Bruno J. Gonzalez

PACAP protects cerebellar granule neurons against oxidative stress-induced apoptosis

Oxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role in neuronal cell death associated with neurodegenerative diseases and stroke. In the present study, we have investigated the potential neuroprotective effect of pituitary adenylate cyclase‐activating polypeptide (PACAP) on oxidative stress‐induced apoptosis. Incubation of cerebellar granule cells with PACAP inhibited hydrogen peroxide‐evoked cell death in a concentration‐dependent manner. The effect of PACAP on granule cell survival was not mimicked by vasoactive intestinal polypeptide and was blocked by the antagonist PACAP6‐38. The protective action of PACAP upon hydrogen peroxide‐induced neuronal cell death was abolished by the MAP‐kinase kinase (MEK) inhibitor U0126 and mimicked by the caspase‐3 inhibitor Z‐DEVD‐FMK. PACAP markedly inhibited hydrogen peroxide‐evoked caspase‐3 activation and DNA fragmentation. Taken together, these data indicate that PACAP, acting through PACAP receptor type 1, exerts a potent protective effect against neuronal degeneration induced by hydrogen peroxide. The anti‐apoptotic effect of PACAP is mediated through the MAP‐kinase pathway and can be accounted for by inhibition of caspase‐3 activation resulting from oxidative stress.

Pituitary adenylate cyclase-activating polypeptide promotes cell survival and neurite outgrowth in rat cerebellar neuroblasts.

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors have been detected in the rat cerebellum during ontogenesis. In particular, PACAP receptors are actively expressed in immature granule cells, suggesting that PACAP may act as a neurotrophic factor in the developing rat cerebellum. In the present study, we have investigated the effect of PACAP on cell survival and neurite outgrowth in cultured immature cerebellar granule cells. In control conditions, cultured granule cells undergo programmed cell death. Exposure of cultured cells to PACAP for 24 and 48 h provoked a significant increase in the number of living cells. The effect of PACAP on cell survival was inhibited by the PACAP antagonist PACAP(6-38). Vasoactive intestinal polypeptide was approximately 1000 times less potent than PACAP in promoting cell survival. PACAP also induced a significant increase in the number of processes and in the cumulated length of neurites borne by cultured neuroblasts. The present results demonstrate that PACAP promotes cell survival and neurite outgrowth in cultured immature granule cells. Since PACAP and its receptors are expressed in situ in the rat cerebellar cortex, these data strongly suggest that PACAP plays a physiological role in the survival and differentiation of cerebellar granule cells.

Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death

Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoked apoptotic cell death. Incubation of granule cells with PACAP prevented ethanol-induced apoptosis, and this effect was not mimicked by vasoactive intestinal polypeptide, suggesting that PAC1 receptors are involved in the neurotrophic activity of PACAP. Ethanol exposure induced a strong increase of caspase-2, -3, -6, -8, and -9 activities, DNA fragmentation, and mitochondrial permeability. Cotreatment of granule cells with PACAP provoked a significant inhibition of all of the apoptotic markers investigated although the neurotrophic activity of PACAP could only be ascribed to inhibition of caspase-3 and -6 activities. These data demonstrate that PACAP is a potent protective agent against ethanol-induced neuronal cell death. The fact that PACAP prevented ethanol toxicity even when added 2 h after alcohol exposure, suggests that selective PACAP agonists could have potential therapeutic value for the treatment of fetal alcohol syndrome.

