water maze. J. Pharmacol. Exp. Ther. 293, 1091–1098.
Gozes I., McCune S. K., Jacobson L., Warren D., Moody T. W., Fridkin M., and Brenneman D. E. (1991) An antagonist to vasoactive intestinal peptide affects cellular functions in the central nervous system. J. Pharmacol. Exp. Ther. 257, 959–966.
Grimaldi M. and Cavallaro S. (1999) Functional and molecular diversity of PACAP/VIP receptors in cortical neurons and type I astrocytes. Eur. J. Neurosci. 11, 2767–2772.
Hashimoto H., Kunugi A., Arakawa N., Shintani N., Fujita T., Kasai A., et al. (2003) Possible involvement of a cyclic AMP-dependent mechanism in PACAP-induced proliferation and ERK activation in astrocytes. Biochem. Biophys. Res. Commun. 311, 337–343.
Hernandez A., Kimball B., Romanchuk G., and Mulholland M. W. (1995) Pituitary adenylate cyclase-activating peptide stimulates neurite growth in PC12 cells. Peptides 16, 927–932.
Jamen F., Bouschet T., Laden J. C., Bockaert J., and Brabet P. (2002) Up-regulation of the PACAP type-1 receptor (PAC1) promoter by neurotrophins in rat PC12 cells and mouse cerebellar granule cells via the Ras/mitogen-activated protein kinase cascade. J. Neurochem. 82, 1199–1207.
Journot L., Villalba M., and Bockaert J. (1998) PACAP-38 protects cerebellar granule cells from apoptosis. Ann. N. Y. Acad. Sci. 865, 100–110.
Kao S., Jaiswal R. K., Kolch W., and Landreth G. E. (2001) Identification of the mechanisms regulating the differential activation of the MAPK cascade by epidermal growth factor and nerve growth factor in PC12 cells. J. Biol. Chem. 276, 18169–18177.
Kienlen Campard P., Crochemore C., Rene F., Monnier D., Koch B., and Loeffler J. P. (1997) PACAP type I receptor activation promotes cerebellar neuron survival through the cAMP/PKA signaling pathway. DNA Cell Biol. 16, 323–333.
Kim W. K., Kan Y., Ganea D., Hart R. P., Gozes I., and Jonakait G. M. (2000) Vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide inhibit tumor necrosis factor-alpha production in injured spinal cord and in activated microglia via a cAMP-dependent pathway. J. Neurosci. 20, 3622–3630.
Kong L. Y., Maderdrut J. L., Jeohn G. H., and Hong J. S. (1999) Reduction of lipopolysaccharide-induced neurotoxicity in mixed cortical neuron/glia cultures by femtomolar concentrations of pituitary adenylate cyclase-activating polypeptide. Neuroscience 91, 493–500.
Lazarovici P., Jiang H., and Fink D. Jr. (1998) The 38-aminoacid form of pituitary adenylate cyclase-activating polypeptide induces neurite outgrowth in PC12 cells that is dependent on protein kinase C and extracellular signal-regulated kinase but not on protein kinase A, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase. Mol. Pharmacol. 54, 547–558.
Leker R. R., Teichner A., Grigoriadis N., Ovadia H., Brenneman D. E., Fridkin M., et al. (2002) NAP, a femotomolar-acting peptide, protects the brain against ischemic injury by reducing apoptotic death. Stroke 33, 1085–92.
May V., Beaudet M. M., Parsons R. L., Hardwick J. C., Gauthier E. A., Durda J. P., and Braas K. M. (1998) Mechanisms of pituitary adenylate cyclase activating polypeptide (PACAP)-induced depolarization of sympathetic superior cervical ganglion (SCG) neurons. Ann. N. Y. Acad. Sci. 865, 164–175.
Miyata A., Arimura A., Dahl R. R., Minamino N., Uehara A., Jiang L., et al. (1989) Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem. Biophys. Res. Commun. 164, 567–574.
