Pituitary Adenylate Cyclase-Activating Polypeptide and Sonic Hedgehog Interact to Control Cerebellar Granule Precursor Cell Proliferation

Abstract

Although positive and negative signals control neurogenesis in the embryo, factors regulating postnatal proliferation are less well characterized. In the vertebrate cerebellum, Sonic Hedgehog (Shh) is an efficacious mitogen for cerebellar granule neuron precursors (GNPs), and mutations activating the Shh pathway are linked to medulloblastoma, a tumor derived from GNPs. Although the mitogenic effects of Shh can be blocked by increasing cAMP or protein kinase A activity, the physiological factors antagonizing this stimulation are undefined. In the embryo, pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1) signaling regulates neural precursor proliferation. We now show that in the developing cerebellum, PAC1 mRNA colocalizes with gene transcripts for Shh receptor Patched 1 and target gene Gli1 in the external germinal layer. We consequently investigated the interactions of PACAP and Shh in proliferation of purified GNPs in culture. Shh exhibited mitogenic activity in both rat and mouse cultures, stimulating DNA synthesis approximately 10-fold after 48 hr of exposure. PACAP markedly inhibited Shh-induced thymidine incorporation by 50 and 85% in rat and mouse GNPs, respectively, but did not significantly affect the stimulation induced by other mitogens. This selective effect was reproduced by the specific PAC1 agonist maxadilan, as well as by the adenylate cyclase activator forskolin, suggesting that PAC1 provides a potent inhibitory signal for Shh-induced proliferation in developing cerebellum. In contrast, in the absence of Shh, PACAP and maxadilan modestly stimulated DNA synthesis, an effect reproduced by activating protein kinase C. These observations suggest that G-protein-coupled receptors, such as PAC1, serve as sensors of environmental cues, coordinating diverse neurogenetic signals.

Pituitary adenylate cyclase-activating polypeptide prevents the effects of ceramides on migration, neurite outgrowth, and cytoskeleton remodeling

“…PACAP and its receptors are actively expressed in the central nervous system during development, notably in the germinative neuroepithelia and migratory zones (9, 10), suggesting that PACAP could be involved in the regulation of neurogenesis and͞or cell differentiation (11). In agreement with this hypothesis, in vitro studies have shown that PACAP exerts growth cone attraction in cultured neural tube precursors from Xenopus laevis (12) and promotes survival and differentiation of rat cerebellar granule cells (13)(14)(15). In vivo, PACAP injection at the surface of the cerebellum of 8-day-old rats increases the number of granule neurons in the internal granule cell layer (16).…”

Cyclic AMP-mediated Regulation of Transcription Factor Lot1 Expression in Cerebellar Granule Cells

“…Recent findings in animal models of excitotoxic injuries go in the same direction by linking Lot1 expression to neuroprotection and brain plasticity (10,60,61). Interestingly, in these models, a relationship between Lot1 expres- The role of cAMP in the maturation of newborn neurons is well accepted (13,14), and both PKA-dependent and PKA-independent pathways have been implicated in cAMP-mediated effects. Our study has shown that, in CGC, cAMP activation of Lot1 is PKA-and MEK/ ERK-dependent, as it was counteracted by specific pathway inhibitors.…”

“…Approximately 5 g of RNA were digested with 10 units of RNase-free DNase (Promega) for 30 min at 37°C. After DNase inactivation, RNA was retrotranscribed with an oligo(dT) [12][13][14][15][16][17][18] primer (0.5 M) and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the manufacturer's instructions. For real-time PCR, the following Lot1, glyceraldehyde-3-phosphate dehydrogenase, and c-fos primers were used: rat Lot, 5Ј-TTAGCTGCGTAGTTGCGTGTTA-3Ј (sense) and 5Ј-CGGGTCCCTGAAAAGAACACA-3Ј (antisense); glyceraldehyde-3-phosphate dehydrogenase, 5Ј-GAACATCATCCCTGCATCCA-3Ј (sense) and 5Ј-CCAGTGAGCTTCCCGTTCA-3Ј (antisense); and c-fos, 5Ј-TGGACCTGTCTGGTTCCTTC-3Ј (sense) and 5Ј-ATG-CACCAGCTCAGTCAGTG-3Ј (antisense).…”

“…Recent studies have reported that the cAMP-PKA cascade is one of the key signal transduction pathways involved in neuronal proliferation (13,14). To define the effects of adenylate cyclase activation on CGC proliferation, we analyzed BrdUrd incorporation in cells exposed to PACAP or forskolin for 24 h after cell plating.…”

“…Therefore, identifying factors that are involved in the regulation of Lot1 expression during CGC differentiation could represent an important step toward deciphering a possible relationship between Lot1 expression and neurogenesis. Evidence from both in vivo and inculture studies suggests that several steps of neurogenetic process are crucially linked to an increase in intracellular cAMP (13,14). cAMP is a second messenger that regulates a striking number of physiological processes, including intermediary metabolism, cell proliferation, and neuronal signaling, by altering the basic pattern of gene expression (15)(16)(17).…”