The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are closely related members of the secretin superfamily of neuropeptides expressed in both the brain and peripheral nervous system, and they exhibit neurotrophic and neurodevelopmental effects in vivo. Like the index member of the Trk receptor ligand family, nerve growth factor (NGF), PACAP promotes the differentiation of PC12 cells, a well‐established cell culture model, to investigate neuronal differentiation, survival and function. Stimulation of catecholamine secretion and enhanced neuropeptide biosynthesis are effects exerted by PACAP at the adrenomedullary synapse in vivo and on PC12 cells in vitro through stimulation of the specific PAC1 receptor. Induction of neuritogenesis, growth arrest, and promotion of cell survival are effects of PACAP that occur in developing cerebellar, hippocampal and cortical neurons, as well as in the more tractable PC12 cell model. Study of the mechanisms through which PACAP exerts its various effects on cell growth, morphology, gene expression and survival, i.e. its actions as a neurotrophin, in PC12 cells is the subject of this review. The study of neurotrophic signalling by PACAP in PC12 cells reveals that multiple independent pathways are coordinated in the PACAP response, some activated by classical and some by novel or combinatorial signalling mechanisms.

VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells.

The capacity of embryonic stem cells (ES cells) to differentiate into neuronal cells represents a potential source for neuronal replacement and a model for studying factors controlling early stages of neuronal differentiation. Various molecules have been used to induce such differentiation but so far neuropeptides acting via functional G‐protein‐coupled receptors (GPCRs) have not been investigated. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP) are neuropeptides expressed in early development which affect neuronal precursor proliferation and neuronal differentiation. VIP and PACAP share two common receptors (VPAC1 and VPAC2 receptors) while only PACAP binds with high affinity to PAC1 receptors. The aim of the study was to determine whether VIP and PACAP could produce functional neuronal differentiation of ES cells. Mouse ES cells were allowed to aggregate in embryoid bodies (EBs) in the presence or not of VIP and PACAP for 1 week. VIP and PACAP potently increased the proportion of EB‐derived cells expressing specifically a neuronal phenotype shown by immunocytochemistry and neurite outgrowth without altering glial cell number. Binding and RT‐PCR analyses demonstrated the presence of VPAC2 and PAC1 receptors on ES cells. Accordingly, both peptides increased cyclic AMP and intracellular calcium. In contrast, EB‐derived cells only expressed a functional PAC1 receptor, suggesting a switch in GPCR phenotype during ES cell differentiation. These original data demonstrate that functional GPCRs for VIP and PACAP are present on ES cells and that these neuropeptides may induce their differentiation into a neuronal phenotype. It opens an exciting new field for neuropeptide regulation of tissue ontogenesis.

Pituitary adenylate cyclase-activating polypeptide prevents C2-ceramide-induced apoptosis of cerebellar granule cells

The sphingolipid metabolites, ceramides, are critical mediators of the cellular stress response and play an important role in the control of programmed cell death. In particular, ceramides have been shown to induce apoptosis of cerebellar granule cells. We show that pituitary adenylate cyclase‐activating polypeptide (PACAP) prevents C2‐ceramide‐induced apoptosis. The neuroprotective effect of PACAP was dose‐dependent and blocked by its antagonist, PACAP6‐38, whereas the PACAP‐related peptide VIP was inactive. The effect of PACAP on cell survival was mimicked by dibutyryl‐cAMP (dbcAMP) and forskolin and prevented by the MEK inhibitor U0126, indicating that both the adenylyl‐cyclase and MAP‐kinase pathways contribute to the neuroprotective action of the peptide. C2‐ceramide and PACAP induced opposite effects on phosphorylated forms of ERK and JNK without affecting the total amounts of ERK and JNK, suggesting that a balance between these two MAP‐kinases is critical for the cell survival/death decision. The effect of PACAP on ERK phosphorylation was blocked by U0126, but was not affected by H89 or chelerythrine indicating that PACAP activates ERK through a PKA‐ and PKC‐independent mechanism. C2‐ceramide induced a time‐dependent activation of caspase‐3, enhanced the amount of cleaved caspase‐3 and stimulated the DNA fragmentation process, while PACAP strongly inhibited the C2‐ceramide‐induced activation of caspase‐3, reduced the expression of cleaved caspase‐3 and blocked DNA fragmentation. Taken together, the present results show that C2‐ceramide induces apoptosis of cerebellar granule cells through a mechanism involving activation of caspase‐3. Our data also demonstrate that PACAP is a potent inhibitor of C2‐ceramide‐induced apoptosis.