Miyata A., Jiang L., Dahl R. D., Kitada C., Kubo M., Fujono M., et al. (1990) Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38). Biochem. Biophys. Res. Commun. 170, 643–648.
Morio H., Tatsuno I., Hirai A., Tamura Y., and Saito Y. (1996a) Pituitary adenylate cyclase-activating polypeptide protects rat-cultured cortical neurons from glutamate-induced cytotoxicity. Brain Res. 741, 82–88.
Morio H., Tatsuno I., Tanaka T., Uchida D., Hirai A., Tamura Y., and Saito Y. (1996b) Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic factor for cultured rat cortical neurons. Ann. N. Y. Acad. Sci. 805, 476–481.
Moroo I., Tatsuno I., Uchida D., Tanaka T., Saito J., Saito Y., and Hirai A. (1998) Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates mitogenactivated protein kinase (MAPK) in cultured rat astrocytes. Brain Res. 795, 191–196.
Morozov A., Muzzio I. A., Bourtchouladze R., Van-Strien N., Lapidus K., Yin D., et al. (2003) Rap1 couples cAMP signaling to a distinct pool of p42/44MAPK regulating excitability, synaptic plasticity, learning, and memory. Neuron 39, 309–325.
Offen D., Sherki Y., Melamed E., Fridkin M., Brenneman D. E., and Gozes I. (2000) Vasoactive intestinal peptide (VIP) prevents neurotoxicity in neuronal cultures: relevance to neuroprotection in Parkinson’s disease. Brain Res. 854, 257–262.
Ohtaki H., Dohi K., Yofu S., Nakamachi T., Kudo Y., Endo S., et al. (2004) Effect of pituitary adenylate cyclaseactivating polypeptide 38 (PACAP38) on tissue oxygen content-Treatment in central nervous system of mice. Regul. Pept. 123, 61–67.
Onoue S., Endo K., Ohshima K., Yajima T., and Kashimoto K. (2002) The neuropeptide PACAP attenuates betaamyloid (1–42)-induced toxicity in PC12 cells. Peptides 23, 1471–1478.
Pinhasov A., Mandel S., Torchinsky A., Giladi E., Pittel Z., Goldsweig A.M., et al. (2003) Activity-dependent neuroprotective protein: a novel gene essential for brain formation. Brain Res. Dev. Brain Res. 144, 83–90.
Reglodi D., Fabian Z., Tamas A., Lubics A., Szeberenyi J., Alexy T., et al. (2004) Effects of PACAP on in vitro and in vivo neuronal cell death, platelet aggregation, and production of reactive oxygen radicals. Regul. Pept. 123, 51–59.
Romano D., Magalon K., Ciampini A., Talet C., Enjalbert A., and Gerard C. (2003) Differential involvement of the Ras and Rap1 small GTPases in vasoactive intestinal and pituitary adenylyl cyclase activating polypeptides control of the prolactin gene. J. Biol. Chem. 278, 51386–51394.
Sandgren K., Lin Z., and Ekblad E. (2003) Differential effects of VIP and PACAP on survival of cultured adult rat myenteric neurons. Regul. Pept. 111, 211–217.
Seidel M. G., Klinger M., Freissmuth M., and Holler C. (1999) Activation of mitogen-activated protein kinase by the A(2A)-adenosine receptor via a rap1-dependent and via a p21(ras)-dependent pathway. J. Biol. Chem. 274, 25833–25841.
Shioda S., Ohtaki H., Suzuki R., Nakamachi T., Takenoya F., Dohi K., and Nakajo S. (2004) Prevention of delayed neuronal cell death by PACAP and its molecular mechanism. Nippon Yakurigaku Zasshi 123, 243–252.
Sigalov E., Fridkin M., Brenneman D. E., and Gozes I. (2000) VIP-related protection against iodoacetate toxicity in pheochromocytoma (PC12) cells: a model for ischemic/hypoxic injury. J. Mol. Neurosci. 15, 147–154.