Comparative Distribution of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Binding Sites and PACAP Receptor mRNAs in the Rat Brain During Development

The distribution and density of pituitary adenylate cyclase‐activating polypeptide (PACAP) binding sites as well as PACAP‐specific receptor 1 (PAC1‐R), vasoactive intestinal polypeptide/PACAP receptor 1 (VPAC1‐R), and VPAC2‐R mRNAs have been investigated in the rat brain from embryonic day 14 (E14) to postnatal day 8 (P8). Significant numbers of binding sites for the radioiodinated, 27‐amino‐acid form of PACAP were detected as early as E14 in the neuroepithelia of the metencephalon and the myelencephalon. From E14 to E21, the density of binding sites in the germinative areas increased by 3‐ to 5‐fold. From birth to P12, the density of binding sites gradually declined in all neuroepithelia except in the external granule cell layer of the cerebellum, where the level of binding sites remained high during the first postnatal weeks. Only low to moderate densities of PACAP binding sites were found in regions other than the germinative areas, with the exception of the internal granule cell layer of the cerebellum, which contained a high density of sites. The localization of PACAP receptor mRNAs was investigated by in situ hybridization using [35S] uridine triphosphate‐specific riboprobes. The evolution of the distribution of PAC1‐R and VPAC1‐R mRNAs was very similar to that of PACAP binding sites, the concentration of VPAC1‐R mRNA being much lower than that of PAC1‐R mRNA. In contrast, intense expression of VPAC2‐R mRNA was observed in brain regions other than germinative areas, such as the suprachiasmatic, ventral thalamic, and dorsolateral geniculate nuclei. The discrete localization of PACAP binding sites as well as PAC1‐R and VPAC1‐R mRNAs in neuroepithelia during embryonic life and postnatal development strongly suggests that PACAP, acting through PAC1‐R and/or VPAC1‐R, may play a crucial role in the regulation of neurogenesis in the rat brain. J. Comp. Neurol. 425:495–509, 2000.

[Pituitary adenylate cyclase-activating polypeptide].

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been originally isolated from the sheep hypothalamus on the basis of its ability to stimulate cAMP formation in anterior pituitary cells. Post-translational processing of the PACAP precursor generates two biologically active molecular forms, PACAP38 and PACAP27, and a novel peptide called PACAP-related peptide whose activity remains unknown. The primary structure of PACAP has been remarkably conserved during evolution, from protochordates to mammals, suggesting that the peptide exerts important activities throughout the vertebrate phylum. The sequence of PACAP27 exhibits substantial similarities with those of vasoactive intestinal polypeptide (VIP), glucagon and secretin. The gene encoding the PACAP precursor is widely expressed in the brain and in various peripheral organs, notably in endocrine glands, the gastro-intestinal and uro-genital tracts and the respiratory system. In vivo and in vitro studies have shown that PACAP exerts multiple activities as a hormone, neurohormone, neurotransmitter or trophic factor. For instance, PACAP triggers the release of insulin and glucagon, activates steroidogenesis in the adrenal gland and gonads, and stimulates the secretion of most hypophysial cells. PACAP exerts a potent relaxant activity on smooth muscle fibers in blood vessels, lung and gut. In the brain, PACAP stimulates the electrical activity of various populations of neurons and increases tyrosine hydroxylase gene expression. Recent studies have shown that PACAP exerts a trophic activity during ontogenesis, notably in the adrenal medulla and in the central nervous system. The biological effects of PACAP are mediated through three distinct receptor subtypes which exhibit differential affinities for PACAP and VIP. The PAC1 receptor, which shows high selectivity for PACAP, is coupled to several transduction systems. In contrast, VPAC1 and VPAC2, which bind with the same affinity PACAP and VIP, are mainly coupled to the adenylyl cyclase pathway. The bronchodilatator and vasorelaxant effects of PACAP, as well as the antiproliferative and neuroprotective actions of the peptide, make it a valuable target for new drug development.

Localization and characterization of PACAP receptors in the rat cerebellum during development: Evidence for a stimulatory effect of PACAP on immature cerebellar granule cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are abundant in the brain and particularly in the cerebellum of adult rats. In contrast, the occurrence of PACAP binding sites has not been investigated during ontogenesis. The aim of the present study was to determine the distribution and biochemical characteristics of PACAP binding sites in the rat cerebellum during postnatal development, and to examine the effect of PACAP on immature cerebellar granule cells. Autoradiographic studies revealed that PACAP binding sites are transiently expressed in a germinative matrix of the cerebellar cortex, the external granule cell layer, and in the medulla, from postnatal days 8 to 25. A population of PACAP binding sites persisted in the internal granule cell layer in the mature cerebellum. Emulsion-coated cytoautoradiography, performed on cultured immature granule cells from eight-day-old rat cerebellum, demonstrated that transient PACAP binding sites are expressed by cerebellar immature granule cells. Biochemical characterization of binding revealed the occurrence of two classes of PACAP recognition sites exhibiting, respectively, high (Kd = 0.39 +/- 0.08 nM) and low (Kd = 21.2 +/- 9.4 nM) affinity for PACAP27. The two naturally occurring forms PACAP38 and PACAP27 were equipotent in competing for [125I]PACAP27 binding. In contrast, the [Des-His1]PACAP38 analog was eight times less efficient and vasoactive intestinal polypeptide only induced weak displacement of the binding. Exposure of cultured immature granule cells to PACAP27 resulted in a dose-dependent stimulation of the production of cAMP, indicating that PACAP binding sites represent authentic receptors positively coupled to adenylate cyclase. These results show that PACAP receptors are actively expressed in the cerebellum of rats during postnatal development. The presence of functional PACAP receptors in the external granule cell layer suggests that PACAP may play a role in the control of proliferation and/or differentiation of granule cells.

Opposite regulation of the mitochondrial apoptotic pathway by C2-ceramide and PACAP through a MAP-kinase-dependent mechanism in cerebellar granule cells

The sphingomyelin‐derived messenger ceramides provoke neuronal apoptosis through caspase‐3 activation, while the neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP) promotes neuronal survival and inhibits caspase‐3 activity. However, the mechanisms leading to the opposite regulation of caspase‐3 by C2‐ceramide and PACAP are currently unknown. Here, we show that PACAP prevents C2‐ceramide‐induced inhibition of mitochondrial potential and C2‐ceramide‐evoked cytochrome c release. C2‐ceramide stimulated Bax expression, but had no effect on Bcl‐2, while PACAP abrogated the action of C2‐ceramide on Bax and stimulated Bcl‐2 expression. The effects of C2‐ceramide and PACAP on Bax and Bcl‐2 were blocked, respectively, by the JNK inhibitor L‐JNKI1 and the MEK inhibitor U0126. L‐JNKI1 prevented the alteration of mitochondria induced by C2‐ceramide while U0126 suppressed the protective effect of PACAP against the deleterious action of C2‐ceramide on mitochondrial potential. Moreover, L‐JNKI1 inhibited the stimulatory effect of C2‐ceramide on caspase‐9 and ‐3 and prevented C2‐ceramide‐induced cell death. U0126 blocked PACAP‐induced Bcl‐2 expression, abrogated the inhibitory effect of PACAP on ceramide‐induced caspase‐9 activity, and promoted granule cell death. The present study reveals that C2‐ceramide and PACAP exert opposite effects on Bax and Bcl‐2 through, respectively, JNK‐ and ERK‐dependent mechanisms. These data indicate that the mitochondrial pathway plays a pivotal role in the pro‐ and anti‐apoptotic effects of C2‐ceramide and PACAP.