Stork P. J. (2003) Does Rap1 deserve a bad Rap? Trends Biochem. Sci. 28, 267–275.
Suk K., Park J. H., and Lee W. H. (2004) Neuropeptide PACAP inhibits hypoxic activation of brain microglia: a protective mechanism against microglial neurotoxicity in ischemia. Brain Res. 1026, 151–156.
Tatsuno I., Morio H., Tanaka T., Hirai A., Tamura Y., Saito Y., and Arimura A. (1996a) Astrocytes are one of the main target cells for pituitary adenylate cyclase-activating polypeptide in the central nervous system. Astrocytes are very heterogeneous regarding both basal movement of intracellular free calcium ([Ca2+]i) and the [Ca2+]i response to PACAP at a single cell level. Ann. N. Y. Acad. Sci. 805, 613–619.
Tatsuno I., Morio H., Tanaka T., Uchida D., Hirai A., Tamura Y., and Saito Y. (1996b) Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulator of astrocytes: PACAP stimulates proliferation and production of interleukin 6 (IL-6), but not nerve growth factor (NGF), in cultured rat astrocyte. Ann. N. Y. Acad. Sci. 805, 482–488.
Vaudry D., Falluel-Morel A., Basille M., Pamantung T.F., Fontaine M., Fournier A., et al. (2003) Pituitary adenylate cyclase-activating polypeptide prevents C2-ceramide-induced apoptosis of cerebellar granule cells. J. Neurosci. Res. 72, 303–316.
Vaudry D., Gonzalez B. J., Basille M., Pamantung T. F., Fontaine M., Fournier A., and Vaudry H. (2000) The neuroprotective effect of pituitary adenylate cyclaseactivating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32. Proc. Natl. Acad. Sci. U. S. A. 97, 13390–13395.
Vaudry D., Pamantung T. F., Basille M., Rousselle C., Fournier A., Vaudry H., et al. (2002a) PACAP protects cerebellar granule neurons against oxidative stress-induced apoptosis. Eur. J. Neurosci. 15, 1451–1460.
Vaudry D., Stork P. J., Lazarovici P., and Eiden L. E. (2002b) Signaling pathways for PC12 cell differentiation: making the right connections. Science 296, 1648,1649.
Villalba M., Bockaert J., and Journot L. (1997) Pituitary adenylate cyclase-activating polypeptide (PACAP-38) protects cerebellar granule neurons from apoptosis by activating the mitogen-activated protein kinase (MAP kinase) pathway. J. Neurosci. 17, 83–90.
York R. D., Yao H., Dillon T., Ellig C. L., Eckert S. P., McCleskey E. W., and Stork P. J. (1998) Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392, 622–626.
Zaltzman R., Alexandrovich A., Beni S. M., Trembovler V., Shohami E., and Gozes I. (2004) Brain injury-dependent expression of activity-dependent neuroprotective protein. J. Mol. Neurosci. 24, 181–187.
Zamostiano R., Pinhasov A., Gelber E., Steingart R. A., Seroussi E., Giladi E., et al. (2001) Cloning and characterization of the human activity-dependent neuroprotective protein. J. Biol. Chem. 276, 708–714.
Zemlyak I., Furman S., Brenneman D. E. and Gozes I. (2000) A novel peptide prevents death in enriched neuronal cultures. Regul. Pept. 96, 39–43.
Zusev M. and Gozes I. (2004) Differential regulation of activity-dependent neuroprotective protein in rat astrocytes by VIP and PACAP. Regul. Pept. 123, 33–41.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, M., David, C., Kikuta, T. et al. Signaling cascades involved in neuroprotection by subpicomolar pituitary adenylate cyclase-activating polypeptide 38. J Mol Neurosci 27, 91–105 (2005). https://doi.org/10.1385/JMN:27:1:091
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1385/JMN:27:1:091